You are on page 1of 104

ISSN: 1524-4636

Copyright 2007 American Heart Association. All rights reserved. Print ISSN: 1079-5642. Online
7272 Greenville Avenue, Dallas, TX 72514
Arteriosclerosis, Thrombosis, and Vascular Biology is published by the American Heart Association.
2007;27;e35-e137 Arterioscler Thromb Vasc Biol
Arteriosclerosis, Thrombosis, and Vascular Biology Annual Conference 2007
http://atvb.ahajournals.org
located on the World Wide Web at:
The online version of this article, along with updated information and services, is
http://www.lww.com/reprints
Reprints: Information about reprints can be found online at

journalpermissions@lww.com
410-528-8550. E-mail:
Fax: Kluwer Health, 351 West Camden Street, Baltimore, MD 21202-2436. Phone: 410-528-4050.
Permissions: Permissions & Rights Desk, Lippincott Williams & Wilkins, a division of Wolters

http://atvb.ahajournals.org/subscriptions/
Biology is online at
Subscriptions: Information about subscribing to Arteriosclerosis, Thrombosis, and Vascular
by on March 25, 2011 atvb.ahajournals.org Downloaded from
Abstracts
Arteriosclerosis, Thrombosis, and
Vascular Biology Annual Conference 2007
April 19-21, 2007
Palmer House Hilton
Chicago, IL
Sponsored by the American Heart Association Council on Arteriosclerosis, Thrombosis,
and Vascular Biology and the Council on Nutrition, Physical Activity, and Metabolism.
Co-sponsored by the National Heart, Lung, and Blood Institute.
Conference Program Committee
Chair: Martha K. Cathcart, PhD, FAHA
William C. Aird, MD; Alan Daugherty, PhD, DSc, FAHA; George E. Davis, MD, PhD; William
P. Fay, MD, FAHA; Ronald M. Krauss, MD, FAHA; Mark W. Majesky, PhD; Gwendalyn J.
Randolph, PhD; Lawrence L. Rudel, PhD, FAHA; Jonathan Smith, PhD, FAHA; Nancy R.
Webb, PhD
Abstracts for the oral and poster presentations are provided in this special on-line supplement.
by on March 25, 2011 atvb.ahajournals.org Downloaded from
2007 ATVB Oral Presentations
1
Pharmacological Inhibition of PCSK9 in Hyperlipidemic Mice Significantly
Reduces Serum LDL-C While Increasing Hepatic Low-Density Lipoprotein
Receptor Protein Abundance
Mark Graham, Kristina M M Lemonidis, Charles Whipple, Amuthakannan Subramaniam,
Brett P Monia, Rosanne M Crooke; Isis Pharmaceuticals, Carlsbad, CA
Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a member of the proprotein convertase
family of proteases that has been previously demonstrated to promote the degradation of the
low density lipoprotein receptor (LDLr) through an undefined mechanism. Mutational analysis
in humans has demonstrated that genetic polymorphisms which inactivate PCSK9 produce
dramatic reductions in serum LDL-C and further, appear to reduce the risk of coronary artery
disease (CAD). These encouraging epidemiological findings have been corroborated using
PCSK9 knockout mice, which exhibit the same phenotypic profile (i.e. decreased LDL-C and
increased hepatic LDLr). For this reason, we have developed second generation antisense
oligonucleotide inhibitors (ASOs) which target both human and mouse forms of PCSK9 mRNA
to define their hypolipidemic effects both in vitro and in vivo. Due to their optimal
pharmacokinetic/pharmacodynamic properties, 2MOE-modified ASOs have been extensively
exploited to inhibit a broad range of therapeutically attractive liver gene targets such as apoB,
Lp(a) and ACAT2. Administration of the PCSK9 ASO (ISIS 394814) to high fat fed mice for 6
weeks (i.p., 100mg/kg/wk) resulted in reductions in both total cholesterol and LDL-C, 53% and
38%, respectively. In addition, hepatic RNA and protein analysis revealed that ISIS 394814
reduced PCSK9 mRNA expression by 92% while increasing LDLr protein levels greater than
2-fold relative to controls. The magnitude of these effects is consistent with results previously
reported in PCSK9 knockout mice. Based on these data, additional studies are in progress in
LDLr deficient and additional mouse models to further demonstrate the specificity and
pharmacological efficacy of this drug. These promising in vivo results suggest that inhibiting
PCSK9 may indeed represent a novel therapeutic approach for reducing LDL-C in man.
2
Intestinal Cholesterol Absorption Is Required for the LXR Agonist to
Increase Plasma HDL Cholesterol in Mice
Weiqing Tang, Yinyan Ma, Lin Jia, Liqing Yu; Wake Forest Univ Sch of Medicine,
Winston-Salem, NC
Genetic manipulation has established the liver as the major source of HDL in normal mice.
However there is about 30% of plasma HDL contributed to the small intestine. It has been
reported that liver X receptor (LXR) agonist GW3965 raises plasma HDL cholesterol levels in
wildtype and liver-specific ABCA1 knockout mice but not in mice lacking intestinal ABCA1,
indicating that the intestine plays an important role under circumstance in the biogenesis of
plasma HDL cholesterol. The source of cholesterol for intestinal HDL formation is unclear. We
hypothesize that cholesterol absorption from the gut lumen plays a role in the LXR
agonist-stimulated HDL formation. To test our hypothesis, mice lacking Niemann-Pick C1-Like
1 (NPC1L1) (L1-KO mice), a gene that is essential for intestinal cholesterol absorption, were
treated with the LXR agonist T0901317 at 25 mg/kg BW/day for 7 days. As expected,
T0901317-treated wildtype mice showed a dramatic increase in the plasma HDL cholesterol
level but this effect was almost abolished in the L1-KO mice in which a much greater fecal
cholesterol excretion was observed instead. The intestinal ABCA1 mRNA level was about 4-fold
lower in the untreated L1-KO versus wildtype mice, and increased 4.4-fold and 7.8-fold in the
T0901317-treated wildtype and L1-KO mice, respectively. Hepatic ABCA1 failed to respond to
T0901317 in both wildtype and L1-KO mice although hepatic ABCG5/G8 mRNA levels were
higher in the T0901317-treated versus untreated animals. In conclusion, intestinal cholesterol
absorption is required for LXR agonist to increase plasma HDL cholesterol in mice.
3
HDL Transport Through Endothelial Cells Is Mediated by ABCG1 and SR-BI
Lucia Rohrer, Madeleine Zemp, Iris Lorenzi, Marc Lehnert, Arnold von Eckardstein; Univ
Hosp, Zurich, Switzerland
High density lipoproteins (HDL) and their main protein constituent, apolipoprotein A-I (apoA-I),
exert potentially anti-atherogenic properties within the arterial wall. However, it is not clear how
they are transported from the blood stream into the vascular wall. Previously we have shown
that ABCA1 mediates apoA-I transcytosis in endothelial cells. Here we investigated the
interaction of HDL with endothelial cells. Endothelial cells bound and cell associate both
125
I-apoA-I and
125
I-HDL with high affinity and in a saturable manner. Binding and cell
association of HDL was only competed by excess HDL not by apoA-I and albumin. In contrast
binding and cell association of apoA-I is competed by excess apoA-I and HDL and not by
albumin. Biotinylation experiments showed that endothelial cells internalize labeled HDL and
only minor amounts of the internalized HDL was degraded. Cultivated in a Transwell system,
the cells transported a fraction of
125
I-HDL from the apical to the basolateral compartment as
an intact protein in a competable and temperature sensitive manner. Moreover, RNA
interference to modulate ATP binding cassette G1 (ABCG1) and SR-BI resulted in reduced HDL
cell surface binding, internalization and transport. We conclude that endothelial cells
transcytose HDL in an ABCG1 and/or SR-BI dependent process.
4
Impact of Apolipoprotein M Expression on Nascent Pre- HDL Formation
by ATP Binding Cassette Transporter A1 (ABCA1)
Anny Mulya, Amanda Wibley, Soon-Kyu Chung, Abraham K Gebre, Gregory S Shelness,
John S Parks; Wake Forest Univ Sch of Med, Winston-Salem, NC
ApoM is a novel apolipoprotein that mainly associates with high density lipoproteins (HDLs) in
plasma and has been reported to play a role in pre-beta (preb) HDL formation. We investigated
the role of apoM expression on the initial steps of nascent preb HDL particle assembly by
ABCA1 using HEK293 cells. Neither control nor ABCA1 expressing HEK293 cells expressed
detectable apoM mRNA or protein based on real time PCR and Western blot analysis,
respectively. Control and ABCA1-expressing cells were transfected with apoM C-FLAG and
radiolabeled with
35
S-Met/Cys prior to immunoprecipitation of apoM from medium and cell
lysates. Results showed that only 1% of the total cellular apoM was secreted both in the
absence or presence of ABCA1 expression. Immunofluorescence with anti-FLAG antibody
demonstrated that apoM protein was located primarily at intracellular sites and not at the
plasma membrane of transfected cells. To investigate the role of apoM in nascent preb HDL
formation, ABCA1-expressing or control cells, transfected with empty vector, apoM WT or apoM
C-FLAG, were incubated with
125
I-lipid-free apoA-I for 24 hours. Conditioned media were
harvested and fractionated by FPLC to observe HDL particle size distribution as monitored by
elution of
125
I apoAI. Preb HDL particles were formed in the absence of apoM expression (WT
or C-FLAG); however, apoM expression promoted formation of larger-sized nascent preb HDL.
Immunoprecipitation of the different sized FPLC-isolated preb HDL with anti-apoA-I antibody
followed by apoM Western blot analysis of pellet and supernatant showed that very little
secreted apoM associated with preb HDL particles. Our results suggest that apoM is poorly
secreted by HEK293 cells, is not required for preb HDL assembly by ABCA1, and interacts
poorly with secreted nascent preb HDL. However, apoM expression results in the appearance
of larger preb HDL particles in media. We propose that apoM may function catalytically at an
intracellular site to transfer lipid onto preb HDL during or after their formation by ABCA1.
5
Docosahexaenoic Acid Impairs the Formation of Fully Lipidated VLDL by
Oxidative Modification, Aggregation, and Degradation of the Precursor
Particles
Vatsala Maitin, NYU Sch of Medicine, New York, NY; Kevin J Williams, Thomas Jefferson
Univ, Philadelphia, PA; Carly St. Germain, Zemin Yao, Univ of Ottawa, Ottawa, Canada;
Edward A Fisher; NYU Sch of Medicine, New York, NY
Background: One mechanism of the lipid-lowering effects of n-3 fatty acids including
docosahexaenoic acid (DHA) is reduced hepatic VLDL secretion by promoting intracellular
apolipoprotein B (apoB) degradation. We reported that this is a non-proteasomal post-ER
process; activated by intracellular conversion of DHA to lipid peroxides. DHA-treatment of
hepatocytes also resulted in the intracellular formation of malondialdehyde-modified high
molecular weight apoB aggregates. Objective: To determine the stage in the VLDL secretory
pathway where oxidative modification and degradation of apoB occurs. Results: In pulse-chase
studies, initial stages (up to 30 min of chase, C30) of lipoprotein assembly were not significantly
different between oleic acid (OA) and DHA treated cells, with similar luminal quantities of total
apoB, pre-VLDL and VLDL. At C45, compared to OA, a decrease in luminal apoB was observed
in DHA-treated cells, but in contrast to OA, this apoB was not recovered in the medium.
Interestingly, the C45 time point in case of DHA coincided with accumulation of apoB-
aggregates, partially accounting for the loss of intracellular apoB and reduced formation of
luminal VLDL. In experiments carried out at 20
o
C to block protein exit from the Golgi,
comparable amounts of pre-VLDL particles accumulated in OA vs. DHA-treated cells, but with
DHA, only 50% of these were able to form fully lipidated VLDL, consistent with the amount
recovered in the medium. The same trend was observed for lipoproteins extracted from
Golgi-specific microsomes. Co-administration of desferrioxamine (DFX), an iron-chelator that
prevents lipid peroxidation, caused DHA-treated cells to assume an OA-like profile, accumu-
lating luminal VLDL-apoB that was secreted. Using an antibody for light-chain 3 (LC3), a
specific autophagosomal marker, apoB aggregates were shown to be enriched in cells treated
with DHA compared to OA or DHADFX. Conclusions: (1) DHA allows step I (pre-VLDL) particle
formation and their transport to the Golgi (2) Oxidative damage of the pre-VLDL apoB and/or
its association with peroxidized lipids in the Golgi results in reduced luminal formation of
secretion-competent VLDL particles (3) Aggregated apoB is co-localized with autophagosomes.
6
12-Lipoxygenase Deficiency Inhibits Angiotensin II-induced Abdominal
Aortic Aneurysm Rupture and Incidence in Apolipoprotein E-deficient Mice
Nicholas Hatch, Fjoralba Babamusta, Victoria L King; Univ of Kentucky, Lexington, KY
Objective: Arachidonic acid is metabolized through the cyclooxygenase-1 and -2 (COX-1 and
-2), 5-lipoxygenase (5-LO) and 12/15-lipoxygenase (12-LO) pathways which produce a host of
biologically active fatty acid derivates that affect cardiovascular disease. We have recently
demonstrated that selective pharmacological inhibition of the COX-2 pathway markedly
attenuated angiotensin II (AngII)-induced AAA formation. Interestingly, 5-LO deficiency also
attenuated AAA formation in an experimental animal model of AAAs. Genetic or pharmacolog-
ical inhibition of one of these pathways may result in the shunting of arachidonic or linoleic acid
down the12-LO pathway. Therefore, we sought to determine the effect of 12-LO deficiency on
AngII-induced AAA formation. Methods: Age matched male C57BL/6 apolipoprotein E (apoE)
deficient (12-LO/) and C57BL/6 12-LO deficient apoE-/- (12-LO-/-) mice were infused with
e-36 Arterioscler Thromb Vasc Biol June 2007
by on March 25, 2011 atvb.ahajournals.org Downloaded from
AngII (1,000 ng/kg/min) for 28 days. Results: 12-LO deficiency in apoE-/- mice did not alter
AngII induced increases in systolic blood pressure ( Saline: 12-LO/: 117 6 vs 12-LO-/-:
125 6 mmHg; AngII: 12-LO/: 160 5 vs 12-LO-/-: 154 6 mmHg) or plasma total
cholesterol concentrations. Interestingly, 12-LO deficiency markedly decreased AngII-induced
aneurysmal mortality (12-LO/: 41% vs 12-LO-/-: 3%; P 0.001). The decrease in
mortality was related to a significant reduction in abdominal aortic rupture (12-LO/: 23 %
vs 12-LO-/-: 3%; P 0.016) and an ablation of aortic arch rupture (12-LO/: 18% vs
12-LO-/-: 0.0%; P 0.013). 12-LO deficiency also modestly decreased AngII-induced
abdominal aortic expansion (12-LO/: 1.82 0.15 vs 12-LO-/-:1.44 0.11 mm; P
0.045) resulting in an reduction in the incidence (12-LO/: 81.25% vs 12-LO-/-: 50% ; P
0.018) and severity (P 0.015) of AngII-induced AAAs. Plasma 11-dehydro thromboxane B
2
concentrations were increased in 12-LO-/- mice in response to 28 days of AngII infusion
(12-LO/: 192 29 vs 12-LO-/-: 406 73 pg/ml, P 0.011). Conclusion: These findings
suggest that 12-LO generated eicosanoids play a fundamental role in AngII-induced aneurys-
mal rupture leading to an increase in mortality, as well as, contribute to the incidence and
severity of AngII-induced AAA formation.
7
Tetrahydrobiopterin Reverses Preexisting Hypertension-induced Ventricular
Remodeling by Recoupling eNOS
An L Moens, Carlo Tocchetti, Elizabeth Ketner, Azeb Haile, Champion Hunter, Robert Weiss,
Johns Hopkins Med Insts, Baltimore, MD; Pawel Kaminski, Michael S Wolin, New York Med
College, Valhalla, NY; David A Kass, Jeffrey Rade; Johns Hopkins Med Insts, Baltimore, MD
Background: Pressure overload triggers eNOS as a prominent source of myocardial ROS that
contribute to dilatory remodelling and cardiac dysfunction. Administration of tetrahydrobiopterin
(BH4) can prevent pressure-load remodelling. The aim of this study was to investigate that BH4
can reverse established non-decompensated heart failure by interacting with uncoupled eNOS,
and on this way prevent the evolution to end-stage heart failure. Methods: Compensated
cardiac remodelling was induced in 60 mice by transverse aortic constriction (TAC). After 4wks,
mice were randomized to receive BH4 (200mg/kg/d, n30) or placebo (n30) for the following
5wks. Echocardiography, MRI and PV-loop analysis were performed. Cold SDS-PAGE was
performed to evaluate eNOS dimer/monomer. ROS generation was evaluated with dihydro-
ethidium (DHE) confocal staining and chemiluminescence. NOS activity and downstream NO
products (PKG and P-VASP) were determined. Isolated myocyte studies were performed.
Myocyte dimensions and fibrosis (score 0: abscent - 3 pronounced) were histologically
evaluated. Overexpression of endothelial GTPCH, the rate limiting enzyme of BH4 synthesis,
was evaluated in this TAC model (n15 mice). Results: BH4 significantly reversed cardiac
hypertrophy (heart weight 24017 mg at 4wks, 32418 mg at 9wks and 212 11mg at
9wks with BH4 p0.001, idem for myocyte dimensions, wall thickness and calculated LV
mass) and diminished fibrosis (score 2.10.4 vs. score 0.60.4 with BH4, p0.05). BH4
prevented the evolution towards cardiac decompensation (ejection fraction: 45.71.6% at
4wks, 34.72% at 9wks and 53.44.5% at 9wks with BH4, p0.001 and confirmed by MRI
and PV loop analysis). BH4 recoupled the already uncoupled eNOS and increased its activity
back to the normal level. Superoxide generation (total and NOS-dependent) was markedly
reduced by BH4. BH4 improved fractional shortening and calcium-kinetics in isolated myocytes.
Endothelial upregulation of BH4 by GTPCH-Tg had no beneficial effect on remodeling.
Conclusion: BH4 can reverse established cardiac remodelling by re-coupling uncoupled eNOS
and as a consequence less NOS dependent ROS is generated, leading to less hypertrophy and
fibrosis and an amelioration of cardiac function.
8
New Pathway to Gene Expression in Human Endothelial Cells: Filamin B
Translation Is Preserved by Internal Ribosome Entry in Virally Infected EC
Huimiao Jiang, Brandt B Jones, John Kriesel, Douglas I Schmid, Andrew S Weyrich, Guy A
Zimmerman, Larry W Kraiss; Univ of Utah, Salt Lake City, UT
Introduction: Eukaryotic translation initiation occurs either at the 5 cap of the untranslated
region (UTR) or at an internal ribosome entry site (IRES). Messenger RNA containing IRES may
still be translated despite cellular stress that impairs cap-dependent translation. Whether
IRES-mediated translation occurs in stressed EC is unknown. Hypothesis: IRES-containing
mRNAs are selectively enriched in polyribosomes from human EC when cap-dependent
translation is impaired by viral infection. Methods: Cap-dependent translation in primary human
umbilical vein EC was disabled by Poliovirus Type 1 infection. Polysome-associated RNA from
virus- or mock-treated EC was analyzed by whole genome microarray with later verification by
real-time RT-PCR. The presence of an IRES in the 5UTR of candidate mRNA was confirmed
using a dicistronic luciferase reporter assay. Results: Infection of human EC was verified by
immunochemistry (poliovirus I antigen). Four hours post-infection, loss of polysome peaks in
ribosomal profiles and detection of eIF4G cleavage products on western blots indicated
repression of cap-dependent translation. On microarray analysis, 277 mRNAs were enriched
2-fold in polysome fractions from infected v control EC including several known to contain
an IRES (Cyr 61, Pim1). Sequences enriched in polysomes from infected EC with 5UTR 100
bp are more likely to contain an IRES; 20 were selected for further analysis, including filamin
B (not previously known to have an IRES). Real-time PCR confirmed the microarray findings for
18/20 mRNAs. The filamin B 5UTR (165 bp) was cloned into a dicistronic luciferase reporter
plasmid. Translation of the downstream luciferase product after transfection of the reporter
construct into EAHY surrogate EC confirmed the presence of an IRES in the filamin B 5UTR.
Conclusions: (1) Translation of filamin B, an actin-binding protein implicated in mechanotrans-
duction and cell signaling, is preserved in virally infected EC via an IRES-mediated mechanism,
identifying another mechanism by which EC regulate the pattern of expressed protein in
response to microbial challenge and stress; (2) Microarray analysis of poliovirus-infected EC is
an effective large-scale method to screen for IRES-containing mRNAs in EC.
9
Reoxygenation Leads to Dissociation of Histone Deacetylase 7 from
Hypoxia-inducible Factor-1a
Hiroyuki Kato, Shiori Tamamizu-Kato, Vasanthy Narayanaswami, Bruce N Ames; Childrens
Hosp, Oakland Rsch Institute, Oakland, CA
Interruption of cerebral blood flow causes hypoxia leading to the death of neurons and glia.
Hypoxia-inducible factor-1a (HIF-1a) plays an important role in cell survival under oxygen
deprivation by regulating the transcription of genes involved in glucose metabolism, cell
proliferation, and angiogenesis. Products of genes targeted by HIF-1a include vascular
endothelial growth factor (VEGF), erythropoietin, and glucose transporter-1, which play a role
in improved cell survival under hypoxia. When oxygenation is normal, HIF-1a is rapidly
degraded through the ubiquitin-proteasome pathway, which is triggered by the
oxygena dependent hydroxylation of proline residues (Pro402 and Pro564) in HIF-1a. These
hydroxylated proline residues are recognized by the von-Hippel-Lindau tumor suppressor
protein (pVHL), a component of an E3 ubiquitin ligase complex (pVHL, Elongin B, and Elongin
C). The E3 ubiquitin ligase complex promotes ubiquitination of HIF-1 leading to degradation of
HIF-1a in 26S proteasome in mammalian cells in normal oxygen concentrations. Under hypoxia,
this oxygen dependent-degradation system is repressed. Previously, we identified histone
deacetylase (HDAC)7 as a binding protein of HIF-1a using yeast two-hybrid system. HDAC7 was
also shown to increase HIF-1a transcriptional activity under hypoxia. In this study, we
investigated the roles of HDAC7 in degradation of HIF-1a under re-oxygenation. We found that
degradation of HIF-1a was correlated with translocation of HDAC7 from the nucleus to the
cytoplasm upon re-oxygenation. Using a mutant of HIF-1a (Pro402A/P564A), we also found that
the stabilized HIF-1a mutant localized in the nucleus under re-oxygenation whereas HDAC7
was exported to the cytoplasm. The amino acids substitution mutations of nuclear export
sequences (NES) in HDAC7 (NES mut.) blocked translocation of HDAC7 to the cytoplasm and
stabilized HIF-1a in the nucleus upon re-oxygenation. Moreover NES mut HDAC7 bound HIF-1a
and suppressed ubqutination of HIF-1a upon re-oxygenation. Taken together, these results
suggest that the dissociation of HDAC7 from HIF-1a and translocation of HDAC7 to the
cytoplasm may lead to degradation of HIF-1a upon re-oxygenation.
10
Role of Autotaxin, a Plasma Lysophospholipase D, in Hemostasis and
Thrombosis
Zehra Pamuklar, Univ of Kentucky, Lexington, KY; Shuying Liu, The Univ of Texas M D
Anderson Cancer Cntr, Houston, TX; Anping Dong, Univ of Kentucky, Lexington, KY; Gordon
B Mills, The Univ of Texas M D Anderson Cancer Cntr, Houston, TX; Andrew J Morris,
Susan S Smyth; Univ of Kentucky, Lexington, KY
Lysophosphatidic acid (LPA) is a bioactive lipid mediator produced by platelets and found in
plasma and atherosclerotic plaques. LPA stimulates platelets, leukocytes, endothelial cells, and
smooth muscle cells to regulate cell growth, differentiation, survival, motility, and contractility.
LPA is thus poised to serve as a key regulator of vascular cell function. LPA is generated in
large part by the secreted lysophospholipase D autotoxin (ATX; enpp2). Mice with only one ATX
gene (enpp2/-) have plasma LPA levels that are 50% of wild type mice. ATX deficiency in
mice (enpp2-/-) results in embryonic lethality due to vascular defects, implicating ATX and
potentially LPA in vascular development. We observed excessive bleeding in three founder lines
of transgenic FVB mice globally overexpressing ATX (ATX-TG). To characterize the bleeding
defect further, tail vein bleeding times were performed. The mean bleeding time in control FVB
mice was 3 2.7 min (n 6), whereas none of the ATX-TG mice (n 10) stopped bleeding
within 10 min (p 0.001). Platelet counts were similar in control (928 184 x 10
3
/mm
3
) and
ATX-TG (808 156 x 10
3
/mm
3
), and platelets from control and ATX-TG mice displayed similar
levels of platelet membrane glycoproteins (GPIIb/IIIa, GPIb, GPVI, GPIa/IIa) as measured by flow
cytometry. Upon stimulation by different agonists (ADP, collagen, thrombin), platelets from
ATX-TG mice also exposed P-selectin and bound fibrinogen as did control platelets. No
differences in shear-induced platelet aggregation in whole blood from control and ATX-TG mice
were observed (surface coverage 10.25 1.73% and 9.21 1.6%, respectively, p 0.595).
Thus, ex vivo studies were not able to recapitulate a platelet function defect. Additionally,
clotting times were normal in the ATX-TG mice. To determine if thrombus formation was altered
in the ATX-TG mice, mice were studied in the ferric chloride-induced carotid artery thrombosis
model. In control mice, thrombus formation occluded the vessel in 10 1.4 min (n 3);
whereas none of the ATX-TG mice (n 3) formed occlusive thrombus within 30 min
(p0.001). In summary, our results suggest that ATX, and potentially LPA, regulate hemostasis
and thrombosis through effects on vascular function.
11
Variable Growth Rates and Diameter-Dependent Expression of Vascular
Endothelial Growth Factor Receptors in Experimental Abdominal Aortic
Aneurysms
Maureen M Tedesco, Masahiro Terashima, Francis G Blankenberg, Zoia Levashova, Stanford
Univ Med Cntr, Stanford, CA; Marina Backer, Joseph Backer, SibTech, Inc, Newington, CT;
Mien Sho, Eiketsu Sho, Michael V McConnell, Ronald L Dalman; Stanford Univ Med Cntr,
Stanford, CA
Purpose: Transmural inflammation and adventitial neovascularization are important patho-
physiologic correlates of both human and experimental abdominal aortic aneurysm disease
progression. We hypothesized that adventitial VEGF receptor expression would be increased in
diseased aortic segments of the Apoprotein E deficient angiotensin II AAA murine model.
Methods: Apoprotein E deficient mice with a C57/Bl6 background were infused with
Angiotensin II (1000 ng/kg/min) via a subcutaneous osmotic pump. The mice were maintained
on a high fat diet. Luminal diameter was determined in vivo via serial transabdominal
ultrasound imaging examinations (n 22). Greatest lumen diameter was recorded. Near-
infrared fluorescent imaging of VEGF receptors with single chain VEGF-Cy5.5 conjugate injected
2007 ATVB Oral Presentations e-37
by on March 25, 2011 atvb.ahajournals.org Downloaded from
intravenously (10g/mouse) was performed on select mice (n 3) once AAA diameter reached
at least 175% of baseline aortic diameter. Results: All AAAs identified were suprarenal and
exhibited a variable growth rate. By postoperative day 12, 45% of the mice demonstrated a
large AAA (defined as 175% or greater lumen diameter dilation compared to normal) with
transabdominal ultrasound. We visualized large AAAs in 82% of the mice by postoperative day
24, and 95% of the mice by postoperative day 30 with ultrasound. In vivo and ex vivo
fluorescence imaging of VEGF receptors with scVEGF-Cy5.5 in select mice demonstrated
increased signal in the larger AAA (245% dilated) compared to the smaller AAA (183% dilated).
Conclusion: We have demonstrated a variable growth pattern and size dependent enhance-
ment in VEGFR-2 expression in a mouse model of AAA disease. VEGF receptors may prove
useful as a clinical marker of AAA progression.
12
Sustained Angiogenic Activity of Thrombin-Resistant Mutants of FGF-1 on
Human Endothelial Cells in Fibrin Hydrogel
Luke P Brewster, Loyola Univ Med Cntr, Oak Park, IL; Eric M Brey, Illinois Institute of
Technology, Chicago, IL; Dominick Buffalino, Howard P Greisler; Loyola Univ Med Cntr,
Maywood, IL
Objective: To design a thrombin resistant mutant of FGF-1 (R136K), ligate it to a collagen
binding domain (CBD) to create R136K-CBD, and then to test their angiogenic activity on human
endothelial cells (EC) in vitro using a 3-D fibrin hydrogel matrix. Methods: Thrombin resistance
of growth factors were quantified by SDS-PAGE analysis after incubating the growth factors in
thrombin (2U/mL) at 37 degrees Celsius. Angiogenic activity was tested in our 3-D
angiogenesis assay, which incorporates growth factors into a fibrin hydrogel matrix wherein
ECs are clustered as a multicellular aggregate; the pellets are incubated in media and the
length of tubule invasion is quantified over time; gels without growth factors served as control.
Results: R136K and R136K-CBD exhibited thrombin resistance that was superior to that of
FGF-1 (Figure A,B). By day 2, R136K-CBD and R136K, but not FGF-1, stimulated significantly
greater lengths of EC sprouting than the untreated control group (R136K-CBD: 28744,
P.007; R136K: 29547, P.005; FGF-1: 24768, P.14; control: 18857). At
3 days, all treatment groups reached statistical significance over the untreated control
(P.0001), but after day 3, FGF-1 treated ECs sprouts regressed, while both R136K and
R136K-CBD continued to demonstrate sprout lengthening which was statistically significant
(P.0002) compared to control (Figure C,D). Conclusion: R136K-CBD is resistant to thrombin
proteolysis and demonstrates angiogenic activity superior to that of FGF-1. This combination
growth factor/matrix delivery vehicle may provide sustained angiogenic activity for application
in vascular tissue engineering.
13
Critical Role of Endothelial Notch1 Signaling in Postnatal Angiogenesis
Kyosuke Takeshita, Nagoya Univ, Nagoya, Japan; James K Liao; Brigham and Womens
Hosp, Harvard Med Sch, Cambridge, MA
Notch receptors are important mediators of cell fate during embryogenesis, but their role in
adult physiology, particularly in postnatal angiogenesis, remains unknown. Of the Notch
receptors, only Notch1 and Notch4 are expressed in vascular endothelial cells. Here we show
that blood flow recovery and postnatal neovascularization in response to hindlimb ischemia in
haploinsufficient global or endothelial-specific Notch1/- mice, but not Notch4-/- mice, were
impaired compared with wild-type mice. The expression of vascular endothelial growth factor
(VEGF) in response to ischemia was comparable between wild-type and Notch mutant mice,
suggesting that Notch1 is downstream of VEGF signaling. Treatment of endothelial cells with
VEGF increases presenilin proteolytic processing, gamma-secretase activity, Notch1 cleavage,
and Hes-1 (hairy enhancer of split homolog-1) expression, all of which were blocked by treating
endothelial cells with inhibitors of phosphatidylinositol 3-kinase/protein kinase Akt or infecting
endothelial cells with a dominant-negative Akt mutant. Indeed, inhibition of gamma-secretase
activity leads to decreased angiogenesis and inhibits VEGF-induced endothelial cell prolifera-
tion, migration, and survival. Overexpression of the active Notch1 intercellular domain rescued
the inhibitory effects of gamma-secretase inhibitors on VEGF-induced angiogenesis. These
findings indicate that the phosphatidylinositol 3-kinase/Akt pathway mediates gamma-
secretase and Notch1 activation by VEGF and that Notch1 is critical for VEGF-induced postnatal
angiogenesis. These results suggest that Notch1 may be a novel therapeutic target for
improving angiogenic response and blood flow recovery in ischemic limbs.
14
A Novel Translational Pathway Represses Vascular Endothelial Growth
Factor Expression and Angiogenic Activity by Activated Monocytes
Partho Sarothi Ray, Paul L Fox; Lerner Rsch Institute, Cleveland Clinic, Cleveland, OH
Post-transcriptional regulation of gene expression in macrophages is a principal mechanism
limiting inflammation. Early pro-inflammatory actions of the cytokine interferon- (IFN-) later
switch to anti-inflammatory, contributing to inflammation resolution. In activated monocytes,
IFN- elicits formation of the IFN--activated inhibitor of translation (GAIT) complex that inhibits
translation of ceruloplasmin, an acute phase inflammatory protein. Bioinformatic and
riboimmunoprecipitation-microarray analyses reveal multiple transcripts as potential targets of
GAIT-mediated translational silencing. Our identification of the angiogenic factor VEGF as a
candidate target was particularly significant because it is a potent angiogenic factor mediating
inflammatory and tumor angiogenesis. VEGF synthesis by macrophages is induced in tumors
and in chronic inflammatory conditions such as atherosclerosis; however, no negative
regulatory mechanisms are known. We show that translation of VEGF mRNA in activated
monocytes is silenced by GAIT complex-binding to a defined 3UTR RNA element. IFN-
increases VEGF mRNA transcription but causes delayed silencing of VEGF protein synthesis and
suppression of macrophage angiogenic activity as shown by endothelial cell proliferation and
tube-formation assays. Our results are the first to show negative regulation of VEGF expression
under inflammatory conditions. GAIT-mediated translation repression of VEGF, and other
inflammatory genes, might constitute a post-transcriptional operon that limits the response to
inflammatory stimuli and contributes to the resolution of chronic inflammation.
15
Atox1 as a Novel Copper-dependent Transcription Factor for Cell
Proliferation: Role in Neovascularization
Ha Won Kim, Masuko Ushio-Fukai, Tohru Fukai; Univ of Illinois at Chicago, Chicago, IL
Copper plays a fundamental role in regulating cell proliferation involved in angiogenesis.
Recently, we found that antioxidant-1 (Atox1), previously appreciated as a copper chaperone,
functions as a novel copper dependent transcription factor that mediates copper-induced cell
proliferation. We performed present study to test the hypothesis that Atox1 play an important
role in postnatal neovascularization. Using mouse ischemia hindlimb model, we found that
neovascular formation in the ischemic hindlimb was significantly impaired in Atox1-/- mice as
compared with WT (35.96.7% decrease) as evaluated by laser Doppler blood flow. Capillary
density in ischemic hindlimb at 7 days after ischemia was markedly reduced in Atox1-/- mice
as compared to WT mice (27.84.7% decrease). In addition, superoxide production which
plays a critical role in angiogenesis was significantly decreased in ischemic hindlimb of
Atox1-/- mice as examined by lucigenin assay (33.55.2% decrease). Interestingly, the
numbers of CD34/CD31- and c-kit/CD31- double positive endothelial progenitor cells (EPCs)
derived from bone marrow and peripheral blood were markedly decreased in Atox1-/- mice
after ischemia as compare to WT (37.5- and 22.4% decrease, respectively) as analyzed by
FACS. To gain insight into the molecular mechanism, we performed EMSA and CHIP assays and
found that Atox1 binds to the promotor region of cyclin D1 in a copper dependent manner.
Moreover, copper-induced increase in cell proliferation and protein levels of cyclin D1 is
markedly inhibited in Atox1 deficient mouse fiblobrasts and by knockdown of Atox1 using
siRNA. Taken together, we provide the first evidence that Atox1 functions as a copper
dependent transcription factor for cyclin D1 and thus stimulates cell proliferatioin, thereby
promoting neovascularization induced by tissue ischemia.
16
Candidate Susceptibility Loci for Vascular Remodeling Identified Through a
Genome-wide Association Study of In-stent Restenosis
Santhi K Ganesh, NHGRI/NIH, Bethesda, MD; Kimberly Skelding, Mayo Clinic, Rochester,
MN; Jungnam Joo, Gang Zheng, NHLBI, Bethesda, MD; Adong Yu, NHGRI/NIH, Bethesda,
MD; Nancy Geller, NHLBI, Bethesda, MD; Stephen Pan, NHGRI/NIH, Bethesda, MD; Eric
Billings, NHLBI, Bethesda, MD; Laxmi Mehta, William Beaumont Hosp, Royal Oak, MI;
Kathleen ONeill, NHLBI, Bethesda, MD; Robert Simari, David Holmes Jr, Mayo Clinic,
Rochester, MN; William ONeill, William Beaumont Hosp, Royal Oak, MI; Giulia Kennedy,
Affymetrix, Inc, Santa Clara, CA; Elizabeth G Nabel; NHLBI/NHGRI/NIH, Bethesda, MD
Introduction: Risk-conferring genes responsible for complex diseases are characterized by
variable expressivity, low penetrance, epistasis and locus heterogeneity, making the analysis
of complex genetic traits challenging. For complex cardiovascular diseases, candidate gene
approaches have achieved limited success, making genome-wide association study (GWAS)
designs appealing. Molecular and genetic studies suggest that in-stent restenosis (ISR) is
primarily an inflammatory and proliferative disease, with distinct roles for cell cycle proteins,
growth factors, and inflammatory cytokines. Methods: To investigate the genetic basis of ISR,
we designed a case control GWAS on a dataset of 116,000 single nucleotide polymorphisms
(SNPs) assayed in 407 patients (150 cases, 257 controls). We undertook a haplotype analysis
of regions highlighted by the presence of two or more SNPs within 250 kb of one another and
with p0.001 (unadjusted) in univariate tests of allelic association. Haplotypes were defined
in the regions and tested for association with ISR, using a Bonferroni correction for all
haplotypes tested. Results: We identified eight candidate susceptibility loci containing five
genes: NOV, ARNTL, TAF4B, PKP4 and a hypothetical protein FLJ21986. We observed
expression of these genes by RNA and protein analysis in normal, atherosclerotic and restenotic
human coronary arteries, supporting the hypothesis that these genes may mediate vascular
homeostasis and adverse vascular remodeling. We estimated each subjects haplotypes and
examined the time to development of ISR as a quantitative trait in a Cox regression model and
demonstrated a significant effect of allele dose for each of the eight regions identified
(p0.00038 to 0.035, after Bonferroni correction), suggesting that allele copy number
contributes to ISR. We additionally analyzed the data using the updated BRLMM algorithm to
call genotypes and identified some of the same genes and EPHB1 and ST18. The additional
e-38 Arterioscler Thromb Vasc Biol June 2007
by on March 25, 2011 atvb.ahajournals.org Downloaded from
regions demonstrate significant allele dose effect as well (p0.003). Conclusions: Haplotype
analysis of GWAS using SNP markers is a useful approach to identify candidate genes for
complex vascular diseases. These areas of association with ISR warrant further investigation.
17
Ambient Particulate Pollutants in the Ultrafine Range Promote
Atherosclerosis and Systemic Oxidative Stress
Jesus A Araujo, Berenice Barajas, UCLA Sch of Medicine, Los Angeles, CA; Michael
Kleinman, Univ of California, Irvine, Irvine, CA; Xuping Wang, Brian J Bennett, Ke Wei Gong,
Mohamad Navab, UCLA Sch of Medicine, Los Angeles, CA; Jack Harkema, Michigan State
Univ, Lansing, MI; Constantinos Sioutas, Univ of Southern California, Los Angeles, CA;
Aldons J Lusis, Andre Nel; UCLA Sch of Medicine, Los Angeles, CA
Background - Air pollution has been associated with significant adverse health effects leading
to increased cardiovascular morbidity and mortality. Cumulative evidence supports the
pathogenic role of particulate matter (PM) in atherosclerosis. While EPA is currently regulating
particles with an aerodynamic diameter of 2.5 m (PM
2.5
), there is increasing evidence that
the smaller particulate components shed into polluted urban air from vehicular emissions may
even be more dangerous due to their high content of pro-oxidative chemicals. Methods and
Results - To test our hypothesis that the smaller the particles, the larger the cardiovascular
effect, we used particle concentrator technology to compare the atherogenic effects of ambient
particles with aerodynamic diameter 0.18 m (ultrafines) vs. fine particles 2.5 m (PM
2.5
)
in downtown Los Angeles. Two experimental protocols were used. In the first, 6-week-old male
C57BL/6J apoE null mice were placed on a chow diet and exposed to concentrated ambient
PM (CAPs) for a combined total of 75 hours over a period of 40 days; while in the second,
2-month-old male apoE null mice were fed a high fat diet (HFD) and exposed to CAPs for a
combined total of 120 hours over a period of 58 days. Control groups included mice exposed
to filtered air (FA) or left non-exposed (NE). Chow-fed mice exposed to concentrated ultrafines
developed 25%, 55% and 91% greater aortic atherosclerotic lesions than animals exposed to
PM
2.5
, FA or NE mice, respectively (n1417/group, p0.05). Exposure to ultrafine particles
resulted in an inhibition of the antiinflammatory capacity of plasma high density lipoproteins
and increased systemic oxidative stress markers as evidenced by a significant (i) increase in
liver malondialdehyde levels, (ii) upregulation of Nrf2 and Nrf2-related phase-2 response
antioxidant genes (e.g, catalase, superoxide dismutase, NQO-1). HFD-fed apoE null mice
exposed to concentrated ultrafines also exhibited evidence of increased systemic oxidative
stress but not of greater atherosclerosis. Conclusions - Ultrafine particles concentrate the
proatherogenic effects of ambient PM and constitute a significant cardiovascular risk factor that
may need to be considered in air quality regulation, similar to PM
2.5
.
18
Induction of Apoptosis in Established Atherosclerotic Lesions Promotes
Inflammation and Monocyte Recruitment in Apoe-/- Mice
Emmanuel Gautier, Thierry Huby, Betty Ouzilleau, John Chapman, Phillipe Lesnik; INSERM
U551, Paris, France
Introduction: The impact of apoptosis on atherosclerosis progression may be deleterious as
apoptotic cell clearance appears to be defective in atherosclerotic plaques. We evaluated the
impact of the induction of lesional apoptosis on intraplaque inflammation and monocyte
recruitment in a new genetically-modified mouse model. Methods: Mice expressing the
Diphteria Toxin Receptor (DTR) under the control of the CD11c promoter were crossed with
Apoe-/- mice. Induction of apoptosis in CD11c lesional cells was achieved by injection of
Diphteria Toxin (DT, 4ng/g). Apoe-/- and DTR Apoe-/- mice were fed a Western diet for 8 weeks
and divided into 3 groups: Apoe -/- DT (control), DTR Apoe-/- PBS (control) and DTR
Apoe-/- DT. Two days after injection, chemokines and monocyte markers expression was
evaluated in the aorta, while apoptosis and newly recruited macrophage detection were
assessed in aortic root lesion. Results: Increased TUNEL staining in the aortic tissue of DTR
Apoe-/- DT mice as compared to control mice (p0.05) was associated with increased
mRNA expression for the small inducible chemokines MCP-1, MIP-1, MIP-1 and MIP-2
(p0.05 versus controls). Concomitantly, mRNA expression of monocyte markers (CD11b,
CCR2, CX3CR1) was significantly increased in the aorta of DTR Apoe-/- DT mice as
compared to controls. Moreover, analysis of aortic root lesions revealed the presence of newly
recruited macrophages in areas of apoptotic cell accumulation, which was indicative of
monocyte recruitment. Monocyte tracing using fluorescent beads confirmed that apoptotic cell
deposition promoted recruitment of circulating monocytes in the aorta of DTR Apoe-/- DT
mice. Conclusions: These data suggest that apoptosis associated with impaired apoptotic cell
clearance may promote inflammation and recruitment of monocytes in established atheroscle-
rotic lesions.
19
Distinctive Expression of Chemokines and Transforming Growth Factor-
Signaling in Human Arterial Endothelium During Atherosclerosis
Oscar L Volger, Joost O Fledderus, Academic Med Cntr, Univ of Amsterdam, Amsterdam,
The Netherlands; Natasja Kisters, Cardiovascular Rsch Institute Maastricht, Univ of
Maastricht, Maastricht, The Netherlands; Ruud D Fontijn, Perry D Moerland, Academic Med
Cntr, Univ of Amsterdam, Amsterdam, The Netherlands; Johan Kuiper, Theo J van Berkel,
Gorlaeus Laboratories, Leiden Univ, Leiden, The Netherlands; Ann-Pascale J Bijnens, Mat J
Daemen, Cardiovascular Rsch Institute Maastricht, Univ of Maastricht, Maastricht, The
Netherlands; Hans Pannekoek, Anton J Horrevoets; Academic Med Cntr, Univ of Amsterdam,
Amsterdam, The Netherlands
Objectives: Knowledge about the in vivo role of endothelium in chronic human atherosclerosis
has mostly been derived by insights from mouse models. Therefore, we set out to establish by
microarray analyses the gene expression profiles of endothelium from human large arteries, as
isolated by Laser Microbeam Microdissection, having focal atherosclerosis of the early or the
advanced stage. Within individual arteries the endothelial transcriptomes of initial (N7) and
advanced (N6) lesions were compared pairwize to the unaffected sides, thus limiting genetic
and environmental confounders. Results: Specific endothelial signature gene sets with
changed expression levels in either early (N718) or advanced atherosclerosis (N403),
relative to their paired plaque-free controls, were identified (paired Cyber-T test: FDR-corrected
p0.05). Gene Set Enrichment Analysis identified distinct sets of chemokines and differential
enrichments (p0.05) of NF-kappa B-, p53- and TGF--related genes in advanced plaques,
compared with initial plaques. Immunohistochemistry on a separate set of human arteries with
focal atherosclerosis of the early- (N6) or the advanced-stage (N5) validated the
discriminative value of corresponding endothelial protein expression between early (fractalkine/
CX3CL1, IP10/CCL10, TBX18) or advanced (BAX, NFKB2, phosphorylated SMAD2) stages of
atherosclerosis and versus their plaque-free controls. Conclusions: The functional involvement
of TGFbeta-signaling in directing its downstream gene repertoire was substantiated by a
consistent detection of activated endothelial SMAD2 in advanced lesions. Thus, we identified
truly common, local molecular denominators of pathological changes to vascular endothelium,
with a marked distinction of endothelial phenotype between early and advanced plaques.
20
Inactivation of Macrophage Fatty Acid Synthase Decreases Atherosclerosis
Jochen G Schneider, Manu V Chakravarthy, Kara N Standley, Clay F Semenkovich;
Washington Univ, St. Louis, MO
Fatty acid metabolism is disturbed in atherosclerotic lesions but whether it affects lesion
formation is unknown. To test the hypothesis that fatty acid synthesis affects atherosclerosis,
we inactivated macrophage fatty acid synthase (FAS) in apoE-deficient Cre-Lox mice. FASKOM
(FAS KnockOut in Macrophages) animals showed essentially no FAS mRNA or enzyme activity
in macrophages. FASKOM mice and their wild type littermates on the apoE-null background
were fed a Western diet for 8 weeks. There was no effect of genotype on body weight, glucose
metabolism or serum lipids in either sex. FASKOM mice compared to controls had significantly
lower systolic and diastolic blood pressures both at baseline on chow diet (928/737 vs
1027/8310, p0.05) and with high fat feeding (1008/756 vs 10812/829,
p0.05). Compared to littermate controls (n17), FASKOM mice (n21) had 40% less
atherosclerosis in the abdominal aorta (p0.0001), 20% less in the aortic arch (p0.05), and
23% less in the thoracic aorta (p0.05). In other tissues, FAS is necessary for endogenous
activation of the nuclear receptor PPARalpha and expression of its target genes. In elicited
macrophages from FASKOM mice, there was no effect on PPARalpha mRNA (as expected), but
expression of the PPARalpha target genes CPT1 and ACO was decreased compared to
macrophages from control mice, consistent with decreased activation of PPARalpha in the
absence of FAS. There was also a robust induction of mRNA for the antiatherosclerotic oxysterol
receptor LXRalpha as well as its targets ABCA1 and SREBP1c in FASKOM as compared to
control macrophages. Treatment of FASKOM macrophages with a PPARalpha agonist normal-
ized the expression of PPARalpha target genes as well as LXRalpha. These results suggest that
macrophage FAS promotes atherosclerosis in apoE-deficient mice by decreasing LXRalpha
expression through endogenous activation of PPARalpha.
21
A Cluster of Basic Residues Within the Factor V B-domain Contributes to
Preserving the Procofactor State
Mettine H A Bos, Michael Boltz, Rodney M Camire; The Childrens Hosp of Philadelphia,
Philadelphia, PA
Blood coagulation factor V (FV) circulates as an inactive procofactor protein and must be
proteolytically processed to express procoagulant activity. Thrombin is the principal activator of
FV and cleaves three peptide bonds, thereby removing a large central B-domain in order to
generate active FV (FVa). Mechanistic explanations as to how B-domain release facilitates FV
activation are not adequately developed. Recent studies from our laboratory using a panel of
B-domain truncated FV variants suggest that B-domain sequences within Asp902-Glu1033
contribute to preserving the FV procofactor state. Interestingly, the Lys963-Lys1008 portion of
this sequence is very basic (18 out of 46 residues are Arg or Lys) and is highly conserved, even
though most of the B-domain is poorly conserved within the vertebrate lineage. The aim of the
current study was to examine whether this basic region is sufficient to maintain the FV
procofactor state. Using the previously characterized and constitutively active FV-810 derivative
as scaffold (B-domain sequence Pro811-Gly1491 deleted), we expressed and purified three FV
variants with portions of the basic region inserted: FV-1007BR with Lys963-Glu1007 inserted,
FV-1015BR with Lys963-Leu1015 inserted, and FV-1033BR with Lys963-Glu1033 inserted.
Each of the proteins migrated as a single band on SDS-PAGE and was processed by thrombin
to yield the expected heavy and light chains. Using both a one-stage PT-based clotting assay
and a purified prothrombinase assay, we were able to demonstrate that insertion of the basic
region into FV-810 converted this cofactor-like species back to the procofactor state.
Furthermore, additional experiments showed that all derivatives exhibited full cofactor activity
following treatment with thrombin. These observations indicate that the highly conserved basic
region (Lys963-Lys1008) is able to maintain the procofactor state, even when assembled into
a B-domain that lacks 75% of the full length B-domain sequence and structure. Based on
our data, we speculate that the basic sequence might serve an inhibitory function, possibly by
directly concealing binding interactions that govern the function of the active cofactor species.
22
The Utility of Quantitative Calf Muscle Near-infrared Spectroscopy in the
Follow-up of Acute Deep Vein Thrombosis
Takashi Yamaki, Motohiro Nozaki, Hiroyuki Sakurai, Masaki Takeuchi, Kazutaka Soejima,
Taro Kono; Tokyo Womens Med Univ, Tokyo, Japan
BACKGROUND: To investigate patterns of venous insufficiency and changes in calf muscle
deoxygenated hemoglobin (HHb) levels after an acute deep vein thrombosis. METHODS: A total
2007 ATVB Oral Presentations e-39
by on March 25, 2011 atvb.ahajournals.org Downloaded from
of 78 limbs with an acute deep vein thrombosis (DVT) involving 156 anatomic segments were
evaluated with duplex scanning and near-infrared spectroscopy (NIRS) at 1 month, 3 months,
6 months, and 1 year. Venous segments were examined whether they were occluded, partially
recanalized, and totally recanalized, and the development of venous reflux was noted. The NIRS
was used to measure calf muscle HHb levels. Calf venous blood filling index (HHbFI) was
calculated on standing, then the calf venous ejection index (HHbEI) and the venous retention
index (HHbRI) were obtained after exercise. RESULTS: The segments investigated were the
common femoral vein (CFV; 38 segments), femoral vein (FV; 37), popliteal vein (POPV; 44), and
calf veins (CV; 37). At 1 year, thrombi had fully resolved in 67% of the segments, 27% remained
partially recanalized, 6% were occluded. The venous occlusion was predominant in the FV
(24%) at 1 year. On the contrary, rapid recanalization was obtained in CV than proximal veins
at each examination (p0.01). Venous reflux was predominant in POPV (55%), followed by FV
(19%), and no reflux was found in CV. At 1 year, the HHbFI in POPV reflux patients was
significantly higher than these with resolution (0.190.14, 0.110.05mol/lsec, p0.009,
respectively). Similarly, there was a significant difference in the HHbRI between the two groups
(3.081.91, 1.421.56, p0.002, respectively). In patients with FV occlusion, the value of
HHbRI was significantly higher than these with complete resolution (2.591.50, 1.421.56,
p0.011, respectively). CONCLUSIONS: The lower extremity venous segments show different
proportions of occlusion, partial recanalization, and total recanalization. The CV shows more
rapid recanalization than proximal veins. The NIRS-derived HHbFI and HHbRI could be
promising parameters as the overall venous function in the follow-up of acute DVT. These
findings might be very helpful for physician in detecting patients who require much longer
follow-up studies.
23
A Unique Function for Low-density ReceptorRelated Protein-1: A
Component of a 2-Receptor System Mediating Specific Endocytosis of
Plasma-derived Factor V by Megakaryocytes
Beth A Bouchard, Natalie T Meisler, Univ of Vermont, Burlington, VT; Michael E Nesheim,
Queens Univ, Kingston, Canada; Dudley K Strickland, Univ of Maryland Sch of Medicine,
Baltimore, MD; Paula B Tracy; Univ of Vermont, Burlington, VT
Factor V is endocytosed by megakaryocytes from plasma via a specific, receptor-mediated,
clathrin-dependent mechanism to form the functionally and physically unique platelet-derived
factor V pool. Because of its ability to endocytose proteins involved in hemostasis, the role of
low density lipoprotein receptor-related protein-1 (LRP-1) in factor V endocytosis by the
megakaryocyte-like cell line, CMK, was examined. Equilibrium binding of
125
I-factor V to CMK
cells is defined by a sigmoidal binding isotherm, suggesting that factor V binding is cooperative
and mediated by a two-receptor system. Furthermore,
125
I-factor V binding is reversible and
partially sensitive to receptor associated protein (RAP), a known LRP-1 ligand. Based on these
observations a two-receptor model for factor V binding to megakaryocytes was hypothesized.
In this model, factor V binds to a specific receptor facilitating binding of another factor V
molecule to LRP-1 or a like molecule, which subsequently endocytoses factor V. The purpose
of the current study was to identify which member of the LRP-1 receptor family is involved.
Using RT-PCR, expression of an LRP-1 transcript in CMK cells was demonstrated. In contrast,
transcripts representing other LRP family members could not be identified. Cell surface
expression of LRP-1 antigen by CMK cells was confirmed by flow cytometry using polyclonal
anti-LRP-1 antibodies. Greater than 70% of the CMK cells expressed LRP-1 on their cell
surface. Co-localization of endocytosed AlexaFluor488-factor V and LRP-1 demonstrated that
all of the factor V positive cells expressed LRP-1. These same anti-LRP-1 antibodies were used
to displace 40% of the bound
125
I-factor V from the surface of the cells. Furthermore, factor
VIII, a known ligand of LRP-1, inhibited
125
I-factor V endocytosis equally as well as factor V
when present at a 25-fold molar excess. These combined observations confirm our model of
the cell surface events regulating factor V binding to megakaryocytes, and represent a novel
paradigm whereby an essential coagulation protein is endocytosed from plasma and modified
intracellularly to yield a functionally distinct molecule. Furthermore, a role for LRP-1 in
endocytosis of a protein not destined for lysosomal degradation is unique.
24
Induction of Tissue Factor and Loss of Thrombomodulin Activities upon
Inflammatory Stimulation in Vivo
Hugo ten Cate, Kim Frederix, Ingeborg Kooter, Rene van Oerle, Karly Hamulyak, Henri M
Spronk; Academic Hosp Maastricht, Maastricht, The Netherlands
Introduction: Inflammation induces organ specific changes in expression of tissue factor (TF)
and thrombomodulin (TM) antigen, but it is unknown whether this translates to a net
procoagulant phenotype in vivo. Hypothesis: induction of TF and suppression of TM activities
contribute to organ-associated thrombogenicity in response to specific inflammatory stimuli
including endotoxin (LPS) and particulate matter (PM). Design and results: in C57BlJ/6 mice
TFactivity in the lungs was induced more by intraperitoneal LPS (25.8 5.3 pM) than by saline
(12.7 1.7 pM, p0.05); TF activities in other organs (brain, heart, spleen, liver, kidney) were
comparable for LPS and saline. Addition of lung homogenate from control mice to plasma
markedly attenuated the plasma endogenous thrombin potential (ETP) (175 61 vs. 1437
112 nM. min; p0.01). This inhibitory effect was due to TM in the lungs, because I) it was
absent in protein C deficient plasma; II) lungs from TM
pro/pro
mice with a 100-fold reduced
potential to activate protein C did not inhibit thrombin generation in plasma (ETP: 1686 209
nM.min). LPS challenge abolished the inhibitory activity of the lungs, indicated by a significant
increase in ETP (941 523 vs.194 159 nM.min); LPS did not affect other organs in their
effect on thrombin generation. We applied these assays to analyze the consequences of
intratracheal instillation of increasing concentrations of PM, an environmental determinant of
chronic lung- and cardiovascular disease, to spontaneous hypertensive rats PM(road tunnel
dust: 0.31310 mg/kg bodyweight) dose-dependently induced an increase in TF activities
after 4 and 48 hours of exposure compared to controls (1045 472, 1021 62 for 10 mg/kg
dose vs. 397 231 pM for control, p0.02). At the highest dose of PM the ETP increased
significantly after 4 hours (390 148 vs. 175 61 for saline, p0.05) and after 48 hours
(1441 289 nMmin vs. saline: 164 64 nMmin, p0.0001), while TF activity remained
stably elevated in this period, suggesting progressive loss of TM activity. Conclusion:
inflammation-induced prothrombotic activity is established by induction of TF in an organ
specific manner and the loss of TM activity is a major determinent of this effect in vivo.
25
Modulation of LPS-induced Inflammation and Coagulation by the PI3K-Akt
Pathway
James P Luyendyk, Michael Tencati, Todd Holscher, Nigel Mackman; Scripps Rsch Institute,
La Jolla, CA
Lipopolysaccharide (LPS) stimulation of monocytes/macrophages activates various intracellular
signaling pathways, including the mitogen-activated protein kinases (MAPKs) and the
phosphatidylinositol-3-kinase (PI3K)-Akt pathway. LPS activation of the MAPK pathways is
required for the expression of inflammatory cytokines and tissue factor (TF), the principal
activator of blood coagulation. We have shown that pharmacologic inhibition of PI3K enhances
LPS signaling and the induction of inflammation and coagulation, both in monocytic cells and
in endotoxemic mice. To extend these findings, we determined the effect of genetically
reducing or enhancing activation of the PI3K-Akt pathway on LPS induction of inflammatory
mediators and TF in peritoneal macrophages (PMs) and in mice. We utilized PMs lacking p85,
which have decreased PI3K-Akt activity and PMs lacking the phosphatase PTEN, which have
increased Akt activity. LPS activation of the MAPKs and expression of TF, IL-6 and TNF was
enhanced in p85
-/-
PMs. Furthermore, inflammation and coagulation were enhanced in
endotoxemic wild type mice lacking p85
-/-
in hematopoietic cells. LPS activation of the MAPKs
and the expression of TF, IL-6 and TNF were reduced in PTEN
-/-
PMs. Our results indicate that
LPS activation of the PI3K-Akt pathway in macrophages inhibits the MAPK signaling pathways
and reduces inflammation and coagulation. Activation of PI3K or inhibition of PTEN may
represent novel strategies to reduce inflammation and coagulation in endotoxemia and sepsis.
26
Angiotensin II-induced Hypertrophy and Fibrosis Is Prevented by
Angiotensin 17 Overexpression in the Heart
Alvaro Yogi, Univ of Ottawa, Ottawa, Canada; Chantel Mercure, Clinical Rsch Institute of
Montreal, Montreal, Canada; Augusto C Montezano, Glaucia E Callera, Univ of Ottawa,
Ottawa, Canada; Rita C Tostes, Univ of Sao Paulo, Sao Paulo, Brazil; Rhian M Touyz, Univ
of Ottawa, Ottawa, Canada; Timothy L Reudelhuber; Clinical Rsch Institute of Montreal,
Montreal, Canada
Background: In vitro studies demonstrate that Ang-17 opposes many actions of Ang II.
Whether similar effects occur in vivo remains unclear. Objectives: We tested the hypothesis
that Ang17 protects the heart from damage induced by Ang II and assessed some signaling
pathways whereby this occurs. Methods and results: Transgenic mice over-producing
Ang17 (TG/Ang17) exclusively in the heart (8-fold) were generated. Basal blood pressure and
cardiac contractility were similar between groups. To evaluate whether Ang17 exerts direct
cardioprotective effects, TG/Ang17 and control mice were infused with Ang II (350ng/kg/min,
19 days). Ang II-infused TG/Ang17 mice developed less ventricular hypertrophy and fibrosis
versus controls, in spite of developing similar levels of hypertension (p0.05). Increased
membrane translocation of the NAD(P)H oxidase subunit p47phox, an index of activity, was
observed in Ang II-infused controls. This was blunted in TG/Ang17 mice. Phosphorylation of
cardiac c-Src was increased by Ang II in controls (2.5-fold) but not in TG/Ang17 mice.
Activation of redox-sensitive growth signaling molecules, Akt and p38MAPK, was increased by
Ang II in controls (23-fold, p0.001) without effect in TG/Ang17 mice. Ang II increased
cardiac ERK 1/2 phosphorylation in control and TG/Ang17 mice. Effects of Ang17
overproduction on cardiac cell cycle regulatory proteins were also assessed. Expression of
Cdk2 /cyclin E and Cyclin D/Cdk4 was increased in Ang II-infused controls (12-fold) and
abrogated in TG/Ang17. Increased expression of cell cycle inhibitors p21 (1-fold) and p53
(1.5-fold) by Ang II was observed in controls but not in TG/Ang17 mice. p27 expression was
not different between groups. Conclusion: Our data indicate that Ang17 protects the heart
from an Ang II-dependent hypertensive challenge. This is associated with downregulation of
NAD(P)H oxidase, c-Src, Akt and p38MAPK, but not with ERK1/2. Moreover Ang(17) influences
cell cycle regulatory proteins. Our findings suggest that the negative modulatory effects of
Ang-(17) may represent a protective mechanism whereby potentially deleterious actions of
Ang II are counterbalanced.
27
Requirement of RhoA Serine 188 Phosphorylation for cGMP
Kinase-mediated Vascular Protection
Naoki Sawada, Hiroshi Itoh, Kazutoshi Miyashita, Hirokazu Tsujimoto, Kazuwa Nakao; Kyoto
Univ Graduate Sch of Medicine, Kyoto, Japan
[Background] cGMP agonists such as natriuretic peptides dilate blood vessels and inhibit
vascular remodeling. Small GTPase RhoA and its effector ROCK, crucial mediators of
e-40 Arterioscler Thromb Vasc Biol June 2007
by on March 25, 2011 atvb.ahajournals.org Downloaded from
vasocontraction and vascualr growth, antagonize cGMP effects. We previously demonstrated
that cGMP-dependent protein kinase type I (cGK I), the direct cGMP effecter, phosphorylates
RhoA at Ser188 and inhibits its downstream signaling. However, the in vivo relevance of this
interaction remains elusive. This study assessed our hypothesis that cGK I phosphorylation of
RhoA mediates vascular protection by cGMP agonists. [Results] Ser188 phosphorylation of
RhoA, as assessed by phospho-specific antibodies, was diminished in the aortas of cGK I
haploinsufficient mice (cGK I/-), whereas RhoA/ROCK activity was enhanced. Brain natriuretic
peptide transgenic mice (BNP-Tg), with 3- to 4-fold increase in plasma cGMP levels, showed
augmented phosphorylation of RhoA with less ROCK activity as compared to control. cGK I/-
mice treated with angiotensin II (Ang II) developed exaggerated BP elevation, cardiac
hypertrophy, coronary artery medial thickening (MT) and perivascular fibrosis (PVF), and
expression of fibrogenic cytokines and extracellular matrix (ECM) proteins. ROCK inhibitor
Y-27632 markedly normalized these changes except elevated BP. To assess the role of Ser188
phosphorylation of RhoA in cGK I-mediated vascular protection, we generated transgenic mice
that express cGK I-unphosphorylatable mutant RhoA (A188RhoA) or wild-type RhoA (wtRhoA)
in arterial smooth muscle using SM22alpha promoter. A188RhoA-Tg exhibited 2.7-fold
augmented ROCK activity and 2-fold increase in MT and PVF compared to non-Tg, whereas
these changes were milder in wtRhoA-Tg mice. While wtRhoA/BNP double Tg (DTg) displayed
normalized MT and PVF, those of A188RhoA/BNP DTg remained worse (1.5-fold) than non-Tg.
Ang II treatment induced comparable increase (1.5- to 4-fold) in MT, PVF and ECM gene
expression in A188RhoA/BNP DTg and non-Tg mice. By contrast, these changes induced by
Ang II were blunted in wtRhoA/BNP DTg and BNP-Tg mice. [Conclusion] Our data suggest that
Ser188 phosphorylation and inhibition of RhoA by cGK I is required for cGMP/cGK I-mediated
protection against vascular remodeling.
28
Cyclooxygenase-2 Expression Increases Vascular Inflammation and
Abdominal Aortic Aneurysm Formation in Mice
Jonathan M Gitlin, Darshini B Trivedi, Charles D Loftin; Univ of Kentucky, Lexington, KY
Abdominal aortic aneurysms (AAAs) are associated with a profound inflammatory response
within the vessel wall throughout propagation of the disease. Increased expression of
cyclooxygenase-2 (COX-2) is suggested to contribute to the disease in humans. We examined
the hypothesis that COX-2-dependent proliferation or inflammation in the abdominal aorta
contributes to AAA formation. AAAs were induced by angiotensin II infusion and compared
between COX-2-deficient mice and their wild-type littermate controls. COX-2-deficient mice
showed significantly reduced AAA incidence at multiple time-points following angiotensin II
infusion (day 3, 31%
5
16 for COX-2
/
versus 6%
1
16 for COX-2
-/-
, P 0.07; day 7, 43%
15
35
for COX-2
/
versus 12%
3
26 for COX-2
-/-
, P 0.01; day 21, 37%
11
30 for COX-2
/
versus
4%
1
28 for COX-2
-/-
, P 0.01; day 28, 54%
13
24 for COX-2
/
versus 0%
0
23 for COX-2
-/-
, P
0.01). COX-2 has previously been shown to be required for angiotensin II-induced smooth
muscle cell proliferation. To examine the role of proliferation in COX-2-dependent AAA
formation, we determined the effect of COX-2 expression on activation of AKT and ERK1/2. As
determined by western blot, the levels of phosphorylated AKT or ERK1/2 were not significantly
different between angiotensin II-infused COX-2
/
and COX-2
-/-
mice, suggesting that COX-2
does not contribute to altered proliferation during AAA formation. Angiotensin II-induced AAA
formation is initially characterized by macrophage infiltration into the wall of the abdominal
aorta. Therefore, we examined macrophage infiltration as a potential mechanism by which
COX-2 contributes to AAA formation. At time-points where angiotensin II was shown to induce
COX-2, mRNA expression of the macrophage marker CD68 was significantly attenuated in the
abdominal aorta of COX-2
-/-
mice, as compared to wild-type controls (for COX-2
/
versus
COX-2
-/-
: day 3 was 0.6 0.05 versus 0.3 0.03, n 9, P 0.01; day 7 was 0.7 0.1
versus 0.2 0.05, n 10, P 0.01). Furthermore, abdominal aortas of COX-2
-/-
mice
showed attenuated expression of the chemokines MCP-1 and MIP-1alpha. In conclusion,
increased COX-2 expression in the abdominal aorta may contribute to AAA formation by
enhancing macrophage recruitment.
29
Integrin Signaling Is Critical for Pathological Angiogenesis
Ganapati H Mahabeleshwar, Weiyi Feng, David Phillips, Tatiana Byzova; Lerner Rsch
Institute, Cleveland, OH
The process of postnatal angiogenesis plays a crucial role in pathogenesis of numerous
diseases, including but not limited to tumor growth/metastasis, diabetic retinopathy, and in
tissue remodeling upon injury. However, the molecular events underlying this complex process
are not well understood and numerous issues remain controversial, including the regulatory
function of integrin receptors. To analyze the role of integrin phosphorylation and signaling in
angiogenesis, we generated knock-in mice that express a mutant beta3 integrin unable to
undergo tyrosine phosphorylation. Two distinct models of pathological angiogenesis revealed
that neovascularization is impaired in mutant beta3 knock-in mice. In an ex vivo angiogenesis
assay, mutant beta3 knock-in endothelial cells did not form complete capillaries in response
to vascular endothelial growth factor (VEGF) stimulation. At the cellular level, defective tyrosine
phosphorylation in mutant beta3 knock-in cells resulted in impaired adhesion, spreading, and
migration of endothelial cells. At the molecular level, VEGF stimulated complex formation
between VEGF receptor-2 and beta3 integrin in wild-type but not in mutant beta3 knock-in
endothelial cells. Moreover, phosphorylation of VEGF receptor-2 was significantly reduced in
cells expressing mutant beta3 compared to wild type, leading to impaired integrin activation in
these cells. These findings provide novel mechanistic insights into the role of integrin-VEGF axis
in pathological angiogenesis.
30
Matrix Metalloproteinase-1: Role in Aneurysm Formation in Vivo and
Regulation by Nicotine in Vascular Smooth Muscle Cells
Vincent Lemaitre, Jeanine DArmiento; Columbia Univ, New York, NY
The role of matrix metalloproteinase-1 (MMP-1) and the molecular mechanisms regulating its
expression in aneurysm remain unknown. Transgenic mice expressing human MMP-1 in
macrophages were crossed into the ApoE-null / Timp-1 (Tissue inhibitor of
metalloproteinases-1) knockout background, which develops a high number of micro-
aneurysms associated with atherosclerosis. Transgenic mice (n11) and their littermate
controls (n10) were given a high-fat diet for ten weeks and sacrificed. Transgenic mice had
larger aneurysms (45,21628,111 m
2
, n33) compared to the controls (26,18522,026
m
2
, n28; P0.005). These aneurysms were also characterized by the bulging of the
destroyed media into the adventitia and an increased deposition of fibrillar collagen (transgenic
: 3214% of the aneurysm area, controls : 125%, n11 in each group; P0.008), a marker
of severity in the human disease. Since smoking is a major risk factor for aneurysm
development and rupture, we tested the hypothesis that nicotine could modulate the expression
of MMP-1 in vascular cells. Human aortic vascular smooth muscle cells were treated with
nicotine, at concentrations found in the circulation of moderate smokers (10 nM to 1000 nM).
After 24 hours, nicotine increased the expression of MMP-1, both at the mRNA and protein
levels (P0.05). This up-regulation was abrogated by specific inhibitors of p38 and ERK,
suggesting that nicotine induces MMP-1 through the MAP kinases signaling pathway.
Western-blot analysis of cell lysates showed that nicotine activates the Jak/STAT kinase
pathway, leading to increased phosphorylation of Jak2, ERK, p38, Jnk, and Stat3, and
subsequent induction of MMP-1 expression. Our data demonstrates that MMP-1 enhances the
severity of mouse aneurysm in vivo, and that nicotine induces MMP-1 expression in smooth
muscle cells through the Jak/Stat kinase pathway. This study suggests that increased
susceptibility to aneurysm formation and rupture in smokers might result from augmented
collagenase activity in the vessel wall, due to a chronic exposure to circulating nicotine
31
Notch Signaling Negatively Regulates Endothelial Lineage Specification
Rhiannon Penkert, UNC-Chapel Hill, Chapel Hill, NC; Kevin Vogeli, Univ of Southern
California, Los Angeles, CA; Jessica Alsio, UNC-Chapel Hill, Chapel Hill, NC; Didier Stainier,
UCSF, San Francisco, CA; Suk-Won Jin; UNC-Chapel Hill, Chapel Hill, NC
Endothelial cells and hematopoietic cells are thought to arise from a common progenitor, the
hemangioblast. This notion was initially suggested as a result of the observation that these two
cell types develop in close proximity during embryonic development. Although the data
acquired from cell culture experiments suggest the existence of the hemangioblast, the
presence of bipotential progenitors has yet to be demonstrated in vivo until our recent study.
We have reported the first in vivo evidence for the existence of hemangioblast during embryonic
development using zebrafish gastrula as a model system. As the initial step to delineate the
molecular signaling pathways that regulate hemangioblast lineage specification, we analyzed
the function of Notch signaling pathway in this process. Previous study indicated that Notch
could function as a bi-modal switch in the hemangioblast equivalent cell population in
Drosophila. We find that Notch signaling appears to promote hematopoietic fate over
endothelial fate. Embryos with reduced Notch activity show a significant increased number of
angioblasts at the expense of hematopoietic progenitors. This early requirement of Notch
activity can be separated from the previously described function of Notch in promoting arterial
endothelial fate. Furthermore, by using lineage tracing we show that a certain population of
cells which would give rise to primitive erythrocytes transfate into endothelial precursors in
embryos with compromised Notch signaling. Although further analyses need to be done to test
whether the increased number of angioblasts is due to the fate changes within the
hemangioblast lineage or the direct transformation of hematopoietic progenitors, our data
suggest that Notch signaling might functions as a cell fate switch by negatively regulating
endothelial fate.
32
The Atheroprotective Effect of Flk-1-based DNA Vaccination in LDL
ReceptorDeficient Mice Is Enhanced by Coexpression of CCL21
Ramona J Petrovan, Audrey S Black, Joshua Bulgrien, David J Bonnet, Linda K Curtiss; The
Scripps Rsch Institute, La Jolla, CA
Suppressors of angiogenesis can reduce plaque neovascularization and inhibit atherosclerotic
lesion growth. Vaccination of hyperlipidemic LDL receptor-deficient (LDLr-/-) mice with an oral
DNA vaccine specific for the murine VEGF receptor 2 (Flk-1) evoked a CD8 T cell-mediated
response that suppressed atherosclerosis-related angiogenesis and attenuated lesion progres-
sion. Appropriate adjuvants can improve effector T cell function and increase the efficacy of
DNA vaccines. We therefore tested the hypothesis that co-expression of the secretory
chemokine CCL21 with Flk-1 can enhance the atheroprotective effect of the Flk-1 vaccination.
Empty expression vector, Flk-1 or Flk-1/CCL21 vaccinated LDLr-/- mice (n 8/group) were fed
a high fat diet for 16 weeks. Consistent with previous findings, there was no difference in total
cholesterol levels between all groups of mice, despite the expected increase in plasma
cholesterol after high fat diet consumption. Compared to both Flk-1 and control immunized
animals, immunization with the vector encoding Flk-1/CCL21 led to increased in vitro lysis of
murine endothelial target cells expressing Flk-1 by T cell-enriched splenocytes. Co-expression
of CCL21 led to increased protection against diet-induced atherosclerosis in both male and
female mice, as demonstrated in both cross sections of the aortic sinus and in lipid stained en
face preparations of the aorta. A time course study of lesion development revealed that the
extent of aortic sinus lesions was substantially decreased in LDLr-/- mice vaccinated with the
Flk-1/CCL21 construct after only 4 weeks of consuming the high fat diet. Both foam-cell lipid
accumulation and macrophage infiltration in aortic segments within areas of disturbed blood
flow were altered by the Flk-1-based DNA vaccination at early stages of the disease. In
2007 ATVB Oral Presentations e-41
by on March 25, 2011 atvb.ahajournals.org Downloaded from
conclusion, these studies demonstrated that co-expression of CCL21 with Flk-1 improved T cell
mediated immune responses against Flk-1 and reduced lesion progression in hyperlipidemic
mice.
33
Diet-induced Dyslipidemia Is Associated with Accleration of Disease and
Mortality in Lupus-susceptible LDLr
-/-
Mice
Amy S Major, Nicole A Braun, Adam C Morgan; Vanderbilt Univ Med Cntr, Nashville, TN
Individuals suffering from systemic lupus erythematosus (SLE) are predisposed to accelerated
atherosclerosis. Unfortunately, the underlying mechanisms for increased vascular disease in
lupus are not well understood. Our laboratory has recently developed an animal model of
SLE-accelerated atherosclerosis. We have shown that radiation chimeras consisting of
SLE-derived hematopoietic cells transferred to LDLr
-/-
mice have increased atherosclerosis.
Surprisingly, feeding mice high-fat diet for 8 weeks resulted in significant mortality in
SLE-susceptible mice compared to controls. Based on these data, we hypothesized that
increased dyslipidemia, characterized by an accumulation of non-HDL lipoproteins, is
associated with increased autoimmunity. To test this hypothesis, we created radiation chimeras
of LDLr
-/-
mice that were either SLE-susceptible (LDLr.Sle) or resistant (LDLr.B6). Eight weeks
following bone marrow reconstitution, mice were placed on a normal chow or high fat (21%
fat, 0.15% cholesterol) diet for eight weeks. All animals fed a high-fat diet had significantly
increased total cholesterol and triglycerides compared to chow fed mice, however there were
no significant differences within groups between LDLr.B6 control and LDLr.Sle mice. Compared
to all chow-fed animals and high-fat fed LDLr.B6 controls, high-fat fed LDLr.Sle mice exhibited
increased mortality (37%) and were mildly, but significantly hypertensive. In addition, ECHO
analyses showed that 60% (3 of 5 mice) of the LDLr.Sle mice fed high fat diet had increased
left ventricular mass compared their LDLr.B6 counterparts. Increased blood pressure did not
overtly appear to be due to advanced renal disease as serum creatinine and urea levels
between LDLr.Sle mice on chow or high-fat diet did not differ significantly. These data
demonstrate that increased dyslipidemia resulting from feeding a high-fat diet can exacerbate
autoimmunity and associated vascular complications.
34
Chemical Genetic Analysis Reveals the Central Role of
Phosphatidylinositol-3 Kinase and MAP Kinase/ERK Signaling Pathways in
Artery/Vein Specification
Charles C Hong, Vanderbilt Univ Sch of Medicine, Nashville, TN; Quinn P Peterson, Ji-Young
Hong, Randall T Peterson; Massachusetts General Hosp, Boston, MA
How the endothelial progenitor cells are specified to either the arterial or venous fates is a
fundamental biological question with significant clinical implications. We reasoned that the
artery/vein specification during development is amenable to chemical genetic analysis, in a
manner analogous to the classical genetic analyses, which have been instrumental in
elucidation of numerous biological pathways in prokaryotes and invertebrates. Specifically, we
hypothesized that small molecules found to suppress zebrafish model of arterial deficiency will
function by augmenting the signaling for arterial specification. In a high-throughput chemical
screen, we discovered two classes of small molecules that rescued the defective arteriogenesis
caused by a genetic mutation. Using these compounds as chemical tools to dissect the
signaling pathways involved in artery/vein specification, we made the following important and
surprising observations: 1) p42/44 MAP kinase (ERK) activation is a specific and early marker
of arterial progenitors, 2) ERK activation is a key determinant of arterial specification, and 3)
the phosphatidylinositol-3 kinase (PI3K) has the opposite effect on artery-vein specification. In
summary, our chemical genetic approach has revealed opposing roles of PI3K and ERK in
artery/vein specification, a better understanding of which will lead to novel therapies for
numerous conditions, such as ischemic heart/limb diseases and vascular malformations.
35
ER Stressors Promote TLR4/SRA-dependent Macrophage Apoptosis
Through Ca
2
-mediated CaMKII Activation
Tracie A Seimon, Wahseng Lim, Janelle Timmins, Columbia Univ, New York, NY; Kathryn J
Moore, Harvard Med Sch, Boston, MA; Douglas T Golenbock, Univ of Massachusetts Med
Sch, Worcester, MA; Ira A Tabas; Columbia Univ, New York, NY
The macrophage (M) scavenger receptor-A (SRA) and toll-like receptor 4 (TLR4) are pattern
recognition receptors (PRRs) of the innate immune system. In vivo data suggest that the SRA
and TLR4 contribute to the development of atherosclerosis, although the mechanistic links
between these PRRs and atherosclerosis have not been elucidated. Our laboratory has focused
on a critical event in the formation of necrotic atherosclerotic plaques, namely, M death. We
recently discovered that SRA ligands trigger M apoptosis in an SRA- and TLR4-dependent
manner. SRA ligands activate the pro-apoptotic TLR4-JNK pathway but, unlike LPS, silence the
pro-survival TLR4-IFN pathway. We also found that ER stressors that induce Ca2
mobilization enhance TLR4-dependent signaling and that chelating Ca2 inhibits TLR4-
dependent JNK activation and apoptosis. In the current study we show that LPS, which
normally leads to cell survival, induces apoptosis in the presence of various SRA ligands such
as fucoidan, -amyloid, and advanced glycation endproducts (AGEs). These SRA ligands do not
induce apoptosis when added in the absence of LPS. Moreover, M apoptosis induced by LPS
and SRA ligands was markedly amplified in the setting of Ca2-releasing ER stressors like
thapsigargin. To probe mechanism, we investigated the role of Ca2-regulated calmodulin-
dependent protein kinase II (CaMKII). We found that apoptosis-enhancing Ca2-releasing ER
stressors are potent activators of CaMKII. Most importantly, TLR4-dependent JNK activation
and apoptosis were blocked by the CaMKII inhibitor KN93. These data and our previous studies
suggest that Ca2-releasing ER stressors contribute to two events necessary for M
apoptosis: enhancement of TLR4-dependent JNK activation through activation of CaMKII; and
induction of the pro-apoptotic CHOP branch of the ER stress pathway through depletion of ER
Ca2 stores. These findings reveal a novel link between ER stress, Ca2 mobilization, and
PRR signaling and have potential implications for M death and plaque necrosis in advanced
atherosclerosis and perhaps other diseases where PRRs, ER stress, and cell death are known
to play a role.
36
Signal-Dependent Splicing of Tissue Factor Pre-mRNA Modulates the
Thrombogenicity of Human Platelets: A New Mechanism Linked to
Disordered Coagulation in Sepsis
Hansjorg Schwertz, Neal D Tolley, Jason M Foulks, Univ of Utah, Salt Lake City, UT; Rachel
E Tilley, The Scripps Rsch Institute, La Jolla, CA; Estelle S Harris, Matthew T Rondina, Guy
A Zimmerman, Univ of Utah, Salt Lake City, UT; Nigel Mackman, The Scripps Rsch Institute,
La Jolla, CA; Andrew S Weyrich; Univ of Utah, Salt Lake City, UT
Introduction: We recently demonstrated that platelets use a previously-unrecognized pre-
mRNA splicing pathway to generate tissue factor (TF)-dependent procoagulant activity. Here,
we hypothesize that TF pre-mRNA splicing events are activated in patients with sepsis, a
syndrome of dysregulated coagulation. Methods: Patients meeting consensus criteria for
sepsis were prospectively enrolled. Platelets were freshly-isolated from whole blood within the
first 24 hours of admission to the ICU. Platelets from non-medicated healthy controls were
assayed in parallel to eliminate differences due to inter-assay variability. TF mRNA expression
patterns and procoagulant activity was measured in both groups. Platelets were also incubated
with bacteria from septic patients or bacterial-derived products. Results: Without exception,
platelets from healthy controls (n54) expressed TF pre-mRNA. In contrast, platelets from 26
of 46 septic patients expressed spliced TF mRNA. The incidence of TF pre-mRNA splicing
increased with increasing severity of illness as measured by APACHE II scores. Consistent with
TF mRNA expression patterns, procoagulant activity in platelets from septic patients was
significantly (p0.05) higher than in healthy controls (45.913.3 vs. 11.12.8 pM). In a
subgroup of patients (n16), we also assessed TF pre-mRNA splicing in serial samples. We
found progressive splicing in the platelets over time. Altogether, 81% of these patients
expressed spliced TF mRNA at some point during their ICU stay. Gram
-
(E. coli) or gram

(S.
aureus) bacteria isolated from septic patients also induced TF pre-mRNA splicing. Similarly,
products of E. coli (lipopolysaccharide) or S. aureus (-toxin) induced TF pre-mRNA splicing in
platelets, resulting in accelerated clot formation. Conclusions: These data demonstrate that
circulating platelets from septic patients spontaneously splice TF pre-mRNA and generate
procoagulant activity. TF pre-mRNA splicing by platelets may contribute to abnormal
coagulation in sepsis and may be a target of future therapeutics.
37
Targeting Coagulation Factor XII Provides Protection from Pathological
Thrombosis in Myocardial Infarction and Cerebral Ischemia Without
Interfering with Hemostasis
Thomas Renne, Ulrich Walter, Christoph Kleinschnitz, Guido Stoll, Stefan Frantz, Johann
Bauersachs, Univ of Wurzburg, Wurzburg, Germany; Hans-Ulrich Pauer, Univ of Gottingen,
Gottingen, Germany; David Gailani; Vanderbilt Univ Sch of Medicine, Nashville, TN
Formation of fibrin is critical for limiting blood loss at a site of blood vessel injury (hemostasis),
but may also contribute to vascular thrombosis. Hereditary deficiency of factor XII (FXII), the
protease that triggers the intrinsic pathway of coagulation in vitro, is not associated with
spontaneous or excessive injury-related bleeding, indicating FXII is not required for hemostasis.
We demonstrate that deficiency or inhibition of FXII protects mice from ischemic brain injury
and cardiac ischemia/reperfusion damage. Following transient middle cerebral artery occlu-
sion, the volume of infarcted brain in FXII deficient and FXII inhibitor-treated mice was
significantly less than in wild type controls, without an increase in infarct-associated
hemorrhage. FXII-null and inhibitor treated mice were largely protected from ischemia/
reperfusion injury and survival rate was significantly higher in myocardial infarction models.
Targeting FXII reduced fibrin formation in ischemic vessels, and reconstitution of FXII deficient
mice with human FXII restored fibrin deposition. Mice deficient in the FXII substrate factor XI
were similarly protected from vessel-occluding fibrin formation in brain and heart, suggesting
that FXII contributes to pathologic clotting through the intrinsic pathway. These data
demonstrate that some processes involved in pathologic thrombus formation are distinct from
those required for normal hemostasis. As FXII appears to be instrumental in pathologic fibrin
formation, but dispensable for hemostasis, FXII inhibition may offer a selective and safe
strategy for preventing stroke, myocardial infarction and other thromboembolic diseases.
38
The Antithrombotic Potential of Selective Blockade of Talin-dependent
Integrin IIb3 (Platelet GPIIb-IIIa) Activation
Brian G Petrich, Univ of California, San Diego, La Jolla, CA; Susan J Monkley, Univ of
Leicester, Leicester, United Kingdom; Anthony W Partridge, Nima Yousefi, Sanford J Shattil,
Univ of California, San Diego, La Jolla, CA; David R Critchley, Univ of Leicester, Leicester,
United Kingdom; Mark H Ginsberg; Univ of California, San Diego, La Jolla, CA
Studies in vitro and with cultured cells indicate that talin binding to the 3 cytoplasmic domain
is a final step in platelet integrin IIb3 (GPIIb-IIIa) activation. We tested the significance of
talin-mediated integrin activation by generating platelet-specific talin1 knock-out mice (PKO).
Platelets from PKO mice showed a dramatic reduction in agonist-induced llb3 activation as
determined by soluble fibrinogen binding. In addition, more than 90% of PKO mice showed
pathological bleeding that was associated with reduced survival. To determine whether the
phenotype of PKO mice was due to lack of talin binding to integrin 3, we generated 3
integrin knock-in mice harboring point mutations in the 3 integrin cytoplasmic domain that
disrupt talin- integrin interactions. We introduced a 3(Y747A) substitution that disrupts the
e-42 Arterioscler Thromb Vasc Biol June 2007
by on March 25, 2011 atvb.ahajournals.org Downloaded from
binding of talin, filamin, and other cytoplasmic proteins and a 3(L746A) substitution that
selectively disrupts interactions only with talin. Analysis of platelets from animals homozygous
for each mutation showed decreased agonist-induced fibrinogen binding, providing proof that
that inside-out signals that activate IIb3 require talin binding to the 3 tail. Injection of
collagen and epinephrine resulted in the death of 62% of wild type mice from pulmonary
thromboembolism; in sharp contrast, both 3(Y747A) and 3(L746A) mice were resistant to
this form of thrombosis. Pathological bleeding, as measured by the presence of fecal blood and
development of anemia, occurred in 53% of 3(Y747A) and virtually all 3 integrin knock-out
and PKO mice examined. Remarkably, none of the 3(L746A) animals exhibited this form of
bleeding. These results establish that IIb3 activation in vivo is dependent on the interaction
of talin with the 3 integrin cytoplasmic domain. Furthermore, they suggest that modulation of
3 integrin-talin interactions may provide an attractive target for anti-thrombotics with reduced
risk of pathological bleeding.
39
Mapping Structure-Function Relationships of ABCA1
Dmitri Sviridov, Nigora Mukhamedova, Ying Fu, Baker Heart Rsch Institute, Melbourne,
Australia; Michael Bukrinsky; George Washington Univ, Washington, DC
ABCA1 is a key element of cholesterol efflux, but the mechanisms of ABCA1-dependent lipid
efflux and structure-function relationships of ABCA1 are still unclear. Three monoclonal
antibodies against ABCA1 were developed and used to map functional domains of ABCA1. Two
antibodies were directed against a fragment of the first extracellular loop of ABCA1 between
amino acids 602620, the third antibody was directed against a fragment of its fourth
extracellular loop between amino acids 1311 -1450. One antibody against the first loop (AB1)
inhibited cholesterol efflux from human macrophages without inhibiting apolipoprotein A-I
(apoA-I) binding and internalization. Another antibody against the first loop (AB2) inhibited
apoA-I binding and internalization without inhibiting cholesterol efflux. The antibody against the
fourth loop (AB3) inhibited apoA-I binding, but enhanced cholesterol efflux from macrophages
and reduced intracellular cholesterol content. This antibody also increased cholesterol efflux
from HeLa cells transfected with ABCA1 elevating it to the level of that from cells transfected
with PEST-ABCA1. The mechanism of the stimulating effect of this antibody on cholesterol
efflux was found to be stabilization of ABCA1 in macrophages leading to the increase in
abundance of cell-surface ABCA1. We are proposing the following model of ABCA1/apoA-I
mediated lipid efflux. Three events can happen as a result of apoA-I binding to ABCA1. First,
binding of apoA-I to ABCA1 may induce changes in the plasma membrane enabling cholesterol
efflux. The antibody AB1 blocks this process and reduces cholesterol efflux, without affecting
binding of apoA-I. Second, binding of apoA-I to ABCA1 may lead to the internalization of
apoA-I/ABCA1 complex, this event is blocked by the antibody AB2 and may not be related to
cholesterol efflux. The third event is binding of apoA-I to the fourth extracellular loop of ABCA1
preventing its calpain-mediated proteolysis. The antibody AB3 is likely binds to this site and
acts as an agonist preventing ABCA1 degradation.
40
PGC-1 Strongly Stimulates VLDL Secretion While Suppressing Triglyceride
Synthesis: Evidence for an Important Role for PGC-1 in Regulating VLDL
Assembly
Zhouji Chen, Brian N Finck, Jin Y Norris; Washington Univ Sch of Med, St. Louis, MO
Dysregulation of hepatic VLDL production is a major cause of dyslipidemia associated with
several pathophysiologic conditions including type II diabetes and obesity. Mechanisms
regulating hepatic VLDL synthesis and secretion remain to be fully understood. The peroxisome
proliferator-activated receptor- coactivator-1 (PGC-1) has emerged as an important
regulator for hepatic triglyceride metabolism. This study was carried out to explore its potential
role in regulation of VLDL secretion. Adenovirus-mediated overexpression of PGC-1 in HepG2
cells increased fatty acid oxidation and decreased triglyceride synthesis by 40%. In contrast,
rates of triglyceride secretion were induced by 2- and 4-fold under basal and oleic
acid-stimulated conditions, respectively. Secretion of VLDL-apoB100 was coordinately in-
creased. Similarly, overexpression of PGC-1 in cultured mouse hepatocytes also suppressed
triglyceride synthesis and induced VLDL secretion. Furthermore, infusion of PGC-1-
adenoviruses to mice resulted in a 30% decrease in hepatic triglyceride contents and a 50%
increase in VLDL-triglyceride secretion. In subcellular fractionation analyses, PCG-1 overex-
pression increased microsomal triglyceride contents by 2.5 fold while reducing cytosolic
triglycerides by 45%. PCG-1 decreased mRNA for the mitochondrial GPAT mRNA by 70% but
did not affect the expression of the key genes involved in VLDL assembly, including MTP, apoB,
apoE, Dgat1, and Dgat2. While PGC-1 strongly induced apoA4 gene expression, adenoviral
overexpression of mouse apoA4 did not induce triglyceride secretion by HepG2 and mouse
hepatocytes. Together, these results demonstrate that PGC-1 has an important role in
regulation of partitioning of cellular triglycerides for VLDL assembly. Elucidating the underlying
mechanism may provide novel information for understanding the regulation of hepatic VLDL
secretion.
41
Expression of Lipin-1 in Transfected Hepatic Cells Stimulates VLDL
Assembly and Secretion
Maroun Bou Khalil, Philip Links, Khai Tran, Meenakshi Sundaram, Univ of Ottawa Heart
Institute, Ottawa, Canada; Karen Reue, Univ of California, Los Angeles, CA; Zemin Yao; Univ
of Ottawa Heart Institute, Ottawa, Canada
Lipin-1 plays a key role in the metabolism of triacylglycerol (TG) in the adipose tissue and the
liver, functioning both in TG biosynthesis and induction of adipogenic gene expression. The
yeast and mammalian lipins have recently been shown to be phosphatidate phosphatase-1
(PAP-1), the enzyme that catalyzes the formation of diacylglycerol (DG) from phosphatidate. In
primary rat hepatocytes, PAP-1 activity could be increased (8-fold) by dexamethasone in a
cycloheximide- and alpha-aminitin-sensitive manner. Using differential display PCR, we
determined that the dexamethasone-induced PAP-1 activity was attributable to lipin-1.
Transient expression of mouse lipin-1alpha (driven by the CMV promoter) in rat hepatoma
McA-RH7777 cells resulted in increased synthesis and secretion (2- to 3-fold) of [3H]glycerol-
labeled DG, TG, phosphatidylcholine (PC) and phosphatidylethanolamine (PE), in an oleate
dose-dependent (0.1 - 0.4 mM) manner. Fractionation of secreted lipoproteins by cumulative
rate flotation revealed that the increased 3H-TG secretion from cells transiently expressing
lipin-1alpha was mainly attributable to that of VLDL1 (Sf 100) and VLDL2 (Sf 20 - 100).
Preliminary experiments showed that expression of lipin-1alpha resulted in increased (3-fold)
incorporation of [35S]methionine into cell-associated and secreted apoB100, the major protein
constituent of VLDL. Increased TG secretion (3- to 4-fold) was also observed in the cells stably
expressing moderate levels of lipin-1alpha or lipin-1beta (lipin-1beta is a splice variant that
contains an insertion of 33 amino acids). As observed in primary hepatocytes, exogenous oleate
treatment promoted translocation of PAP-1 activity from cytosol to microsomal membranes,
and also induced the association of recombinant lipin-1alpha or lipin-beta with the microsomes
and the nucleus in transfected McA-RH7777 cells. Specifically, oleate treatment stabilized
recombinant lipin-1alpha and lipin-1beta, which was associated with their increased associ-
ation with lipid droplets. These results confirm that lipin-1 has PAP-1 activity in hepatic cells,
and that the overexpression of lipin-1 increases hepatic lipogenesis and VLDL assembly/
secretion.
42
Carboxyl Ester Lipase Affects Hepatic HDL Selective Uptake and
Metabolism and Reverse Cholesterol Transport in Mice
Lisa Camarota, Philip Howles; Univ of Cincinnati College of Medicine, Cincinnati, OH
Introduction: Selective uptake of HDL cholesterol by the liver is an important source of biliary
cholesterol and bile salts, and thus, may contribute significanlty to reverse cholesterol
transport. Most HDL cholesterol is esterified and must be hydrolyzed to free cholesterol during
this process. Carboxyl ester lipase (CEL) is a broad spectrum lipase made and secreted by the
liver as well as pancreas and other tissues. Previous work has shown that CEL affects SR-BI
mediated HDL-cholesteryl ester (CE) selective uptake and hydrolysis by hepatocytes. Hypoth-
esis: We have used CEL -null and -transgenic mice to test the hypothesis that this enzyme is
important for normal trafficking of HDL-CE to bile and thereby affects overall reverse cholesterol
transport. Methods: Effects on circulating HDL were determined by FPLC analysis of plasma
lipoproteins and slective uptake of radiolabeled HDL-cholesteryl ether into liver. Hepatic bile
was collected to measure hydrolysis and secretion of radiolabeled CE from HDL into bile. Gall
bladder and fecal sterols were quantitated to assess reverse cholesterol transport. All groups
were n 8 mice. Results: Selective uptake and hydrolysis of HDL-CE is reduced in CEL-/-
mice, resulting in elevated plasma cholesterol (89.9 17.2 mg/dL vs. 74.6 12.6 for
controls, P 0.012) and larger HDL particles. Secretion into bile of hydrolyzed CE from HDL
is reduced from 81.4 13.4 % in controls to 26.0 12.8 % in CEL-/- mice (P 0.001),
confirming a primary role of this enzyme in hydrolysis of CE after selective uptake. Gall bladder
cholesterol concentration is reduced from 2.70 0.73 mM in controls to 2.12 0.50 mM in
CEL-/- mice (P 0.012). Fecal cholesterol excretion is reduced from 28.5 3.2 mg/d/g body
weight in controls to 22.7 4.8 mg/d/g body weight in CEL-/- mice. Hepatic overexpression
of a CEL transgene reduces plasma cholesterol (196.6 29.8 mg/dL, controls vs. 166.7
17.3 mg/dL, transgenics, P 0.015). This is primarily due to reduced cholesteryl esters
(137.7 22.8 mg/dL, controls vs. 110.8 15.3 mg/dL, transgenics, P 0.009).
Conclusions: The data strongly suggest that CEL plays a physiologically significant role in
hepatic HDL metabolism and reverse cholesterol transport.
43
Inactivation of ABCG1 in C57Bl/6 Mice Results in Reduced Adipose Tissue
Mass Development When Challenged with a High-fat/High-cholesterol Diet
Elke M Wagner, NHLBI/NIH, Bethesda, MD; Matthew A Kennedy, Columbus Childrens Rsch
Institute, Columbus, OH; Young-Hun Choi, NHLBI/NIH, Bethesda, MD; Dan M Schimel, NIH,
Bethesda, MD; Ashley Dulin, Columbus Childrens Rsch Institute, Columbus, OH; Silvia
Santamarina-Fojo, Alan T Remaley; NHLBI/NIH, Bethesda, MD
Adipose tissue is a unique storage site for large amounts of cholesterol, and the regulation of
the cholesterol content in adipocytes is a keyfactor to size-dependent fat cell metabolism,
signal transduction, gene regulation and plasma cholesterol levels. The lipid transporter ABCG1
has been described to facilitate cellular cholesterol transport in mouse macrophages, thereby
playing a crucial role in the maintenance of normal lipid levels in tissue macrophages and foam
cells. Furthermore, Buchmann et al. have recently reported the impact of ABCG1 whole body
ablation on adipose tissue development of C57BL/6 mice where exons three through five were
targeted for deletion. In parallel studies, our group has investigated fat tissue development in
a different ABCG1-KO mouse model which deleted the Walker A region of the ATP-binding site
of exon three of C57BL/6. We found significantly reduced body weights of male and female
ABCG1-KO vs. WT mice when mice were placed on a high cholesterol (HC, 1.2% w/w) and/or
a high fat (HF, 21% w/w) diet (1325% less body weight in males and 514% in females
depending on time, (p0.05)). Using Micro-CT imaging and gravimetric measurement of
isolated tissues we show a 40% (p0.05) decrease in abdominal and subcutaneous white
adipose tissue, as well as a 25% (p0.05) decrease in brown adipose tissue. Cell diameter
measurements of isolated abdominal fat cells using light microscopy revealed a significant
decrease in the median cell diameter of female ABCG1-KO (82.5 um; n8) mice on a HC/HF
diet compared to WT mice (112.5um; n8; p0.001). ABCG1-KO mice exhibited increased
mRNA levels of expression of LXRa, PPARg, PPARd, and ABCA1 by 1.52-fold as early as 6
weeks on a HF/HC diet in white and 12 weeks in brown adipose tissue as measured by
Real-time PCR. Of interest, plasma lipids changed in male but not female ABCG1-KO mice vs.
WT mice on a HF/HC diet. The rise of total plasma cholesterol and phospholipids during 6
weeks on diet was lower in ABCG1-KO vs. WT mice (Total Cholesterol 188 vs. 210%; n6;
p0.05, Phospholipids 154 vs. 172%; p0.05). In summary, our results suggest that ABCG1
controls adipose tissue development, possibly by altering intracellular adipocytes as well as
plasma cholesterol levels to regulate lipid storage and energy balance.
2007 ATVB Oral Presentations e-43
by on March 25, 2011 atvb.ahajournals.org Downloaded from
44
-Adrenergic Receptors Modulate Tissue-type Plasminogen Activator
Release Through Nitric Oxide Synthase-dependent Pathway in
Normotensive Subjects and Hypertensive Patients
Chiara Giannarelli, Agostino Virdis, Ferdinando De Negri, Emiliano Duranti, Lorenzo Ghiadoni,
Armando Magagna, Antonio Salvetti, Stefano Taddei; Univ of Pisa, Pisa, Italy
Introduction: In essential hypertension, reduced tissue-type plasminogen activator (t-PA)
release is a resultance of impaired nitric oxide availability (Hypertension, 2007). Sympathetic
system, mainly acting via beta receptors, modulates both endogenous fibrinolysis and nitric
oxide release. Hypothesis: Thus, nitric oxide deficiency might contribute to the impaired t-PA
release in hypertensive patients. Methods: In 9 healthy men (age4910) and 5 hypertensive
patients (age5211), the t-PA release (ELISA method), during intra-arterial infusion of
isoprenaline (0.3 g/min/100 ml) was evaluated. Isoprenaline was repeated in the presence of
L-NMMA (100 g/min/100 ml). The isolated forearm model was used to measure forearm
blood flow changes (FBF) by strain gauge venous plethysmography. The net balance of t-PA
from venous (V) and arterial (A) blood samples (V-A plasma difference x FBF) was calculated.
Results: In normotensive subjects, vasodilation induced by isoprenaline (from 3.70.2 to
17.12.5 ml/100ml/min; p0.01 vs basal levels) was significantly blunted by L-NMMA (from
3.70.4 to 12.9 ml/100ml/min; p0.05 vs isoprenaline alone). In hypertensive patients,
vasodilation to isoprenaline was slightly reduced, although not statistically different, as
compared to normotensive subjects (from 3.50.4 to 13.73.2 ml/100ml/min; pNS vs
controls). In these patients, response to isoprenaline was unaffected by L-NMMA (from
3.60.4 to 12.3 ml/100ml/min; pN.S. vs isoprenaline alone). In normotensive subjects
isoprenaline infusion significantly increased t-PA balance (from 0.130.1 to 2.40.8
ng/100ml/min; p0.01 vs basal levels). This effect was almost abolished by L-NMMA (from
-0.10.1 to 0.10.3 ng/100ml/min). In contrast, in hypertensive patients no t-PA release was
observed, either in the absence (from 0.020.2 to 0.780.4 ng/100ml/min; p0.05 vs
controls) or in the presence of L-NMMA (from 0.50.5 to 0.30.4 ng/100ml/min; p0.01 vs
controls). Conclusions: In conclusion, these data indicate that in healthy conditions beta-
adrenergic stimulation induces t-PA release via a nitric oxide dependent-mechanism. The
reduced nitric oxide availability contributes to the impaired t-PA release in essential
hypertension.
45
Expression and Characterization of Mutants of Human TAFI Resistant to
Activation by Specific Proteases
Michael B Boffa, Bradley Poulsen, Kyra Nabeta; Queens Univ, Kingston, Canada
Thrombin-activatable fibrinolysis inhibitor (TAFI) is a human plasma zymogen that functions as
a molecular connection between the coagulation and fibrinolytic cascades. Activated TAFI
(TAFIa) is formed by cleavage at Arg92, a reaction that can be catalyzed by several proteases
including thrombin and plasmin; thrombin in complex with thrombomodulin (TM) is the most
potent activator of TAFI. However, the relative role of each of these activators in different
physiological contexts remains unknown. We have designed mutants of TAFI with the aim of
creating variants that are resistant to activation by either thrombin or plasmin. Substitution of
serine for proline at position 91 (P921S) yielded a variant of TAFI that could not be activated
by thrombin and was only very slowly activated by thrombin-TM; this variant was activated
normally by plasmin. The P91S variant was expressed in mammalian cells, purified, and its
ability to inhibit lysis of clots made from TAFI-deficient plasma was compared to that of
wild-type (wt) recombinant TAFI. TAFI (P91S) was markedly impaired in its antifibrinolytic
activity, both in the presence or absence of TM (see Figure). Thus, activation by thrombin and
thrombin-TM predominates under these circumstances. Plasmin is a promiscuous enzyme, but
is unable to cleave its own activation site; accordingly, introduction of residues from this site
into peptide substrates for plasmin prevents their cleavage. However, introduction these
residues into analogous positions in TAFI (specifically, valine at positions 93 and 94) failed to
affect plasmin activation of TAFI suggesting that exosite interactions are more critical for
recognition of TAFI by plasmin.
46
The Molecular Basis for Fibrin Clot Elasticity
Bernard Lim, Arshad Jahangir; Mayo Clinic, Rochester, MN
Fibrinogen when activated by thrombin forms a thrombus having elasticity that buffers bloods
shear forces. The molecular basis of this elasticity is unknown. Fibrinogen has coiled coils
which are structural motifs that have been demonstrated in myosin to have perfect elasticity
by atomic force microscopy (AFM). Signature phases of the coiled coils force extension
curves have been defined that can distinguish it from other protein domains. Furthermore,
polymers of fibrinogen molecules could be created for force spectroscopy experiments to
amplify the signal from pulling single fibrinogen molecules. Fibrinogen is thus ideal for pulling
experiments to evaluate the candidacy of the coiled coil region for fibrins elasticity. To
generate linear polymeric fibrin strands, 70 l of purified fibrinogen (0.1 mg/ml) was reacted
with 10 l of CaCl2 (2.5 mmol), 10 l of FXIII (0.002 mg/ml) and 10 l of thrombin (0.2 U/ml)
for 3.5 minutes. These were then imaged with the AFM in liquid tapping mode. Linear strands
of 7 to 10 fibrin monomers with lengths of 250 /- 75 nm were observed. Fibrinogen and fibrin
polymers were deposited onto gold-sputtered coverslips and adsorbed for 5 minutes. AFM in
force spectroscopy mode was used to stretch the fibrinogen molecules and fibrin polymers.
When the fibrinogen molecule was stretched beyond 20 nm, there was an abrupt increase in
force of about 60 pN, followed by a plateau phase during which the force was relatively
constant. This intermediate conformational transition likely contributes to fibrin elasticity. The
magnitude of the plateau force changed with pH and calcium ion concentration. Stretching
fibrin polymers to 400 nm revealed a blunted sawtooth pattern of consecutive force peaks,
each demonstrating the triphasic coiled coil signature. Increasing the stretch to 3 times their
length unfolded the globular domains. Repeated extension and relaxation cycles demonstrated
that the coiled coil regions were able to refold at high rates. Our data provide the first evidence
for the coiled coils of fibrinogen being the origins of fibrin elasticity. Alterations to the coiled
coils through mutations or disease may interfere with its function, changing the elasticity of the
fibrin clot and cause thromboembolism.
47
Polymorphisms in Coagulation Factors Are Not Associated with Risk of
Recurrent Major Adverse Cardiovascular Events in Men
Marieke D van der Krabben, Carine J M Doggen, Johanneke G van der Bom, Frits R
Rosendaal; Leiden Univ Med Cntr, Leiden, The Netherlands
Introduction: Most myocardial ischemic events occur through plaque ruptures that precipitate
formation of an occluding thrombus. The growth of a thrombus may be affected by an
increased activity of the coagulation system. Therefore, it has been suggested that plasma
levels of coagulation factors affect the risk of recurrent cardiovascular events. Objective: The
aim of this study was to examine whether genetic predisposition to high levels of coagulation
factors influence the risk of developing fatal and non-fatal recurrent major arterial cardiovas-
cular events in men with a first myocardial infarction. Methods: We performed a cohort study
among 547 myocardial infarction patients, with a mean age of 56 years (range 32 to 70). All
men had a first myocardial infarction between 1990 and 1996 and were followed until
September 1
st
2004. DNA was analysed for polymorphisms of fibrinogen, prothrombin (factor
II), factor V, factor VII and plasminogen activator inhibitor type-1 (PAI-1), which are all
associated with gain of function (either in plasma concentration or stability). We collected
information from hospital files and general practitioners on the occurrence of recurrent major
arterial cardiovascular events. Results: In total, 259 recurrent events occurred. The point
estimates of the relative rates (RR) of major cardiovascular events for the genotypes were all
between 0.7 and 1.1 except for prothrombin 20210A mutation: RR 1.8 (95%CI 0.84.1).
Adjustment for traditional risk factors of cardiovascular disease did not alter the findings.
Conclusion: These findings suggest that there is no association between genetically
determined high levels or stability of fibrinogen, factor V, factor VII and PAI-1, and the risk of
recurrent major cardiovascular events. Increased prothrombin levels, however, may be
associated with the risk of recurrent events.
48
The Effect of Factor XIII Levels and Factor XIII A Subunit V34L
Polymorphism on the Risk of Coronary Artery Disease: Novel Clinical
Aspects
Laszlo Muszbek, Zsuzsanna Bereczky, Emilia Balogh, Zoltan Voko, Eva Katona, Istvan
Czuriga, Levente Karpati, Zsuzsa Pocsai, Roza Adany, Istvan Edes; Univ of Debrecen, Med
and Health Science Cntr, Debrecen, Hungary
Studies on the association of polymophisms in factor XIII A subunit (FXIII-A) with coronary artery
disease (CAD) are rather inconclusive. The original paper and confirmatory reports demon-
strated that FXIII-A V34L polymorphism exerted a protective effect against CAD, but a number
of reports demonstrated the lack of protection. Only a few studies with mainly negative
outcome investigated the effect of plasma FXIII level on the risk of CAD. In this study FXIII
activity and antigen levels and FXIII-A V34L polymomorphism were determined in 1010
consecutive patients admitted for coronary angiography. Patients were classified according to
the presence of significant (50%) coronary sclerosis (CS) and the history of myocardial
infarction (MI). In females, but not in males, adjusted FXIII activity and antigen levels were
significantly elevated in the CSMI group compared to the CSMI- group. When CSMI
patients were compared to clinical controls, FXIII levels in the upper tertile were associated with
significantly increased risk in females, but not in males. Elevated FXIII levels also conferred
significantly increased risk of MI to females with CS. In neither sex did elevated FXIII levels
increase the risk of CS. To reveal the cumulative effect of FXIII-A V34L polymorphism on the
risk of CAD first a meta-analysis of 16 studies on 5346 cases and 7053 controls that
investigated the association between V34L polymorphism and CAD was performed. Because of
the heterogeneity of the study specific results, the pooled effect estimates were calculated by
a random-effects empirical Bayes model. The combined odds ratios for CAD were 0.82 (95%
CI: 0.730.94) for the heterozygotes (V34L) variant, and 0.81 (95% CI: 0.700.92) for the
heterozygotes and homozygotes combined. The heterogeneity of individual studies suggested
that gene-gene and/or gene-environmental interactions might significantly influence the
protective effect of the polymorphism. This hypothesis was supported by results showing that
in the Hungarian population the Leu34 allele provided a statistically significant protection
against MI only in patients with fibrinogen level in the upper quartile.
e-44 Arterioscler Thromb Vasc Biol June 2007
by on March 25, 2011 atvb.ahajournals.org Downloaded from
49
Role of LIM Kinase 1 in Endothelial Function
Tatyana Voyno-Yasenetskay, Matvey Gorovoy; Univ of Illinois, Chicago, IL
Microtubule (MT) destabilization promotes the formation of actin stress fibers and enhances the
contractility of cells; however, the mechanism involved in the coordinated regulation of MTs
and the actin cytoskeleton is poorly understood. LIM kinase 1 (LIMK1) regulates actin
polymerization by phosphorylating the actin depolymerization factor, cofilin. We have shown
that LIMK1 is involved in the MT destabilization. Overexpression of wild type LIMK1 resulted in
MT destabilization, whereas the kinase-dead mutant of LIMK1 (KD) did not affect MT stability.
Importantly, down-regulation of endogenous LIMK1 by small interference RNA resulted in
abrogation of the thrombin-induced MTs destabilization and the inhibition of thrombin-induced
actin polymerization. Expression of Rho kinase 2, which phosphorylates and activates LIMK1,
dramatically decreases the interaction of LIMK1 with tubulin but increases its interaction with
actin. Interestingly, expression of KD-LIMK1 or small interference RNA-LIMK1 prevents
thrombin-induced microtubule destabilization and F-actin formation, suggesting that LIMK1
activity is required for thrombin-induced modulation of microtubule destabilization and actin
polymerization. We examined pulmonary vascular permeability in LIMK1 knockout mice. We
found that endothelial permeability in the lungs of LIMK1 -/- mice was lower than that of wild
type mice. Perfusion of the lungs of wild type mice with PAR1 peptide showed significant
increase of endothelial permeability. Notably, the endothelial permeability of the lungs of LIMK1
-/- mice after PAR1 peptide perfusion was significantly lower than that of wild type. Acute lung
injury (ALI) is a syndrome of acute respiratory failure that results from acute pulmonary edema
and inflammation. Using lipopolysaccharide (LPS) injection as a model of ALI, we have shown
that LIMK1 (-/-) mice did not develop lung edema and showed significantly reduced mortality
as compared with wild type mice. Our findings indicate that LIMK1 coordinates microtubules
and actin cytoskeleton. We suggest that the loss of LIMK1 protein leads to less permeable
pulmonary blood vessels.
50
Distinct eNOS Regulation by Protease-activated Receptors Involving G12/13
and Rho-kinase
Heigoro Shirai, Hiroyuki Suzuki, Kunie Eguchi, Sadaharu Higuchi, Temple Univ Sch of
Medicine, Philadelphia, PA; Evangeline D Motley, Meharry Med College, Nashville, TN;
Satoru Eguchi; Temple Univ Sch of Medicine, Philadelphia, PA
Protease-activated receptors (PARs), such as PAR-1 and PAR-2 have been implicated in the
regulation of endothelial nitric oxide (NO) production. We hypothesized that PAR-1 and PAR-2
distinctly regulate the activity of endothelial NO synthase (eNOS) through the selective
phosphorylation of a positive regulatory site, Ser1179 and a negative regulatory site, Thr497.
In bovine aortic endothelial cells (BAECs), a PAR-1 ligand, TFLLR, phosphorylated eNOS at
Thr497. It had no effect on Ser1179 phosphorylation or cyclic GMP (cGMP) production. In
contrast, a PAR-2 ligand, SLIGRL, phosphorylated Ser1179 with no noticeable effect on Thr497.
SLIGRL stimulated cGMP production that was blocked by L-NAME. Neither eNOS phosphory-
lation nor cGMP production was observed by a PAR-4 agonist, AY-NH2. Thrombin has been
shown to transactivates PAR-2 through PAR-1. Thus, thrombin stimulates eNOS phosphoryla-
tion at both sites as well as cGMP production in BAECs. Importantly, SLIGRL-induced Ser1179
phosphorylation and cGMP production were inhibited by a Gq inhibitor, YM-254890. YM-
254890 also blocked thrombin-induced cGMP production and eNOS phosphorylation at
Ser1179 but not Thr497. In addition, eNOS phosphorylation was not affected by Gi inhibitors
(over-expression of GRK2 C-tail and pertussis toxin pretreatment). By contrast, TFLLR-induced
Thr497 phosphorylation was selectively inhibited by a Rho-kinase (ROCK) inhibitor, Y-27632
and over-expression of p115RhoGEF RGS domain, a selective G12/13 inhibitor. Thus, TFLLR but
not SLIGRL stimulated Y-27632-inhibitable phosphorylation of MYPT and MLC, the downstream
components of Rho/ROCK in BAECs. We also confirmed TFLLR-induced activation of G12/13
and Rho by pull-down assays. From these data, we conclude that PAR-1 and PAR-2 distinctly
regulate eNOS activity through Gq and G12/13/ROCK, respectively, delineating the novel
signaling pathways by which proteases regulate eNOS activity.
51
-Arrestin-2 Is Required for B1 Kinin Receptor-dependent Activation of
iNOS
Frank Kuhr, Univ of Illinois College of Medicine, Chicago, IL; Yongkang Zhang, Viktor
Brovkovych, Fulong Tan, Randal A Skidgel; Univ of Illinois College of Medicine, Chicago, IL
Kinins have wide ranging effects on vascular homeostasis, especially in inflammatory
conditions. Des-Arg
9
-bradykinin and des-Arg
10
-kallidin (DAKD) are endogenous peptide B1
receptor (B1R) agonists and angiotensin converting enzyme (ACE) inhibitors can also directly
activate B1Rs. We recently identified a novel endothelial B1R signaling pathway that activates
the extracellular-signal regulated kinase (ERK), which then phosphorylates inducible nitric oxide
synthase (iNOS). iNOS phosphorylation activates it beyond its normal high output mode to
generate super-high output NO that enhances endothelial barrier function. It was recently
reported that prolonged G protein-coupled receptor activation of ERK can depend on
-arrestin-mediated signaling and -arrestin is known to bind ERK. We tested the hypothesis
that B1R-dependent NO production is dependent on -arrestin-2 recruitment and scaffolding
of ERK and iNOS. HEK293 cells stably expressing B1Rs were transfected with iNOS cDNA and
arrestin-2 specific siRNA and NO production in response to B1R activation was measured in
real time with a porphyrinic electrode. -arrestin-2 siRNA (40 - 180 pmol) inhibited NO
production stimulated by 100nM DAKD by up to 85% in a concentration-dependent manner.
iNOS and -arrestin-2 were colocalized in the cells as determined by confocal imaging and
colocalization intensified after 5 or 10 min stimulation with 100 nM DAKD. Immunoprecipitation
of iNOS co-immunoprecipitated -arrestin-2 in resting cells and the coimmunoprecipitation
increased after B1R stimulation with 100 nM DAKD. These data show that the B1R signaling
pathway leading to iNOS activation is dependent on -arrestin-2. The results are consistent
with the function of -arrestin-2 as a scaffolding protein, bringing ERK and iNOS together to
facilitate iNOS phosphorylation, activation and production of super-high output NO. This B1R
signaling pathway may be important in regulating endothelial barrier function during
inflammation and may also play a role in the beneficial therapeutic effects of ACE inhibitors,
which can also act as direct agonists of B1Rs.
52
Rap1b-deficiency Leads to Decreased Angiogenesis, Endothelial Cell
Proliferation, Migration, and MAPK Signaling
Magdalena Chrzanowska-Wodnicka; Blood Rsch Institute, Milwaukee, WI
Introduction Small GTPase Rap1 regulates basic cellular functions: cell-matrix and cell-cell
adhesion, migration, differentiation and growth. We described murine Rap1b-knockout (KO)
adult phenotype, which includes a mild platelet aggregation defect and protection from
thrombosis in vivo. More striking is the embryonic phenotype, as the majority of Rap1b KO mice
suffer from embryonic bleeding resulting in embryonic and perinatal mortality. Hypothesis The
severity of the embryonic defect, a smaller size of surviving KO mice and the fact that platelets
are not required for embryo hemostasis indicated a defect in vascular development. However,
gross vascular patterning of KO embryos was normal. Thus, we assessed the hypothesis that
Rap1b-KO mice suffer a defect in angiogenesis, the main mechanism of vascular remodeling
during late development and after birth. Methods Angiogenesis in vivo was analyzed in a
neonatal retinal model. To determine if the defect is inherent in blood vessels, aortic ring
sprouting assay was performed. Angiogenic responses of endothelial cells (ECs) were
assessed: proliferation in vivo and migration and biochemical activation of primary lung ECs in
response to VEGF and bFGF in vitro. Specifically, phosphorylation-dependent activation of p38
MAPK and p42/44 ERK, two kinases critical for EC migration and growth, was analyzed. Results
Vascularization of KO retinas was decreased to 76.7 8.4% of the littermate controls (n5).
Ex vivo, the number of microvessels was dramatically reduced in KO aortas compared to
controls in response to both, VEGF (1.2 1.1 vs.7.7 4.3; n8) and bFGF (1.7 2.2 vs.
13.4 6.9; n6), indicating an endothelial defect. The number of dividing ECs was
significantly reduced in KO vs. normal retinas (284 75 vs. 543 101; n6; p0.067). In
vitro migration of KO LECs was significantly delayed (p0.0234, 0.025, n9; paired
t-test). p38 MAPK phosphorylation was decreased in KO LECs in response to both growth
factors and ERK phosphorylation was decreased in response to VEGF. Conclusions In
conclusion, Rap1b deficiency leads to decreased proliferation and angiogenesis in vivo. Rap1b
is required for normal migration of endothelial cells and, at the molecular level, regulation of
p38MAP and p42/44pERK activation.
53
Defining the Role of Sprouty1 as an Inhibitor of Angiogenesis
Sangjin Lee, Tri M Nguyen, Sundaram Ramakrishnan, Univ of Minnesota, Minneapolis, MN;
Robert Friesel, Maine Med Cntr Rsch Institute, Scarborough, ME; Jennifer L Hall; Univ of
Minnesota, Minneapolis, MN
The overall goal of this study was to identify novel conserved tyrosine kinase signaling
pathways inhibiting angiogenesis. Our laboratory identified the receptor tyrosine kinase
Sprouty1 from a set of gene expression profiles in human tissue. We screened this
compendium for targets containing hypoxia inducible factor-1 alpha (HIF-1a) binding sites. A
promoter analysis revealed three putative HIF-1a binding sites in the Sprouty1 gene. Using
human umbilical vein endothelial cells (HUVECs), Sprouty1 protein expression was significantly
increased after exposure to 0.2% oxygen for 24 hours. Sprouty1 overexpression significantly
inhibited tubule formation of HUVECs on matrigel vs. adenoviral infected controls (n6,
p0.01). Similarly, there was a significant decrease in cellular proliferation in Sprouty1
infected HUVECs, as determined by Brdu incorporation (n6, p0.01). Interestingly, Sprouty1
is sufficient to increase p21 protein expression, a well-known cell cycle inhibitor. HUVECs under
hypoxic conditions (0.2% oxygen) demonstrated a significant increase in p21 protein
expression at 24 hours. In summary, we have identified a novel conserved tyrosine kinase
signaling pathway that inhibits angiogenesis and may have important therapeutic implications
in cardiovascular disease.
54
OxLDL-pulsed Dendritic Cells: An Immunotherapy in Atherosclerosis
Johan Kuiper, Kim L.L. Habets, Gijs H.M. van Puijvelde, Eva J.A. van Wanrooij,
Biopharmaceutics, Leiden, Leiden, The Netherlands; Rene E. Toes, LUMC, Leiden, Leiden,
The Netherlands; Theo J.C. van Berkel; Biopharmaceutics, Leiden, Leiden, The Netherlands
Modification of lipoproteins plays an important role in the development of atherosclerosis.
Oxidatively modified LDL (oxLDL) has a number of pro-inflammatory effects, whereas
immunization with various forms of oxLDL is able to reduce atherosclerosis. The uptake of
modified LDL by dendritic cells (DCs) and the presentation of epitopes thereof may form an
important step in the immunomodulatory effects of modified LDL. In this study we transferred
oxLDL-pulsed dendritic cells (DCs) to LDLr
-/-
mice and examined the effects of these cells on
atherosclerosis. Bone marrow derived DCs (DCs) were cultured for 10 days in the presence of
granulocyte/macrophage-colony stimulating factor (GM-CSF). Immature DCs were pulsed with
cupper oxidized LDL (Cu-oxLDL) and further matured by TLR4 activation. LDLr
-/-
mice were
injected (3 times, every other day) with 1.5 x10
6
mature DCs (n11), oxLDL-pulsed mature
DCs (n11) or were treated with saline (n8) before induction of atherosclerosis by Western
type diet feeding and subsequent perivascular collar placement. Mice were sacrificed 8 weeks
after collar placement. Transfer of oxLDL-pulsed DCs resulted in a 91.7 % and 87.5 %
reduction in carotid artery lesion size and 87.4% and 84.7 % reduction of intima/lumen ratio
compared to the PBS-treated or mature DCs-treated groups, respectively (p0.01). The
reduction in atherosclerosis was accompanied by a 3.8 fold increase (p0.05) in Cu-oxLDL
specific IgG titers in mice treated with oxLDL-pulsed DCs, whereas the levels of anti-MDA-LDL
IgG and IgM were not significantly affected. Initially, treatment with oxLDL-pulsed DCs did not
2007 ATVB Oral Presentations e-45
by on March 25, 2011 atvb.ahajournals.org Downloaded from
affect serum cholesterol levels up to 3 weeks of diet. However, after 10 weeks of diet there
was a significant 23.7% reduction in serum cholesterol levels compared to the groups treated
with mDCs (p0.05). We conclude that oxLDL-pulsed DCs induce the proliferation of memory
cells which are responsible for the enhanced production of anti-oxLDL IgG. Due to these higer
titers, the immunostimulatory effects of oxLDL on the arterial wall are inhibited as shown by
the highly significant reduction in lesion size. These data indicate that vaccination with
oxLDL-pulsed DCs provides a novel and powerful strategy in the treatment of atherosclerosis.
55
Differential iPLA
2
and cPLA
2
Signaling Regulates Directed Migration of
Monocyte to MCP-1: Validation in a Novel Mouse Model
Ravi S Mishra, Cleveland Clinic, Cleveland, OH; Katherine Preston, Univ of Cambridge,
Cambridge , United Kingdom; Martha K Cathcart; Cleveland Clinic, Cleveland, OH
Monocyte chemoattractant protein-1 directs migration of monocytes from the peripheral blood
to sites of inflammation or injury. Contributions of known signaling molecules to the kinetics of
directed migration have not been rigorously addressed and few studies address whether in vitro
observations obtained under controlled conditions can indeed be substantiated in dynamic in
vivo situations. Earlier we reported that iPLA
2
and cPLA
2
are both required for monocyte
chemotaxis to MCP-1 and act in parallel. We also proposed that due to the identity of different
end products and inability to influence each others activity, these enzymes might regulate
distinct characteristics of migrating monocytes, probably, from different intracellular locations.
In this study, we report that MCP-1 induces recruitment of these two phospholipases to
different intracellular locations; iPLA
2
is preferentially recruited to the pseudopod and cPLA
2
to the endoplasmic reticulum. This differential spatial distribution is manifested also in their
functional independence. Monocytes deficient in cPLA
2
displayed reduced speed, whereas in
contrast, iPLA
2
-deficient monocytes make wider and more frequent turns as well as exhibiting
reduced speed. Thus, iPLA
2
provides a directional clue or compass supporting migration
toward the MCP-1. We further validated the important roles of these phospholipases in
migration of monocytes in vivo using a newly developed mouse model. Adoptively transferred
murine monocytes, if rendered deficient in either of these phospholipases by their specific
antisense oligonucleotides, displayed profound defects in migration to the peritoneum in
thioglycolate-induced peritonitis, a MCP-1-dependent process. We have identified a previously
unknown function of iPLA
2
as a cellular compass and present a new approach for evaluating
the relevant contributions of signaling molecules in regulating monocyte chemotaxis to MCP-1,
in vivo.
56
Macrophage Phenotype in Atherosclerotic Human Coronary Arteries Is
Different from Macrophage Phenotype in Normal Human Coronary Arteries
Chiara Buono, NIH, Bethesda, MD; Stephen W Waldo, NIH/Howard Hughes Med Institute,
Bethesda, MD; Janet Chang, Yifu Li, Howard S Kruth; NIH, Bethesda, MD
We previously characterized the divergent atherogenic potential of human monocyte-derived
macrophages differentiated with either M-CSF (M-Mac) or GM-CSF (GM-Mac) in vitro. Gene
expression analysis and cytokine secretion assays indicated significant differences between the
two macrophage phenotypes related to inflammation and cholesterol homeostasis. M-Mac
appear to be more inflammatory while GM-Mac show increased expression of genes related to
cholesterol efflux. Immunocytochemistry showed that both macrophage phenotypes express
the pan-macrophage marker CD68 while only M-Mac express the pro-inflammatory marker
CD14. In the present study, fluorescence double immunostaining of CD68 and CD14 was used
to determine the macrophage phenotype in normal and atherosclerotic human coronary
arteries. In accordance with our in vitro findings, CD68/CD14cells were considered similar
to the M-Mac phenotype and CD68/CD14- cells were considered similar to the GM-Mac
phenotype. Both GM-Mac and M-Mac phenotypes were present in normal intimal regions and
atherosclerotic lesions of coronary arteries. Quantification of immunostained cells showed that
M-Mac were more prevalent in atherosclerotic lesions (75 4%) compared with normal
intimal regions of coronary arteries (39 6%). In contrast, GM-Mac were more prevalent in
normal intimal regions (54 1%) compared with atherosclerotic lesions of coronary arteries
(21 5%). The immunostaining also revealed the presence of CD68/CD14 cells
throughout the adventitia in both normal and diseased regions of the coronary arteries. Cells
with CD68/CD14 immunolabeling were not present within the media of normal coronary
artery. However, media of human coronary arteries with advanced atherosclerotic lesions
showed apparent migration of CD68/CD14cells from the adventitia through the media into
the intima. The predominance of M-Mac in atherosclerotic lesions suggests that this
macrophage subpopulation promotes development of these lesions. The identification of
macrophage phenotypes with different gene expression patterns and different potentials for
promoting atherosclerosis has important experimental and clinical implications.
57
CXCR6 Promotes Atherosclerosis by Supporting T-cell Homing, Interferon
Production, and Macrophage Accumulation in the Aortic Wall
Elena Galkina, Brian Harry, Univ of Virginia, Charlottesville, VA; Andreas Ludwig, Elisa A
Liehn, Christian Weber, Institute of Molecular Cardiovascular Rsch, Aachen, Germany; Klaus
Ley; Univ of Virginia, Charlottesville, VA
T cells are important in atherosclerosis, but very little is known about the mechanisms behind
lymphocyte recruitment into atherosclerosis-prone aortas. We tested the hypothesis that
CXCR6, a chemokine receptor that is expressed on a subset of T cells and natural killer T cells,
is involved in lymphocyte homing into the aortas and modulates atherosclerosis. To investigate
the role of CXCR6 in atherosclerosis, we bred CXCR6-deficient (CXCR6
GFP/GFP
) mice with
apolipoprotein E-deficient (ApoE
-/-
) mice. CXCR6
GFP/GFP
/ApoE
-/-
mice fed a western diet for 17
weeks or a chow diet for 56 weeks had decreased atherosclerosis compared with ApoE
-/-
controls. Flow cytometry analysis of the aortas from CXCR6
GFP/GFP
/ApoE
-/-
mice showed that the
reduction of atherosclerosis was accompanied by a decreased percentage of CXCR6

T cells
within the aortas. Short-term homing experiments demonstrated that CXCR6 is involved in the
recruitment of CXCR6

leukocytes into the atherosclerosis-prone aortic wall. The reduced


percentage of CXCR6

T cells within the aortas was associated with diminished production of


IFN and reduced accumulation of CD11b

/CD68

macrophages in the aortas. To address the


potential importance of our finding for human atherosclerosis, we examined the expression of
CXCR6 in human carotid endarteriectomy specimens and found CXCR6 expressing T cells and
some Moma-2

/CXCR6

within the analyzed carotid tissues. These data provide evidence for
a functional role of CXCR6 in atherosclerosis by altering of the recruitment of CXCR6

leukocytes and modulating of the local immune response within the aorta.
58
Serum Amyloid A Is an Endogenous Ligand for Toll-like Receptor 2
Ni Cheng, Rong He, Richard D Ye; Univ of Illinois at Chicago, Chicago, IL
Serum amyloid A (SAA) is an apolipoprotein produced by hepatocytes during acute-phase
response and by other inflammatory cells such as macrophages. SAA is an important clinical
indicator of atherosclerosis and other inflammatory diseases, and contributes to cholesterol
metabolism through its binding to high-density lipoprotein (HDL) and scavenger receptors. In
phagocytes, SAA is a potent inducer of proinflammatory cytokines such as MCP-1, IL-1beta,
IL-8 and IL-12p40. The observation that SAA could induce cytokine production independently
of formyl peptide receptor-like 1 and the scavenger receptor SR-BI led us to propose the
presence of another receptor. To assess this hypothesis, we analyzed the cytokine production
profiles of SAA-stimulated monocytic cells and identified similarities with the gene induction
profile of Toll-like receptors (TLRs). Evidence for the involvement of a TLR includes the findings
that expression of a dominant negative MyD88 significantly reduced SAA-stimulated NF-B
activation in monocytic cells, and that an antibody recognizing the extracellular domain of TLR2
significantly reduced SAA-induced cytokine production. In transfection experiments, cells that
fail to respond to SAA acquired the ability to activate NF-B upon SAA stimulation after
exogenous expression of TLR2. Finally, incubation with HDL reduced SAA-stimulated transcrip-
tional activation and cytokine production. In conclusion, these results indicate that SAA is an
endogenous ligand for TLR2. Given the increased production of SAA at the site of
atherosclerotic lesion and the potential involvement of TLR2 in atherogenesis, we predict that
SAA plays a role in the development of atherosclerosis in part through TLR2 activation.
59
Glutamate Mediates Platelet Activation Through the AMPA Receptor
Craig N Morrell, Henry Sun, Nauder Faraday, Oguz Gozen, Anne Marie Swaim, Tanika
Martin, Richard Huganir, Charles J Lowenstein; Johns Hopkins, Baltimore, MD
Glutamate is an excitatory neurotransmitter in the central nervous system (CNS) that binds to
the kainate receptor, the N-methyl-D-aspartate (NMDA) receptor, and the a-amino-3-hydroxy-
5-methyl-4-isoxazolepropionic acid (AMPA) receptor (AMPAR). Each receptor was first char-
acterized and cloned in the CNS. Glutamate is also present in the periphery, and glutamate
receptors have been identified in non-neuronal tissues, including bone, heart, kidney, pancreas,
and platelets. Platelets have a central role in normal thrombosis and hemostasis as well as
contributing to disease processes such as stroke and myocardial infarction. But the role of
glutamate during hemostasis is unknown. We now show that glutamate binding to the AMPAR
on platelets modulates platelet activation and thrombosis. Platelets contain and release
glutamate, and glutamate increases agonist induced platelet activation. AMPAR subunits GluR1
and GluR2 are expressed on platelets. Glutamate or AMPA increases intracellular sodium
concentration, an important step in platelet activation. In contrast, platelets treated with the
AMPAR antagonist CNQX, or platelets derived from GluR1 knockout mice are resistant to AMPA
actions ex vivo. Furthermore, mice lacking GluR1 have a prolonged time to platelet adhesion
and thrombosis in vivo. Our data identify glutamate as a regulator of platelet activation, and
suggest that the AMPA receptor is a novel anti-thrombotic target.
60
Hematopoietic Lineage Cell-specific Protein-1 (HS1) Is an Important
Signaling Molecule Downstream from Protease-activated Receptors and Is
Involved in Multiple Signaling Pathways of Platelet Activation
Bryan N Kahner, Robert T Dorsam, Soochong Kim, Satya P Kunapuli; Temple Univ Sch of
Medicine, Philadelphia, PA
Injury to a blood vessel exposes subendothelial collagen and initiates the coagulation cascade
producing thrombin. We hypothesize that hematopoietic lineage cell-specific protein-1 (HS1),
a 75 kDa tyrosine phosphorylated adapter protein, expressed in cells exclusively of hemato-
poietic lineage, is a critical regulator of platelet activation. Here we investigate the role of HS1
downstream of GPCRs in PAR receptor signaling pathway. We observed both a bleeding
diathesis and an inhibition of thrombus formation by the in vivo FeCL
3
thrombosis model,
indicating HS1s involvement in multiple pathways. HS1 phosphorylation occurs downstream of
both PAR-1 and PAR-4, in a Gq dependent manner, however, ADP secretion has no affect on
HS1 phosphorylation. HS1 is phosphorylated initially by Syk tyrosine kinase on tyrosine residues
397 and 378. Syk then dissociates from HS1, allowing for docking and subsequent
phosphorylation of HS1 by Src family kinases on tyrosine residue 222. We demonstrate that Src
family kinase inhibitors abolish the activation of Syk, as measured by its phosphorylation on
tyrosine residues 525/526, and also completely block HS1 phosphorylation. This is in contrast
to the previously reported results indicating that inhibition of Src family kinases results in partial
HS1 phosphorylation, possibly on the Syk phosphorylated residues. We propose that Src is
directly downstream of PAR receptors and phosphorylates Syk, which in turn phosphorylates
HS1. Further, studies with HS1 null mouse platelets show an inhibition of aggregation, secretion
and thromboxane A2 generation compared to their wild type littermates. HS1 null mice also
display a decrease in Akt and Erk phosphorylation, signaling molecules shown to be important
in aggregation and thromboxane generation. Taken together with our previous results,
e-46 Arterioscler Thromb Vasc Biol June 2007
by on March 25, 2011 atvb.ahajournals.org Downloaded from
indicating that HS1 is an important modulator in GPVI activation, these data shows that HS1 is
a functionally important adapter molecule involved in multiple pathways of platelet activation.
61
LPS Promotes Platelet Activation via the TLR4 and cGMP-dependent
Protein Kinase II Pathway
Guoying Zhang, Univ of Illinois at Chicago, Chicago, IL; Franz Hofmann, Institute for
Pharmakologie und Toxikologie der Technischen Univ, Munchen, Munchen, Germany;
Xiaoping Du, Zhenyu Li; Univ of Illinois at Chicago, Chicago, IL
Bacterial lipopolysaccharide (LPS) activates Toll-like receptor 4 (TLR4) leading to rapid
thrombocytopenia, hypotension and sepsis. Although growing evidence suggests that platelet
activation plays important roles in LPS-induced thrombocytopenia and tissue damage, the
effect of LPS on platelet activation and the signaling mechanisms of LPS-induced platelet
activation have not been defined. Here we show that LPS significantly enhances platelet
aggregation and secretion induced by low concentrations of platelet agonists. The enhance-
ment of platelet aggregation and secretion by LPS is abolished by an anti-TLR4 blocking
antibody, and platelets express important molecular components of TLR4 signaling, CD14,
MD2, and the adaptor protein MyD88, as well as TLR4, suggesting that the effect of LPS on
platelet aggregation requires TLR4 pathway. LPS alone induces ATP release and P-selectin
expression in human platelets and promotes FeCl3-injured carotid artery thrombus formation.
Furthermore, the enhancement of platelet aggregation by LPS is inhibited in PKG II deficient
platelets as well as specific PKG inhibitor-treated platelets. LPS induces significant increase of
intracellular cGMP levels in platelets. Thus, our data indicate that LPS induces platelet secretion
and promotes platelet aggregation and thrombosis by a TLR4 and PKG II dependent signaling
pathway.
62
CD36 Modulates Platelet Reactivity in Vivo During Hyperlipidemia: A
Mechanism Linking Hyperlipidemia, Oxidant Stress, and a Prothrombotic
Phenotype
Eugene A Podrez, Maria Febbraio, Cleveland Clinic, Cleveland, OH; Robert G Salomon, Case
Western Reserve Univ, Cleveland, OH; Yi Ma, Cleveland Clinic, Cleveland, OH; Eugenia
Poliakov, Case Western Reserve Univ, Cleveland, OH; Manojkumar Valiyaveettil, Cleveland
Clinic, Cleveland, OH; Mingjiang Sun, Case Western Reserve Univ, Cleveland, OH; Brian R
Curtis, Blood Cntr of Wisconsin, Milwaukee, WI; Paula J Finton, Juhua Chen, Renliang
Zhang, Roy L Silverstein, Tatiana V Byzova, Stanley L Hazen; Cleveland Clinic, Cleveland,
OH
Enhanced platelet reactivity is critical to the pathophysiology of occlusive arterial thrombotic
disease. Despite the strong clinical associations between hyperlipidemia, a major risk factor for
atherosclerosis, and a pro-thrombotic phenotype, the mechanisms responsible for enhanced
platelet reactivity during hyperlipidemia remain unknown. Pro-atherosclerotic lipid abnormal-
ities such as hypercholesterolemia are associated with both enhanced oxidant stress and
generation of biologically active oxidized lipids, including potential ligands for the scavenger
receptor CD36, a major platelet surface glycoprotein. Using multiple murine in vivo thrombosis
models and hyperlipidemic atherosclerosis-prone apo E-deficient or LDL receptor-deficient
mice we now demonstrate that these mice form occlusive intravascular thrombi faster than
wildtype mice, and that genetic deletion of CD36 protects mice from hyperlipidemia-associated
enhanced platelet reactivity and accompanying pro-thrombotic phenotype. Structurally defined
oxidized choline glycerophospholipid molecular species that serve as endogenous high affinity
ligands for CD36 are shown to be present in significant amount in human plasma, markedly
increased in plasma of hyperlipidemic mice and to promote platelet activation and alpha-
granule release via CD36 at pathophysiological levels. These studies thus demonstrate that
platelet CD36 interactions with specific endogenous oxidized lipids play a heretofore
unrecognized role in the well-known clinical associations between hyperlipidemia, oxidant
stress and a pro-thrombotic phenotype.
63
NGR: A Novel Fibrinogen Adhesion Motif Mediating Platelet Activation
Roisin D Moriarty, Royal College of Surgeons in Ireland, Dublin, Ireland; Ciara McManus,
Univ College of Dublin, Dublin, Ireland; Dermot Cox; Royal College of Surgeons in Ireland,
Dublin, Ireland
The integrin
IIb

3
on resting platelets can bind to immobilised fibrinogen resulting in platelet
spreading and activation through a process known as outside-in signalling, but requires
activation to bind to soluble fibrinogen.
IIb

3
is known to interact with the general integrin
recognition motif RGD (arginine-glysine-aspartate) as well as the fibrinogen -specific -chain
dodecapeptide. It is not known how fibrinogen binding triggers platelet activation. Recently the
amino acid sequence NGR (asparagine-glysine-arginine) has been identified as an integrin
recognition motif. Since NGR is present on both the 413415 and 254256 chains of
fibrinogen we investigated the potential role of NGR to interact with
IIb

3
. We carried out site
directed mutagenesis to replace each NGR motif with three alanines. Chinese hamster ovary
cells were stably transfected and the recombinant fibrinogens were purified by immunopre-
cipitation. Resting platelet adhesion assays showed that while the mutant fibrinogens
supported platelet adhesion, this was to a reduced extent compared to normal fibrinogen
(-chain: 57.9% 19.0; -chain: 32.3% 20.4 of the control platelet adhesion, p0.01).
Confocal microscopy analysis of platelet adhesion to the mutant fibrinogens showed a distinct
difference in platelet morphology compared to the control fibrinogen. Adherent platelets were
classified as resting (round), activated (spiked) or spread (pancake). On normal fibrinogen
54.8 15.0% platelets spread fully, while platelets that adhered to the mutant fibrinogens
showed reduced levels of spreading (-chain: 11.3% 5.3 and -chain: 23.0% 9.9,
p0.0001). These differences were accounted for by increased levels of resting and activated
platelets with both mutant fibrinogens. Kinetic studies of platelet spreading confirmed these
results at all time points up to 45 min. However, when TRAP-activated platelets were used
morphology analysis showed no difference in platelet spreading between the control and
mutant fibrinogens. Thus, we can conclude that fibrinogen lacking either NGR sequence
supports reduced resting platelet adhesion, however, subsequent activation does not occur.
This suggests a role of the NGR motif in the activation of adherent platelets.
64
Role of the NR4A Orphan Nuclear Receptor NOR1 in Neointima Formation
and Vascular Smooth Muscle Cell Proliferation
Takashi Nomiyama, Florence Gizard, Elizabeth B Heywood, Karrie L Jones, Dennis
Bruemmer; Univ of Kentucky, Lexington, KY
Nuclear hormone receptors comprise a large superfamily of ligand-activated transcription
factors and have emerged as key regulators of glucose metabolism and inflammation in
diabetes and cardiovascular diseases. The neuron-derived orphan receptor-1 (NOR1) belongs
to the ligand-independent NR4A receptor subfamily which has previously been characterized as
important transcriptional regulator of hepatic gluconeogenesis and inflammation. We have
recently demonstrated NOR1 expression in vascular smooth muscle cells (SMC) of atheroscle-
rotic lesions and outlined a key role for NOR1 to regulate SMC proliferation in vitro. In the
present study, we demonstrate that SMC isolated from NOR1-deficient mice exhibit decreased
cell proliferation due to a G2/M arrest of the cell cycle. NOR1-deficiency results in diminished
phosphorylation of the retinoblastoma protein, cyclin D1 and D2 expression and mitogen-
induced degradation of the cyclin-dependent kinase inhibitor p27. Using microarray technology
we further characterize Skp1, Cul1, and Nedd8, key proteins of the ubiquitin ligase complex
required for degradation of p27 through the proteasome pathway, as NOR1 target genes.
Finally, using a model of guide-wire induced arterial injury we observed decreased neointima
formation in NOR1-deficient mice. These experiments demonstrate that NOR1 functions as a
key transcriptional regulator of SMC proliferation and neointima formation by inducing cyclin D1
and the expression of genes required for the ubiquitination and degradation of p27. Therefore,
NOR1 is a key regulator of SMC proliferation and may provide an important molecular target
for the treatment of cardiovascular diseases.
65
Impaired ABCA1 Expression in Intimal-phenotype and Human
Atherosclerotic Lesion Smooth Muscle Cells
Hong Y Choi, Univ of Alberta, Edmonton, Canada; Maziar Rahmani, Brian W Wong, Bruce M
McManus, Univ of British Columbia, Vancouver, Canada; Lindsay Lane, Daniel S Ory,
Washington Univ, St. Louis, MO; J Geoffrey Pickering, Robarts Rsch Institute, London,
Canada; Gordon A Francis; Univ of Alberta, Edmonton, Canada
Smooth muscle cells (SMC) represent the major cell type in human atherosclerotic intima, and,
like intimal macrophages, accumulate excess cholesterol. Epithelioid SMC are the major SMC
phenotype in atherosclerotic intima, while medial arterial SMC have a predominantly spindle
morphology. We tested the hypothesis that epithelioid SMC have an impaired ability to release
lipids to apolipoproteins to form high-density lipoproteins (HDL), resulting in intimal SMC
cholesterol accumulation. Epithelioid (intimal-phenotype) and spindle (medial-phenotype)
morphology SMC from Wistar Kyoto rat arteries were tested for their ability to express
ATP-binding cassette transporter AI (ABCA1), the rate-limiting mediator of HDL formation, and
to bind and release lipids to apolipoprotein A-I (apoA-I). ABCA1 mRNA and protein as well as
apoA-I binding was much lower in basal and cholesterol-loaded epithelioid compared to spindle
SMC. The low expression of ABCA1 in epithelioid SMC was explained by poor production of the
physiological ABCA1 inducer, oxysterols (25- and 27-hydroxycholesterol). Incubation of spindle
morphology SMC with 10 g/mL apoA-I for 16 h removed 70 5% (mean SD) of
cholesterol available for esterification, and after 24 h removed 1012% of total cell plus
medium [
3
H]cholesterol and [
3
H]phosphatidylcholine to the medium of these cells. Generation
of HDL particles by spindle SMC was confirmed by two-dimensional gel electrophoresis. The
same incubations of epithelioid SMC resulted in little or no removal of these lipids to apoA-I,
and very poor generation of HDL particles. Consistent with low ABCA1 expression in rat
intimal-phenotype SMC, in situ hybridization in human coronary artery specimens showed low
levels of ABCA1 mRNA in atherosclerotic coronary artery intima compared to media generally.
Immunohistochemical staining for smooth muscle alpha actin and ABCA1 showed significantly
lower levels of ABCA1 protein in human intimal SMC compared with medial SMC. These
combined in vitro and in vivo results suggest ABCA1 expression and activity is impaired in
intimal SMC, providing a novel explanation for the accumulation of excess cholesterol in the
main cell type of human atherosclerotic lesions.
66
Vascular Smooth MuscleDerived Tissue Factor Is Critical for Arterial
Thrombosis After Fecl3 Injury
Li Wang, Christine Miller, Mohan Rao, Joseph M Miano, Univ of Rochester, Rochester, NY;
Nigel Mackman, The Scripps Rsch Institute, La Jolla, CA; Mark B Taubman; Univ of
Rochester, Rochester, NY
Tissue factor (TF) is a glycoprotein that initiates coagulation, regulates hemostasis and plays
a critical role in mediating arterial thrombosis. In this study, we determined the importance of
vascular smooth muscle cell (VSMC) TF in mediating thrombosis. We also measured levels of
circulating TF in the form of microparticles (MPs). We generated VSMC-specific TF deficient
mice by breeding TF
flox/flox
mice with SM22-Cre
/-
mice. TF mRNA was selectively decreased
in the aorta media of TF deficient mice by 88% as determined by real time PCR, whereas TF
activity was reduced by 70.5% (TF
flox/flox
: 225.67 72.67 pM Xa / min, n 6;
TF
flox/flox
/SM22Cre
/-
: 66.59 54.87 pM Xa / min, n 6). Circulating TF activity was
estimated from MPs prepared by pelleting plasma from the two groups of mice at 200,000 x
g for 1.5 hrs. There were no differences in circulating TF activity (TF
flox/flox
: 12.07 9.5 pM Xa
/ min, n 4; TF
flox/flox
/SM22Cre
/-
: 9.21 5.52 pM Xa / min, n 6; C57BL/6: 10.42 3.12,
2007 ATVB Oral Presentations e-47
by on March 25, 2011 atvb.ahajournals.org Downloaded from
n5; by ANOVA, p 0.76). Both groups of mice also had similar levels of adventitial TF
antigen as measured by immunohistochemistry and had similar tail bleeding time (TF
flox/flox
:
52.8 17.4 sec., n 5; TF
flox/flox
/SM22Cre
/-
: 50.7 18.8 sec., n 6; by t-test, p 0.86).
To examine the role of VSMC-specific TF in mediating arterial thrombosis, we employed ferric
chloride injury of the carotid artery. Occlusion was defined as a stable reduction in flow of
75% for 3 min. 11 / 12 TF
flox/flox
occluded, with a median time of 304 sec. In contrast, only
6 / 16 TF
flox/flox
/SM22Cre
/-
occluded (by Logrank analysis, p 0.001 with hazard ratio
0.2262 for non-occlusion). These data indicate that in a macrovascular model of arterial
thrombosis, such as that seen in human coronaries, VSMC-derived TF is critical for initiation
of arterial thrombosis. In contrast, circulating TF does not appear to be important. This mouse
model should be valuable in determining the relative contribution of VSMC-derived and
circulating TF in other TF-mediated phenomena, such as restenosis.
67
Regulation of Smooth Muscle Cell Proliferation by Hyaluronan and CD44
Devashish Kothapalli; Univ of Pennsylvania Sch of Medicine, Philadelphia, PA
High molecular weight hyaluronan (HMW-HA) is a widely distributed component of the ECM, but
its biological activities remain incompletely understood. We previously reported that HMW-HA
binding to CD44 antagonizes mitogen-induced S phase entry in cultured vascular smooth
muscle cells; we now characterize the underlying molecular mechanism and document its
relevance during vascular injury in vivo. In particular, we show that HMW-HA inhibits the
mitogen-dependent induction of cyclin D1 and degradation of p27kip1 in vascular smooth
muscle cells. These effects were associated with an inhibition of Rb phosphorylation, cyclin A
induction, and S phase entry. p27kip1 mRNA levels were unaffected by HMW-HA, but the
expression of Skp2, the rate-limiting component of the SCF complex that degrades p27kip1,
was reduced. Rescue experiments identified cyclin D1 as the primary target of HMW-HA.
Similar effects were detected in fibroblasts. These effects were not detected in vascular smooth
muscle cells isolated from CD44-null mice. Moreover, analysis of arteries from wild-type and
CD44-null mice showed that the effects of HMW-HA/CD44 on cyclin D1 and Skp2 expression
are detected in vivo and associated with altered smooth muscle cell proliferation after vascular
injury. Our data indicate that HMW-HA is anti-mitogenic for multiple mesenchymal cell types
and identify cyclin D1 as a major target of HMW-HA binding to CD44.
68
The CX
3
C Chemokine Fractalkine Acts via an Extracellular Regulated
Kinase-Dependent Mechanism to Induce Human Coronary Artery Smooth
Muscle Cell Proliferation
Gemma E White, Alison E John, David R Greaves; Univ of Oxford, Oxford, United Kingdom
Chemokines are important mediators of cell adhesion and migration that are expressed in both
normal and pathological conditions and signal through G protein-coupled receptors. Fractalkine
(CX
3
CL1) is an atypical membrane-bound chemokine that signals through its receptor CX
3
CR1
and has been implicated in the development and progression of atherosclerosis. We have
previously reported that CX
3
CR1 is expressed by smooth muscle cells in human coronary artery
atherosclerotic plaques and by primary human coronary artery smooth muscle cells (CASMC),
where it mediates chemotaxis towards CX
3
CL1. We assessed the hypothesis that CX
3
CL1 may
also function as a mitogen for human CASMC in vitro. Experiments were replicated using cells
from three independent donors and a CASMC line, HCM-601EB. Using three independent
methods to measure cell proliferation, we now demonstrate that CX
3
CL1 induces primary
human CASMC proliferation via a G
i
dependent pathway in vitro. CX
3
CL1 induces phosphor-
ylation of Extracellular Regulated Kinase (ERK) 1/2 and Akt in CASMC and specific inhibitors of
either the ERK or phosphoinositide-3-kinase (PI3K) pathways abrogate CX
3
CL1-induced
proliferation. Furthermore, CX
3
CL1 synergises with submaximal concentrations of Platelet
Derived Growth Factor-BB (PDGF-BB) to induce CASMC proliferation. A pro-inflammatory role
for CX
3
CL1 in monocyte recruitment in atherosclerosis is well documented. However, our data
suggest that CX
3
CL1 may have an additional role in CASMC proliferation, which could
potentially lead to the formation of more stable plaques with higher smooth muscle cell content.
This may have relevance in other vascular pathologies including restenosis and transplant
vascular disease.
69
Adipocyte-specific Low-density Lipoprotein ReceptorRelated Protein-1 as
a Novel Regulator of Adiposity, Energy Expenditure, and Glucose
Metabolism
Susanna Hofmann, Univ of Cincinnati, Cincinnati, OH; Li Zhou, Univ of Texas Southwestern
Med Ctr, Dallas, TX; Diego Perez-Tilve, Todd Greer, Erin Grant, Lauren Wancata, Andrew
Thomas, Paul Pfluger, Joshua Basford, Univ of Cincinnati, Cincinnati, OH; Joachim Herz,
Univ of Texas Southwestern Med Ctr, Dallas, TX; Matthias H Tschoep, David Y Hui; Univ of
Cincinnati, Cincinnati, OH
Mouse models resulting from tissue-specific gene disruption have established adipose tissue
as an essential endocrine organ for the control of glucose homeostasis and energy balance. The
multifunctional receptor LRP-1 is expressed in adipose tissue where it mediates cellular
cholesterol uptake. Herein we used the adipose tissue-specific LRP-1 knockout mouse model
(adLRP1KO) to test the hypothesis that adipocyte LRP-1 plays an essential role in lipid storage
and energy metabolism. Adipocyte-specific LRP1 inactivation in mice (n12) resulted in
delayed postprandial lipid clearance (area under the curve (AUC): 31925 /- 6397 vs. 16612
/- 1949, P 0.05), lower body weights (BW: 22.4 /- 1.4 vs. 28.3 /- 0.8 g, P 0.001),
smaller fat stores (4.3 /- 0.5 vs. 9.5 /- 1.4 % fat of BW, P 0.002), lipid-depleted brown
adipocytes, improved glucose tolerance (AUG: 20016 /- 517 vs. 27251 /- 1681, P 0.05),
elevated energy expenditure (1007 /- 44 vs. 863 /- 25 kcal/kg/96hr, P 0.002), and
increased food intake (1.5 /- 0.2 vs. 1.1 /- 0.1 g/g BW/day, P 0.05). The slightly higher
caloric intake may represent a compensatory phenomenon. Intriguingly, such increased
thermogenesis in adLRP1KO mice was confirmed by increased body temperature and
paralleled by muscle shivering, quantified using a multi-dimensional light beam system to
analyze motor activity (7102 /- 389 in KO vs. 4013 /- 333 WT beam strokes/36h, P
0.001). Additional radiolabeled studies revealed that glucose and lipid uptake were significantly
increased in skeletal muscle of adLRP1KO mice compared to WT mice (glucose: 12830 /-
651 vs. 8835 /- 1203 dpm/lean mass, P 0.01; oleic acid: 6824 /- 662 vs. 4791 /- 595
dpm/lean mass, P 0.05), suggesting that skeletal muscle compensates for decreased
thermogenic function of BAT in adLRP1KO mice, which may be reflected in the shivering
phenotype. When placed for 4 weeks on high fat diet adLRP1KO mice were resistant to diet
induced obesity (11.2 /- 1.5 vs. 22.2 /- 2.7 % fat of BW, P 0.002) and glucose
intolerance (AUC: 23150 /- 1295 vs. 30920 /- 2074, P 0.005). We conclude that
reducing lipid transport to adipocytes and enhancing muscular energy expenditure via
adipocyte LRP1 inhibition may be an efficient strategy to prevent diet-induced obesity and
diabetes.
70
In Vivo Kinetic of C-Reactive Protein and Its Relationship with Features of
the Metabolic Syndrome in Men and Women
Jean-Francois Mauger, Patrick Couture, Marie-Eve Paradis, Jose e Le vesque, Laval Univ,
Ste-Foy, Canada; Nathalie Bergeron, Touro Univ, Cypress, CA; Benot Lamarche; Laval Univ,
Ste-Foy, Canada
BACKGROUND Individuals with elevated (3.0 mg/l) plasma C-reactive protein (CRP) levels, a
key feature of the metabolic syndrome, are at greater risk for cardiovascular disease than
individuals with low levels of CRP (1.0 mg/l). The in vivo kinetics of CRP and the physiological
mechanisms responsible for the observed sub-acute-phase circulating CRP levels in the
metabolic syndrome and obesity are virtually unknown. Here we describe for the first time the
intravascular kinetic of CRP and its relationship with features of the metabolic syndrome.
METHODS Sixteen men and 16 women (aged 499 years, BMI28.74.5 kg/m
2
) underwent
a 12-hour primed constant infusion of D3-L-leucine in the constant fed state. Blood samples
were drawn at pre-determined time points. CRP was purified from the plasma fraction d1.25
g/ml at each time point by affinity chromatography followed by SDS-PAGE. Isotopic enrichment
was determined by GC-MS. Plasma CRP levels were measured with high sensitivity using a
commercial ELISA. RESULTS Mean CRP production rate (PR) and pool size (PS) were similar
between men and women (0.030 0.026 vs. 0.032 0.039 mg/d and 4.66 3.39 vs.
4.64 3.94 mg respectively). However, the fractional catabolic rate (FCR) of CRP in men was
60% higher than in women (0.57 0.29 vs. 0.35 0.20 pool/day, P0.05). Circulating CRP
concentrations were more strongly correlated with its PR (r0.91, P0.0001) than its FCR
(r0.55, P0.005). PR of CRP was directly correlated with BMI (r0.42, P0.02), waist girth
(r0.44, P0.02), and with plasma LDL apoB-100 (r0.38, P0.05), triglyceride (r0.35,
P0.05) and interleukine-6 levels (r0.50, P0.05). An inverse trend was also observed
between the PR of CRP and HDL-C (r-0.34, P0.06) while LDL-C and blood pressure showed
no association with CRP kinetics. CONCLUSIONS Men were characterised by a 60% greater
FCR of CRP compared with women. However, sub-acute-phase CRP levels appeared to be
mainly explained by the PR of CRP rather than its FCR. Our results also suggest that features
of the metabolic syndrome may be more predictive of CRP kinetics than traditional risk factors
like LDL-C and hypertension.
71
Reduced Macrophage Infiltration in Visceral Adipose Tissue of
12-Lipoxygenase Knockout Mice
Jerry L Nadler, Hong Pei, Melissa Bevard, Anthony Bruce; Univ of Virginia Sch of Medicine,
Charlottesville, VA
Obesity induces the accumulation of macrophages in adipose tissue and generates a state of
low-grade inflammation which is associated with the development of type 2 diabetes and
release of pro-atherogenic cytokines such as tumor necrosis factor alpha (TNF-) and
interleukin 6 (IL-6). Our recent studies indicate that 12-Lipoxygenase (12-LO) expression is
increased in isolated visceral adipocytes in models of insulin resistance and that deletion of
12-LO prevents the increases in TNF- and IL-6 during high fat feeding. The aims of this study
were to examine the role of 12-LO pathway in the accumulation of macrophages in adipose
tissue of mice fed a high-fat diet. C57BL/6J (B6) and 12-LO knockout (12-LO KO) mice on the
B6 background were fed chow diet or Western diet for 12 and 24 weeks. The macrophages
in visceral fat pad were examined by FACS and immunohistochemistry. Macrophage content in
fat with chow feeding was similar in 12-LO KO and B6 mice. Western diet significantly
increased macrophage content in fat tissue compared with chow diet in B6 mice. However,
there was significantly reduced macrophage content in 12-LO KO mice compared to B6 mice
as shown in the table below. After the mice were fed Western diet for 24 weeks, the percentage
of positive Mac2 staining within visceral fat pad was markedly increased in B6 mice compared
to 12-LO KO mice (0.58 0.07 % vs. 0.26 0.03 % in male mice, respectively, P 0.01;
0.32 0.04 % vs. 0.18 0.02 % in female mice, respectively; P 0.01). These data suggest
that 12-LO plays a key role in regulating macrophage trafficking and inflammation in visceral
fat in states of obesity. Therefore blockade of 12-LO could provide a novel therapeutic approach
to reduce the inflammatory state with visceral adiposity.
MACROPHAGES (X1000) PER GRAM ADIPOSE TISSUE
Chow diet
(12 weeks)
Western diet
(12 weeks)
Chow diet
(24 weeks)
Western diet
(24 weeks)
B6 mice Male 12.451.82 38.645.64 15.892.56 54.327.21
12-LO KO mice Male 11.481.96 21.543.98* 14.932.57 31.335.13*
*P0.05 vs. B6 mice
e-48 Arterioscler Thromb Vasc Biol June 2007
by on March 25, 2011 atvb.ahajournals.org Downloaded from
72
Fatty Acid Desaturase Gene Expression in Human Adipose Tissue Is
Regulated by Dietary Composition Independent of Energy Restriction and Is
Correlated with Plasma Triglyceride Response
Lara M Mangravite, Childrens Hosp of Oakland Rsch Institute, Oakland, CA; Kevin Dawson,
Univ of California, Davis, Davis, CA; Ryan R Davis, Jeffrey P Gregg, Univ of California, Davis
Sch of Medicine, Sacramento, CA; Ronald M Krauss; Childrens Hosp of Oakland Rsch
Institute, Oakland, CA
Atherogenic dyslipidemia, associated with elevated triglycerides and reduced HDL, is
independently improved by dietary energy restriction and reduced carbohydrate consump-
tion in an equivalent but non-additive manner. We assessed the hypothesis that total
energy restriction and isoenergetic alteration in dietary composition regulate common
molecular pathways involved in lipid metabolism by monitoring transcriptional expression
in human adipose tissue. Subcutaneous adipose biopsies were obtained from 131
moderately overweight, otherwise healthy men (BMI, 29.22.0 kg/m
2
) following (1) one
week on basal diet (54:15:30[7], carbohydrate:protein:fat[saturated fat]), (2) three weeks
on randomized diet differing in nutritional composition (basal, 39:29:31[8], 26:29:46[9] or
26:29:46[15]), (3) five weeks of acute weight loss on randomized diet (-1103.0216.5
kcal/d resulting in -10.03.3 lb), and (4) four weeks stabilized at reduced weight.
Transcriptional responses were characterized using genome-wide expression array
analysis on samples from thirteen subjects and findings for the most responsive genes
were confirmed using real time PCR across all subjects. Energy restriction resulted in
significantly reduced expression of 1473 transcripts and, of these, 30 were also responsive
to isoenergetic alterations in dietary composition. Twelve of these genes are involved in
energy metabolism including four in lipogenesis and five in lipid metabolism. Significant
responses were confirmed for four top-changing genes (p0.003): stearoyl CoA desatu-
rase (SCD), fatty acid desaturases 1 and 2 (FADS1, FADS2), and diacylglycerol transferase
2 (DGAT2). SCD response was strongly correlated with carbohydrate intake (p0.019) and,
on a low carbohydrate diet, was inversely correlated with saturated fat intake (p0.05).
Moreover, plasma triglyceride responses to changes in dietary composition were
independently correlated with SCD (p0.003) and DGAT2 (p0.05) response. In
conclusion, fatty acid desaturases in human adipose tissue are independently regulated by
energy restriction and dietary composition and may be involved in dietary regulation of
systemic triglyceride metabolism.
73
Effect of Protein, Monounsaturated Fat, and Carbohydrate Intake on
Plasma Apolipoprotein B and VLDL and LDL Particles Containing apoCIII:
Results from the OmniHeart Trial
Jeremy D Furtado, Hannia Campos, Harvard Sch of Public Health, Boston, MA; Lawrence J
Appel, Johns Hopkins Univ, Baltimore, MD; Vincent Carey, Brigham and Womens Hosp,
Boston, MA; Frank M Sacks; Harvard Sch of Public Health, Boston, MA
Plasma apoB and VLDL and LDL particles with apoCIII are independent risk factors for cardiovascular
disease. We examined the effect of three healthy diets modeled after the DASH diet on these
apolipoproteins and lipoproteins. All diets were high in fruits, vegetables, and whole grains and low in
saturated fat, but differed by emphasis of either carbohydrate (CARB), monounsaturated fat (MONO), or
protein (PROT). In the setting of a controlled, 3 period cross-over feeding study, healthy subjects, N164,
consumed each diet for 6 weeks. As shown in the table below, all three diets similarly lowered plasma
apoB and VLDLLDL cholesterol compared to baseline when the participants ate their own diet. Only the
PROT diet significantly lowered plasma triglyceride (TG). LDL particles without apoCIII (the major LDL type)
were reduced equally by all diets, with an accompanying reduction in their TG and cholesterol
concentrations. The PROT diet reduced LDL with apoCIII compared to baseline and CARB. In contrast,
compared to baseline, CARB and MONO diets but not the PROT diet increased VLDL with apoCIII. In VLDL
without apoCIII, the PROT diet reduced TG compared to baseline and CARB and cholesterol compared to
baseline. The diets did not affect the molar ratios of TG to apo B and cholesterol to apo B in any of the
particle type suggesting that the diets did not alter particle composition. In conclusion, substituting protein
for carbohydrate reduced atherogenic apoCIII-containing LDL and had the most favorable effects on the
VLDL particle types, resulting in the least atherogenic lipoprotein profile.
Change from Baseline Between Diet Differences
CARB PROT MONO
MONO-
CARB
PROT-
CARB
MONO-
PROT
apoB in plasma -6%* -10%* -7%* -2% -4% -3%
TG in plasma -5% -14%* -7% -2% -9%* 8%*
chol in VLDLLDL -8%* -13%* -11%* -3% -6%* 3%
apoB in LDL with no apoCIII -8%* -10%* -8%* -1% -2% 2%
TG in LDL with no apoCIII -8%* -19%* -13%* -5% -11%* 7%
chol in LDL with no apoCIII -9%* -14%* -12%* -3% -6%* 3%
apoB in LDL with apoCIII 5% -12%* -3% -7% -16%* 10%
apoB in VLDL with apoCIII 34%* 13% 25%* -7% -16% 11%
TG in VLDL with no apoCIII -3% -20%* -10% -7% -18%* 13%
chol in VLDL with no apoCIII -6% -18%* -9% -4% -13% 11%
*p0.05, TG - triglyceride, chol - cholesterol
2007 ATVB Oral Presentations e-49
by on March 25, 2011 atvb.ahajournals.org Downloaded from
2007 ATVB Poster Presentations
P74
Factor XIII Levels Are Normal in Afibrinogenemic Plasma
Faisal I Ahmad, Susan T Lord; Univ of North Carolina at Chapel Hill, Chapel Hill, NC
Blood coagulation factor XIII (FXIII) is found in plasma and platelets. During the final stages of
coagulation, FXIII catalyzes the cross-linking of adjacent fibrin monomers through the formation
of -glutamyl--lysyl isopeptide bonds. Prior studies have shown plasma FXIII is associated
with fibrinogen, forming a non-covalent FXIII:fibrinogen complex. This finding suggests that
FXIII levels in blood are dependent on circulating fibrinogen levels. To determine whether the
absence of fibrinogen affects circulating FXIII, we measured FXIII levels in an afibrinogenemic
(fibrinogen-deficient) individual. We used gel electrophoresis and immunoblot procedures to
determine FXIII levels in plasma from an afibrinogenemic individual and plasma pooled from
normal individuals. We analyzed platelet-poor plasma in order to eliminate the cellular FXIII.
Samples were prepared at fifty-, one-hundred-, and two-hundred- fold dilutions. Immunoblot
analysis was performed using an affinity-purified, sheep anti-FXIII subunit A specific antibody.
We found FXIII levels in the afibrinogenemic and normal plasma samples were indistinguishable
at all dilutions, indicating comparable physiological concentrations of FXIII in the fibrinogen-
deficient and normal plasmas. These results show that the concentration of FXIII in plasma is
independent of the presence or absence of fibrinogen. Furthermore, these data indicate FXIII
can stably circulate in plasma even in the absence of fibrinogen and fibrinogen is not required
as a carrier for circulating plasma FXIII.
P75
Regulation of Factor Xa Mediated Signal Transduction by Annexin 2
Gourab Bhattacharjee, The Scripps Rsch Institute, La Jolla, CA; Jasimuddin Ahamed,
Rockefeller Univ, New York, NY; Cheng Liu, Nigel Mackman, Wolfram Ruf, Thomas S
Edgington; The Scripps Rsch Institute, La Jolla, CA
The serine protease zymogen factor X is converted to its catalytically active form factor Xa (Xa)
by the binary complex of factor VIIa bound to its cell surface receptor tissue factor (TF) or
alternatively by the intrinsic Xase complex which consists of active factors VIII (VIIIa), IX (IXa),
factor X, and Ca2. Xa has procoagulant activity to convert prothrombin to thrombin and also
induce cell signal transduction, either alone or in the ternary TF:VIIa:Xa complex. Xa cleaves
and activates protease activated receptor (PAR)1 or 2, but Xa signaling efficiency varies
between cell-types . We now observe that Annexin 2 acts as a receptor for Xa on the surface
of human endothelial cells (HUVEC) and that Annexin 2 association facilitates Xa activation of
PAR-1, but not the coagulant function of Xa. Over-expression of TF abolishes Annexin
2-dependence of Xa signaling and diminishes binding to cell surface Annexin 2. We propose
that Annexin 2 serves to regulate Xa cell signaling specifically in the absence of cell surface
TF and may thus play physiological or pathological roles when Xa is generated by the intrinsic
coagulation pathway.
P76
Effect of Exercise Training on Endothelium-dependent Vasodilatation in
Aged Rat Is Associated with Reduced Caveolin Status
Dong-Ju Choi, Young-Seok Cho, Hyeok-Jae Chang, Eun-Ji Kim, Woo-Young Chung, Tae-Jin
Yon, In-Ho Chae, Cheol-Ho Kim; Seoul National Univ, Seongnam, Republic of Korea
Ageing impairs endothelial-dependent vasodilatation in humans and animals. In the endothe-
lium, caveolin-1 regulates nitric oxide signaling by binding to and inhibiting endothelial nitric
oxide synthase(eNOS). The purpose of this study was to examine whether exercise training
alleviates impaired endothelial-dependent dilatation of aorta in aged rats by 12 weeks of
treadmill exercise and to determine their mechanisms. Three- and twenty-two-month-old male
Fischer344 rats were assigned to young sedentary, young exercise-trained, old sedentary, or
old exercise-trained groups. Abdominal aortic rings were prepared and vascular responses to
acetylcholine(10
-9
10
-4
M) were determined in vitro. To determine the potential role for nitric
oxide and caveolin-1 in vasodilatation in sedentary and exercised old rats, we examined serum
activity of NOx and expression of eNOS and caveolin-1 in rat aorta. Training improved the
ageing-induced reduction in endothelium-dependent vasodilation in aortic preparations.
Expression of eNOS RNA in aorta was unchanged by exercise training, whereas serum NOx
level was increased by 3 times, while caveolin-1 expression was decreased evaluated by
Wester blot and immunostaining. We conclude that (1) exercise can improve impaired
endothelium-dependent dilatation of aorta by ageing, (2) exercise can restore age-dependent
loss of NO by altering eNOS subcellular distribution and its association with inhibitory proteins,
caveolin-1, and (3) exercise may modify vascular reactivity in old subjects by altering levels of
eNOS protein in the large artery. These results imply the design of clinical strategies that
approach the age-associated loss of endothelial NO availability and through targeting these
pathways.
P77
Anemia Has Been Shown to Be an Adverse Indicator in Patients with Acute
Coronary Syndrome and in Chronic Heart Failure
Giorgio Corinaldesi, Medicina Generale ASL 7, Ancona, Italy; Christian Corinaldesi; Universita`
Politecnica Delle Marche, Ancona, Italy
Anemia is associated with an increased number of adverse cardiovascular events (CVD) in
particular with coronary artery disease (CAD), and chronic heart failure (CHF), and it is also
correlated with gender, aging, renal insufficiency, low BMI. Anemia involves inflammatory
cytokines (C-reactive protein, IL-6), it reduces marrow response to erythropoietin (EPO) and
heme-oxygenases-1(HO-1), it also reduces red cells life span and it may impairs reuse of iron,
it mostly reduces, the peak VO-2 (peak aerobic power). The latter appears to be an independent
factor that may be associated with an adverse outcome, in fact, for a reduction of one gram
of heamoglobin (Hb) the risk of morbility and mortality increase respectively by 32% and 18%.
The aim of the study was to determine the clinical implication of anemia in patients with CHF
or CAD; we have studied 38 patients (24 male, 14 female) with CHF, and 42 patients (28 male,
14 female) with CAD, with a range of Hb concentration included between 9.4gr/dl and
12.6gr/dl. We have evaluated moreover the tolerance to exercise on a treadmill and six minute
walk distance (21032 m in CAD), (18028 m in HF), the presence of rest dyspnea, the
presence of supraventricular or ventricular arrhytmias (atrial/or ventricular premature beats,
sinus tachycardia, or ventricular tachycardia, atrial fibrillation); lower levels of Hb, Fe, TIBC
correlate with a greater tendency to develop ventricular arrhytmias instead of supraventricular
arrhytmias. Anemia management included erythropoietin stimulating protein , blood transfu-
sion; we have used darbopoietin 50 mcg every week, and this treatment is associated to a
significant improvement in functional class and cardiac and renal function. Our data also
confirm the link between an increased tendency to develop CVD and a decreased level of Hb.
TABLE 1
RISK FACTORS CAD CHF
Sex (M/F) 24/14 28/14
Age (Years) 606 644
Hb (g/dl) 10.81.8 10.61.2
MCV (fl) 82.64.4 774.2
Iron (mg/dl) (Fe) 42.810.2 369.4
Total Iron binding capacity (TIBC) 31668.8 28062.8
P78
Identification of a Molecular Switch That Controls Cell Spreading and
Retraction
Panagiotis D Flevaris, Aleksandra Stojanovic, Emily Welch, Michael Burleigh, Xiaoping Du;
Univ of Illinois at Chicago, Chicago, IL
The dynamic process of cell membrane movement of blood platelets and other cells is initiated
and regulated by integrin-mediated signaling, and is important in thrombosis, hemostasis,
wound repair and cancer development. The ability of integrins to produce both cell spreading
and retraction remains a long standing paradox. In this study, we establish how integrin
cleavage by calpain controls the direction of cell membrane movement. The calcium-
dependent protease calpain cleaves the cytoplasmic domain of the integrin beta 3, mainly at
Y759. A calpain cleavage-resistant mutant of beta 3, R760E, when co-expressed with wild type
alpha IIb in Chinese hamster ovary (CHO) cells, enhanced cell spreading, but reduced the ability
of these cells to mediate integrin-dependent fibrin clot retraction. Indeed, calpain cleaves beta
3 at Y759 during clot retraction, and this cleavage is diminished in R760E cells. Furthermore,
inhibition of calpain cleavage of beta 3 is associated with reduced integrin-dependent Rho
activation and localized inhibition of myosin light chain phosphorylation. Conversely, cells
expressing a cleavage- mimicking mutant beta 3 spread poorly on fibrinogen, but this defect
is corrected by inhibition of Rho-dependent kinase, suggesting that truncation at Y759 causes
activation of RhoA-dependent retractile mechanisms and inhibits cell spreading. Thus, timed
calpain cleavage of beta 3 switches the direction of integrin signaling from promoting cell
spreading to promoting cell retraction.
P79
Thrombospondin-1 and CD47 Promote Platelet Aggregation by Opposing NO
Signaling
William A Frazier, Washington Univeristy, Saint Louis, MO; Jeff S Isenberg, David D Roberts;
National Institutes of Health, Bethesda, MD
Since its discovery over 35 years ago, the physiological role of platelet thrombospondin-1
(TSP1) has remained elusive in spite of its high level in platelet alpha granules from which it
is released upon activation. We reported earlier that monoclonal antibodies vs. TSP1 could
inhibit secretion-dependent platelet aggregation, but this result has been disputed and the
TSP1 knockout mouse was reported to have no platelet phenotype. We have recently found that
TSP1, acting via CD47, can inhibit NO-stimulated cGMP production and physiological responses
in endothelial and vascular smooth muscle cells. Another well-known role of NO signaling is its
antithrombotic inhibition of platelet activation and aggregation. Therefore, we hypothesized that
platelet TSP1 might act via CD47 to suppress NO-cGMP signaling in platelets thereby promoting
their aggregation. We first compared aggregation of wild type, TSP1- and CD47-null mouse
platelets in response to increasing doses of thrombin and found that platelets lacking either
TSP1 or CD47 require 2 to 4 fold more thrombin than WT platelets for full activation. TSP1-null
platelets have higher resting cGMP levels and a more robust elevation of cGMP in response to
e-50 Arterioscler Thromb Vasc Biol June 2007
by on March 25, 2011 atvb.ahajournals.org Downloaded from
NO donors and L-arginine than WT platelets. Low levels of exogenous NO (100 nM) stimulate
intraplatelet cGMP up to 4 fold and completely inhibit thrombin-induced aggregation of human
platelets. These effects of NO on aggregation and cGMP levels are both reversed by added
TSP1 (0.2 to 2 nM) or the recombinant C-terminal domain of TSP1 (2 nM) and CD47 agonist
peptides (1 to 10 uM) derived from it. These results suggest that earlier attempts to assess the
role of TSP1 in platelet aggregation did not adequately take into account the ephemeral nature
of NO. In conclusion, TSP1 and CD47 have a discernable and significant role in promoting
platelet aggregation, and perhaps stabilizing aggregates, under more physiological conditions
of nitric oxide tone. (Supported by the National Institutes of Health.)
P80
Predictors of Left Atrial Thrombus in Atrial Fibrillation
Henna Kalsi, Naser Ammash, Robert D McBane, David Hodge, Waldemar Wysokinski; Mayo
Clinic, Rochester, MN
Background: Stroke in atrial fibrillation (AF) encompasses mechanisms other than emboliza-
tion. The CHADS-2 score is highly predictive of stroke in this setting yet its specificity for left
atrial thromboembolism is not clear. Whereas warfarin is more effective for cardioembolic
compared to non-cardioembolic stroke prevention, we sought to assess the correlation
between left atrial thrombus (LAT) assessed by transesophageal echocardiography (TEE) and
CHADS-2 score. Methods: In a retrospective case-control study, Mayo Clinic Echocardiography
Laboratory and Cardioversion Unit Database were used to identify AF patients not previously
treated with warfarin who were found to have LAT by TEE. Variables of CHADS-2 system were
compared for both cases and controls. Results: Between 20002005, 179 cases with LAT
(mean age 7012 years; 46% women) and 440 controls (7113 years; 38% women) without
LAT were identified. Clinical and echocardiographic variables are summarized (Table). The
mean CHADS-2 score was low, but significantly higher for cases (average 2.41.6; median
2.0; range 06) compared to controls (average 1.61.3; median 1.0; range 06) (Figure).
However, for those patients with TEE confirmed LAT, the CHADS-2 score varied considerably
with 34% of CHADS-2 scores between 0 - 1. Furthermore, high scores (5 or 6) were uncommon
(12.9%). Conclusions: The prevalence of LAT confirmed by TEE is not reliably predicted by the
CHADS 2 score system. Left atrial size and left ventricular function remain the strongest risk
variables for presence of LAT and may provide mechanistic insight into thrombogenesis in atrial
fibrillation.
LATn179 No LAT n440 p
CHADS Score 2.41.6 1.61.3 0.001
CHF (clinical) 59% 22% 0.0001
Hypertension 56% 53% 0.54
Age 75 years 36% 44% 0.11
Diabetes 26% 17% 0.01
Stroke / TIA 36% 12% 0.0001
LVEF (%) 3624 5115 0.0001
LVEF30% 42% 24% 0.0001
LAD mm 5611 488 0.0001
P81
Platelet Activity, Coagulation, and Fibrinolysis During Exercise in Healthy
Males: Effects of Thrombin Inhibition by Argatroban and Enoxaparin
Nailin Li, Shu He, Margareta Blomback, Paul Hjemdahl; Karolinska Univ Hosp (Solna),
Stockholm, Sweden
BackgroundRelationships between exercise-induced activation of platelets, blood coagulation
and fibrinolysis, and the importance of thrombin for responses to exercise are not clear.
Methods and ResultsEffects of thrombin inhibition on haemostatic parameters were examined
in a double-blind, cross-over study comparing the direct thrombin inhibitor argatroban (350
ug/kg i.v. bolus followed by 25 ug/kg/min infusion), the indirect thrombin inhibitor enoxaparin
(0.75 mg/kg, i.v. bolus), or placebo (saline) in 21 healthy males. Measurements were made at
rest, before and during/after thrombin inhibitor treatment, and immediately after exhaustive
exercise. At rest argatroban abolished, and enoxaparin attenuated platelet activation by
thrombin, but not by ADP. Argatroban and, even more so, enoxaparin decreased thrombin
generation (F12) and the coagulation potential, and increased the fibrinolytic potential.
Exercise increased circulating activated platelets (from 5.50.3 to 9.40.9x10^9/L;
P0.001), circulating platelet-platelet microaggregates, the platelet responsiveness to in vitro
stimulation, leukocyte activation (leukocyte CD11b expression and plasma elastase), and
platelet-leukocyte aggregation (P0.01 for all). Exercise increased coagulation (F12;
P0.01) and fibrinolysis, but did not alter the balance between them; fibrin gel permeability
increased (P0.01). Neither argatroban nor enoxaparin counteracted exercise-induced platelet
or leukocyte activation. Both thrombin inhibitors augmented exercise effects on fibrinolysis.
ConclusionsStrenuous exercise enhances platelet and leukocyte activation independently of
thrombin. Exercise augments both coagulation and fibrinolysis, but the balance between them
appears to be maintained. At therapeutic dosages argatroban counteracted thrombin-induced
platelet activation most efficiently, while enoxaparin had somewhat stronger anticoagulant and
profibrinolytic effects.
P82
Regulated Expression of the Human Thrombin-activable Fibrinolysis
Inhibitor Gene in Nonhepatic Cell Types
Joellen H Lin, Michael B Boffa, Marlys L Koschinsky; Queens Univ, Kingston, Canada
The balance between coagulation and fibrinolysis is critical for the hemostatic response upon
injury. The carboxypeptidase thrombin-activable fibrinolysis inhibitor (TAFI) plays a key role in
controlling this balance by attenuating fibrinolysis; high plasma TAFI antigen levels have been
associated with increased risk of thrombotic diseases. Activated TAFI (TAFIa) can also
inactivate pro-inflammatory peptides such as the anaphylatoxins and bradykinin, suggesting a
role for the TAFI pathway as a link between coagulation and inflammation. TAFI expression in
HepG2 cells is decreased by interleukins -1 and -6 and plasma TAFI concentrations in humans
are decreased in experimental endotoxemia. Although liver is presumably the main source of
plasma TAFI, TAFI has also been identified in platelets, and TAFI mRNA has been detected in
the Dami (megakaryoblastic) cell line, human umbilical vein endothelial cells, and adipocytes
of patients with type 2 diabetes. Using RT-PCR and real-time RT-PCR, we now report the
detection of TAFI mRNA in the human monocytic cell line THP-1 as well as THP-1 cells that
have been differentiated into macrophage-like cells (THP-1ma) by treatment with phorbol
esters. We found no evidence of TAFI gene expression in human coronary artery smooth muscle
cells. It has been hypothesized that platelet TAFI arises from TAFI gene expression in
megakaryocytes. Accordingly, Dami cells were treated with phorbol esters for up to 72 hours
to differentiate them along the megakaryocyte/platelet lineage. Using real-time RT-PCR
analysis, we found that TAFI mRNA abundance was increased throughout Dami differentiation
(up to 9-fold after 50 hours). Using real-time RT-PCR analysis, we also found that TAFI mRNA
abundance was decreased when THP-1 and THP-1ma were treated with bacterial lipopoly-
saccharide for 24 hours (2-fold and 16-fold, respectively). Extra-hepatic expression of TAFI,
such as in platelets, monocytes and macrophages, suggests a novel role for localized TAFI
expression in regulation of hemostasis and inflammation.
P83
Paclitaxel, but Not Zotarolimus or Dexamethasone, Inhibits Human
Coronary Artery Endothelial Cell Serum-induced Migration in Vitro: Role of
p70S6K Activation in Endothelial Cell Migration
Matthew M Mack, Anton Stetsenko, Manisha Jhala, Sandra E Burke; Abbott Vascular Inc,
Abbott Park, IL
Introduction: Drug-eluting stents have reduced restenosis rates but elute agents which could
interfere with endothelial cell (EC) migration and re-endothelialization of lesions, leading to
thrombosis. Previous studies have shown that sirolimus blocks smooth muscle migration in
response to individual growth factors such as PDGF-BB or sphingosine-1-phosphate an effect
which may be mediated by inhibition of p70S6K activation. This study reports the effects of the
anti-restenotic agents zotarolimus, paclitaxel and dexamethasone on activation of p70S6K and
its role in the migration of human coronary artery EC (hEC) induced by serum. Hypothesis:
Paclitaxel but not zotarolimus or dexamethasone is hypothesized to inhibit hEC migration.
Reduced p70S6K activation alone will not result in anti-migratory activity. Methods: hEC
migration in response to serum growth factors was determined using a modified Boyden
chamber. hEC were synchronized and treated for 0 or 24 hrs in basal or growth media. After
migration (24 hrs) cells were stained with calcein-AM and fluorescence measured. Phosphor-
ylated p70S6K (T389) was measured by Western blot. Results and Conclusions: Paclitaxel but
not zotarolimus or dexamethasone blocks migration, however, all agents reduce p70S6K(T389)
phosphorylation (fig). These data suggest that in the presence of multiple chemotactic factors,
inhibition of p70S6K alone is insufficient to reduce migration. Dexamethasone and zotarolimus
should not impair, and the former may promote, re-endothelialization of vascular lesions. In
contrast, paclitaxel is predicted to potently attenuate re-endothelialization by blocking hEC
migration.
P84
WITHDRAWN
2007 ATVB Poster Presentations e-51
by on March 25, 2011 atvb.ahajournals.org Downloaded from
P85
Comparison of Short- and Mid-term Outcomes of Patients with Coronary
Artery Disease Treated with Drug-eluting Stents and Coronary Artery
Bypass Grafting
Muhammad S Munir, Amany H Ahmed, Craig M DeLaughter, KJ Shankar, Alan M Brewer,
Vei-Vei Lee, MacArthur A Elayda, Texas Heart Institute, Houston, TX; S W Casscells, Univ of
Texas Health Science Cntr, Houston, TX; James M Wilson; Texas Heart Institute, Houston,
TX
Background The use of stents in current clinical practice has evolved to include complex
lesions and high-risk patients who may be candidates for CABG. In such patients, DES is being
used with increasing frequency. However, the comparative performance of DES and CABG is
unknown. Methods We examined in-hospital and long-term follow-up data from unselected
patients who underwent isolated primary revascularization by DES or CABG at our institution.
Two case-matched groups were created using the DES and CABG patients propensity score.
Early (30 day) major adverse cardiac events (MACE), including death, myocardial infarction,
stroke and mid-term survival, using the Kaplan-Meier method, are reported. Outcomes were
compared using Chi-Square and Log-rank statistics. Results Of the patients who underwent
revascularization, 1598 were identified with matched characteristics (DES, n 799, CABG,
n799). Follow-up was available for three years. Early MACE was similar for both populations
(DES 5.01% vs CAB 3.78%; pNS). The risk of myocardial infarction and death were
respectively similar (CABG vs DES; pNS); (CABG vs DES, pNS). However, stroke was more
common in patients undergoing CABG (DES 0.11% vs CABG 1.11%; p0.01). Estimated
three-year mortality after revascularization was not different (CABG 6.6% vs DES 9%; pNS).
Conclusions The data suggest that the risk of early MACE and late mortality after
revascularization is similar for patients undergoing DES implantation or CABG.
P86
Aspirin Resistance in Stable Coronary Artery Disease Patients Is
Associated with Increased Platelet Turnover but Not Oxidative Stress
Chantal Pharand, Marie Lordkipanidze , Donald A Palisaitis, Hopital Sacre -Coeur, Montreal,
Canada; Jacques Turgeon, Universite de Montreal, Montreal, Canada; Erick Schampaert,
Jean G Diodati; Hopital Sacre -Coeur, Montreal, Canada
Introduction: Several patient characteristics have been shown to increase the risk of aspirin
resistance, yet underlying mechanisms remain unknown. It has been suggested that oxidative
stress may be involved. Hypothesis: We evaluated the hypothesis that increased oxidative
stress, as assessed by urinary isoprostane concentration, is associated with aspirin resistance.
Methods: Two hundred and one consecutive subjects suffering from stable CAD and under
daily aspirin therapy were enrolled. Platelet aggregation was measured by optical aggregom-
etry after stimulation with 1.6 mM of arachidonic acid. Aspirin resistance was defined as
residual platelet aggregation 20%. Blood samples were used for routine clinical testing and
morning urinary isoprostane (8-iso-PGF
2
) concentrations were measured using an enzyme
immunoassay. Results: Eight subjects were found to be aspirin resistant. They had higher
platelet counts (318 73 vs. 224 53 x 10
9
/L, p 0.0001) and lower daily aspirin dose
(80 0 vs. 187 145 mg daily, p 0.038) than aspirin sensitive subjects, suggesting
increased platelet turnover and sub-therapeutic aspirin therapy. They also tended to present
higher levels of C-reactive protein (CRP, 5.4 3.3 vs. 3.5 2.7 mg/mL, p 0.065), possibly
indicating an underlying inflammatory process. However, oxidative stress levels, as measured
by urinary 8-iso-PGF
2
, were similar between aspirin-resistant and sensitive subjects (86.4
33.4 vs. 81.4 72.9 ng/mmol of creatinine, p 0.847). In a simple univariate logistic
regression model, alcohol use, platelet count and CRP level were found to be predictive of
aspirin resistance (p 0.1), while prior revascularization was protective. Only platelet count
remained significant in a multiple logistic regression model (p 0.001). Conclusion: While
oxidative stress was present in all subjects, as expected in patients suffering from stable CAD,
it did not appear to contribute to aspirin resistance. Aspirin resistant subjects presented higher
platelet counts, potentially via increased platelet turnover. Future research should evaluate
whether increasing aspirin administration to twice daily would counteract apparent platelet
resistance to aspirin.
P87
Lack of Agreement Between 6 Major Platelet Function Assays Commonly
Used to Assess the Prevalence of Aspirin Resistance in Stable Coronary
Artery Disease Patients
Chantal Pharand, Marie Lordkipanidze , Donald A Palisaitis, Hopital Sacre -Coeur, Montreal,
Canada; Jacques Turgeon, Universite de Montreal, Montreal, Canada; Erick Schampaert,
Jean G Diodati; Hopital Sacre -Coeur, Montreal, Canada
Background: Platelet response to aspirin is variable. The unstandardized use of platelet
function tests has yielded aspirin resistance prevalences varying from 0.4 to 83.3% in the
literature. Hypothesis: We assessed the hypothesis that aspirin resistance prevalence was
dependent upon the platelet function test used. Methods: Two hundred and one consecutive
subjects suffering from stable coronary artery disease and taking daily aspirin were enrolled.
To evaluate the aspirin-sensitive platelet aggregation pathway, optical aggregometry (LTA) after
stimulation with 1.6 mM of arachidonic acid (AA) was used as the gold standard to measure
platelet aggregation; resistance was defined as residual platelet aggregation 20%. Other
platelet function assays evaluated included LTA with adenosine diphosphate (ADP) as the
agonist (5, 10 and 20 M; resistant if 70%), whole blood impedance (AA 1.6 mM; resistant
if 3 ), PFA-100

(resistant if 190 sec), VerifyNow Aspirin

(resistant if 550 ARU) and


urinary 11-dehydrothromboxane B
2
(d-TxB
2
, resistant if 67,9 ng/mmol of creatinine).
Results: Of the 201 subjects, 8 were aspirin resistant by LTA-AA (prevalence 4%,
CI
0,95
[0,01:0,07]). When assay-specific cut-off values were applied to the above-specified tests,
aspirin resistance prevalence varied. *: p0.05. Conclusion: Aspirin resistance remains rare
when evaluated with the current gold standard (LTA-AA) in stable coronary artery disease
patients. Platelet function tests are not equally effective in measuring aspirins antiplatelet
effect and correlate poorly with one another, which may explain the discrepancies reported in
the literature. Given the low agreement between the various assays, their usefulness to detect
appropriately aspirin resistant patients remains questionable.
PLATELET FUNCTION ASSAYS
Platelet
function test
Aspirin Resistance
Prevalence
Coefficient o
f correlation with LTA-AA
Agreement with
LTA-AA
LTA-AA 4% - -
LTA-ADP 5 M 10% 0.06 0.25*
LTA-ADP 10 M 18% 0.09 0.17*
LTA-ADP 20 M 52% 0.06 0.02
Impedance, AA 18% 0.24* 0.17*
PFA-100

60% -0.12 0.02


VerifyNow Aspirin

7% 0.13 0.25*
Urinary d-TxB
2
23% 0.18* -0.03
P88
Impaired Endothelial Function in Human C-reactive Protein Transgenic
Mice
Adrian Quan, Hwee Teoh, Sam Tirgari, St. Michaels Hosp, Toronto, Canada; Alexander J
Szalai, Univ of Alabama at Birmingham, Birmingham, AL; Michael E Ward, Subodh Verma;
St. Michaels Hosp, Toronto, Canada
BACKGROUND: Increasing evidence suggests that the inflammatory biomarker, CRP, may play
a causal role in the development and progression of atherothrombosis. Since endothelial
dysfunction is an early and integral component of atherosclerosis, we hypothesized that
endothelial homeostasis would be impaired in-vivo in human CRP-transgenic mice. METHODS
AND RESULTS: Male CRP transgenic (CRP Tg) and wild-type mice were injected (s.c.) thrice
over two weeks with either peanut oil vehicle or turpentine to induce the inflammation-sensitive
CRP transgene. Serum CRP was undetectable in wild-type mice, while levels in turpentine- and
vehicle-treated CRP Tg were 276.2895.7 g/mL and 1.410.2 g/mL (n68), respec-
tively. Endothelium-dependent and -independent vascular responses were studied using an
isolated tissue bath technique. Aortae isolated from mice with elevated CRP levels demon-
strated profound impairment in endothelium-dependent responses to acetylcholine (57.19.5
vs. 85.05.0, p0.05, n6), without affecting endothelium-independent vasoreactivity to
SNP. Massons trichrome staining revealed increased perivascular fibrosis in turpentine-treated
CRP Tg mice compared to vehicle-treated CRP Tg mice. Furthermore, CRP overexpression
in-vivo promoted an increased expression of MCP-1 and macrophage infiltration in mouse
aortic tissue. CONCLUSIONS: We demonstrate that human CRP transgenic mice exhibit
endothelial dysfunction in-vivo with resultant changes in vascular structure and endothelial
responses to injury. These data strengthen the role of CRP as a partaker of vascular risk.
e-52 Arterioscler Thromb Vasc Biol June 2007
by on March 25, 2011 atvb.ahajournals.org Downloaded from
P89
Hsp72 Increases Endothelial Survival by Inhibitory Interaction with Mst1
Aixia Ren, Guijun Yan, Bei You, Qian Wang, Jianxin Sun; UMDNJ-New Jersey Med Sch,
Newark, NJ
BACKGROUND: Apoptosis of vascular endothelial cells (ECs) is observed in many inflammatory
and immune disorders and is believed to contribute to the pathogenesis of diverse vascular
diseases. Mammalian sterile 20-like kinase 1 (Mst1) is a ubiquitously expressed serine/
threonine kinase known to be activated in response to a wide variety of apoptotic stimuli.
However, its role in vascular endothelial cells and the mechanism underlying its regulation are
not well understood. METHODS AND RESULTS: Treatment of Umbilical Vein Endothelial cells
(HUVEC) with TNF and H
2
O
2
induced apoptosis and cleavage of Mst1, which is a marker of
its activation, as well as activation of caspase 3. Furthermore, caspase-3 inhibitor and
N-acetylcysteine suppressed both TNF- and H
2
O
2
-induced Mst1 activation, suggesting that
both caspase-3 and reactive oxygen species are involved in Mst1 activation in HUVECs.
Adenovirus-mediated overexpression of wild-type Mst1 (AdMst1) in HUVECs increased
apoptotic cells with activation of caspase 3, whereas overexpression of dominant negative
Mst1 (K59R) attenuated TNF- and H
2
O
2
-induced apoptosis. To further understand the
mechanism underlying the regulation of Mst1 in HUVECs, a yeast two-hybrid screening strategy
was conducted to identify interacting molecules that associate with Mst1. Binding studies
indicated that Hsp72 boud directly to the inhibitor domain of Mst1, as demonstrated by
co-immunoprecipitation and GST pulldown assays. The colocalization assay showed that both
Mst1 and Hsp72 colocalized in the cytoplasm. Furthermore, we showed the overexpression of
Hsp72 or mild heat shock inhibited TNF-induced activation of Mst1 as well as its downstream
targets p38 MAPK, and JNK (c-Jun N-terminal kinase), thereby blocking Mst1 dependent
apoptosis in HUVECs. In addition, Hsp72 antisense oligonucleotides prevented the inhibitory
effects of mild heat shock on TNF and H
2
O
2
-induced Mst1 activation and apoptosis in HUVECs.
CONCLUSIONS: Our results suggest that Mst1 plays an important role in the induction of
apoptosis of ECs and Hsp72 functions as an endogenous inhibitor of Mst1. Mst1 may be a
potential therapeutic target for vascular diseases associated with endothelial apoptosis.
P90
Rapid Inhibition of Platelet Reactivity with AZD6140 in Rats with
Streptozotocin-induced Diabetes
Andreas Scha fer, Corinna Scho pp, Stephanie Pfo rtsch, Jutta Neumu ller, Stefanie Fiedler,
Juliane Jaitner, Christian Vogt, Ulrike Flierl, Johann Bauersachs; Univ of Wu rzburg,
Wu rzburg, Germany
Background. Excessive platelet activation fundamentally contributes to cardiovascular events
and mortality in patients with diabetes mellitus. We investigated whether acute administration
of AZD6140, a reversible oral P2Y
12
antagonist with rapid onset of action, would beneficially
modulate platelet reactivity in diabetic rats. Methods. Diabetes had been induced by
intravenous injection of streptozotocin in male Wistar rats. After 4 weeks of diabetes,
single-dose treatment with AZD6140 (5 mg/kg) was given by gavage. Plasma samples were
collected and functional assays assessing platelet reactivity to ADP and vascular function were
performed at 0.5, 1, 2, 4, 6, 8, 12, and 24 hours post application. Results. Sufficient plasma
levels of AZD6140 were achieved within 30 minutes. CD62P surface expression in response to
ADP was significantly reduced 0.54 hours after single administration of AZD6140. ADP-
induced platelet aggregation was rapidly inhibited by single-dose administration of AZD6140
within 0.5 hours and lasted over the complete observation period of 24 hours. Washed
ADP-stimulated platelets were perfused through a perfusion flow-chamber over a fibrinogen-
coated membrane. Acute administration of AZD6140 significantly reduced platelet adhesion to
fibrinogen. Conclusions. Acute administration of AZD6140 resulted in rapid absorption in
diabetic rats. AZD6140 rapidly inhibited platelet reactivity. Platelet inhibition by AZD6140 could
provide a new useful tool for positive modulation of the cardiovascular system in diabetes.
P91
Impaired Bioactive Nitric Oxide Despite Higher Basal Nitric Oxide Synthase
Activity in Platelets from Patients with Heart Failure
Ashish Shah, Eugenia Gkaliagkousi, Kings College London, London, United Kingdom; Julia
DeCourcey, Ruth Buckley, Kings College Hosp, London, United Kingdom; James Ritter,
Albert Ferro; Kings College London, London, United Kingdom
Platelet derived nitric oxide (NO) plays an important role in prevention of thrombosis. Heart
failure (HF) is characterised by increased thrombotic events. Impaired endothelium-derived NO
is believed to contribute to this; however the role of platelet-derived NO is unclear. We therefore
sought to determine whether platelet NO production is impaired in patients with HF. Thirty four
HF subjects (55.1 2.8 yrs; 26 male, 8 female) and 22 healthy controls (56.6 2.7 yrs;16
male, 6 female) were studied. Platelet NO synthase (NOS) activity (both basal & after stimulation
with albuterol 10
-5
mol/l or collagen 8g/ml) was determined in gel-filtered platelets from the
rate of conversion of L-[
3
H] arginine to L-[
3
H] citrulline. Intraplatelet cGMP was determined by
radioimmunoassay under same conditions. Data were expressed as mean SEM, and were
analyzed by ANOVA with or without repeated measures as appropriate, p 0.05 taken as
significant. Basal platelet NOS activity was higher in HF subjects than controls (0.15 0.02
vs.0.09 0.02 pmol L-citrulline / 10
8
plts respectively, p 0.035). By contrast, stimulated
platelet NOS activity in response to either albuterol or collagen was impaired in HF subjects
compared to controls; percentage increases for albuterol were -11.77 5.08% vs. 39.47
16.33% respectively, p 0.001, and for collagen were -10.18 5.33% vs. 23.40 9.08%
respectively, p 0.001. On other hand, basal cGMP was lower in HF subjects than in controls
(36.37 21.01 vs. 104.7 22.01 fmols/10
8
plts respectively, p0.033). Furthermore,
albuterol- and collagen-stimulated cGMP were attenuated in HF subjects compared to controls
(albuterol: 36.69 19.49 fmols/10
8
plts vs.120.7 20.99 fmols/10
8
plts respectively,
p0.006; collagen: -6.05 22.63 fmols/10
8
plts vs.107.50 21.39 fmols/10
8
plts
respectively, p0.001). Our results demonstrate that platelets from patients with HF in the
basal state exhibit lower cGMP than controls, despite higher basal NOS activity, suggesting
impaired availability or responsiveness to NO. Moreover, stimulated NOS and cGMP production
in platelets from HF subjects are blunted compared to controls. These abnormalities in platelet
NO signaling could contribute to the pathophysiology of thrombotic events in patients with HF.
P92
Myocardial Ischemia Produces Detectable Temperature Changes in the
Swine Myocardium That Precede Electrocardiographic Changes
Muhammad S Munir, KJ Shankar, Alan M Brewer, Igor V Stupin, Tuzun Egemen, Marlos R
Fernandes, G G Costas, Mello E Oliveira, Texas Heart Institute, Houston, TX; Naushad A
Shaik, S W Casscells, Univ of Texas Health Science Cntr, Houston, TX; Amany H Ahmed;
Texas Heart Institute, Houston, TX
Background We used 5 juvenile pigs in a model of acute experimental coronary occlusion to
determine the effect of myocardial ischemia on intramyocardial temperature (T), and its
temporal relationship to the electrocardiographic (EKG) manifestations of ischemia. Methods
Three thermosensors were placed approximately 4mm intramyocardially (one-half the typical
mural thickness), corresponding to the areas perfused by the right coronary artery (RCA), Left
anterior descending (LAD) and Left Circumflex Coronary Arteries (LCX) respectively. EKG and T
data were acquired in real time by cardiac functional data software. Serial balloon occlusions
of the respective coronaries were performed, employing 1 minute of ischemic preconditioning,
followed by 3 and 5 minute balloon occlusions, each separated by 3 minutes of reflow.
Subsequent to this, sustained ischemia, lasting for upto an hour was created by occlusion of
the angiographically dominant coronary artery. We measured intramyocardial T in the area at
risk relative to T in the non-ischemic zones. Results We performed a total of 7 coronary
occlusions in each animal. Thirty-three balloon occlusions were included in the analysis, with
5 occlusions being in the dominant coronary artery. Due to absence of ischemic EKG changes,
possibly due to collateralization, 15 occlusions were excluded. Of the remaining occlusions
(n18), 12 (66.6%) [(RCA2); (LAD6); (CX1); (DV3)] demonstrated progressive T
changes. LAD and LCX occlusions were associated with a mean T of -0.410.39
o
C. A rise
in T was observed in both RCA occlusions with a mean T of 0.25 0.21
o
C. Time to onset
of T changes was 23.70.6s, whereas EKG changes were apparent after 81.712.3s.
Changes in T significantly preceded EKG chanhes of ischemia by 58 seconds (p0.01). Time
to peak drop in T was 9013s. EKG and T changes began reverting to pre-ischemic baseline
levels within 20s of reflow. With sustained ischemia, intramyocardial T began to rise slowly
after 20 to 30 minutes. Conclusion T of acutely ischemic myocardium changed relative to
baseline. T changes appeared to precede EKG changes of ischemia. Acute myocardial ischemia
appears to produce a detectable T change in the swine myocardium. T may be used as a novel
and surrogate marker for myocardial ischemia.
P93
Localization of Coagulation Proteins Within the Arterial Vessel Wall in
Relation to Progression of Atherosclerosis
Henri M H Spronk, Sylvia Heeneman, Peter Kassak, Karly Hamulyak, Mat J A P Daemen,
Hugo ten Cate; Univ of Maastricht, Maastricht, The Netherlands
Introduction: Thrombogenicity of the atherosclerotic plaque depends in part, on the quantity and
activity of tissue factor localized in the arterial vessel wall. Recent experiments indicate that in
addition to local tissue factor (TF), also factor (F) VII is expressed by cells in human
atherosclerotic plaques. Hypothesis: Vitamin K dependent proteins including prothrombin and
FX, are synthesized by cells within the arterial vessel wall and contribute to the thrombogenicity
of atherosclerotic plaques. Methods and Results: Immunohistochemical staining of TF and
coagulation factors II, VII, IX, X, XI, and XII revealed the presence of these proteins in all three
stages of atherosclerosis. In arterial vessels characterized by intimal thickening, TF was
demonstrated in association with medial and intimal smooth muscle cells (SMCs), whereas in
advanced lesions TF and FVII colocalized in association with SMCs and macrophages, both in
intima and media. FX mainly showed a cytoplasmatic localization, while thrombin stained
positive in the extracellular matrix of adventitia, media, and necrotic core of the plaque. In
thrombotic lesions, macrophages and polymorphonuclear leukocytes contained TF, whereas
FVII was localized in macrophages and in regular and migrating SMCs of plaque cap shoulder
region. Factors IX and XI were mainly associated with macrophages in all three stages of
atherosclerosis and SMCs in thrombotic lesions were positive for factor IX. Factors XI and XII
were dispersed positive in the lipid/necrotic cores of lesions in association with macrophages
and sometimes present in the media. TF activity in atherosclerotic lesions assessed using an
in house assay ranged between 9 and 133 pM and correlated with thrombin generating
potential (endogenous thrombin potential) and lag time derived from calibrated automated
thrombograms. Conclusions: Co-localization of TF, FVII and FX on macrophages and SMC
suggests the presence of procoagulant complexes in all stages of atherosclerosis. The
presence of procoagulant proteins suggests that they contribute to thrombin generation. In
addition to being determinants in thrombogenicity, these proteins may be involved in other
pathophysiological processes like cell migration and inflammation.
P94
Interactions of mCRP and pCRP with Members of the Fibrinolytic Family of
Proteins
Meredith J Stevenson, Tammy Strawn, William P Fay; Univ of Missouri, Columbia, MO
Background: Increased plasma concentration of C-reactive protein (CRP), an acute phase
reactant protein, is associated with increased risk of myocardial infarction in humans and
CRP-transgenic mice exhibit accelerated thrombosis after vascular injury compared to
wild-type mice. As the plasminogen activation (PA) system plays a major role in modulating
thrombosis, we examined potential interactions of CRP (both monomeric and pentameric forms)
with Glu-plasminogen and fibrin, key components of the PA system. Methods: Monomeric
human CRP (mCRP) was prepared by denaturing pentameric CRP (pCRP) with urea, which was
2007 ATVB Poster Presentations e-53
by on March 25, 2011 atvb.ahajournals.org Downloaded from
subsequently removed by dialysis. Binding of CRP to immobilized Glu-plasminogen and fibrin
was studied by ELISA, and degradation of mCRP and pCRP by plasmin was tested by
SDS-PAGE. Rate of tissue plasminogen activator (t-PA)-mediated lysis of
125
I-labeled fibrin clots
suspended in plasma was determined by measuring radioactive counts of buffer aliquots over
4 hours. Results: Both mCRP and pCRP bound to immobilized plasminogen, with an estimated
Kd of approximately 1.70.5 g/mL vs. 3.70.6 g/mL. However, only mCRP catalyzed
plasminogen activation by t-PA, producing a cofactor effect similar in potency to that of soluble
fibrin fragments. Plasmin efficiently degraded mCRP, while pCRP was resistant to plasmin
degradation. Both mCRP and pCRP bound to fibrin at pathophysiologically relevant concentra-
tions (0.130 g/mL). However, the rate of lysis of
125
I-labeled fibrin clots was unaffected by
addition of either mCRP or pCRP (10 g /mL). Conclusions: Binding of CRP to plasminogen and
fibrin may serve to target CRP to sites of vascular injury, where it may modulate inflammatory
processes. The distinctly different effects of mCRP and pCRP on plasminogen activation could
potentially help to explain the differential effects of mCRP and pCRP on vascular disease
progression that have been observed in prior in vivo studies.
P95
Prevalence and Distribution of Deep Vein Thrombosis in Patients with
Symptomatic Pulmonary Embolism
Takashi Yamaki, Motohiro Nozaki, Hiroyuki Sakurai, Masaki Takeuchi, Kazutaka Soejima,
Taro Kono; Tokyo Womens Med Univ, Tokyo, Japan
BACKGROUND: To investigate the prevalence and distribution of deep vein thrombosis (DVT) in
patients with symptomatic pulmonary embolism (PE), and to compare characteristics between
patients with PE and these without PE. MATERIALS AND METHODS: A total of 420 consecutive
patients with DVT were included. The distribution of DVT was evaluated with duplex scanning,
and patients with clinical suspicion of PE were investigated using ventilation/perfusion lung
scintigraphy. The risk factors for DVT and markers of thrombophilia were also investigated.
RESULTS: Of 420 patients evaluated, PE was found in 82 (20%) patients. There were no
significant differences in mean age, gender, and laterality of leg involvement between patients
with DVT combined with PE and these with DVT alone. Proximal DVT was found 39 (48%)
patients with PE and 282 (83%) with no PE, and there was a significantly higher proportion of
proximal DVT in patients with DVT alone (p0.0001). In patients with PE, the DVT was found
most predominantly in soleal vein (53%) followed by popliteal (37%), peroneal (32%), common
femoral (27%), femoral (27%), gastrocnemius vein (21%). The significantly higher proportion of
DVT was found in gastrocnemius vein in patients with PE (p0.018). In contrast, the proportion
of common femoral vein thrombosis was found to be significantly higher in patients who had
DVT alone (p0.049). The operation and trauma (43%), immobilization (22%), and active
cancer (16%) were the main risk factors for DVT related to patients with PE, and there were
similar tendencies in the proportions of risk factors for patients with DVT alone. In thrombophilia
testing, only proportion of positive antiphospholipid syndrome was significantly higher in
patients with PE (p0.011). CONCLUSIONS: The lower extremity venous duplex scanning
demonstrates that distal DVT was more predominant in patients who had PE compared to these
with DVT alone. Thrombus in the gastrocnemius vein was frequently found in patients with PE.
Positive antiphospholipid syndrome was more often found in patients with PE. However, there
were similar tendencies in age, gender, laterality of leg involvement, and risk factors for DVT
between patients with PE and these with DVT alone.
P96
Localization of Classical Complement Components and C1 Inhibitor on
Platelet Microparticles
Wei Yin, Ellinor I B Peerschke; Weill Med College of Cornell Univ, New York, NY
Platelet microparticles (PMP) play an important role in hemostasis and thrombosis, and are
released from activated platelets. We previously demonstrated in situ classical complement
pathway activation on stimulated platelets. The present study extends these observations to
PMP. PMP were generated by treating platelets with 10 M A23187 (37C, 5 min). PMP were
characterized by flow cytometry based on size, expression of phosphatidylserine (Annexin V
binding), P-selectin (anti CD62P), and GPIIb (anti CD41). Complement activation was assessed
as a function of C4d and iC3b deposition following PMP exposure to 1/10 citrated normal
human plasma (37 C, 45 min). Samples were washed and monoclonal anti human C4d and
iC3b antibodies added. Flow cytometry showed an approximately 2-fold increase in mean peak
fluorescence intensity (MPF) over background for anti C4d and anti iC3b. Since C1 inhibitor
(C1-INH), the major classical pathway complement inhibitor, has been described to bind
P-selectin, we also measured PMP C1-INH expression. Using rabbit anti human C1-INH
antibody, PMP surface C1-INH expression increased approximately 10-fold over background
following exposure to plasma. To understand the mechanism of complement activation, PMP
were examined for the expression of known activators of classical (IgG, gC1qR) and alternative
(P-selectin) pathways. Increases in MPF of approximately 50, 5 and 10 fold over background
were observed for IgG (mouse anti human IgG), gC1qR (monoclonal 74.5.2) and P-selectin (anti
CD62), respectively. In addition, complement activation on PMP was examined in Factor B
(alternative pathway) depleted serum. iC3b deposition was not affected by inhibition of the
alternative pathway. In summary, our findings provide the first evidence for in situ complement
activation on PMP and suggest regulation of both alternative and classical complement
activation by C1 INH.
P97
Serum Glutamyltransferase Level Is Increased in Patients with Metabolic
Syndrome Undergoing Coronary Artery Bypass Graft Surgery
Ayse Baysal, Sabri Dagsali; Dr Siyami Ersek Hosp, Istanbul, Turkey
Introduction: Gamma-glutamyltransferase (GGT) is an enzyme responsible for the extracellular
catabolism of glutathione. Modest increases of serum GGT may be an early marker of cellular
oxidative stress. The aim of this study is to evaluate the level of this enzyme in patients that
are undergoing coronary artery surgery and demonstrate the relation with the presence of
metabolic syndrome criterias. Methods: 118 patients were evaluated retrospectively. The
metabolic syndrome criterias including the presence of three of the five parameters were
considered in the division of groups. While group 1 had metabolic syndrome (MS), group 2
had no metabolic syndrome (MS-). All patients had blood withdrawn for GGT, other hepatic
enzymes, uric acid, creatinine, and lipid levels before surgery. Patients were diagnosed MS ()
if they had 3 of the following 5 abnormalities: (1) abnormal girth in men 102 cm, in women
88cm; (2) triglycerides 150 mg/dl; (3) high-density lipoprotein (HDL) cholesterol in men
40 mg/dl or in women 50 mg/dl; (4) fasting blood glucose 110 mg/dl; (5) systolic blood
pressure 130 mmHg, diastolic blood pressure 85 mmHg, or treated hypertension. The data
compared includes; age, sex, body mass index, cigarette smoking and metabolic syndrome
citerias. Results: The demographic data including age and sex were not different in both
groups. The serum GGT levels rise more dramatically in patients with MS (group 1; 26 3.6
U/mg) than patients without MS (group 2; 212.2 U/mg, p 0.05). The serum uric acid,other
hepatic enzymes,creatinine, and lipid levels did not show significant changes between groups.
Conclusions: Our study demonstrated that elevated serum GGT levels are closely associated
with the presence of metabolic syndrome in patients undergoing coronary artery bypass graft
surgery. GGT can be a diagnostic marker for the presence of coronary atherosclerosis and
metabolic syndrome.
P98
Identification of Platelet Proteins Involved in Platelet Structure and
Inflammation That Are Regulated by Fish Oil Supplementation in Healthy
Volunteers
Baukje de Roos, Emma Massie, Karen Ross, Garry Rucklidge, Rowett Rsch Institute,
Aberdeen, United Kingdom; Frank Thies, Univ of Aberdeen, Aberdeen, United Kingdom;
Niamh OKennedy; Provexis, Aberdeen, United Kingdom
Long chain polyunsaturated fatty acids, present in fish and fish oil, lower the risk of coronary
heart disease. Beneficial effects may include regulation of anti-thrombotic and anti-
inflammatory pathways, but mechanisms are not well understood. We studied the effects of
fish oil supplementation on platelet function and platelet proteomics in healthy volunteers.
Fifteen male and female subjects received 1 g/day of fish oil (n8) or 1 g/day of placebo oil
(n7) for three weeks. Fasted blood samples were obtained before and on the last day of the
intervention period for the measurement of platelet aggregation induced by collagen, plasma
levels of hsCRP and sP-selectin, and platelet glutathione peroxidase 1 activity. Non-activated
platelets were processed for proteomics by separating proteins by 2-dimensional gel
electrophoresis. Proteins that were significantly up-or down-regulated were cut, trypsinized
and identified by MALDI-TOF and LC mass spectrometry methods. Fish oil, as compared with
control oil, did not significantly affect platelet aggregation, plasma hsCRP, and sP-selectin, nor
did it affect platelet glutathione peroxidase 1 activity. Proteomic analysis identified 45 platelet
proteins that were significantly up-or down regulated by fish oil, as compared with control oil,
after three weeks of intervention. Principal component analysis revealed that the treatment
effect of fish oil was mainly explained by the up-regulation of tubulin, filamin, vinculin, and
thrombospondin protein (all p0.05) in platelets. In addition, fish oil lowered levels of the
pro-inflammatory platelet protein -parvin (p0.05). In conclusion, consumption of a moderate
amount of fish oil caused significant changes in the platelet proteome after three weeks,
without affecting platelet aggregation. Our proteomics approach provided new insights into the
mechanisms of fish oil in platelets, involving the up-regulation of several structural proteins,
possibly promoting the cytoskeletal stability of the resting platelet, and reducing the incidence
of shape-change in response to external stimuli. In addition, fish oil contributed to the increased
levels of anti-inflammatory and decreased levels of pro-inflammatory platelet proteins.
P99
The Effects of Weight Loss on Cholesteryl Ester Transfer Protein and
Expression of the Metabolic Syndrome in Obese Individuals
Ron C Hoogeveen, Baylor College of Medicine, Houston, TX; Helena Svobodova, Charles
Univ, Prague, Czech Republic; Maushmi T Savjani, Peter H Jones, Christie M Ballantyne;
Baylor College of Medicine, Houston, TX
Background: Metabolic syndrome (MS) is a clustering of risk factors, including insulin
resistance, abdominal obesity, hypertension, low HDL cholesterol (HDL-C) and elevated
triglycerides (TG). Moderate weight loss markedly improves many of these abnormalities.
Cholesteryl ester transfer protein (CETP) is a key enzyme involved in HDL metabolism and
although high levels of CETP activity are often associated with low HDL-C and high TG, recent
studies have shown that diabetics have low CETP activity levels. Therefore, we tested the
hypothesis that insulin resistance regulates CETP expression in obese individuals. Methods:
We analyzed the relation of plasma CETP concentration and activity with adiposity, dyslipidemia
and insulin resistance in obese subjects with (MS, n40) and without (MS-, n40) the
metabolic syndrome according to NCEP ATP III criteria and examined the acute effects of weight
loss at 48 weeks and 34 months of very-low-calorie diet (VLCD). Baseline measurements
for a group of 20 healthy lean subjects were included for comparison. Results: At baseline, we
found a strong positive correlation between plasma CETP concentration and activity (r0.71,
P0.0001). Mean CETP activity was significantly lower in lean controls compared to obese MS-
subjects (37.2 vs. 46.4 pmol/0.5 l/3 hr, p0.05), but not to obese MS subjects (37.2 vs.
40.4 pmol/0.5 l/3 hr, p0.34). Subsequent analyses showed that in obese individuals with
impaired fasting blood glucose (fasting blood glucose 100 mg/dL) compared to obese
individuals with normal blood glucose, CETP activity was significantly lower (35.8 vs. 46.9
pmol/0.5 l/3 hr, p0.001) and CETP mass showed a trend towards being lower (0.83 vs. 0.96
g/mL, p0.10). Acute weight loss (48 weeks of VLCD) resulted in significant 33% and 27%
reductions in plasma CETP activity and mass, respectively (p0.001), in obese MS subjects,
which were associated with an 8% decrease in HDL-C (p0.001). Conclusions: Although
obesity is associated with increased plasma CETP activity, in obese individuals with impaired
fasting blood glucose, plasma CETP activity is significantly reduced.These findings suggest that
insulin resistance modulates CETP expression in obese individuals.
e-54 Arterioscler Thromb Vasc Biol June 2007
by on March 25, 2011 atvb.ahajournals.org Downloaded from
P100
High Glucose Augments Interferon -Stimulated Matrix Metalloproteinase-1
Expression in U937 Macrophages by Enhancing STAT-1 Activity
Alena Nareika, Med Univ of South Carolina, Charleston, SC; Bryan A Game, Ralph H
Johnson VA Med Cntr, Charleston, SC; Maria F Lopes-Virella, Yan Huang; Ralph H Johnson
VA Med Cntr and Med Univ of South Carolina, Charleston, SC
Plaque rupture is a principal cause of luminal thrombosis in acute coronary syndromes
occurring in 75% of patients who die of acute myocardial infarction. Recent studies called
Diabetes Control and Complications Trial and the Epidemiology of Diabetes Interventions and
Complications Research (DCCT /EDIC) have shown that rigorous glycemic control in diabetic
patients leads to a significant reduction of cardiovascular events, suggesting that hyperglyce-
mia may contribute to destabilization of atherosclerotic plaques. Although mechanisms such as
protein glycosylation by which hyperglycemia promotes atherosclerosis have been established,
it is not well understood how hyperglycemia interacts with inflammatory cytokines such as
interferon (IFN) gamma, a key cytokine involved in atherosclerosis, in inflammation-related
plaque destabilization. In this study, U937 macrophages cultured in medium containing either
normal (5 mM) or high glucose (25 mM) were treated with 100 units/ml of IFN gamma for 24 h.
After treatment, matrix metalloproteinase (MMP)-1 expression was examined. Results from
real-time PCR showed that high glucose and IFN gamma increased MMP-1 mRNA level by
100% and 150%, respectively, while the combination of high glucose and IFN gamma resulted
in a 340% increase, suggesting a synergistic effect between high glucose and IFN gamma.
Data from ELISA also demonstrated the similar synergistic effect on MMP-1 secretion. Besides
MMP-1, high glucose and IFN gamma also synergistically stimulate MMP-9 and IL-1 beta, but
had no effect on tissue inhibitor of metalloproteinase (TIMP)-1 and -2. Furthermore, the
molecular mechanism underlying the synergistic effect was explored. Results from Western
blot and electrophoretic mobility shift assay (EMSA) showed that high glucose augmented IFN
gamma-stimulated MMP-1 expression by increasing phosphorylation and DNA-binding activity
of signal transducing and activator of transcription (STAT)-1, a major transcription factor
involved in IFN gamma-regulated gene expression. Given that hyperglycemia is a major
abnormality in diabetes and diabetic patients have increased cardiovascular events, this study
revealed a novel mechanism involved in diabetes-promoted cardiovascular disease.
P101
Effect of Chronic Insulin Plus Atorvastatin Therapy on Mitochondrial
Function in an Ex Vivo Animal Model of Diabetes and Hypercaloric Diet
Submitted to Global Myocardial Ischemia-Reperfusion
Pedro Monteiro, Marta Paiva, Raquel Carreira, Lino Goncalves, Luis A Provide ncia; Coimbra
Univ Hosp, Coimbra, Portugal
Introduction: Obese diabetics have more ischemic heart disease; this may be improved by
insulin (INS) and atorvastatin (ATV). If these subjects are treated with INSATV, do they have
better mitochondrial function? Aim: To evaluate, in a model of diabeteshypercaloric
dietischemia-reperfusion (IR), if therapy with INSATV improves mitochondrial function.
Material and methods: Goto-Kakizaki (GK) diabetic rats (fed with an hypercaloric diet), divided
in 4 groups(n10/group):GKHD control (no medication/no IR);INS control (Insulin bid -as
needed- and ATV 10 mg/kg/day between month 5 and 6/no IR);GKHD IR (as GKHD control and
then IR);INS IR (as INS control and then IR). At 6 months, hearts were submitted to 165 min
perfusion (control) or 10 min perfusion35 min ischemia120 min reperfusion (IR).
Parameters assessed were: oxidative stress (colorimetric tiobarbituric acid colourimetry
test-TBARS), mitochondrial swelling and calcium uptake (fluorimetry). Results: INSATV-
treated rats had significantly lower oxidative stress levels, both in control (0.700.01 vs.
0.810.04 nmol TBARS/mg protein;p0.05) and IR (0.940.04 vs. 1.050.02 nmol
TBARS/mg protein;p0.05)-Fig. 1. INS-treated animals also showed a significant decrease in
mitochondrial swelling, both in IR (52.72.1 vs. 68.11.8 arbitrary units-AU;p0.05), and
control (29.13.2AU vs. 37.04.3AU;p0.05). INS therapy showed a significant improvement
in calcium uptake in IR (59.81.0 vs. 53.90.8 nmol/mg protein;p0.05). Conclusion: In our
model, INSATV improves cardiac mitochondrial function, by lowering oxidative stress and
improving ischemia tolerance.
P102
New Insights into VLDL Triglyceride Metabolism in Obese Subjects with
Nonalcoholic Fatty Liver Disease
Bruce W Patterson, Bernard V Miller, Kevin Korenblat, Elisa Fabbrini, Samuel Klein;
Washington Univ Sch of Medicine, Saint Louis, MO
Elevated levels of VLDL triglyceride (TG) are associated with obesity, diabetes, non-alcoholic
fatty liver disease (NAFLD) and increased cardiovascular risk. Metabolic tracer kinetics studies
are used to evaluate the mechanisms that regulate VLDL-TG levels (production vs. clearance).
We have previously used a bolus of [
2
H
5
]glycerol to determine the fractional catabolic rate (FCR)
of VLDL-TG, and, using an independent analysis, a constant infusion of [
2
H
2
]palmitate to
determine the proportion of VLDL-TG palmitate derived from systemic (labeled) vs. non-
systemic (unlabeled) sources. We have developed a new model that for the first time combines
the glycerol and palmitate data into a single analysis to provide a better definition of the
VLDL-TG FCR, the size and turnover rate of the pool responsible for tracer recycling, and
resolution of the latter from the direct incorporation of plasma palmitate into VLDL-TG. VLDL-TG
kinetics studies were performed in 21 obese subjects without and 27 obese subjects with type
II diabetes mellitus; intrahepatic fat content (IFC) measured by magnetic resonance spectros-
copy ranged 0.736% across all subjects. The tracer recycling pool turnover rate averaged
0.100.05 (SD) pools/h (negatively correlated with IFC, P0.001) and had an average mass
of 7.86.3 mmol TG (positively correlated with IFC, P0.01), which was less than 10% of IFC.
The percents of VLDL-TG palmitate derived directly from plasma fatty acids, the tracer recycling
pool, and other sources of unlabeled palmitate averaged 28%, 40%, and 32%, respectively; the
percent from unlabeled palmitate increased with IFC (P0.001) whereas the percent from
plasma decreased with IFC (P0.01). VLDL-TG concentration decreased with VLDL-TG FCR
(P0.001), increased with IFC (P0.02), increased with the production rates from unlabeled
palmitate sources (P0.02) and from plasma palmitate (P0.005), and was not significantly
correlated with plasma FFA concentration (P0.25). We conclude that both metabolically active
(responsible for tracer recycling) and inactive (unlabeled by tracer) hepatic fat depots provide
more than two-thirds of the fatty acids for VLDL-TG production in obese subjects with NAFLD.
P103
Diet-induced Hyperlipidemia Worsens Vascular Compliance and Function of
Resistance Vessels in Type 2 Diabetes
Kamakshi Sachidanandam, Univ of Georgia, Augusta, GA; Jim Hutchinson, Erin Muller, Med
College of Georgia, Augusta, GA; Mostafa Elgebaly, Univ of Georgia, Augusta, GA; Adviye
Ergul; Med College of Georgia; Univ of Georgia, Augusta, GA
Diabetes causes both micro and macrovascular complications leading to a 24 fold increased
risk of cardiovascular mortality and morbidity. Diet-induced obesity and related manifestations
such as insulin resistance and hypertension oftentimes cluster with diabetes and form the
metabolic syndrome that poses a major challenge on the vasculature. However, the relative
roles of diabetes and diet-induced hyperlipidemia in mediating microvascular compliance and
function have not been well understood. Thus the aims of this study were to study the individual
and combined effects of two in eliciting 1) vascular compliance and 2) vascular function. Wistar
rats and spontaneously diabetic Goto-Kakizaki (GK) rats fed control or equal amounts of high-fat
diet were used for the studies. Body weight, blood glucose, plasma lipids, insulin and adiposity
levels were increased in GK rats with high-fat diet treatment (*p0.05 vs GK). Third order
mesenteric arteries were evaluated for myogenic tone, stiffness and vascular relaxation using
the pressurized arteriograph. Vessel stiffness was increased in both the regular or high fat diet
GK groups compared to the Wistar groups with an interaction between diabetes and the
high-fat diet in increasing stiffness (*p0.05 vs Wistar). Myogenic tone evaluated at 60 mmHg
showed increased tone in the GK rats (*p0.05 vs Wistar), which was not present with high-fat
diet treatment. Vascular response to both acetylcholine (ACh) and sodium nitroprusside (SNP)
(0.1nm-1m) were impaired as seen by decreased relaxation to ACh and decreased sensitivity
to SNP respectively (*p0.05 vs Wistar) in GK rats on a high-fat diet, but not in Wistar rats.
These results suggest that diabetes decreases vascular compliance, increases vascular tone
and impairs vasorelaxation in GK rats and high fat diet exacerbates vascular dysfunction, which
may contribute to increased and accelerated vascular disease in diabetes. Thus, both
hyperglycemia and hyperlipidemia need to be targeted for effective prevention and treatment
of diabetic vascular disease.
P104
Advanced Lipoxidation End Products Induce the Expression of
Proinflammatory Cytokines and Chemokines in Human THP-1 Monocytes
Narkunaraja Shanmugam, James L Figarola, Yan Li, Piotr M Swiderski, Samual Rahbar,
Rama Natarajan; Beckman Rsch Institute of City of Hope, Duarte, CA
Reactions of carbohydrates or lipid-derived intermediates with proteins lead to advanced
glycation or -advanced lipoxidation end products respectively (AGE/ALEs). AGE/ALE levels
increase with age and in diseases like diabetes and atherosclerosis. Although the cellular
effects of AGEs have been extensively studied, ALE effects have not yet been reported. In this
study, we tested our hypothesis that ALE (malondialdehyde-lysine, MDA-Lys) can induce the
expression of proinflammatory genes in monocytes and thereby lead to monocyte activation.
We first adopted a profiling method using cytokine antibody arrays containing antibodies to 120
known human cytokines. THP-1 monocytes were cultured either under normal glucose
conditions (NG, 5.5 mM) or treated with in synthesized ALE ( MDA, Lys, 10ug/ml) or MDA alone
(MDA, 10ug/ml) for 8hrs and the conditioned media hybridized to the arrays. Specific targets
were induced in MDA-Lys treated cells relative to NG or MDA alone. In particular, there were
significant increases in monocyte chemotactic protein-1&2 (MCP-1, 2), TNF ligand superfamily
member 14 (TNFSF14), Chemokine CC motif Ligand 11 (Eotaxin), Growth Related Oncogene
& (GRO), and Chemokine CXC motif Ligand 13 (BLC). In the next approach, RT-PCR and
qPCR analyses of total RNA showed that, similar to AGEs, MDA-Lys also significantly increased
the mRNA expressions of receptor for AGEs (RAGE) as well as Interferon-g-inducible protein
(IP)-10, MCP-1, 1 , 2-Integrin, Cyclooxygenase (COX)-2 and iNOS. Furthermore, MDA-Lys
was a potent inducer of oxidant stress in monocytes (4-fold increase in ROS and superoxide
production). MDA-Lys significantly increased the activities of p38MAPK and transcriptional
factors NF-kB and AP1, and also induced 4-fold increase in monoctye binding to vascular
smooth muscle and endothelial cells. Interestingly, there were significantly increased levels of
MDA-Lys in the serum of diabetic rats relative to control (by ELISA) and this correlated with
increased serum Eotaxin in the diabetic rats. These new results demonstrate that ALEs formed
under diabetic and atherosclerotic conditions can lead to monocyte activation by inducing
oxidant stress and key inflammatory genes.
2007 ATVB Poster Presentations e-55
by on March 25, 2011 atvb.ahajournals.org Downloaded from
P105
Dietary Cholesterol Selectively Increases Visceral Adipose Tissue
Macrophage Accumulation in Obese LDL ReceptorDeficient Mice
Savitha Subramanian, Elizabeth A Kirk, Kevin D OBrien, Alan Chait; Univ of Washington,
Seattle, WA
In obesity, macrophages accumulate in adipose tissue and contribute to insulin resistance.
Obesity also results in chronic low-grade systemic inflammation that is associated with insulin
resistance, type 2 diabetes and contributes to atherosclerosis. We previously showed that
dietary cholesterol induces low-grade inflammation and increased atherosclerosis in LDLR-/-
mice fed a Western diet. To study the interaction of dietary cholesterol and obesity on chronic
inflammation, LDLR-/- mice were fed either a rodent chow diet (control) or a diabetogenic diet
(high in fat and CHO), without or with 0.15% cholesterol (D-C & DC respectively). Weight gain
was similar in D-C and DC mice, and significantly more than controls. Insulin levels were
higher and responsiveness to insulin was lower in the DC as compared to the D-C group,
suggesting an additive effect of dietary cholesterol on insulin resistance. As compared to
controls, circulating levels of the inflammatory protein, serum amyloid A, were 2-fold higher in
D-C animals and 5-fold higher in DC animals, suggesting chronic inflammation. Morpho-
metric analysis of epididymal (visceral) adipose tissue (AT) showed adipocyte hypertrophy and
greatest macrophage infiltration in the stroma in the DC group compared to the chow and
D-C groups (p0.001). The DC group also showed the greatest stromal expansion and
proteoglycan area compared to chow-fed mice (p0.01). Visceral AT mRNA levels of the
chemokine, MCP-1, and of the macrophage marker, F4/80, were highest in the DC group
(p0.05 & 0.001 respectively). As compared to chow-fed animals, visceral AT mRNA levels
were increased for TNF- and decreased for adiponectin in both D-C and DC diet-fed
animals (both p0.05). While adipocyte hypertrophy was noted in subcutaneous AT, no
significant macrophage accumulation was seen in any of the groups, and there were no
differences between groups in adiponectin, F4/80 or TNF- mRNA levels. These findings
indicate that dietary cholesterol induces a chronically inflamed state, with increased
macrophage infiltration and TNF- production confined to visceral, but not subcutaneous AT.
The exact mechanism whereby dietary cholesterol leads to macrophage recruitment into
visceral AT in obesity needs further elucidation.
P106
Effect of Increased Consumption of Whole Grain Foods on Markers of
Cardiovascular Disease Risk in Middle-aged Healthy Volunteers
Paula Tighe, Nicholas Vaughan, Julie Brittenden, William G Simpson, William Mutch, Univ of
Aberdeen, Aberdeen, United Kingdom; Graham Horgan, Garry Duthie, Rowett Rsch Institute,
Aberdeen, United Kingdom; Frank Thies; Univ of Aberdeen, Aberdeen, United Kingdom
Epidemiological studies suggest that high intakes of whole grain foods may lower cardiovas-
cular disease (CVD) risk. However, current recommendations that consumption of three
servings of whole grain foods daily may be cardio-protective have not been validated. The aim
of this on going study is to assess the effects of increased consumption of whole grain foods
on markers of CVD risk in relatively high-risk individuals. Volunteers (n 65; 4065y, 38
males and 27 females), recruited from the local community, were randomised into one of three
dietary intervention groups and asked to consume three portions per day of either refined,
wheat-based or oat wheat-based foods for 12 weeks. Blood samples were collected at
baseline and post-intervention and analysed for lipid profiles, lipoproteins and high-sensitivity
C-reactive protein. Blood pressure, body mass index (BMI) and arterial stiffness (determined by
pulse contour analysis) were also measured. None of the blood bio-markers assessed differed
significantly after dietary intervention. Arterial stiffness was also unaffected by dietary
treatment. However, after adjustment for age, sex and BMI, both systolic and diastolic blood
pressure decreased significantly in the wheat-based and oat wheat-based groups compared
to the refined group (table). This initial data indicates that the daily consumption of 3 portions
of either wheat-based or oat wheat-based whole grain foods may protect against CVD via
a blood pressure-lowering mechanism.
Refined (n14) Wheat (n27) Oat wheat (n24)
Systolic BP (mmHg)
Pre-intervention 127 (2.6) 128 (2.3) 131 (2.2)
Post-intervention 127 (2.8) 122 (2.1) 126 (1.5)
% change
*
-0.3 (1.0)
a
-4.6 (1.1)
b
-3.8 (1.2)
b
Diastolic BP (mmHg)
Pre-intervention 76 (1.4) 77 (1.7) 79 (1.1)
Post-intervention 77 (1.3) 75 (1.5) 77 (0.9)
% change
*
1.6 (1.1)
a
-2.3 (1.2)
b
-2.9 (1.1)
b
Mean (SEM).
a,b
Unlike superscripts are significantly different (ANOVA)(P0.05;
LSD).
*
% change to intervention was calculated from post-treatment minus
pre-treatment values expressed as a percentage of pre-treatment value.
P107
Lipid and Glucose Optimization Using Phytonutrient Combination Therapy
in Diabetes
Peter J Verdegem, Unicity International, Orem, UT; Bobbi Horne, Isabel Martinez; Blue Mesa
Med Associates, Katy, TX
Introduction: Dietary approaches to management of lipid and glucose parameters in diabetes
is gaining popularity among patients. Monotherapy with dietary ingredients has shown positive
effects, but with limited clinical relevance. Our research focuses on using a phytonutrient
combination in optimizing lipid and glucose parameters. All four ingredients have individual
data supporting their use for optimizing lipoprotein fractions in hypercholesterolemia. This pilot
study evaluates their combined efficacy in type-II diabetes. Methods: A group of 34 subjects
with established type-II diabetes and hypercholesterolemia added the product to their diet. The
drink was taken twice daily 1520 minutes before meals. The fiber drink consists of viscous
soluble fiber, minerals, vitamins, policosanol, phytosterols, and an aqueous Chrysanthemum
morifolium extract. Lipid and glucose parameters were measured at baseline, 4 and 8 weeks.
Results:
Parameter
Inclusion criteria
at BL (mg(dL)
B.L.
(mg/dL)
t8 weeks
(mg/dL) % p-value
TC All 208 178 -14.2 0.01
TC 200 245 195 -21.5 0.001
LDL-c All 127 104 -18.3 0.05
LDL-c 160 197 141 -28.9 0.005
HDL-c All 46 48 3.5 n.s.
HDL-c 40 35 40 14.4 n.s.
TG All 182 143 -21.3 n.s.
TG 150 242 163 -32.5 0.05
Glu All 162 134 -17.3 0.05
Glu 175 218 155 -28.9 0.05
HbA1c All 7.2
a
6.6
a,b
-9.4 0.05
HbA1c 8
a
9.2
a
7.8
a,b
-15.8 0.05
a
in %;
b
measurement at 12 weeks.
Conclusion: BiosLife, a phytonutrient combination drink, shows potential in optimizing
parameters associated with cardio vascular disease risk in type-II diabetes. These findings are
well in line with previously reported clinical results. The fiber component has reduced the
post-prandial glucose levels and the resulting lower HbA1c levels indicate that BiosLife
provides a natural option to improve diabetes management.
P108
Impact of Omega-6 Polyunsaturated Fatty Acid:Eicosapentaenoic acid
Docosahexaenoic Acid Ratios in LDL-receptor Knockout (LDLr-/-) Mice on
Atherosclerotic Lesion Formation and Elicited Peritoneal Macrophages
Inflammatory Response
Shu Wang, Dayong Wu, Nirupa R Matthan, Alice H Lichtenstein; Tufts Universtiy, Boston,
MA
Objective Very long chain omega-3 fatty acids have been associated with decreased risk of
CVD. LDL receptor knockout mice were used to assess the effect of different omega-
6:EPADHA ratios on atherosclerotic lesion formation and elicited peritoneal macrophage
inflammatory response. Methods and Results Mice (n10/group) were fed atherogenic
diets for 32 weeks: high fat (20% w/w) without EPADHA (HF omega-6), and high fat with
omega-6:EPA DHA ratios of 20:1 (HF R20:1), 4:1 (HF R4:1), and 1:1 (HF R1:1). Aortic total
cholesterol was 2.6%, 19.8% and 24.3% lower in the HF R20:1, HF R4:1 and HF R1:1 fed mice,
respectively, compared with HF omega-6 fed mice. Elicited peritoneal macrophage cholesteryl
ester content (mg/100mg protein) was 4.3 1.3
a
, 3.9 1.1
ab
, 2.6 0.7
b
, and 2.7
0.7
b
(values with different superscripts are significantly different at P0.05) in mice fed HF
omega-6, HF R20:1, HF R4:1, and HF R1:1 diets, respectively. Peritoneal macrophage
membrane fatty acid profile reflected dietary treatment. Elicited peritoneal macrophages
isolated from these mice were stimulated with lipopolysaccharide. Monocyte chemoattractant
protein-1 (MCP-1) release into the culture medium was 28 7
a
, 23 9
ab
, 18 8
ab
, and 17
6
b
ng/mg protein in HF omega-6, HF R20:1, HF R4:1, and HF R1:1 diet groups, respectively.
Gene expression levels of MCP-1, tumor necrosis factor alpha (TNF-), and ATP-transporter
cassette A1 (ABCA1) were significantly lowered in elicited peritoneal macrophages from mice
fed HF R1:1 compared to mice fed HF omega-6 diet. Conclusions These data suggest that
diets with lower omega-6:EPADHA ratios resulted in a lower inflammatory response which
was associated with less aortic lesion formation.
P109
CD36 Modulates Macrophage Spreading and Migration in Response to
Oxidized Low-density Lipoprotein
Young M Park, Case Western Reserve Univ/Lerner Rsch Institute, The Cleveland Clinic
Foundation, Cleveland, OH; Maria Febbraio, Roy L Silverstein; Lerner Rsch Institute, The
Cleveland Clinic Foundation, Cleveland, OH
CD36 is a class B scavenger receptor expressed on mononuclear phagocytes that has been
implicated in many biological processes including atherosclerosis and angiogenesis. Previous
work showed that CD36 binds and mediates uptake of oxidized LDL (oxLDL) and also transmits
intracellular signals through src and MAP kinases. We hypothesized that oxLDL may affect
cellular adhesion and migration via CD36-dependent signalling by modulating cytoskeletal
function. Using a modified Boyden chamber migration assay, we showed that oxLDL but not
native LDL inhibited random and MCP-1 induced mouse peritoneal macrophage migration, an
effect abrogated in macrophages from CD36 null mice. OxLDL but not native LDL induced rapid
spreading on serum-coated glass, and this was delayed in CD36 null cells. There were 2.7 fold
more spread wild type than CD36 null cells 5 minutes after stimulation with oxLDL. This was
also evaluated by measuring cellular area; wild type macrophages had 2 fold greater mean
cellular area compared with CD36 null macrophages at this same time point. Western blots of
extracts from macrophages incubated with oxLDL showed sustained phosphorylation (activa-
tion) of focal adhesion kinase (FAK) and dephosphorylation (inactivation) of src homology
2-containing phosphotyrosine phosphatase, SHP-2. Inactivation of SHP-2 in oxLDL-treated cells
was associated with oxidative modification of cysteine residues, detected by measuring
5-iodoacetamidofluorescein binding. Flow cytometry of macrophages incubated with fluores-
cently labeled phalloidin showed that oxLDL induced actin polymerization. These responses to
oxLDL including activation of FAK, inactivation of SHP-2 and promotion of actin polymerization,
were significantly blunted in macrophages from CD36 null mice. We conclude that CD36
signaling in response to oxLDL contributes to cytoskeletal rearrangement and modulates
macrophage spreading and migration, and suggest that cytoskeletal rearrangements mediated
by CD36 may induce trapping of macrophages in the arterial intima and thus promote
atherosclerotic lesion development.
e-56 Arterioscler Thromb Vasc Biol June 2007
by on March 25, 2011 atvb.ahajournals.org Downloaded from
P110
Sphingosine-1-Phosphate Induces an Anti-inflammatory Phenotype in
Macrophages During Inflammation
Jeniter Hughes, Suseela Srinivasan, Catherine Hedrick; Univ of Virginia, Charlottesville, VA
Activated macrophages acquire a pro-inflammatory (classical) or anti-inflammatory (alternative)
phenotype that influences atherosclerosis. Sphingosine-1-phosphate (S1P), a novel anti-
inflammatory sphingolipid, serves a protective role in inflammation by changing the macro-
phage phenotype from a pro-inflammatory M-1 classical phenotype to an anti-inflammatory
M-2 alternative phenotype. We examined the anti-inflammatory effects of S1P on LPS-
stimulated cytokine secretion in primary C57BL/6J (B6) mouse peritoneal macrophages. LPS
upregulated mRNA expression of the pro-inflammatory cytokines TNF-alpha and MCP-1 by
several-fold. Incubation of macrophages with S1P reduced TNF-alpha mRNA by 3-fold (p
0.0061) and MCP-1 by 2-fold (p0.001). S1P decreased both TNF-alpha and MCP-1 secretion
by LPS-stimulated macrophages by approximately 50%. Alternative macrophage activation is
characterized by induction of the anti-inflammatory Th2 cytokine IL-10 and arginase-1. S1P
triggered a 10-fold increase in IL-10 mRNA expression (p0.001) and a 2-fold increase in
arginase-1 mRNA level (p0.001). Arginase-1 enzyme activity was increased by 35%.
Macrophages were activated by LPS alone to express inducible NO synthase (iNOS), and the
response was significantly decreased by approximately 50% with the addition of S1P. Mouse
peritoneal macrophages express S1P1 and S1P2 receptors. The uncoupling of S1P1 Gi receptor
signaling with pertussis toxin inhibited the anti-inflammatory action of S1P on macrophage
activation. Pertussis toxin blocked the ability of S1P to reduce TNF-alpha and MCP-1 mRNA
expression in LPS-stimulated macrophages. Treatment of B6 macrophages with the selective
S1P2 receptor antagonist JTE-013 did not affect the ability of S1P to reduce LPS-mediated
cytokine expression. Therefore, these results suggest that S1P1 is responsible, at least in part,
for the alternative activation of macrophages during inflammation. We therefore conclude that
S1P promotes the alternative anti-inflammatory macrophage phenotype through action on
S1P1.
P111
Hearts Require Lipoprotein-derived Fatty Acids (FAs) to Compensate for
Chronic Angiotensin II Treatment
Haruyo Yamashita, Shota Ikeda, Tae-Sik Park, Ira Goldberg; Columbia Univ Med Cntr, New
York, NY
Under metabolic conditions found in diabetes and metabolic syndrome, hearts acquire more
relatively more cardiac energy from FA oxidation. Although such cardiac metabolic changes are
thought to play a role in pathogenesis of reduced cardiac function, it is also likely that reduced
cardiac lipoprotein-derived FA metabolism could adversely affect stressed hearts. Because
LpL-derived FAs are a major source of energy in normal hearts, we tested whether cardiac
specific LpL knock-out (hLpL0) mice respond normally to the stress induced by chronic Ang II
treatment. Control and male hLpL0 mice (1012 weeks), whose cardiac morphology and
function are normal under basal conditions, were treated with Ang II (4 mg/kg/day, s.c.) for 2
weeks. In both groups of mice, Ang II treatment similarly increased systolic blood pressure to
170mmHg, heart weight/body weight ratio, and left ventricular mass from 70mg to
125mg. With treatment, control mice showed a significant reduction in left ventricular
diastolic diameter (from 4.1 to 3.8 mm) and preserved % fractional shortening, indicating
compensated concentric hypertrophy. Only in hLpL0 mice, however, the Ang II treatment
significantly decreased % fractional shortening by 10% and significantly increased lung
weight/body weight ratio. In control animals, Ang II treatment increased cardiac Glu uptake
4-fold; hLpL0 mice have elevated basal glucose uptake but with Ang II-treatment glucose
uptake was almost equal between the control and hLpL0 mice. There was no significant
difference in cardiac gene expression of PDK4, fatty acid translocase/CD36, acyl-CoA oxidase
and carnitine palmitoyltransferase I. These results suggest that increased glucose uptake alone
is not sufficient for hearts to compensate for hypertensive stress and that cardiac acquisition
of LpL-derived FA has a critical role to allow normal heart compensation.
P112
The Role of Different Pathways in the Release of Cholesterol from Foam
Cells
Maria Pia Adorni, Francesca Zimetti, George H Rothblat; Childrens Hosp of Philadelphia,
Philadelphia, PA
Introduction: Cholesterol efflux from cells occurs by different pathways, including aqueous
diffusion, and transport mediated by specific proteins, such as SR-BI, ABCA1 and ABCG1. The
level of expression of these proteins is regulated by a number of different factors among which
is intracellular cholesterol content. Previous observation made in Fu5AH hepatoma cells
showed that the fractional (%) efflux of cholesterol to 15 human sera was similar with normal
and enriched cells, suggesting that it was the same mechanism driving efflux, independent of
cholesterol content. In contrast, in mouse peritoneal macrophages (MPM) we observed an
increase in fractional release of cholesterol to serum when cells were enriched by exposure to
acLDL. Aim of study: The aim was to quantitate the contribution of different efflux pathways
using cholesterol-normal and enriched MPM exposed to human serum. This was accomplished
by 1) using MPM from ABCA1 and SRBI KO mice and 2) using the specific ABCA1 and SR-BI
inhibitors Probucol and BLT-1. Methods: MPM were labeled with
3
H-cholesterol with or without
50g/ml of acLDL. After an equilibration period, the cells were incubated in 2.5% human
serum and the efflux of cholesterol was measured after 8h. The inhibitors Probucol and BLT-1
were added to cells in the equilibration phase. The initial cell cholesterol mass was measured
by a fluorimetric assay. Results: In WT MPM loading with cholesterol caused an increase in
efflux by 90100%. The contribution of SR-BI to the total efflux, measured by BLT-1 inhibition,
was very low (1.7%3.2%) in both cholesterol normal and enriched cells. Consistent with this
observation, cholesterol enrichment of SR-BI KO macrophages induced an increase in fractional
release of cholesterol similar to that observed in WT cells. The ABCA1 contribution, as
measured by Probucol inhibition of efflux, increased after cholesterol loading (from 2.4% to
8.5%). When ABCA1 KO cells were enriched there was no increase in the fractional release of
cholesterol. Conclusion: In conclusion, our results demonstrate that the ABCA1 protein plays
the major role in the stimulation of the fractional release of cholesterol upon enrichment of
MPM. Additional experiments are being conducted to asses the role of ABCG1 in this processes.
P113
Acyl-Coa:Cholesterol Acyltransferase 2 and ATP-binding Cassette Half
Transporters G5 and G8 Differentially Alter Hepatic Lipoprotein Secretion
and Composition
Heather M Alger, Wake Forest Univ, Winston-Salem, NC; Paolo Parini, Karolinska Institutet
at Huddinge Univ Hosp, Huddinge, Sweden; Ramesh Shah, Liqing Yu, Lawrence L Rudel;
Wake Forest Univ, Winston-Salem, NC
Three proteins that function in the regulation of cholesterol metabolism and are known to be
expressed primarily in hepatocytes and enterocytes are: ATP-binding cassette half transporters
G5 and G8 (G5G8) and acyl-CoA:cholesterol acyltransferase 2 (ACAT2). G5G8 is responsible for
inside-out transport of cholesterol in cells and ACAT2 is an endoplasmic reticulum-bound
enzyme that esterifies cholesterol with acyl-CoA. One of the common processes that these
proteins appear to regulate in both cell types is lipoprotein particle secretion. To identify the
nature of this regulation, mice bearing gene deletions for G5G8 and/or ACAT2 were created.
G5G8 KO mice had a higher liver-to-body weight ratios than wild type mice, apparently due to
hepatic triglyceride (TG) accumulation. ACAT2 KO mice had lower hepatic TG accumulation but
higher TG secretion. The double KO mice had hepatic triglyceride secretion rates and
accumulation similar to that seen in ACAT2 KO mice. The data suggest that sterol handling
regulates VLDL secretion and composition. Absence of cholesterol esterification resulted in
increased VLDL particle secretion and decreased intracellular triglyceride accumulation while
absence of cholesterol transport out of the cell via G5G8 resulted in triglyceride accumulation
in the cell and decreased VLDL secretion. Thus, the pools of cholesterol accessed by G5G8 and
ACAT2 appear to be different and impose separate outcomes on VLDL particle secretion vs.
intracellular triglyceride accumulation. These effects were effectively balanced out in the
double KO animals.
P114
The Role of the CYP3A4 Gene Variants in Lipid-Lowering Efficacy of
Simvastatin Therapy
Sarah A Alzahrani, Liverpool Univ/King Faisal Specialist Hosp and Rsch Cntr, Riyadh, Saudi
Arabia; Nduna Dzimiri; King Faisal Specialist Hosp and Rsch Cntr, Riyadh, Saudi Arabia
Introduction: Patient responses to drug therapy may vary as a result of intra-individual
difference in their metabolizing enzymes. CYP3A4 is the primary metabolic pathway for
simvastatin. In this study, we tested the hypothesis that genetic variations in the CYP3A4 gene
contribute to the differences in which patients respond to simvastatin therapy. Methods: We
set out to (a) determined the prevalence of the single nucleotide polymorphism (SNP)
rs4986910 in exon 12, rs12721620 in intron 12 and rs2740574 (A-290G) in the promoter
region of the CYP3A4 in 160 angiographed controls, and (b) investigated their possible
relevance for patient responses to simvastatin (20 mg/day) therapy in 133 hyperlipidaemic
patients of Saudi origin. Plasma lipid and lipoprotein levels were measured before treatment
and at 6 month follow-up. Results: The frequencies of the rs2740574 genotypes were 21.1%
for A/G, 78.9% for A/A in patients and 10.8% (A/G), 88.0% (A/A) and 0.6% for G/G in controls.
For the rs12721620 mutation, the frequencies were 6.8% for C/T and 93.2% for the C/C in
patients, and 3.8% (C/T), 95.6% (C/C) and 0.6% for the T/T in controls. No carrier of the
rs4986910 were found among the studied patients or controls. Carriers of the wild type A/A
allele of the rs2740574 had greater reduction in total cholesterol (Chol) (5.3 0.11 vs 4.7
0.11; p 0.001) and in low density lipoprotein (LDL) (3.3 0.10 vs 2.9 0.08; p 0.001),
compared to heterozygous A/G carriers, following treatment with simvastatin. Similarly, for the
rs12721620, the homozygote C/C showed significant reduction in Chol (5.2 0.10 vs 4.6
0.10; p 0.001) and in LDL (3.3 0.09 vs 2.8 0.08; p 0.001) compared to the
heterozygote C/T. However, linear regression analysis suggested an association of heterozygote
C/T of the rs12721620 with reduction in triglycerides (TG) (P 0.041) and Chol (P 0.032),
while no significant associations were observed between either of the SNPs and pretreatment
lipid levels in the patient population. Conclusion: Our preliminary data point to the rs12721620
C/T polymorphism as a predictor of TG and Chol responses to simvastatin treatment.
P115
Severity of Fatty Liver Due to Impaired Lipoprotein Secretion Is Genetically
Determined and Independent of Insulin Resistance
Pin Yue, Robin Fitzgerald, Xiaobo Lin, Zhouji Chen, Gustav Schonfeld; Washington Univ Sch
of Medicine, St. Louis, MO
Patients with Familial Hypobetalipoproteinemia (FHBL) due to apoB truncations have elevated
liver triglyceride (TG) content due to impaired lipoprotein export system. Mouse models of apoB
truncations (apoB38.9 and apoB27.6) mimic the human FHBL phenotype: TG accumulation in
the liver, elevated liver cholesterol, and decreased plasma levels of triglyceride and cholesterol.
However, large variations of liver fat were observed in both, humans and mice of mixed genetic
background bearing the apoB27.6 and apoB38.9 truncations. We hypothesized that the severity
of steatohepatosis due to apoB truncation is genetically determined. To test this hypothesis, we
generated congenic mice carrying apoB38.9 under three genetic backgrounds: SWR/J (low liver
TG strain, 40mg/dL), C57BL/6J (medium liver TG, 140mg/dL), and BALB/cByJ (high liver
TG strain, 200 mg/dL). Liver and plasma lipids (TG, cholesterol, and free fatty acid (FFA))
were measured. Insulin responses were also measured by 2-hour glucose and insulin tolerance
tests (GTT and ITT). Significant interactions between genetic background and apoB genotype
existed for plasma cholesterol (p0.0001), and liver TG (p0.0162), but not for liver
2007 ATVB Poster Presentations e-57
by on March 25, 2011 atvb.ahajournals.org Downloaded from
cholesterol and plasma TG, and FFA. The BALB/cByJ-apoB
/38.9
strain had the largest increase
of liver TG due to the presence of the apoB truncation (100 mg/dL), while SWR/J-apoB
/38.9
had the lowest (40 mg/dL). However, SWR/J-apoB
/38.9
mice showed significant insulin
resistance compared with the cognate wildtype, i.e. glucose concentrations at 30 min, 60 min,
and 120 min and the 30-min insulin values were significantly elevated in SWR/J-apoB
/38.9
mice compared to SWR/J-apoB
/
. There were no significant changes in glucose tolerance test
curve in C57BL/6J and BALB/cByJ apoB
/38.9
mice. Our results showed that the actions of
modifier genes determine the extent of liver fat accumulation and insulin resistance in these
apoB38.9 bearing congenic strains. Molecular mechanisms of these differences are under
further investigation.
P116
Repair of Oxidative Low-density Lipoprotein and High-density Lipoprotein
by Recombinant Human Methionine Sulfoxide Reductase A
Hong Yu, Zhen Wang, Ming Li, Xiaoming Li, Junzhu Wu, Chunyan He; Wuhan Univ, Wuhan,
China
Background Atherosclerosis(As) is characterized by the accumulation of both oxidized lipids
and inflammatory cells in the arterial lesion that caused by a state of heightened oxidative
stress. The existence of oxidized low density lipoprotein (LDL) and high density lipoprotein
(HDL) in circulation could be used as potential markers for coronary artery disease. Methionine
sulfoxide reductase A (MsrA) presents in all living organisms and is one of the most important
antioxidative enzymes which reduce methionine sulfoxide (MetSO) residues in proteins. The
goal of the study was to investigate the ability of recombinant human MsrA to repair oxidative
damage of LDL and HDL in vitro, and protect against damage of cell induced by oxidized
lipoproteins. Methods and Results The recombinant hMsrA was expressed in E. Coli.
M15(pREP4) and purified by Ni-NTA agarose affinity chromatography. Human LDL and HDL
were separated by two-step density gradient ultracentrifugation and oxidized by Cu
2
and
AAPH in vitro. Recombinant hMsrA incubated with lipoproteins could decrease relative
electrophoresis mobility of ox-LDL and ox-HDL in agarose-gel electrophoresis, which denoted
reduction of oxidative extent on LDL/HDL, hMsrA was also identified to protect apolipoprotein
(apo) B100 of LDL against degradation by oxidation and restore the mobility of modification apo
AI of HDL in PAGE, furthermore, hMsrA decreased the amount of MDA of LDL, indirectly
reflecting its inhibitive ability on lipid peroxidation and protected cultured endothelial cells from
impairment induced by ox-HDL. Conclusion hMsrA may play an important role in protecting
against lipoproteins oxidation and cell damage induced by ox-HDL, may account in part for the
repairing function of hMsrA through reducing MetSO of apo AI and apo B100 to methionine
P117
Acetaldehyde Dehydrogenase-2 Gene Polymorphism in Han Chinese People
with Acute Coronary Syndrome
Feng Xu, Yu-guo Chen, Qilu Hosp of Shandong Univ, Jinan, China; Madi Bidya Sagar, Texas
Heart Institute and Univ of Texas-Houston, Houston, TX; He Zhang, Chun-xiao Jiang, Yun
Zhang, Qilu Hosp of Shandong Univ, Jinan, China; Yong-jian Geng; Texas Heart Institute and
Univ of Texas-Houston, Houston, TX
Objective: Acetaldehyde dehydrogenase-2 (ALDH2) gene polymorphism occurs in a significant
number of Asians, leading to an altered metabolism of alcohol, other aldehydes or certain
drogs. In this study we investigated the potential association of the ALDH2 gene polymorphism
with acute coronary events, such as acute myocardial infarction (MI), in Han Chinese. Methods:
DNA was isolated and analyzed by PCR-direct sequencing from peripheral blood samples of
271 Han people with coronary atherosclerotic heart disease (CAD). Coronary function was
determined by coronary angiographic and ultrasound assessment of brachial artery dilation.
The severity of coronary atherosclerosis was evaluated by quantitative angiography with
Gensini score. Results: DNA sequencing identified the missense mutation (
*
1/
*
2
*
2/
*
2) in 98
individuals. The rest of patients have the wild genotype of
*
1/
*
1 (n173). Compared with the
wild-type group, the mutant group had a higher rate of diabetes mellitus, lower prevalence of
drinking and alcohol consumption. The mutant group also showed a reduction in the coronary
diameter and blood flow in response to sublingual administration of nitroglycerin, but the two
groups had a similar coronary dilation response when exposed to reactive hyperemia, and the
almost equal number of lesional coronary arteries and similar Gensini scores. The mutant group
had a significantly increased rate of MI when compared to that in the wild-type group. Multiple
regression analysis by adjustment for conventional risk factors of CAD showed that the ALDH2
missense mutation served as an independent risk factor for acute coronary syndrome in Han
Chinese. Interestingly, this association was weakened when alcohol consumption was put into
consideration as an independent variable. Conclusions: A great portion (36%) of Han Chinese
with coronary heart disease show the ALDH2 missense mutation (genotypes *1/*2*2/*2), and
they have a high incidence of MI and other coronary events. Hence, the mutation may serve
as a potentially independent risk factor for MI in Han Chinese people. ALDH-related changes
in alcohol consumption and drug metabolisms may contribute to impaired endothelial function,
a critical factor for the development of acute coronary events.
P118
Tandem Amphipathic -Helices Influence the Ability of ApoA-I Mimetic
Peptides to Exert Potentially Antiatherogenic Effects
Geoffrey D Wool, Catherine A Reardon, Godfrey S Getz; The Univ of Chicago, Chicago, IL
The apoA-I mimetic peptide 4F has been shown to be anti-inflammatory and anti-atherogenic
in vitro and in vivo. A detailed understanding of its effect on HDL metabolism is lacking,
however. It is also currently unknown if a mimetic better mimicking the punctuated -helical
repeats of full-length apoA-I would show a more anti-inflammatory or anti-atherogenic profile.
This study compares the following mimetics in vitro: L4F (18mer), L4F-proline-L4F (37mer),
and L4F-KVEPLRA-L4F (the human apoA-I 4/5 interhelical sequence (IHS), making a 43mer).
Incubation of peptides with isolated mature mouse HDL (moHDL) caused remodeling. All three
peptides shifted apoA-I to less electronegative pre- particles by agarose electrophoresis as
well as two-dimensional separation. By native PAGE, all three peptides shifted apoA-I from
mature HDL particles (9.8nm) to free protein of 7.2nm. Per gram, the tandem peptides were
more effective at remodeling moHDL than monomeric L4F. Native PAGE separation of
remodeled moHDL revealed not just dissociation of free apoA-I, but also formation of a larger
HDL-like particle which did not contain detectable apoA-I. PeptidemoHDL incubations at the
remodeling threshold were ultracentrifuged to separate these particles. All three peptides
produce A-II/peptide containing HDL-like particles, displacing apoA-I and apoE. The IHS tandem
peptide produced the largest particles (11.5nm) while the Pro tandem and monomeric L4F
produced similarly sized particles (10.75nm). Mimetic-mediated in vitro lipid efflux correlates
with anti-atherogenic reverse cholesterol transport in vivo. Compared to the monomeric L4F,
the Pro tandem showed greater ABCA1-independent and -dependent efflux ability for
3
H-cholesterol loaded J774 murine macrophage foam cells. Peptides also inhibited copper-
oxidation of purified moLDL, as monitored by conjugated diene formation. The peptides
inhibited oxidation based on both lag time and slope in the following rank order: controlPro
tandemIHS tandemL4F monomer. In the presence of both purified moLDL and moHDL, the
peptides showed the following efficacy rank order: controlPro tandemIHS tandemL4F
monomer. These in vitro experiments allow correlative data for future atherosclerosis studies.
P119
Local Variations in Shear and Mural Stress Influence Endothelial
Permeability and Gene Expression in Arterial Segments Exposed to Cyclic
Axial Stretch Ex Vivo
J S VanEpps, Douglas W Chew, David A Vorp; Univ of Pittsburgh, Pittsburgh, PA
Introduction: Certain arteries (e.g., coronary, carotid, etc.) are exposed to cyclic axial
stretching due to their tethering to surrounding tissue beds. It is believed that such stimuli
result in a spatially variable biomechanical stress distribution which has been implicated as a
key modulator of atherosclerotic lesion localization. We have developed a combined ex vivo
experimental / computational methodology to address the hypothesis that local variations in
shear and mural stress associated with cyclic axial stretch of arterial segments influence the
distribution of early markers of atherogenesis. Methods: Bilateral porcine femoral arteries were
surgically harvested and perfused ex vivo under pulsatile arterial conditions. One of the paired
vessels was exposed to cyclic axial stretching of 7% over in vivo length at 1 Hz for 12 hours.
During the last hour, the perfusate was supplemented with Evans blue dye-labeled albumin.
Vessel segments were divided into 4 or 5 portions which could each be individually processed
for both fluorescent microscopy for visualization of albumin permeation and RNA isolation for
DNA microarray analysis. Finite element analysis and computational fluid dynamics techniques
were used to determine the mural and shear stress distributions respectively, for each perfused
segment. Results: On average cyclic stretch caused a 1.4 fold increase in endothelial
permeability. The magnitude of the permeability was directly proportional to the local
circumferential wall stress. DNA microarray analysis revealed a number of genes that were
differentially expressed with respect to circumferential wall stress or shear stress. These
included genes with known roles in the atherogensis including intracellular adhesion
molecule-1 (ICAM-1), inducible nitric oxide synthase (iNOS), matrix metalloproteinase-13
(MMP-13), and monocyte chemotactic protein-1 (MCP-1). Conclusions: We have developed a
unique method for combining ex vivo arterial perfusion culture with computational biome-
chanical simulation. With this tool we have demonstrated that within even a small (7cm)
length of artery there is a complex distribution of atherosclerotic gene expression which
correlates with the biomechanical stress distribution.
P120
The Structure of Lipid-free Apolipoprotein A-IV: Evidence of an Interaction
Between the N- and C-Termini That Modulate Lipid Binding
Matthew R Tubb, Univ of Cincinnati, Cincinnati, OH; Jianwen Fang, Univ of Kansas,
Lawrence, KS; R A Gangani D Silva, W Sean Davidson; Univ of Cincinnati, Cincinnati, OH
Apolipoprotein (apo) A-IV is an intestinally-derived apolipoprotein with potential functions in
reverse cholesterol transport, satiety and chylomicron metabolism. The structural basis for
many of these functions is unknown, due in large part to the lack of three-dimensional structure
information. Our laboratory recently identified a single amino acid in the apoA-IV C-terminus
(F334) that, when mutated to an alanine, permits rapid association of the protein with lipid.
Simultaneous removal of the N-terminus negated this fast-lipid-binding phenotype. We
hypothesize that there is a direct interaction between the N- and C-termini of apoA-IV which
modulates ability of apoA-IV to bind lipid. Using a systematic mutagenesis strategy, we have
identified Trp12 and Phe15 as the two N-terminal amino acids responsible for this effect. To
begin to resolve a structural model for lipid-free apoA-IV, we used chemical cross-linking and
high-resolution mass spectrometry to determine proximal residues in the native structure. We
identified 14 long range and several short range cross-links which we used to construct a
preliminary model of lipid-free apoA-IV. The cross-linking pattern was consistent with a large
helical bundle in which the N- and C-termini where in close proximity. We have begun work
to construct a computer-generated homology model of lipid-free apoA-IV based on sequence
similarity to proteins of known structure. As an independent demonstration of the termini
interaction, a mutant of apoA-IV was constructed that contained Cys residues at position 16 and
336. This mutant failed to bind lipid under oxidizing conditions, but was fully capable when the
disulfide linkage was reduced with 5 mM DTT. We believe that a direct hydrophobic interaction
between specific residues in the extreme ends of apoA-IV stabilizes a helical bundle, thereby
retarding lipid binding. This interaction may toggle the protein between active and inactive lipid
binding states that may dictate its physiological function.
e-58 Arterioscler Thromb Vasc Biol June 2007
by on March 25, 2011 atvb.ahajournals.org Downloaded from
P121
Expression of Type IIA Secretory Phospholipase A
2
Results in Severe
Endothelial Dysfunction in Mice
Markus van der Giet, Markus Tolle, Charite Campus Benjamin Franklin, Berlin, Germany;
Domenico Pratico, Univ of Pennsylvania, Philadelphia, PA; Mirjam Schuchardt, Matthias P
Ho rl, Pauline Lioudmer, Walter Zidek, Charite Campus Benjamin Franklin, Berlin,
Germany; Uwe J F Tietge; Univ Med Cntr of Groningen, Groningen, The Netherlands
Endothelial dysfunction represents an early stage in the development of atherosclerotic
cardiovascular disease, and is also considered as an independent predictor of cardiovascular
disease mortality. The type IIA secretory phospholipase A
2
(sPLA2) may actively contribute to
atherogenesis, acting either locally within the arterial wall or in plasma. In the present study
we tested the hypothesis that vessel wall expression of sPLA2 in transgenic mice results in
endothelial dysfunction. Using the small vessel myograph aortic rings from human sPLA2
transgenic mice displayed severe endothelial dysfunction (maximal vasodilation in response to
10 mol/L ACH in sPLA2 transgenic mice: 368% versus controls: 802% of PE-induced
vasoconstriction), while endothelium-independent vasorelaxation was not impaired. Endothelial
dysfunction was mediated by the increased generation of reactive oxygen species (ROS) within
aortic vascular smooth muscle cells of sPLA2 transgenic aortas. Addition of the free radical
scavenger tiron normalized ROS generation and endothelial dysfunction in sPLA2 transgenic
aortas. sPLA2 transgenic mice also showed increased aortic COX-2 expression and increased
vascular COX-2 activity. Addition of a COX-2 inhibitor normalized endothelial dysfunction in
sPLA2 transgenic mice, while a specific COX-1 inhibitor had no effect. COX-2 inhibition also
suppressed vascular ROS generation in sPLA2 transgenic aortas. In conclusion, our data
demonstrate that sPLA2 expression within the vascular wall results in dramatically decreased
endothelial function by causing oxidative stress in a COX-2-dependent manner. These data
therefore represent an additional pro-atherosclerotic property of sPLA2.
P122
Genome-wide Response of Human Aortic Endothelial Cell to
Triglyceride-rich Lipoprotein Lypolysis Products
Hnin H Aung, Kishorchandra Gohil, Rama S Kota, John C Rutledge; Univ of California at
Davis Sch of Medicine, Davis, CA
The endothelium plays an important role in the control of the vascular system. Triglyceride rich
lipoproteins (TGRL) are an independent risk factor for coronary artery disease and have been
linked to atherosclerosis. We hypothesized that TGRL lypolysis products contribute to
pathogenesis of atherosclerosis by affecting the expression of multiple pro-inflammatory and
pro-coagulant genes. To test this hypothesis, we incubated human aortic endothelial cell
(HAEC) with either TGRL or TGRL lypolysis products for 3 hrs. TGRL were isolated from
postprandial human plasma and incubated with lipoprotein lipase and the resulting lypolysis
products were added to HAEC in culture. Total RNA was extracted from these cells and the
TGRL or TGRL lypolysis products sensitive transcriptomes were obtained using high density
oligonucleotide arrays (U133A 2.0 array). 266 of 285 genes were up-regulated and 19 genes
were down-regulated by TGRL lypolysis products in comparison to TGRL alone. Functional
classification of the affected genes identified transcription factors, cytokines, growth factors,
cell cycle and inflammation-related genes. Specifically, TGRL lypolysis products up-regulated
adhesion molecules (E-selectin and VCAM-1); cytokines (IL6, IL1) and activating transcription
factor 3 gene expressions. Expression of these genes was confirmed by quantitative real-time
RT-PCR. Our results indicate that TGRL lypolysis initiate a stress response in HAEC that has the
molecular signature of inflammation. These preliminary data from in vitro challenged of HAEC
support our hypothesis showing that TGRL lypolysis products modulate multiple pro-
inflammatory genes simultaneously that may contribute to the pathogenesis of atherosclerosis.
P123
Antisense Reduction of Apolipoprotein B-100 Favorably Modifies
Lipoprotein Subclass Distribution in Healthy Human Volunteers
Brenda F Baker, Mark K Wedel, John Su, JoAnn Flaim, Emil Chuang, Diane L Tribble; Isis
Pharmaceuticals, Inc, Carlsbad, CA
Apolipoprotein B (apoB) is a key component of the atherogenic lipoprotein particles VLDL, IDL,
and LDL. Elevated numbers of these atherogenic particles are associated with an increased risk
for cardiovascular disease. An antisense inhibitor of human apoB, ISIS 301012, has
demonstrated a prolonged dose-dependent reduction in apoB in human clinical trials. Favorable
effects on VLDL and LDL particle numbers were observed in a Phase 1 double-blind,
placebo-controlled, dose escalation trial involving 36 healthy volunteers with primary hyper-
cholesterolemia. Subjects received an initial single dose of ISIS 301012 (50 to 400 mg) or
placebo, followed by a 4-week multiple-dosing regimen at the same dose. Fasting lipoprotein
particle numbers were determined by nuclear magnetic resonance spectroscopy (NMR). Large,
medium and small VLDL and HDL, and large and small LDL particle subclasses were assessed.
Wilcoxon rank-sum tests were used to determine significant differences between placebo and
the ISIS 301012-treated groups. A dose-dependent reduction in apoB-containing lipoprotein
particle numbers was observed in subjects treated with ISIS 301012 while HDL particle
numbers were not significantly changed. Total VLDL and LDL particle numbers were
significantly reduced from baseline two weeks post-treatment in the 200 mg dose group (n
8) by a median 36% (p 0.04) and 54% (p 0.0003) respectively. Small VLDL and LDL
particle numbers were also significantly reduced by 61% (p 0.05) and 60% (p 0.004) in
this group. Total LDL particle numbers were significantly reduced in the 100 mg dose group
(n 8). These results demonstrate a substantial reduction in atherogenic lipoprotein particle
numbers by antisense inhibition of apoB. Due to the limited sample size and narrowly defined
subject population, further studies will be required to validate these effects and to determine
whether the results differ depending on the nature of the dyslipidemia.
P124
Statin Therapy Alters the Relationship Between Apolipoprotein B and
Low-Density Lipoprotein Cholesterol and NonHigh-Density Lipoprotein
Cholesterol Targets in High-Risk Patients: MERCURY II Trial
Christie M Ballantyne, Baylor College of Medicine, Houston, TX; Joel S Raichlen, Valerie A
Cain; AstraZeneca, Wilmington, DE
Background: Hypercholesterolemic patients may reduce their LDL-C to a predetermined goal
yet still have an excess of atherogenic lipoproteins. Apolipoprotein B (apo B) provides a
measure of atherogenic lipoproteins and may be a superior predictor for CHD events. An apo
B target of 90 mg/dL has been proposed as an alternative to non-HDL-C 130 mg/dL,
particularly for hypertriglyceridemic (high TG) patients. This analysis examined what levels of
LDL-C and non-HDL-C correspond to an apo B of 90 mg/dL. Methods: The 16-wk MERCURY
II trial examined patients with LDL-C 130 and 250 mg/dL and TG 400 mg/dL at high risk
for CHD (CHD, diabetes or 10-year Framingham risk 20%). LDL-C, non-HDL-C, TG, and apo
B were analyzed at baseline and after statin therapy consisting of rosuvastatin (10 or 20 mg),
atorvastatin (10 or 20 mg), or simvastatin (20 or 40 mg). For these new analyses, data from
all patients were pooled to determine relationships between apo B and LDL-C and non-HDL-C,
and linear regression analyses were done on values from baseline and after 16 wks of statin
therapy. Results: In high TG patients (TG 200 mg/dL; n 656) and in low TG patients (n
1128), baseline apo B was linearly related to baseline non-HDL-C and also linearly related to
baseline LDL-C (Table). Target lipid values calculated from apo B and lipid data from untreated
patients approximated the target values suggested for high-risk high TG patients by the NCEP
in 2001. On statin therapy, apo B also correlated well with non-HDL-C and LDL-C (Table).
However, target lipid values calculated from statin-treated patients were approximately 30
mg/dL lower than those from baseline data.
Linear Regression Slope
Intercept
(mg/dL) r
Lipid value for
apo B 90 mg/dL
Apo B vs non-HDL-C
Low TG baseline 1.02 38.8 0.87 130.6
High TG baseline 1.02 44.1 0.84 135.9
Low TG on statin 1.27 -10.0 0.96 104.3
High TG on statin 1.25 -8.3 0.96 104.2
Apo B vs LDL-C
Low TG baseline 0.89 30.0 0.82 110.1
High TG baseline 0.92 10.4 0.81 93.2
Low TG on statin 1.11 -17.6 0.92 82.3
High TG on statin 1.03 -21.2 0.91 71.5
Conclusion: These data show that before therapy, an apo B 90 mg/dL approximated an LDL-C
100 mg/dL and a non-HDL-C 130 mg/dL. However, during statin therapy, an apo B target
of 90 mg/dL in these high-risk patients corresponded more closely to the optional targets of
LDL-C 70 mg/dL and non-HDL-C 100 mg/dL recommended for very high risk patients.
P125
Novel Variants of Endothelial Lipase in Subjects with Elevated High-Density
Lipoprotein Cholesterol
Vaneeta Bamba, Childrens Hosp of Philadelphia, Philadelphia, PA; Stephanie L
DerOhannessian, Megan L Wolfe, Daniel J Rader; Univ of Pennsylvania, Philadelphia, PA
Background: High levels of high density lipoprotein cholesterol (HDL-C) are associated with
decreased risk of cardiovascular disease. Endothelial lipase (EL) is a recently discovered
phospholipase that influences HDL metabolism in mice: overexpression causes reduced HDL-C
and loss-of-expression causes increased HDL-C. We hypothesized that loss-of-function
mutations in EL might cause high HDL-C levels in humans and therefore sequenced the EL
gene in 96 subjects with HDL-C levels 99th percentile. Methods and Results: All ten exons
and 1 kb of the promoter of the EL gene were sequenced from 96 subjects with high HDL-C
levels (99th percentile). A total of 32 gene variants were found, of which 17 were novel. One
variant, confirmed by restriction analysis, was X501R, resulting in the conversion of the stop
codon to an arginine and a predicted additional 49 amino acids at the carboxyterminus. There
were 4 novel variants in the promoter (-662CG, -655CG, -576AG, -526CT) and 2 novel variants
in the 3 UTR (176GA, 315AG). Several previously undiscovered variants within the intronic
regions were identified as well. Conclusions: In subjects with high HDL-C levels, seventeen
novel variants in the EL gene were identified, of which one is a novel missense mutation of the
stop codon.
P126
Dose-Dependent Effect of Rosuvastatin on Lipoprotein Kinetics in the
Metabolic Syndrome
Esther M Ooi, Gerald F Watts, Dick C Chan, Univ of Western Australia, Perth, Australia; Paul
J Nestel, Dmitri Sviridov, Baker Heart Rsch Institute, Melbourne, Australia; Hugh Barrett;
Univ of Western Australia, Perth, Australia
The dose-dependent effect of rosuvastatin on VLDL, IDL and LDL-apoB-100 and HDL particle
kinetics were studied in a randomized, double-blind, crossover trial of five-week therapeutic
periods with placebo, 10mg/day or 40mg/day rosuvastatin in twelve dyslipidemic men with the
metabolic syndrome. The kinetics apoB-100, apoA-I and apoA-II were measured using
D
3
-leucine, GCMS and multi-compartmental modeling. The kinetics of HDL subpopulations
LpA-I and LpA-I:A-II were also examined using a new compartment model. Compared with
placebo, there was a significant dose-dependent decrease with rosuvastatin in plasma
cholesterol, triglycerides (TG), LDL cholesterol, apoB and apoC-III concentrations and in the
lathosterol:cholesterol ratio. Rosuvastatin significantly increased the fractional catabolic rates
(FCR) of VLDL, IDL and LDL-apoB-100 and decreased the corresponding pool sizes, with
2007 ATVB Poster Presentations e-59
by on March 25, 2011 atvb.ahajournals.org Downloaded from
evidence of a dose-related effect. LDL-apoB production rate (PR) fell significantly with
rosuvastatin 40mg/day, with no change in VLDL and IDL-apoB PR. Changes in TG were
correlated with changes in VLDL apoB FCR and apoC-III, and changes in lathosterol:cholesterol
ratio were correlated with changes in LDL apoB FCR, the associations being more significant
with the higher dose of rosuvastatin. Rosuvastatin dose-dependently increased HDL choles-
terol, LpA-I concentration and HDL particle size. The increase in LpA-I concentration was
associated with a significant dose-related reduction LpA-I FCR with no changes to LpA-I PR.
There was a significant dose-dependent reduction in LpA-I:A-II FCR with concomitant reduction
in LpA-I:A-II PR, and hence no change in LpA-I:A-II concentration. These data suggest that
rosuvastatin decreases the plasma concentration of apoB-containing lipoproteins by a
mechanism that is dose-dependently related to an increase in their rates of catabolism.
Furthermore, rosuvastatin increases HDL cholesterol and LpA-I concentrations and this was
primarily related to reduction in LpA-I fractional catabolic rate. The findings provide a
dose-related mechanism for the benefits of rosuvastatin on cardiovascular disease in the
metabolic syndrome.
P127
Genetic Modifiers Prevent Left Ventricular Hypertrophy and Valvular
Calcification in the Egfr Waved-2 Mouse Model of Aortic Stenosis
Delia J Barrick, Reade Roberts, Mauricio Rojas, David Threadgill, Univ of North Carolina at
Chapel Hill, Chapel Hill, NC; Susan S Smyth; Univ of Kentucky, Lexington, KY
Left ventricular hypertrophy (LVH) is an independent risk factor for cardiovascular morbidity and
mortality. LVH associated with aortic stenosis (AS) is an initial cardiac response to pressure
overload. However, variation exists in the geometric distribution of LVH and cardiac
compensation in patients with similar AS. Moreover, up to 10% of patients with severe AS
never develop LVH. Several clinical risk factors influence LVH in AS, yet few genetic markers
have been identified. Mice homozygous for the hypomorphic Egfr waved-2 (wa2) allele were
reported to have AS on a mixed genetic background. We created C57BL/6J (B6), 129S1 (129),
and B6.129 (F1) Egfr
wa2/wa2
mice to investigate the effect of genetic modifiers on the cardiac
response to chronic AS. At three months of age, all Egfr
wa2/wa2
mice have enlarged aortic valves
relative to Egfr
wa2/
littermates. However, B6 Egfr
wa2/wa2
mice have the thickest valves,
significant transvalvular gradients as assessed by cardiac catheterization (388.70 mmHg),
enlarged, fibrotic hearts, hypertrophied cardiomyocytes and a reduced lifespan. These mice
also have severely reduced systolic function (%FS 263.7 Egfr
wa2/wa2
vs. 412.8 Egfr
wa2/
)
and altered expression of classic cardiac hypertrophy markers. Despite having enlarged valves,
neither 129S1 nor F1-Egfr
wa2/wa2
mice develop histological, functional or molecular evidence
of LVH and maintain normal systolic function (%FS: F1 Egfr
wa2/wa2
403, 129 Egfr
wa2/wa2
41.3 6.5). Histological studies reveal significant cellular changes in cardiac valves unique to
B6 Egfr
wa2/wa2
mice (increased cellular proliferation, ectopic cartilage formation, extensive
calcification and inflammatory infiltrate) which may contribute to altered valve physiology.
These results confirm the usefulness of this model to study AS and LVH. Additionally,
identification of 129 genetic modifiers may uncover novel therapeutic targets for the treatment
of calcific AS.
P128
Granzyme B: A Major Contributor to Atherosclerosis, Xanthomatosis, and
Alopecia in ApoE Knockout Mice
Rani P Cruz, Wendy A Boivin, Jonathan C Choy, Hongyan Zhao, Bruce M McManus, David J
Granville; Univ of British Columbia, Vancouver, Canada
Introduction: Granzyme B (GrB) is a cytotoxic serine protease secreted by cytotoxic T-cells, NK
cells, mast cells and macrophages. GrB is present within lipid-rich regions of human vessels
with advanced atherosclerotic plaques. To assess the role of GrB in a hyperlipidemic
environment, ApoE-/- mice were used. Preliminary studies revealed that GrB strongly
associated with elastin fibres in the blood vessels and skin. Hypothesis: During chronic
hyperlipidemia and inflammation, GrB accumulates on elastin fibres and contributes to elastin
degradation and reduced elasticity in the skin and blood vessels. Methods: To study the role
of GrB in a chronic inflammatory, hyperlipidemic environment, ApoE-/- x GrB-/- double
knockout (ApoE/GrB-DKO) mice were generated and fed a high fat diet for 30 weeks. Tissues
were stained with H&E to assess morphology, oil red O to assess lipid deposition, elastic van
Gieson to visualize elastin, and Movats pentachrome to visualize extracellular matrix
composition and lesion area. GrB co-localization to elastin was also assessed using confocal
microscopy in the skin, hair follicles and aorta. In vitro binding assays were performed to
determine whether insoluble elastin binds directly to GrB. Results: ApoE-/- mice developed
severe xanthomatosis, hair loss and atherosclerotic lesions by 30 weeks. GrB deficiency
completely abolished cutaneous xanthomatosis in addition to significantly reducing atheroscle-
rosis. The absence of GrB resulted in a marked reduction of elastin degradation in both the skin
and blood vessels. When further examined using confocal microscopy, it was observed that GrB
strongly co-localized to the internal elastic lamina and medial elastin fibres. GrB was also
localized in the sheath cells surrounding hair follicles. Further, although ApoE -/- mice had to
be sacrificed by 30 weeks on a high fat diet for humane purposes, the ApoE/GrB DKO mice
were grown for 60 weeks on the same diet with minimal evidence of aging, xanthomatosis, hair
graying or atherogenesis indicating that the absence of GrB may increase longevity.
Conclusions: GrB plays a key role in atherosclerotic plaque formation, loss of cutaneous
elasticity and hair loss in aged, hyperlipidemic ApoE-/- mice.
P129
Reduction of Plaque Size and 5LO, ALOX5P, and LTA4H Gene Expression in
a Dyslipidemic Mouse Model of Atherogenesis by DG-031, an Inhibitor of
Leukotriene Biosynthesis
Jon Mar Bjornsson, Einar Jorundsson, Gudmundur Sigthorsson, Thorkell Andresson, deCODE
genetics, Reykjavik, Iceland; Mark Gurney; deCODE genetics, Woodridge, IL
Background. Previously we reported that DNA variants in the ALOX5P and LTA4H genes
encoding 5-lipoxygenase activating protein (FLAP) and leukotriene A4 hydrolase (LTA4H),
respectively, key enzymes in the leukotriene biosynthetic pathway, are associated with
increased risk of myocardial infarction and stroke (Helgadottir et al., 2005 & 2006). Other
groups independently report that expression of 5-lipoxygenase (5LO), FLAP and LTA4H is
elevated in human atherosclerotic plaque and also in the aorta of dyslipidemic mice. Here we
evaluate the effect of pharmacological inhibition of FLAP in a dyslipidemic mouse model of
atherosclerosis. Methods. Apolipoprotein E-deficient (apoE -/-) mice were administered
DG-031, an inhibitor of FLAP, in a high fat diet from 816 weeks of age. Aortas were harvested
for measurement of mRNA content by TaqMan analysis or atherosclerotic plaque burden in
aortic root sections. Results. By analyzing Oil red O stained cross-sections from the aortic root
we demonstrate that DG-031 causes a dose dependent reduction in atherosclerosis of about
30% (p0.01). Accompanying the reduction in atherosclerotic plaque formation was a
significant reduction in aortic 5LO, ALOX5P, and LTA4H gene expression of greater than 50%
(p0.01). DG-031 also reduced the increase in expression of the metalloproteinase type-3 and
9 (MMP3 and MMP9) genes that normally accompany atherosclerotic lesion formation
(p0.05). Conclusions. DG-031, a FLAP inhibitor, currently being studied in a PhIII clinical trial
for secondary prevention of CV events in post-ACS patients, provides dose-dependent reduction
in atherosclerotic lesion formation and significant decrease in aortic expression of leukotriene
pathway genes in dyslipidemic apoE -/- mice.
P130
The Effect of Sphingomyelin Synthase Gene Knockdown and Knockout on
Plasma Membrane Sphingomyelin Levels and NF-B Activation
Tiruneh K Hailemariam, Zhiqiang Li, Jin Liu, Xian-Cheng Jiang; SUNY Downstate Med Cntr,
Brooklyn, NY
Sphingomyelin (SM) is one of the major lipid components in plasma and on cell membranes.
Two isoforms of SMS (SMS1 and SMS2) are invlolved in the biosynthesis of SM. To investigate
the relationship between SMS inhibition and SM levels, we utilized the gene knockdown and
knockout approaches. We found that SMS1 and SMS2 siRNA significantly diminished cellular
SMS activity by about 55% and 27%, respectively. To investigate the consequence of SMS
inhibition, we incubated the siRNA-transfected Huh7 cells with [
14
C]-L-serine, a precursor of
sphingomyelin and found that both SMS1 and SMS2 siRNA significantly decreased intracellular
[
14
C]-SM levels compared with controls. LC/MS indicated that both SMS1 and SMS2
knockdown significantly reduced cellular SM but not ceramide, S1P and sphingosine levels. In
order to study the impact of SMS knockdown on lipid rafts, we isolated lipid rafts and determine
SM and cholesterol levels in them. We found that SMS1 and SMS2 knockdown significantly
decreased SM but not cholesterol levels in lipid raft fractions. This effect was not observed in
non-lipid raft fractions. Furthermore, SMS siRNA-treated cells exhibited stronger resistance to
cell lyses mediated by lysenin, an SM aggregate binding protein, than did control siRNA-treated
cells. More importantly, complete SMS2 gene knockout (KO), significantly reduced lysenin-
mediated macrophage lysis. These results suggest that SMS plays a significant role in
regulating SM-containing membrane microdomains which play important roles in cell signaling.
Indeed, immunocytochemistry, luciferase and western blot analysis results indicated that the
activation of NF-kappaB was attenuated in SMS knockdown cells treated with TNF, and in
SMS2 KO macrophages treated with LPS. Moreover, there was less induction of the mRNA and
protein levels of iNOS, a NF-kappaB target gene, in LPS treated SMS2 KO macrophages than
cells from wild type mice. Taken together, our data suggest that SMS1 and SMS2 not only
regulate intracellular and plasma membrane SM levels but also play important role in cell
signaling pathways, such as inflammation, which may well have an impact on the development
of atherosclerosis.
P131
Cellular Mechanisms Regulating Cholesteryl Ester Transfer
Protein-mediated Selective Uptake of High Density LipoproteinDerived
Cholesteryl Esters
Meena Na, Ruth McPherson; Univ of Ottawa, Ottawa, Canada
Cholesteryl ester transfer protein (CETP) is a hydrophobic glycoprotein that mediates the
transfer of neutral lipids between lipoproteins. Recently, our laboratory has demonstrated a
novel role for CETP in directly mediating the selective acquisition of CE from HDL by
hepatocytes, indicating a direct and potentially anti-atherogenic function in reverse cholesterol
transport. Further studies have been carried out to address the cellular mechanisms of CETP
mediated selective uptake of HDL-CE. Using biochemical plasma membrane isolation followed
by detergent extraction, we demonstrate that CETP localizes in the low density, detergent-
resistant membrane fractions in both COS-7 cells and primary murine hepatocytes infected
with an adenoviral CETP construct. In an attempt to dissect the intracellular events following
the selective uptake of HDL derived CE mediated by CETP, immunofluorescence confocal
microscopy was used. By incubating HeLa cells and primary hepatocytes overexpressing CETP
with fluorescently labeled HDL and CE, we demonstrate that CETP colocalizes with both CE and
HDL both on the cell surface and intracellularly suggesting internalization of a CETP/HDL
complex. We also note that the CETP/HDL complex colocalizes with a subset of early
endosomes, suggesting a possible raft-mediated endocytic route, consistent with CETPs
non-caveolar raft localization. At 1h post treatment, HDL and CETP were seen to separate from
the early endosomes and segregate in structures that have yet to be identified. We speculate
that HDL and CETP may be recycled through a retroendocytic pathway, during which CE may
e-60 Arterioscler Thromb Vasc Biol June 2007
by on March 25, 2011 atvb.ahajournals.org Downloaded from
be removed from the HDL particle and directed to the lipid droplets for storage. These studies
provide new insight into CETP membrane localization and intracellular trafficking, relevant to
its role in CE selective uptake.
P132
WITHDRAWN
P133
Nuclear Bile Acid Receptor Gene Variant Improves Efficacy of
Lipid-lowering Therapy
Atsushi Nohara, Hayato Tada, Shoji Katsuda, Masa-aki Kawashiri, Akihiro Inazu, Junji
Kobayashi, Masakazu Yamagishi, Hiroshi Mabuchi; Kanazawa Univ Graduate Sch of Med
Science, Kanazawa, Japan
Bile acid receptor FXR has crucial roles in cholesterol conversion into bile acids and in its
recycling through many target genes that may affect cholesterol levels. We have identified
common polymorphism -1g-t in FXR gene in Japanese population, and hypothesized that this
polymorphism could affect lipid-lowering therapy response. Methods and Results: Total 276
patients (M/F147/129) suspected CAD were enrolled. FXR -1g-t genotypes were deter-
mined by PCR-RFLP, and -1t allele frequency was 0.32. Lipid-lowering drugs (statins 92%)
were prescribed in 113 patients (M/F46/67), and baseline Tcho level was not different with
FXR genotype [gg(n65) vs. gttt(n48) 253 42 vs. 251 41(mg/dl; mean SD)].
With lipid-lowering therapy, -1t carriers showed stronger reduction in Tcho [gg vs. gttt
211 33(-16%) vs. 182 31(-27%) mg/dl, p0.0001], and in LDL-C [125 34(-22%) vs.
100 25(-39%) mg/dl, p0.0001]. There was no difference in triglycerides reduction or
HDL-C increase. Conclusion: These results demonstrated that common genetic variant of FXR
gene showed considerable impact on lipid-lowering therapy, probably through modulation of
genes involved in cholesterol metabolism such as CYP7A1. Whether this variant could affect
long-term clinical course should further be sought.
P134
Epoxycholesterol Treatment Increases ABCA1-Mediated Cholesterol Efflux
in LDL- but Not Acetylated LDL-loaded Macrophages
Mireille Ouimet, Ming-Dong Wang, Kenneth Ho, Vivian Franklin, Yves L Marcel; Univ of
Ottawa Heart Institute, Ottawa, Canada
BACKGROUND: We recently showed that LDL cholesterol preferentially effluxed to HDL,
whereas a modified acetylated LDL (AcLDL), primarily effluxed to lipid-poor apoA-I in an ABCA1
dependent fashion in murine bone marrow derived macrophages. While the intracellular
cholesterol trafficking pathways are clearly different, the regulatory mechanisms remain
unclear. Here we studied how the LXR activation regulated cholesterol transport through these
two pathways. METHODS AND RESULTS: LDL and AcLDL labeled macrophages were treated
with the LXR ligand, epoxycholesterol (epoxy), a unique oxysterol derived from a shunt in the
classical cholesterol biosynthesis, and with 22-hydroxycholesterol (22-OH), which is generated
by the oxidation of cholesterol. The results showed that in macrophages loaded with
AcLDL-derived cholesterol, epoxy treatment decreased cholesterol efflux to apoA-I, while in
macrophages loaded with LDL-derived cholesterol it upregulated efflux of both exogenous and
endogenous cholesterol. However, total and cell surface ABCA1 proteins were increased
dramatically after epoxy treatment under all conditions. In contrast, 22-OH significantly
increased cholesterol efflux to apoA-I in AcLDL treated macrophages, but not in LDL treated
cells, and exhibited less potent stimulation of the efflux of newly synthesized cholesterol,
compared to epoxy. Preliminary data indicated that epoxy might fail to mobilize cholesterol from
lipid droplets due to downregulation of neutral cholesteryl ester hydrolase (NCEH) at the
transcriptional level. CONCLUSION: These results suggest that treatment with specific oxysterol
LXR ligands differentially regulate cholesterol homeostasis in distinct cholesterol pools.
P135
Glycoproten IIb/IIIa and Coronary Disease Extension
Susana Gomes, Roberto J Palma dos Reis, Hosp Central do Funchal, Funchal, Portugal; Ana
Isabel Freitas, Madeira Univ, Funchal, Portugal; Ana Ce lia Sousa, So nia Freitas, Hosp
Central do Funchal, Funchal, Portugal; Anto nio Brehm, Madeira Univ, Funchal, Portugal;
Maria Osabel Mendonc a, Hosp Central do Funchal, Funchal, Portugal
Glycoprotein IIb-IIIa plays a pivotal role in the connection to the platelets of fibrinogen and other
adhesion proteins. The gene that codifies sub unit III a, with two alleles PLA1 and PLA2, has
already been identified. The PLA2 allele has been associated to acute coronary syndrome (ACS)
emergence, and individuals possessing the PLA2 allele would bind more fibrinogen to platelets
than do PL A1 homozigotes. The response to thrombin can differ between the two genotypes.
PLA2 allele individuals could have less severe, less complex and less extensive coronary
morphology lesions and more reactive platelets. Aims: to evaluate the influence of polymor-
phism of glycoprotein (GP) IIIa gene (PLA1 and PLA2) in coronary disease extension, in a
Portuguese population. Methodology: In 296 consecutive coronary patients who underwent
coronary angiography for diagnostic purposes, was evaluated the Leaman coronary score in the
GP IIIa polymorphisms according to the presence of genotype PLA1-PLA1; PLA1-PLA2 and
PLA2-PLA2. The average genotype scores were compared by Student T test for independent
samples (unilateral analysis, p 0. 05). Results: In this coronary Portuguese population the GP
IIIa genotype PLA2/ PL A2 presented less severe coronary artery morphology lesions, which
was statistically significant. Conclusion: in this Portuguese population the PLA2 /PLA2
genotype presented less severe coronary morphology lesions than in patients without this
genotype, which was statistically significant. Looking at these results we can conceive that
there are coronary patients with severely altered coronary morphology, in whom the
atherosclerotic factor is predominant and others with relatively preserved coronary morphology
in whom the pro-aggregative and thrombotic factors dominate. In patients with platelet
aggregation alterations, anti-aggregation therapy will be particularly important.
Genotypes and average score
Genotype Average score Standard deviation P value
PLA1-PLA1 10.07 7.36
PLA1-PLA2 9.56 6.08
PLA2-PLA2 5.58 4.32 P0.036
P136
Reduced Hepatic ACAT2 Activity Is Part of the Integrated Response to
Inhibition of Cholesterol Synthesis in Humans
Paolo Parini, Ulf Gustafsson, Karolinska Institute, Stockholm, Sweden; Matt Davis, Wake
Forest Univ, Winston-Salem, NC; Staffan Sahlin, Bo Angelin, Karolinska Institute, Stockholm,
Sweden; Larry L Rudel, Wake Forest Univ, Stockholm, NC; Mats Eriksson; Karolinska
Institute, Stockholm, Sweden
In order to identify how different degrees of cholesterol synthesis inhibition affects human
hepatic cholesterol metabolism, we studied 37 normo-cholesterolemic gallstone patients
randomized to treatment with placebo, 20 mg/d fluvastatin or 80 mg/d atorvastatin for 4 weeks
prior to surgery. Based on serum lathosterol determinations, cholesterol synthesis was reduced
by 42% and 70% in the two groups receiving statins. During gallstone operation a liver biopsy
was taken, and hepatic protein and mRNA expression of several rate-limiting steps of
cholesterol metabolism were assayed and related to the induced serum lipoprotein changes. A
marked induction of LDL receptors and HMG CoA reductase was detected and positively related
to the degree of cholesterol synthesis inhibition. In a corresponding way, we could show that
the activity, protein expression and mRNA for ACAT2 were all decreased in response to
cholesterol synthesis inhibition. The lowering of HDL cholesterol seen in response to high-dose
statin treatment could not be explained by changes in structures such as the HDL receptor
CLA-1, ABCA1 or apoA-I. We conclude that ACAT2 activity in human liver is lowered by
cholesterol synthesis inhibition, and that this effect , in combination with a parallel down
regulation of Apo E expression, may contribute to the favourable lowering of VLDL cholesterol
seen in addition to the LDL lowering during statin treatment.
P137
Aging Increases the Inflammatory Response After Vascular Injury, Leading
to an Exaggerated Neointimal Formation
Roberto I Vazquez-Padron, Yuntao Wei, Dania Mateo, Luis Rodriguez-Menocal, Sen Li, Sashi
Salgar, Si M Pham; Univ of Miami, Miami, FL
Aging is a risk factor for the development of vascular occlusive diseases. The aim of this study
is to determine whether aging prolong the inflammatory reponse after vascular injury , leading
to an exaggerated neointimal development. Using iliac balloon injury model, we found that
arteries from old rats (22 month) developed thicker neointima at 4 weeks after injury than those
from young (4 month) ones (I/M: 0.8 0.2 vs. 0.54 0.15, p0.008). We also found that
old arteries accumulated more alpha-actin vascular smooth muscle cells in the neointima
than their younger counterparts (500 100 vs. 320 120 cell/mm
2
). Although there is similar
number of macrophages (CD68 cells; detected by immunohistochemistry, IHC)- in the
adventisia 24 h after injury in young and aging rats , these cells disappear in the former after
3 days, while remain in the media and neointima of the latter up until 30 days after injury.
Interestingly, the macrophages in old arteries are CD163 negative, indicating they are of
pro-inflammatory phenotype. There are no difference in the number of vascular T cells and
dendritic cells between young and old rats after injury. The vascular cytokines production was
assessed at different time points after balloon injury using the LincoPlex system. Old arteries
contained three folds or more IL18, IL-6, Gro KC, and Leptin than the young ones as early as
three days after injury. These pro-inflammatory cytokines stayed elevated in the old vasculature
for most of the arterial remodeling process. In contrast with the old arteries, young arteries
produced three times or higher levels of anti-inflammatory cytokines -IL-10, IL-17, IL13 and
2007 ATVB Poster Presentations e-61
by on March 25, 2011 atvb.ahajournals.org Downloaded from
IL-9- seven days after injury. Finally, vascular apoptotic cells were determined by ISOL and IHC
for activated Caspace 3. Old arteries had fewer apoptotic cells ( 0.2% 0.05) at any time
after vascular injury when compared with the younger ones which had abundant vascular
apoptotic cells in the media and neointima at seven days after surgery (2.7% 0.45). We
conclude that aging decreased the resolution of the vascular inflammatory response to injury,
leading to exaggerated neointimal formation.
P138
Number of Nitrate Groups Determines Reactivity and Potency of Organic
Nitrates: A Proof of Concept Study in ALDH-2-/- Mice
Philip Wenzel, Ulrich Hink, II. Medizinische Klinik, Johannes-Gutenberg-Univ Mainz, Mainz,
Germany; Moritz Brandt, Lena Steinhoff, Matthias Oelze, Labor f Molekulare Kardiologie,
Mainz, Germany; Tohoyi Isse, Toshihiro Kawamoto, Univ of Occupational and Environmental
Health, Kitakyushu, Japan; Henry Weiner, Purdue Univ, West Lafayette, IN; Andreas Seeling,
Friedrich Schiller Univ Jena, Jena, Germany; Thomas Munzel, II. Medizinische Klinik,
Johannes-Gutenberg-Univ Mainz, Mainz, Germany; Andreas Daiber; Labor f Molekulare
Kardiologie, Mainz, Germany
Background and purpose: Mitochondrial aldehyde dehydrogenase (ALDH-2) has been shown to
provide a pathway for bioactivation of organic nitrates and to be prone to desensitization in
response to highly potent, but not to less potent nitrates. We therefore sought to strengthen the
concept, that bioactivation by ALDH-2 critically depends on the amount of nitrate groups within
the nitrovasodilator. Experimental approach: Nitrates with one (PEMN), two (PEDN; GDN), three
(PETriN; glycerlyl trinitrate, GTN) and four (pentaerithrityl tetranitrate, PETN) nitrate groups were
investigated. Vasodilatory potency was measured in isometric tension studies using isolated
aortic segments of wild type (WT) and ALDH-2-/- mice. Activity of the cGMP-dependent kinase-I
(reflected by levels of phosphorylated VAsodilator Stimulated Phosphoprotein, P-VASP) was
quantified by Western Blot analysis, mitochondrial dehydrogenase activity by HPLC. Following
incubation of isolated mitochondria with PETN, PETriN-chromophore and PEDN, metabolites
were quantified using chemiluminescence nitrogen detection and mass spectrometry. Key
results: Compared to WT, vasorelaxation in response to PETN, PETriN and GTN was attenuated
about 10fold in ALDH-2-/- mice, identical to WT vessels preincubated with inhibitors of ALDH-2.
Reduced vasodilator potency correlated with reduced P-VASP formation and diminished
biotransformation of the tetranitrate- and trinitrate-compounds. Intriguingly, none of these
findings were observed for PEDN, GDN and PEMN. Conclusions and implications: Our results
support the crucial role of ALDH-2 in bioactivating highly reactive nitrates like PETN and PETriN.
ALDH-2-mediated relaxation by organic nitrates therefore mainly depends on the number of
nitrate groups. Less potent nitrates like PEDN, GDN and PEMN are apparently biotransformed
by alternative pathways.
P139
Positive Association of C667T Methylenetetrahydrofolate Reductase Gene
Polymorphism with Acute Ischemic Stroke
Moe K Thu, Limin Sor, Feng Peng Woon, National Heart Cntr, Singapore, Singapore; Deidre
A DeSilva, Li-Hsian Chen, Singapore General Hosp, Singapore, Singapore; Philip Wong, Tian
Hai Koh, National Heart Cntr, Singapore, Singapore; Bronwyn A Kingwell, Baker Heart Rsch
Institute, Melbourne, Australia; Meng Cheong Wong, Singapore General Hosp, Singapore,
Singapore; Jaye Chin-Dusting; Baker Heart Rsch Institute, Melbourne, Australia
BACKGROUND AND PURPOSE: Elevated plasma homocysteine (Hcy) is a risk factor for
ischemic stoke and coronary heart diseases. Polymorphisms of methylenetetrahydrofolate
reductase (MTHFR) gene are associated with high plasma Hcy. Endothelial nitric oxide has been
reported to have a protective effect in endothelial cells. Nitric oxide synthase 3 (NOS3) is a
candidate gene for stroke susceptibility. We investigated the association of C677T mutation of
MTHFR gene and three NOS3 polymorphisms (G894T missense variant in exon 7, variable
number tandem repeats in intron 4, and T-786C in the promoter) with acute ischemic stroke
in a Singapore population. METHODS: Patients with acute ischemic stroke (n120, 60.8%
ethnic Chinese) were studied. Two hundred and seven subjects (74.4% ethnic Chinese) with no
past history of stroke served as controls. Hypertension, diabetes, dyslipidemia and age were
significantly increased among stroke patients as compared to controls (all P0.001). The
specific genotypes for C667T of MTHFR and three NOS3 polymorphisms were isolated using
allele specific gene amplification and restriction fragment length polymorphism (RFLP) analysis
to determine the genotype and allelic frequency in both groups. Plasma Hcy levels were
measured using fluorescence polarization immunoassay (Abbott Diagnostics). RESULTS: TT
genotype in C667T mutation of MTHFR gene showed significant association with ischemic
stroke patients (P0.019). Plasma Hcy levels were significantly higher in patients than controls
(13.3 5.1 vs 9.56 3.0 mol/L, P0.001). Moreover, among patients, TT genotype was
significantly associated with age 60 (P0.03), male (P0.04) and ethnic Chinese
(P0.003). There was no significant association of NOS3 polymorphisms with ischemic stroke
in our population. CONCLUSIONS: We found the TT genotype of C677T mutation in MTHFR
gene to be significantly associated with acute ischemic stroke, particularly in young, male and
ethnic Chinese Singaporeans. We found no association of NOS3 polymorphisms with acute
ischemic stroke. Our ongoing studies are examining potential association of these genotypes
with elevated Hcy levels in our population.
P140
Pioglitazone Increases Macrophage Apoptosis and Plaque Necrosis in
Advanced Atherosclerotic Lesions of LDL ReceptorDeficient Mice
Edward Thorp, George Kuriakose, Ira Tabas; Columbia Univ, New York, NY
Thiazolidinediones (TZDs), whose actions involve both PPAR-dependent and independent effects,
improve insulin sensitivity in type II diabetes and inhibit early foam-cell atherogenesis in mice.
However, the effects of TZDs on advanced lesion progression are unknown. Herein, we explore the
effects of TZDs in vitro on two processes critical in plaque necrosis, namely, macrophage apoptosis
and phagocytic clearance (efferocytosis) of apoptotic macrophages, and in vivo on advanced
atherosclerotic plaque progression. In cell culture studies, the TZDs pioglitazone and rosiglitazone
enhanced FC-induced macrophage apoptosis as well as efferocytosis of apoptotic macrophages by
macrophage phagocytes. Because enhancement of advanced lesional macrophage apoptosis would
be expected to promote plaque necrosis, whereas enhanced efferocytosis would be predicted to be
beneficial, the net effect in vivo was explored. For this purpose, non-diabetic Ldlr-/- mice were first
fed a cholesterol-enriched diet to promote mid-stage lesions, and then pioglitazone was
administered along with diet for an additional 10 weeks. Despite a decrease in plasma non-HDL
cholesterol and an increase in HDL cholesterol, plaques from pioglitazone-treated mice were more
necrotic and displayed increased apoptotic cells in macrophage-rich regions. Thus, the beneficial
effect of commonly used TZDs on insulin resistance may be partially offset by adverse effects of
advanced plaque progression.
P141
Cannabinoid Receptor (CB2) Deficiency Inhibits Oxidized
LDL/Oxysterol-induced Apoptosis in Macrophages
Douglas P Thewke, Natalie E Freeman-Anderson, East Tennessee State Univ, Johnson City,
TN; Nancy E Buckley; California State Polytechnic Univ, Pomona, CA
Apoptosis of macrophages is an important event in the pathophysiology of atherosclerosis. Oxidized
low-density lipoproteins (OxLDL) are a major lipid component of atherosclerotic lesions and
endocytosis of OxLDL is a potent inducer of apoptosis in cultured macrophages. OxLDL-induced
apoptosis in macrophages is largely due to the oxygenated cholesterol derivatives, such as
7-ketocholesterol (7KC), in the OxLDL. Cannabinoids exert their effects though two related G-protein
coupled receptors, CB1 and CB2. CB2 is primarily expressed by cells of the immune system and
several macrophage processes associated with ongoing atherogenesis including proliferation,
secretion of inflammatory cytokines and chemotaxis, are regulated by CB2. Expression of CB2 has
been detected in lesions and a low dose of the cannabinoid,
9
-tetrahydrocannabinol, reduces
plaque progression in hyperlipidemic mice, an effect which is blocked by administration of a CB2
specific antagonist. In the current study we examined the hypothesis that CB2 expression influences
OxLDL-induced macrophage apoptosis. When resident peritoneal macrophages isolated from
CB2(-/-) and CB2(/) mice were cultured for 16h with 50 g/ml OxLDL, in situ terminal
transferase-mediated dUTP nick end labeling (TUNEL) analysis detected significantly less CB2(-/-)
macrophages undergoing apoptosis than CB2(/) controls (27.9 4.7% vs 61.9 8.5%, p
0.001). Treatment of CB2(-/-) macrophages with 7KC for 16h also resulted in significantly fewer
apoptotic cells compared to similarly treated wild type macrophages (18.9 10.5% vs 54.1
6.9%, p0.001). Immunoblots showed reduced cleavage of procaspase-3 and PARP in CB2(-/-)
macrophages treated with 7KC compared to CB2(/) controls, a result which was confirmed by
caspase-3 activity assays. In contrast, staurosporine-induced apoptosis was unaffected by CB2
deficiency consistent with the conclusion that CB2(-/-) macrophages are not generally defective in
apoptosis. From these results we conclude that CB2 deficient macrophages are resistant to
OxLDL-induced apoptosis and suggest that one mechanism by which CB2 influences the
development and progression of atherosclerotic lesions is by mediating the apoptotic response of
macrophages to OxLDL.
P142
New Insights into the Roles of Apolipoprotein C-III in Stimulating the
Production of Hepatic VLDL
Meenakshi Sundaram, Philip Links, Maroun Bou Khalil, Yuwei Wang, Shumei Zhong, Zemin
Yao; Univ of Ottawa, Ottawa, Canada
Apolipoprotein C-III is a constituent of VLDL and plays a role in regulating lipoprotein lipase
activity as well as receptor-mediated lipoprotein endocytosis. We reported previously that
stable expression of apoC-III in McA-RH7777 cells promoted VLDL-triacylglycerol (TG)
secretion, but the effect of apoC-III expression on the synthesis or secretion of apoB100, the
structural protein of VLDL, was not examined. In this study we determined the rate of apoB100
translation and secretion using McA-RH7777 cells transiently transfected with apoC-III. As
observed in stables, transient apoC-III expression increased (by 2-fold) secretion of
[3H]glycerol-labeled TG and phosphatidylcholine (PC) associated with TG-rich VLDL1 (Sf
100). In addition, transient apoC-III expression also resulted in increased incorporation of
[35S]methionine into cell-associated and secreted VLDL/IDL-apoB100 by 2-fold, respectively.
Moreover, transient apoC-III expression increased post-translational stability of 35S-apoB100;
thus pulse-chase analysis showed that the recovery of total 35S-apoB100 (cell medium) was
increased from 59% in control cells to 82% in apoC-III cells. Intracellular degradation of
35S-apoB100 could be blocked by MG132 in control cells, but was less sensitive to MG132 in
apoC-III cells, indicating that apoC-III expression diminished proteosome-mediated apoB100
degradation. The increased apoB100 synthesis/secretion suggested greater lipid availability for
VLDL assembly in apoC-III cells. Indeed, metabolic labeling experiments in conjunction with
subcellular fractionation showed that partitioning of 3H-TG into microsomal lumen was
markedly augmented, and 3H-TG accumulation in cytosolic lipid droplets was decreased in
apoC-III cells. The ability of apoC-III to promote 3H-TG partitioning was tested by expressing an
apoC-III variant (designated apoC-IIISPD) in which the signal peptide sequence was deleted.
Unexpectedly, not only did expression of apoC-IIISPD stimulate 3H-TG secretion (by 1.5-fold)
but also the expressed apoC-IIISPD was secreted normally. Thus, apoC-III possesses a unique
property in stimulating apoB100 synthesis and VLDL assembly/secretion.
P143
Divergence in Vascular Fractalkine Expression and Functional Responses
in Male and Female Spontaneously Hypertensive Rats
Jennifer C Sullivan, Jennifer L Pardieck, Jennifer S Pollock; Med College of Georgia,
Augusta, GA
Men have a greater incidence of cardiovascular disease compared to women, however the
molecular mechanisms responsible for cardiovascular protection in females are unknown.
e-62 Arterioscler Thromb Vasc Biol June 2007
by on March 25, 2011 atvb.ahajournals.org Downloaded from
Using microarray analysis, the chemokine fractalkine (FKN) was found to be more highly
expressed in the mesenteric arterial bed of female SHR compared to males (p0.005). As FKN
has recently been shown to stimulate NO in endothelial cells, we tested the hypothesis that
mesenteric arteries from female spontaneously hypertensive rats (SHR) have greater FKN-
induced NO stimulation. Experiments utilized 13 week-old male and female SHR (n8). Greater
FKN expression in the mesenteric arterial bed of female SHR was verified by RT-PCR (2.21.4
RDU in females vs 0.170.16 RDU in males, p0.05), Western blot analysis (0.140.02 RDU
in females vs 0.100.02 RDU in males, p0.05) and ELISA (6.00.4 ng/mg in females vs
3.80.5 ng/mg in males, p0.005). To assess the ability of FKN to stimulate NO, acetylcholine
(Ach) dose-response curves were performed in isolated third-order mesenteric arteries from
male and female SHR in the absence and presence of the non-selective NOS inhibitor LNAME
(100 M) in arteries pretreated with either vehicle or FKN (1 g/mL). LNAME produced a
comparable decrease in maximal relaxation to Ach in arteries from males and females (%
relaxation: 787, 744, respectively). FKN in combination with LNAME produced a greater
decrease in maximal Ach relaxation in arteries from males compared to arteries from females
(% relaxation: 609, 857, respectively). Since inflammatory mediators stimulate FKN
expression, urinary TGF and MCP-1 were determined. Consistent with increased FKN
expression; excretion of inflammatory markers were greater in females (TGF, pmol/day/g:
509260 in males, 3041745 in females, p0.05; MCP-I, pmol/day/g: 205 in males,
5613 in females, p0.05). In conclusion, despite female SHR having greater FKN expression
in mesenteric arteries compared to males, FKN increased the NOS-sensitive component of
acetylcholine-induced relaxation to a greater degree in arteries from males. Increased FKN
expression in hypertensive females is likely the result of increased levels of inflammation.
P144
Lentiviral Transduction of Human apoA-I and apoA-I Milano into
Hematopoietic Progenitor Cells Reduces Atherosclerosis in apoE-deficient
Mice
Yan Ru Su, John L Blakemore, Youmin Zhang, MacRae F Linton, Sergio Fazio; Vanderbilt
Univ, Nashville, TN
Monocytes/macrophages play an important role in the development of atherosclerosis.
Genetically engineered macrophages can be used as an effective delivery vehicle for cell
therapy of atherosclerosis. We have generated lentiviral constructs expressing the human
apolipoprotein A-I (apoA-I), apoA-I milano (apoA-Im) or green fluorescent protein (GFP) cDNA
under the control of a macrophage specific promoter, CD68, and used them for ex vivo
transduction of mouse hematopoietic progenitor cells (HPC) isolated from bone marrow. When
the transduced HPCs were transplanted into apoE
-/-
mice, human apoA-I and apoA-Im were
detected in the reciepient plasma 16 weeks after transplantation at an average concentration
of 8.41.2 g/dl (apoA-I) and 7.70.8 g/dl (apoA-Im). This represents a significant
improvement in transduction efficiency compared to our previous approach using a retroviral
vector and bone marrow transduction. There was no significant difference in total cholesterol
and HDL levels among groups during the 16 week course of study. There was a 50% and 60%
reduction in en face atherosclerotic lesion area in both apoA-I and apoA-Im recipient mice,
respectively, when compared to the GFP group. There was a significant (31%) reduction in
proximal aortic lesion area in the apoA-Im group when compared to the GFP group; the apoA-I
group had only a 10% reduction. Lentiviral-apoA-I or apoA-Im transduced wild type mouse
peritoneal macrophages showed a significant increase in free cholesterol efflux in both apoA-I
(2.5 fold) and apoA-Im (1.9 fold) when compared to the GFP transduced macrophages. Our data
suggest that lentiviral mediated transduction of apoA-I or apoA-Im into HPC is a viable approach
for cell therapy of atherosclerosis. While apoA-Im is more effective than wild type apoA-I in the
reduction of atherosclerotic lesions in the proximal aorta, the underlying molecular mechanisms
could not be attributed to enhanced cholesterol efflux in macrophages alone.
P145
LXR Activation by 24(S),25-Epoxycholesterol Enhances the Expression of
Niemann-Pick C1 in Macrophages, Leading to Increased Cholesterol Efflux
Michael M Beyea, Cynthia G Sawyez, Jane Y Edwards, Murray W Huff; Robarts Rsch
Institute, London, Canada
Niemann-Pick C (NPC) 1 and NPC2 have been implicated in the trafficking of cholesterol
between lysosomal compartments, the endoplasmic reticulum and the plasma membrane for
efflux to extracellular acceptors. The liver x receptor (LXR) plays an important role in cholesterol
homeostasis and upon activation by oxysterol or synthetic ligands enhances cholesterol efflux
by increasing transcription of genes involved in cholesterol efflux, including ABCA1, ABCG1 and
APOE. The synthetic LXR agonist, TO-901317 was shown to enhance expression of both NPC1
and NPC2 in cultured human macrophages. However, whether this is a direct effect of
LXR-activation and whether the naturally-occurring LXR agonist 24(S),25-epoxycholesterol
(24(S),25-epoxy) enhances macrophage NPC1 and NPC2 expression is unknown. We tested the
hypothesis that 24(S),25-epoxy, either synthesized endogenously by partial inhibition of
oxidosqualene:lanosterol cyclase (OSCi) or added exogenously, increases the expression of
NPC1 and NPC2 and contributes to enhanced cholesterol efflux in THP-1 macrophages. In silico
searches for nuclear receptor binding sites within the upstream promoters of NPC1 (Genbank
#AF157365) and NPC2 (Genbank #AY409809), revealed a DR4 motif for each gene potentially
capable of binding LXR and modulating gene expression. Exposure of THP-1 cells to an OSCi
or exogenous 24(S),25-epoxy increased NPC1 mRNA expression by 1.3 to 1.6-fold (P0.05).
. Similarly, the synthetic LXR-agonist, TO-901317, enhanced NPC1 mRNA expression.
However, NPC2 mRNA was unaffected by any of the LXR agonists. In addition, endogenous
24(S),25-epoxy significantly increased macrophage expression of ABCA1 (1.9-fold), ABCG1
(2.3-fold) and APOE (1.6-fold) and enhanced cholesterol efflux by 1.4-fold (P0.05).
Furthermore, macrophages pre-treated with 24(S),25-epoxy or TO-901317 and incubated with
acetylated LDL demonstrated attenuated intracellular free cholesterol and increased cholesterol
at the plasma membrane as assessed by filipin staining using fluorescence microscopy. These
results suggest that increased NPC1 expression contributes to enhanced LXR-mediated
cholesterol efflux in macrophages.
P146
EP 80317 Stimulates Macrophage Cholesterol Efflux Through ABCA1 and
ABCG1 in a PPAR-dependent Manner
Kim Bujold, Sylvie Marleau, Huy Ong; Universite de Montre al, Montre al, Canada
Cholesterol efflux is essential for macrophage cholesterol homeostasis through reverse
cholesterol transport via extracellular acceptors such as apolipoprotein A-I (apoA-I) and high
density lipoprotein (HDL). We have previously reported that EP 80317 induces a significant
decrease in macrophage foam cell formation. Hypothesis. Our hypothesis is that EP 80317
regulates macrophage cholesterol efflux through the PPAR-LXRa-ABC transporters pathway.
In the present study, the role of nuclear receptors and of the different ABC transporters in EP
80317-mediated cholesterol efflux has been evaluated. Methods. Murine monocytic J774 cells
were loaded with [
3
H]-cholesterol (1 Ci/ml), incubated EP 80317 (100 nM) and exposed to
HDL (50 g/ml) or apoA-I (20 g/ml) in order to promote efflux. Results. With apoA-I as the
cholesterol acceptor, EP 80317 induced a significant increase of cholesterol efflux by 163%
and 95% (p0.001) after 4 and 16 hours, respectively. In contrast, EP 80317-mediated efflux
to HDL increased by only 3226% (p0.001), under the same conditions. The significant
increase of EP 80317-mediated cholesterol efflux was completely inhibited with DIDS, an ABC
transporter inhibitor, with either acceptor. In contrast, no change in EP 80317-elicited
cholesterol efflux was found following the incubation of J774 cells with BLT-1, a SR-B1
inhibitor. To assess the effect of EP 80317 on PPAR in the regulation of cholesterol efflux,
J774 cells were incubated with GW 9662, a PPAR inhibitor. A complete inhibition of EP
80317-mediated efflux was found. The expression of proteins involved in cholesterol efflux, as
assessed by Western blot, was increased by 2.5-, 2.2- and 7.3-fold for LXRa, ABCG1 and
ABCA1, respectively, without significant change in the PPAR protein level. Conclusion. EP
80317 elicits PPAR-dependent cholesterol efflux from J774 cells to HDL and apoA-I as
cholesterol acceptors in the reverse cholesterol transport.
P147
A Specific Role for Cytoplasmic Amino Acid Sequences in Regulating SR-A
Function
Jill Cholewa, Cecelia Gass, Dejan Nikolic, Steven Post; Univ of Kentucky, Lexington, KY
Macrophage class A scavenger receptors (SR-A) are transmembrane receptors that recognize
diverse ligands including modified lipoproteins, bacterial products, and modified extracellular
matrix (ECM). SR-A internalizes soluble ligands and enhances cell adhesion to modified ECM.
The cytoplasmic domains required for mediating SR-A internalization and cell adhesion have
not been defined. In this study, we mutated the cytoplasmic tail of SR-A and stably expressed
the altered receptors in a human embryonic kidney (HEK 293) cell system. Deletion of all but
six cytoplasmic amino acids (SR-A
149
) eliminated receptor internalization while increasing
surface localization and cell adhesion. To examine the importance of specific sequences in the
cytoplasmic tail, we created an SR-A mutant with amide derivatization of 4 highly conserved
cytoplasmic acidic amino acids (SR-A
QNAN
). Similar to SR-A
149,
SR-A
QNAN
displayed increased
surface localization and cell adhesion; however, SR-A
QNAN
retained the ability to internalize
ligand. To create an SR-A receptor that only internalizes ligand, we created a chimeric receptor
in which the cytoplasmic tail of SR-A was replaced by the first 57 amino acids, including the
internalization motif of the transferrin receptor (SR-A
TfR
). In contrast to SR-A
149
, cells
expressing the SR-A
TfR
internalized ligand but did not show enhanced cell adhesion. These
results indicate a strong correlation between surface localization and cell adhesion. Because
PI3-kinase (PI3-K) is involved in receptor trafficking we examined the importance of PI3-K in
SR-A-mediated adhesion. Although PI3-K is activated during SR-A-mediated adhesion, PI3-K
activity is required only for SR-A-mediated adhesion in cells expressing full-length SR-A.
Together, these results indicate that SR-A function can be modulated by the PI3-K-dependent
regulation of receptor localization.
P148
Increases in Lysosomal Cholesterol Correspond with Increased Lysosomal
pH and Decreased Vacuolar-ATPase Activity in Macrophage Foam Cells
Brian E Cox, Jody C Ullery, W G Jerome; Vanderbilt Univ Med Cntr, Nashville, TN
Macrophage foam cells in atherosclerosis develop via massive accumulations of free and
esterified cholesterol, much of which is within lysosomes. In cell culture, macrophages
incubated with mildly oxidized (ox)-LDL, aggregated (agg)-LDL or cholesteryl ester- rich lipid
dispersions (DISP) accumulate cholesterol in lysosomes followed by an inhibition of lysosomal
cholesteryl ester hydrolysis. Lysosome pH is a key mediator of lysosome activity and is thus a
potential mechanism for lipid-induced inhibition. Previously, we showed that treatment of
THP-1 macrophage with ox-LDL, agg-LDL, or DISP resulted in inhibition of the lysosomes
ability to maintain an active pH. In this report, lysosomes from macrophages were isolated
using magnetic beads and the membrane cholesterol was augmented through incubation with
cholesterol enriched methyl--cycylodextrin. Variable increases in membrane cholesterol were
obtained through this method and these increases coincided with a reduction in vacuolar
(v)-ATPase-dependent pumping of H

-ions into lysosomes. This inhibition was not due to


interaction of the isolated lysosomes with cyclodextrin, as -cyclodextrin had no effect on
v-ATPase activation. Lysosomes were also isolated using a sucrose density gradient from either
untreated or agg-LDL treated macrophages. This procedure yielded 3 distinct lysosomal
fractions, a heavy normal fraction, an intermediate fraction, and a light lipid enriched fraction.
Even the normal lysosomes from agg-LDL treated macrophages had reduced v-ATPase
activity when compared to the lysosomes from untreated cells. Lysosome inhibition was not
due to a decrease in the v-ATPase levels, but was concomitant with lysosomal cholesterol
sequestration. Lysosomes from agg-LDL treated cells had a 1.3-fold increase in free
cholesterol levels, were larger, and had a different morphology when compared to identical
density lysosomes from untreated cells. Thus, consistent with our observation of reduced
lysosomal hydrolytic activity in sterol-engorged macrophages, it appears that free sterol in
2007 ATVB Poster Presentations e-63
by on March 25, 2011 atvb.ahajournals.org Downloaded from
lysosomes hampers v-ATPase and inhibits the ability of lysosomes to maintain an active pH.
This could exacerbate foam cell formation and influence atherosclerotic lesion development.
P149
The Association Between Alcohol Consumption and C-Reactive Protein
Levels in College-aged Individuals
Elizabeth Donovan, Amy Olson; College of Saint Benedict, St. Joseph, MN
Purpose. Current screening methods fail to identify over half of individuals at risk for
cardiovascular disease. C-Reactive protein (CRP), an acute phase protein and marker for
inflammation, is highly correlated with cardiovascular disease and is a promising new
screening tool. Many factors, such as alcohol, medications, physical activity, and excess body
fat, affect CRP levels. Alcohol intake results in a J-shaped response curve for CRP in individuals
over forty years. This study examines the effect of alcohol consumption on CRP levels in
college-aged individuals. Binge drinking is prevalent in college-aged individuals potentially
increasing CRP levels. Design. College-aged individuals completed surveys which assessed
factors that can affect CRP levels, such as medication use, smoking habits, recent weight loss,
and alcohol consumption patterns. Three groups, non-drinkers (N6), moderate drinkers
(N10), and heavy drinkers (N9), were matched based on survey responses. C-reactive
protein (hs-CRP) was measured using reflectance photometry. This research was approved by
the institutional review board. Results. The average CRP level across all alcohol consumption
patterns was 0.9 mg/L (low risk for cardiovascular disease). Moderate drinkers had significantly
lower CRP levels (0.58mg/L) than heavy drinkers (1.25 mg/L; p.041), but there was no
significant difference between CRP levels of moderate drinkers and non-drinkers (0.85 mg/L;
p.343). Males had higher CRP levels than females, but this difference did not reach statistical
significance (p.09). Conclusions. There is a J-shaped relationship between alcohol
consumption and CRP levels in college-aged individuals, agreeing with trends found in older
adults. If CRP levels are predictive of future risk for cardiovascular disease, college aged
individuals may be beginning this pattern, which is an additional reason to be concerned about
heavy drinking in college-aged individuals.
P150
Comparison of the Cellular Lipid Efflux Properties of Pre- High-density
Lipoprotein and Lipid-free Apolipoprotein A-I
Phu T Duong, Sissel Lund-Katz, George H Rothblat, Michael C Phillips; The Childrens Hosp
of Philadelphia, Univ of Pennsylvania Sch of Medicine, Philadelphia, PA
The ATP-binding cassette transporter A1 (ABCA1) mediates the efflux of cellular phospholipids
(PL) and free (unesterified) cholesterol (FC) to human apoA-I to form nascent HDL particles.
With J774 macrophages and human skin fibroblasts, the nascent HDL particles present after
24h incubations are discoidal, contain 2, 3 or 4 molecules of apoA-I per particle, possess
-electrophoretic mobility and are about 9 and 12nm in diameter. These particles have
PL/FC/apo A-I molar ratios ranging from 96195/1239/1. Here, we show that human skin
fibroblasts and WI38VA13 (human lung fibroblasts), which release PL and FC to apo A-I
relatively slowly, form precursor particles containing predominantly monomeric apo A-I. These
small particles can be obtained by incubating lipid-free apoA-I (15g/ml) for 12h with either
human skin fibroblasts and or WI38VA13 cells in which ABCA1 is up-regulated. The
hydrodynamic diameter of these particles is 7 nm and they exhibit pre- electrophoretic
mobility as assessed by non-denaturing 2D-PAGE. With both cell types, the PL/apoA-I molar
ratio for these particles, which contain one apo A-I molecule (as assessed by covalent
cross-linking), is in the range of 34/1 and the FC/apoA-I molar ratio is 12/1. SDS-PAGE of
concentrated samples did not reveal any evidence of other proteins beside apoA-I in these
particles. These pre- particles are the lipid-poor apo A-I frequently mentioned in the
literature. When incubated with human skin fibroblasts in which ABCA1 is up-regulated, they
effectively efflux FC with K
m
1 g apoA-I/ml and V
max
67% cellular FC/6h. The equivalent
values for lipid-free apo A-I are very similar indicating that the pre- HDL and lipid-free apo A-I are
equally effective in mediating FC efflux. In both cases, the apo A-I is converted to the 9 and 12nm
discoidal particles upon longer incubation with the cells. The above results suggest that discoidal
HDL particles can be formed similarly by ABCA1-mediated addition of cellular lipids to either
monomeric lipid-free apo A-I molecules or monomeric lipid-poor apo A-I (pre- particles).
Knowledge of the reaction products will aid in the elucidation of the mechanism of ABCA1 action.
P151
The Effect of Selective Inactivation of Macrophage ATP Binding Cassette
Transporter A1 (ABCA1) on Atherosclerosis in LDL-receptor Knockout
(LDLrKO) Mice
MyNgan Duong, Xuewei Zhu, Elena Boudyguina, Wake Forest Univ Sch of Medicine,
Winston-Salem, NC; Nobuyo Maeda, Univ of North Carolina, Chapel Hill, NC; John S Parks;
Wake Forest Univ Sch of Medicine, Winston-Salem, NC
Macrophage ABCA1 is thought to play a critical role in removal of excess cholesterol in
atherosclerotic lesions. Previous studies have shown that transplantation of bone marrow from
ABCA1 KO mice into LDLrKO recipient mice results in more atherosclerosis compared to
transplantation of wild type bone marrow. However, bone marrow transplantation eliminates
ABCA1 in all leukocytes, not just macrophages. We developed LDLrKO mice with targeted
deletion of macrophage ABCA1 (MSKO-LDLrKO) to directly address the role of macrophage
ABCA1 in atherosclerosis development. Mice were fed an atherogenic diet (10% palm oil, 0.2%
cholesterol) for 16 wk before aortic atherosclerosis was quantified as percentage of surface
occupied by lesions as well as cholesterol content. Surprisingly, neither measure of
atherosclerosis was different between the two genotypes of mice. Analysis of plasma revealed
a significant reduction in apolipoprotein B lipoprotein concentration in plasma of MSKO-LDLrKO
mice compared with LDLrKO mice, despite a significant two-fold increase in hepatic cholesterol
in the MSKO-LDLrKO mice. Thioglycolate-elicited peritoneal macrophages from MSKO-LDLrKO
mice on the atherogenic diet responded to lipopolysaccharide stimulation with greater
proinflammatory cytokine (i.e., TNF-alpha, IL-6, IL-1beta) gene expression relative to macro-
phages from LDLrKO mice. These results suggest that deletion of macrophage ABCA1 in the
LDLrKO background has two opposing outcomes with regard to atherosclerosis development,
an increased macrophage proinflammatory response and a novel indirect role in increasing
plasma VLDL and LDL catabolism and/or decreasing hepatic VLDL secretion. These opposing
effects of macrophage ABCA1 may explain the similar atherosclerosis between LDLrKO mice
with or without functional macrophage ABCA1.
P152
Activation of Transient Receptor Potential Vanilloid Receptor Type 1
Inhibits Human Monocyte Migration
D Y Liu, L Q Ma, Z D Luo, T B Cao, L J Wang, Cntr for Hypertension and Metabolic
Disease, Chongqing Institute of Hypertension, Third Military Med Univ, Chongqing, China;
Martin Tepel, Med Klinik IV, Nephrologie, Charite Campus Benjamin Franklin, Berlin,
Germany; Z M Zhu; Cntr for Hypertension and Metabolic Disease, Chongqing Institute of
Hypertension, Third Military Med Univ, Chongqing, China
Monocyte migration play an important role in the development of atherosclerosis (Yvonne
Do rffel, Hypertension,1999).Transient receptor potential (TRP) channels were found to be
involved in atherosclerosis. It is unclear whether activation of vanilloid receptor type 1 (TRPV1)
can influence the monocytes migration. In current study, we investigated the effection of
capsaicin on human monocytes migration. Methods: human peripheral blood monocytes were
isolated by density gradient centrifugation. TRPV1 protein expression was performed using
Western assay. Using the fluorescent dye fura2-AM technique measurements of cytosolic
calcium levels in monocytes. Monocytes migration was measured by stimulation with formyl
methionyl leucyl phenylalanine (fMLP), the chemotactic peptide using Neuro Probe Standard 48
Well Chemotaxis Chamber. Results: The expression of TRPV1 in human monocyte was
significantly increased after treated with 100 nmol/L capsaicin and reduced by capsaicin
antagonist, capsazepine (normalized TRPV1 expression, 23.38 0.29 for control condition;
mean SEM, n 4; 31.69 1.13 for capsaicin treated monocytes; mean SEM, n 4;
p 0.05 compared to control, and 19.01 0.83 for capsaicin combine with capsazepine
treated monocytes; mean SEM, n 4; p 0.05, compared to control). 1 umol/L capsaicin
significantly increased the Thapsigargin-induced sustained Ca
2
influx in human monocytes
from normotensive (0.14 0.06 in control condition, 0.66 0.14 in monocytes after
additional with capsaicin respectively, mean SEM, each n6, p0. 05, compared to control
condition). Capsazepine can inhibit the effection of capsaicin on intracellular calcium in human
monocyte. Upon stimulation with formyl methionyl leucyl phenylalanine (fMLP), the chemotactic
peptide, fMLP-stimulated chemotaxis was partially inhibited by capsaicin (68.46 5.15%,
compared to control condition 100.00 10.21%, mean SEM, each n4, p0. 05).
Capsazepine could partially attenuate the effection of capsaicin in monocytes migration.
Conclusions: This study demonstrated that capsaicin plays an important role in the regulation
of monocytes migration, which may be associated with the activation of TRPV1 in human
monocyte (supported by 973 program 2006CB503804).
P153
SLx-4090, an Enterocyte-specific Microsomal Triglyceride Transport Protein
Inhibitor, Lowers LDL Cholesterol and Triglycerides While Raising HDL
Cholesterol in Apo E -/- Mice Fed a High-fat Diet
James L Ellis, Alessandra Bartolozzi, John Ferkany, Hope Foudoulakis, Enoch Kim, Jay Kuo,
Ruth Ruffing, Olivier Schueller, Eric Wong, Yingfei Yang, Paul Sweetnam; Surface Logix,
Brighton, MA
MTP facilitates the formation and transfer of chylomicrons and VLDL in the intestine and the
liver respectively. Inhibiting both intestinal and hepatic MTP lowers serum levels of total, VLDL
and LDL cholesterol as well as triglycerides but leads to hepatic lipidosis. SLx-4090 was
designed to be enterocyte-specific and non-absorbable thus avoiding mechanism-based liver
toxicity. Male Apo E -/- mice were fed a high fat diet for 10 weeks which contained either no
SLx-4090 (control) or 0.006% (10 mg/kg/day) or 0.019% (30 mg/kg/day) or 0.06% (100
mg/kg/day) SLx-4090. SLx-4090 dose-dependently decreased total cholesterol and LDL levels
while raising HDL levels.
Control
10 mg/kg
SLx-4090
30 mg/kg
SLx-4090
100 mg/kg
SLx-4090
Total Cholesterol
(mg/dL)
163389 1669115 956132*** 52354***
LDL (mg/dL) 157485 1612111 908131*** 47452***
HDL (mg/dL) 161 221* 262*** 352***
Triglycerides (mg/dL) 21625 17824 105D13*** 708***
Body Weight Gain (g) 17.091.22 16.091.47 11.920.90*** 9.951.04***
Fat/Body Wt. Ratio 0.0580.003 0.0530.005 0.0390.007*** 0.0270.0044***
*P0.05, **P0.01, ***P0.001 compared to control. N78 per group, Data are mean SEM
Baseline triglyceride levels were also dose-dependently decreased by SLx-4090. There was a
dose-dependent reduction in body weight gain compared to controls with the mid and high
dose groups receiving SLx-4090 while there was no difference in food consumption among the
4 groups. Correlating with this reduction in body weight gain was a dose dependent reduction
in the fat/body weight ratio with SLx-4090. SLx-4090 had no effect on the levels of the liver
enzymes ALT and AST. Levels of SLx-4090 in the plasma were below the level of quantification
for the 10 and 30 mg/kg groups and 10 ng/ml for the 100 mg/kg group. The ability of the
non-absorbable, enterocyte specific MTP inhibitor, SLx-4090, to effectively reduce body weight
gain, fat/body weight ratios, total and LDL cholesterol and triglycerides while raising HDL levels
indicates the potential clinical utility of this compound in dyslipidemia, atherosclerosis,
metabolic disease and obesity.
e-64 Arterioscler Thromb Vasc Biol June 2007
by on March 25, 2011 atvb.ahajournals.org Downloaded from
P154
Dectin-1, a Non-TLR Pattern Recognition Receptor, Regulates NADPH
Oxidase Activity in Primary Human Monocytes
Deena H Elsori, Martha K Cathcart; Lerner Rsch Institute, Cleveland Clinic, Cleveland State
Univ, Cleveland, OH
Our lab is interested in studying signal transduction pathways that regulate the activity of
NADPH oxidase in primary human monocytes. Zymosan, a yeast cell wall preparation, and its
opsonized form (ZOP) are potent stimulators of NADPH oxidase activity. The activation of this
enzyme complex result in the production of a superoxide anion burst in monocytes.
Monocyte-derived superoxide anion mediates LDL lipid oxidation which is believed to contribute
to chronic inflammation and specifically atherogenesis. Our lab had previously identified and
characterized several pathways that regulate the activity of NADPH oxidase and superoxide
anion production in activated monocytes, however, the receptor(s) responsible for zymosan and
(ZOP) recognition have not yet been determined in primary human monocytes. We hypothesized
that zymosan signals through a pattern recognition receptor for the activation of the NADPH
oxidase enzyme complex. Therefore, to test this hypothesis we examined the role of different
pattern recognition receptors such as Toll-like receptors (TLRs) and the -glucan receptor,
Dectin-1. Our studies show that zymosan binds to Dectin-1 to trigger a superoxide anion burst
in primary human monocytes and this reaction is independent of TLR2 and TLR4. In addition,
we found that (ZOP) activates NADPH oxidase through binding to complement receptor 3 (CR3)
as well as Dectin-1. We are further trying to link zymosan recognition to intracellular signaling
events. Currently, we are investigating receptor phosphorylation and recruitment of intracellular
proteins to the activated receptor upon zymosan stimulation. Taken together, our results
suggest for the first time that Dectin-1 is the predominant receptor for zymosan-mediated
activation of NADPH oxidase in primary human monocytes.
P155
In Vivo Effect of Inflammatory Cytokines on Cystatin C Among Subjects at
Risk for Advanced Atherosclerosis
Luis H Eraso, German Hernandez, Andres Leon; Texas Tech Univ Health Sciences Cntr, El Paso, TX
Background In vitro evidence suggests that inflammatory cytokines augment the secretion of
Cystatin C (CysC), and other cell matrix proteases important in atheroma fibrous cap
degradation, and coronary plaque rupture. Recently, CysC has been proposed as a method to
estimate glomerular filtration rate (eGFR), and as a cardiovascular risk biomarker. Methods We
assessed the relationship between cardiovascular inflammatory biomarkers (IL6 and hsCRP)
and CysC among 18 subjects with CKD not yet on dialysis. GFR was estimated using a
creatinine-based method (MDRD) and a CysC-method. The delta between methods was
calculated (eGFR) and its correlation with IL6 and hsCRP levels was analyzed. Results A
positive Spearman correlation coefficient was noted between CysC and IL-6 (0.658, P0.01),
and between eGFR and IL6 (0.69, p0.01). No correlation was noted with hsCRP, LDL, HDL, TG
or Age. The multivariate linear regression model showed a positive regression coefficient (R
2
0.48).
IL-6 was the only factor contributing to the model (t3.8, p0.003). Conclusions A eGFR change
as serum IL6 increases suggests that in vivo CysC secretion is stimulated by inflammatory cytokines,
potentially limiting the role of CysC as a reliable estimate of GFR. This could explain the excess
cardiovascular risk (beyond traditional risk factors) among patients with CKD.
TABLE 1. LR MODEL. OUTCOME VARIABLE: EGFR.
Standardized Coefficients (B) T P
IL-6 .88 3.80 .003
hsCRP -.25 -1.15 .27
LDL -.18 -.76 .46
HDL .29 .99 .33
TG .07 .28 .78
Age .07 .37 .71
P156
The Functional Significance of a Hydrophobic Patch Identified by Recent
X-ray and Homology Models of Lipid-Free Apolipoprotein A-I
Loren Faulkner, Stacy Martin, W Sean Davidson; Univ of Cincinnati, Cincinnati, OH
Apolipoprotein A-I (apoA-I) is an important component of high density lipoprotein and the
reverse cholesterol transport system. A recently published X-ray crystal structure of lipid free
apoA-I, as well as a cross-linking/homology structure from our laboratory, have identified an
unusually solvent-exposed hydrophobic patch located at one end of a helical bundle formed by
the N-terminus. The patch consists of leucines 42, 44, 46, and 47. To determine any functional
consequences of this site, we generated two apoA-I mutants that replace the hydrophobic
residues with acidic amino acids of roughly similar volume: apoA-I (L42,44D) and apoA-I
(L46,47D). In a liposome clearance assay, both mutants exhibited an enhanced ability to bind
and emulsify dimyristoyl phosphatidylcholine compared to wild type (WT) apoA-I. An
independent vesicle binding assay indicated that apoA-I (L46,47D) was particularly adept at
binding lipid surfaces compared to WT apoA-I. ApoA-I (L42,44D) was capable of stimulating
cholesterol efflux from RAW macrophages in an ABCA1-dependent manner at a level similar to
WT apoA-I. However, apoA-I (L46,47D), despite its apparent increased lipid affinity, was
actually a less efficient stimulator of cholesterol efflux via ABCA1. These data suggest that this
hydrophobic patch may be involved in apoA-I/lipid interactions and is an example of how
detailed structural models of apoA-I may lead to a better understanding of apoA-I function.
P157
Increased Ability of Sera from LCAT-deficient Subjects to Promote
ABCA1-mediated Cholesterol Efflux
Elda Favari, Franco Bernini, Maria Pia Adorni, Univ of Parma, Parma, Italy; Wendy Jessup,
Ingrid C Gelissen, Univ of New South Wales, Sydney, Australia; Elsa Moleri, Guido
Franceschini, Laura Calabresi; Univ of Milan, Milan, Italy
Forty-one carriers of mutant LCAT alleles and 10 non carriers from the same families
volunteered for this study. In homozygotes (n14) plasma HDL-C concentration was markedly
reduced (11.71.7mg/dl) as well as plasma apoA-I (46.24.4mg/dl). The analysis of HDL size
showed a predominance of small HDL
3
particles, with a great proportion of pre-beta HDL.
Heterozygotes (n27) have slightly reduced HDL-C and apoA-I levels (41.22.2mg/dl and
107.04.2mg/dl), with a significant increase (30%) in pre-beta HDL. The capacity of serum
from LCAT deficient subjects and controls to extract cell cholesterol through the various
pathways was tested in different cell models: 1) Fu5aH hepatoma cells, expressing high levels
of SR-BI and low levels of ABCA1, 2) parent and hABCG1-expressing CHO-K1 cells were used
to evaluate the ABCG1-mediated cholesterol efflux and 3) J774 macrophages expressing high
levels of ABCA1, upon treatment with cAMP, and low levels of SR-BI. In Fu5AH cells, cholesterol
efflux to sera from the homozygotes was significantly reduced by 42% compared with
heterozygotes and by 48% compare to control sera with a significant correlation between
SR-BI-mediated efflux and HDL-C serum concentration (R0.699 P0.001). The ABCG1-
mediated cholesterol efflux to LCAT deficient sera was significantly reduced (6.50.5% for
homozygotes carriers and 8.00.2% for heterozygotes) compared to efflux induced by control
sera (9.80.5%). Under basal conditions, the J774 macrophages release membrane choles-
terol to extracellular acceptors mostly by passive diffusion and in such condition, cholesterol
efflux to sera from homozygotes carriers was significantly reduced (6.10.4%) compared to
efflux induced by heterozygous and control sera, which were similar (9.40.4% and
10.50.7%, respectively). On the contrary, ABCA1-mediated efflux to the sera was increased
by 70% in heterozygotes and 106% in homozygotes subjects compared to control sera, and
correlated with the percentage of pre-beta HDL (R0.480 P0.001), suggesting that despite
the dramatic hypoalphalipoproteinemia, LCAT deficient homozygotes have highly efficient HDL
particles in specifically promoting the ABCA1 efflux pathway.
P158
Alternative Transcript Variants of the Mouse Hepatic Lipase Gene in
Macrophages
Lita A Freeman, Zengxuan Nong, Elke M Wagner, Avram Walts, Herminia Gonzalez-Navarro,
Hope Rhodes, Arion J Kennedy, Alan T Remaley, Silvia Santamarina-Fojo; NIH/Natl Heart,
Lung, and Blood Inst, Bethesda, MD
We have recently identified macrophages as a site of synthesis of hepatic lipase (HL) and
demonstrated that macrophage HL is proatherogenic in apoE-KO mice. Here we demonstrate
that HL transcripts in macrophages vs. liver in apoE-KO mice are not identical. 5 RACE analysis
starting from exon 4 indicated that whereas liver and macrophage HL transcripts both contain
sequences coding for exons 14, the 5 ends differ in liver vs. macrophages, indicating that
full-length HL expression is driven by different promoters in liver vs. macrophages. Moreover,
the macrophage HL transcript contains an additional short exon in canonical Intron 2 which is
absent from the liver transcript. RT-PCR analysis with exon-specific primers showed that in
macrophages, exons 59 were more abundant than exons 14, indicative of a second
macrophage-specific HL transcript beginning upstream of exon 5. 5 RACE analysis of
macrophage RNA starting from exon 9 demonstrated a 1.3 kb transcript beginning in Intron 4
and proceeding through exon 9 which potentially encodes an N-terminal-truncated HL protein
lacking the catalytic site but containing heparin binding sites. Northern analysis confirmed that
the major HL transcript is shorter in macrophages than in liver of apoE-KO mice. Thus, in
apoE-KO mice, macrophages synthesize at least two HL transcripts, neither of which is
identical to the HL transcript synthesized by liver. The less abundant HL macrophage transcript
is predicted to encode full-length HL but has a different 5 end and thus a different promoter,
as well as an extra exon, compared to the liver HL transcript. This transcript variant would be
inactivated in E-KOxHL-KO mice. The more abundant macrophage transcript begins in Intron 4,
does not encode full-length HL, and is present in E-KOxHL-KO mice. These findings suggest
that the proatherogenic role of HL in macrophages may require further elucidation.
2007 ATVB Poster Presentations e-65
by on March 25, 2011 atvb.ahajournals.org Downloaded from
P159
Telomerase Reverse Transcriptase Is Induced in Macrophages by
Proinflammatory Stimuli and Mediates Macrophage Survival and
Inflammation
Florence Gizard, Takashi Nomiyama, Elizabeth B Heywood, Karrie L Jones, Dennis
Bruemmer; Univ of Kentucky, Lexington, KY
An emerging consensus underscores the importance of both macrophage inflammation and
apoptosis in the vascular wall for the development of atherosclerosis. Although the intracellular
pathways controlling both processes still remain to be elucidated, a novel area in vascular
biology involves telomerase activation. Telomerase controls key cellular functions including
replicative lifespan, proliferation, differentiation and apoptosis, and activation of telomerase has
been demonstrated in response to injury of the arterial wall. In the present study, we analyzed
the role of telomerase in macrophage biology. We demonstrate expression of the catalytic
subunit of telomerase, telomerase reverse transcriptase (TERT), in macrophages of human
atherosclerotic lesions. Stimulation of primary murine peritoneal macrophages with lipopo-
lysaccaride (LPS) and oxidized LDL (oxLDL) resulted in a significant dose-dependent induction
of TERT mRNA and protein expression. The up-regulation of TERT in response to LPS or oxLDL
was prevented by co-treatment with the SN50 peptide and BAY 117082, two inhibitors of the
NF-B pathway. Consistent with these findings, transient transfection experiments using
5-deletion and site-directed mutagenesis of the TERT promoter identified a proximal NF-B
site which confers the transcriptional induction of TERT expression in response to oxLDL.
Finally, functional experiments demonstrated that TERT-deficient macrophages reveal typical
features of cell senescence and were more prone to oxLDL-induced apoptosis. Furthermore,
oxLDL-induced expression of the pro-inflammatory gene IL-1beta and the matrix-
metalloproteinase-9 (MMP-9) was prevented in peritoneal macrophages isolated from TERT-
deficient mice. In conclusion, these experiments demonstrate that oxLDL induces TERT
expression through an NF-B-dependent pathway and suggest that telomerase mediates
macrophage viability and pro-inflammatory gene expression.
P160
Measuring the Force of Leukocyte Transmigration: New Insights into an
Old Story
Aleksandr Rabodzey, CNRS, Universite of Paris, Paris, France; Pilar Alcaide, Brigham and
Womens, Harvard Med Sch, Boston, MA; Francis W Luscinskas, Harvard Med Sch/BWH,
Boston, MA; Benoit Ladoux; CNRS, Universite of Paris, Paris, France
Background: Leukocyte transmigration across vascular endothelium is a key event during
inflammation, and immune reaction. Currently most of the research is focused at the molecular
mechanisms of cell-cell junction dissolution, while the possibility of junction disruption by the
leukocytes force is overlooked due to the lack of tools to assess those forces. We hypothesized
that leukocyte must exert forces during migration across the endothelium at cell-cell junctions.
Methods: HUVEC were grown in monolayers on a fluorescent micropillar PDMS surface. We
recorded forces exerted by the leukocytes by displacements of pillars and analyzed by our own
software. Results: As a control we observed the same area of HUVEC before transmigration;
force vectors were randomly oriented (1A). Tensile forces generated by monolayer are
homogenous and lower than those generated on the edges or by single cells. We found that
initial endothelial junction disruption is a quick (2030 sec) low-force process (1B); it is
probably governed by biochemically-induced junction dissociation. On the other hand, during
the following stage (5060 sec), leukocyte forms adhesion to the surface and pulls itself in the
intercellular space, exerting large force (1C) that may reach up to 250nN compared to 70nN
force exerted by the cells in the same area. This force is linearly dependent on the rigidity of
the substrate. Conclusion: Leukocyte transmigration involves 3-fold force increase. This
indicates that actin-myosin machinery malfunctioning may impair immune/inflammatory
response. The capability to exert force depends on the rigidity of the substrate and thus may
vary between different tissues and tumors.
P162
Matrix Gla Protein Is Cleared by the Kidney in Hypertensive Humans
Roger Rennenberg, Univ Hosp Maastricht, Maastricht, The Netherlands; Leon Schurgers,
Cees Vermeer, Maastricht Univ, Maastricht, The Netherlands; Jan Scholte, Alphons Houben,
Peter de Leeuw, Bram Kroon; Univ Hosp Maastricht, Maastricht, The Netherlands
Background: Medial vascular calcifications are common among patients with hypertension. The
vitamin K-dependent protein matrix Gla-protein (MGP) plays an important role in preventing
arterial calcification. In this study we investigated the renal clearance of MGP from the
circulation in patients with moderate to severe hypertension. Methods and results: From 90
moderate to severe hypertensive patients scheduled for renal angiography, renal arterial and
renal venous blood was sampled prior to administration of contrast material for measuring the
MGP-clearance by the kidney. Average renal fractional extraction was 12.8%. There was no
significant relationship between creatinine clearance (range 26154) and renal fractional
extraction of MGP in this population. Conclusions: In this study, we demonstrate that the kidney
is able to extract MGP from the plasma. Clearance of plasma MGP was 12.8% and did not
correlate with creatinine clearance.
P163
Ginkgo Biloba (EGb 761) Reduces Arteriosclerotic Nanoplaque Formation
and Size in Cardiovascular High-risk Patients
Petra Scha fer, Charite , Campus Benjamin Franklin, Berlin, Germany; Lovisa Ringstad,
Uppsala Univ Institute of Pharmacy, Uppsala, Sweden; So ren Just, Heart Cntr Brandenburg,
Bernau, Germany; Karl Winkler, Univ Clinic Freiburg, Freiburg, Germany; Gu nter Siegel;
Charite , Campus Benjamin Franklin, Berlin, Germany
Utilizing the isolated lipoprotein receptor syndecan (heparan sulfate proteoglycan (HS-PG) from
arterial endothelium and vascular matrices and coating therewith a silica surface, we were able
to observe the very earliest stages of arteriosclerotic plaque development, the so-called
nanoplaque build-up, by ellipsometric techniques (patent EP 0 946 876). The arteriosclerotic
nanoplaque is represented by the ternary aggregational complex of the HS-PG receptor,
lipoprotein particles and calcium ions. The model was validated in several clinical studies on
cardiovascular high-risk patients applying their blood lipoprotein fractions. In eight high-risk
patients who had undergone aortocoronary bypass operation, the reduction of arteriosclerotic
nanoplaque formation amounted to 11.9% (p 0.0078) and of nanoplaque size to 24.4% (p
0.0234) respectively, in normal blood substitute solution with 2.5 mmol/L [Ca
2
] after a 2
month therapy with 2 x 120 mg Ginkgo biloba extract (EGb 761, Ro kan

novo). Additionally, we
could directly demonstrate and confirm the antioxidative capacity of ginkgo and its oxygen free
radical scavenging effect by disclosing an upregulation of superoxide dismutase (SOD) activity
of 15.7% (p 0.0391) and a lowering of the quotient oxLDL/LDL by 17.0% (p 0.0234) after
the 2 month medication regimen. Furthermore, we measured a significant decrease in
lipoprotein(a) concentration falling from 52.4 8.2 to 42.0 9.9 mg/dL (p 0.0359).
Altogether, these beneficial effects of Ginkgo biloba might have partially repaired endothelial
dysfunction being responsible for the early stages in arteriosclerosis and could present a basis
for a mechanistic explanation of nanoplaque reduction under ginkgo treatment.
P164
Divergent Impact of LDL and Oxidized LDL on CREB Activation and on
ERK-mediated CREB Downregulation
Jane E Reusch, Catherine Gliwa, Jodean Gunther, Matthew Hockin, Univ of Colorado Health
Sciences Cntr, Aurora, CO; Thomas O McDonald, Kevin D OBrien, Univ of Washington,
Seattle, WA; Irene E Schauer; Univ of Colorado Health Sciences Cntr, Aurora, CO
Introduction: LDL is an established mediator of atherosclerosis and is directly toxic to cells of
the vessel wall including vascular smooth muscle cells (VSMC). Our laboratory has identified
the transcription factor CREB, cAMP Response Element Binding protein, as a modulator of
VSMC phenotype. CREB blunts mitogen stimulated VSMC proliferation and protects VSMC from
apoptosis. In numerous models of vascular damage (diabetes, aging, insulin resistance, and
pulmonary hypertension) we observe decreased expression of CREB protein in medial VSMC.
In addition to these published models we observe loss of CREB protein expression in the medial
VSMC of LDL receptor KO mice fed a high fat western diet. We hypothesized that LDL has a
direct effect on VSMC to decrease CREB content and function. Methods: To test this hypothesis
we exposed primary rat aortic VSMC to LDL and oxidized LDL (Intracel, Frederick, MD) at
10100 g/ml, in the presence or absence of kinase inhibitors, for up to 48 hours. Results:
Both LDL and oxidized LDL led to a rapid increase in CREB phosphorylation (4-fold) over 1530
minutes. In contrast to the acute, transient CREB activation by LDL, oxidized LDL resulted in
persistent CREB activation and ultimately in a dramatic, dose-dependent loss of CREB protein
at 2448 hours (90% at 24 hours in 100g/ml oxidized LDL). Though some toxicity was
observed at the higher concentrations, this loss was observed after normalization to total actin
or GAPDH content. Exposure to LDL and oxidized LDL also led to phosphorylation/activation of
P38 MAPK, ERK, and Akt. Pharmacological inhibitors of these pathways were used to identify
the pathway(s) responsible for toxicity and CREB downregulation. Both phenotypes are blocked
by inhibition of ERK, partially blocked by scavenging of reactive oxygen species and inhibition
of PKC, but unaffected by inhibition of JNK, PI3K, P38MAPK, or PKA. Conclusions: LDL
exposure leads to acute CREB activation in VSMC but only with oxidized LDL is CREB protein
loss observed. This protein loss correlates with toxicity and is mediated by reactive oxygen
species and the ERK signaling pathway. One mechanism of oxidized LDL injury to the
vasculature may be CREB downregulation. Studies are underway to define the mechanism of
CREB loss in this model.
P165
Macrophage 3 Integrin Mediates High-Fat Diet-Induced Inflammation
Through TNF
Jochen G Schneider, Yimin Zhu, Clay F Semenkovich; Washington Univ, St. Louis, MO
Integrins are critical effectors of inflammatory cell function, chronic treatment with beta3
integrin inhibitors increases mortality in humans, and beta3 integrin deficiency causes
atherosclerosis and death in certain high fat-fed mice. To test the hypothesis that the beta3
integrin affects diet-induced inflammation, bone marrow from beta3-deficient or beta3-wild
type mice (each appropriately apoE- or LDLR-deficient) was transplanted into apoE- or
LDLR-deficient mice followed by Western diet feeding. At 6 weeks, only 3 of 18 apoE-deficient
(beta3 wild type) mice transplanted with beta3-deficient marrow were alive compared to 19 of
19 mice receiving wild type marrow (p0.0001). In LDLR-deficient (beta3 wild type) mice, 11
of 23 mice transplanted with beta3-deficient marrow survived 12 weeks of Western diet
compared to 19 of 21 mice receiving wild type marrow (p0.001). LDLR-deficient (beta3 wild
type) mice transplanted with beta3-deficient marrow surviving for 12 weeks on Western diet
had more atherosclerosis than wild type-transplanted mice (p0.05). To address the potential
role of non-marrow cells in the inflammatory phenotype, 19 of 19 beta3-deficient mice in the
apoE null model transplanted with beta3 wild type (apoE null) marrow survived the dietary
challenge, compared to 2 of 14 beta3-deficient mice receiving beta3-deficient marrow
(p0.001). Beta3-deficient mice in the LDLR null model were also rescued with wild type
marrow (p0.0001). No phenotype was observed with chow feeding. Pulmonary inflammation
e-66 Arterioscler Thromb Vasc Biol June 2007
by on March 25, 2011 atvb.ahajournals.org Downloaded from
was the cause of death and there was marked induction of lung TNFalpha expression. TNFalpha
was also increased 3-fold in peritoneal macrophages of mice transplanted with beta3-deficient
marrow as compared to wild type-transplanted mice (p0.05), an effect prevented by
reconstitution of beta3 expression in culture. Western diet feeding alone increased beta3
expression 3-fold in wild type macrophages as compared to cells from chow-fed littermates
(p0.0001). Beta3-deficient apoE null mice were rescued from diet-induced death by
treatment with an anti-TNFalpha antibody but not with an isotype-specific control antibody
(p0.01). These data suggest that macrophage beta3 integrin modulates dietary inflammation
through TNFalpha.
P166
Cosegregation of Soat1 Mutations with Dyslipidemia, Body Weight, and
Atherosclerosis in an Intercross Between C57BL/6 and C3H Apolipoprotein
E-Deficient Mice
Weibin Shi, Toru Miyoshi, Zuobiao Yuan, Timothy R Gilbert; Univ of Virginia, Charlottesville,
VA
Background: Dyslipidemia is an integral component of the metabolic perturbations in several
common human disorders, including type 2 diabetes, obesity, and the metabolic syndrome.
Genetic factors are a major determinant for the pathogenesis of dyslipidemia. Sterol
o-acyltransferase 1 (Soat1, also known as ACAT1) is an endoplasmic reticulum enzyme that
catalyzes free cholesterol to cholesterol ester. Methods and Results: We identified four
single-nucleotide polymorphisms (A421C, A439G, C454T, and C613T) within the coding region
of Soat1 between C57BL/6 (B6) and C3H mice and two of the SNPs led to amino-acid
substitutions (Ile147Val and His205Tyr). In an intercross between B6 and C3H apolipoprotein
E-deficient (apoE
-/-
) mice, allelic variation at Soat1 (or a closely-linked gene) explains 3% to
13% of the variations in plasma triglyceride, HDL, non-HDL cholesterol levels, body weight, or
atherosclerotic lesions. Inheritance of the C3H alleles resulted in significant increases in plasma
lipid levels and body weight but decreases in atherosclerotic lesion size compared to
inheritance of the B6 alleles. Soat1 variants were closely associated with variations in plasma
esterified cholesterol levels but not with free cholesterol levels. Real-time PCR revealed that
Soat1 was abundantly expressed in arterial walls. Conclusions: These results support Soat1
to be an important gene contributing to dyslipidemia, body weight, and atherosclerosis in mice.
P167
Microarray Gene Expression Profiling Identifies Candidate Atherosclerosis
Modifier Gene for the Ath26 Locus in Male ApoE-deficient Mice
Jonathan D Smith, Enakshi Chakrabarti, Jeffrey M Bhasin; Cleveland Clinic Lerner College of
Medicine, Cleveland, OH
Objective- To identify genes that modify atherosclerosis severity in a mouse model.Methods
and Results- In a strain intercross between atherosclerosis resistant AKR apoE-deficient mice
and atherosclerosis sensitive DBA/2 apoE-deficient mice, we identified a quantitative trait locus
(QTL) on chromosomes 17 that is associated with lesion severity in the male F
2
cohort. This
QTL, called Ath26, at 34 Mb on chromosome 17, has a LOD score of 4.25, with a genome wide
significance of p0.05. The AKR locus at this QTL has a dominant effect on lesion area, with
the DBA/2 allele associated with increased lesion severity; and, variation at this one locus is
associated with 14.5% of the lesion variation in the male cohort. Bone marrow derived
macrophages were cultured from 114 male F
2
mice, and mRNA was prepared and used for
gene expression analysis on Affymetrix 430v2 arrays. These cells expressed 22,000
transcripts, and the expression level of each was used as a quantitative trait to identify
expression QTLs (eQTLs) with LOD scores 3, defined as loci that are associated with the
expression level of a specific transcript. More than 12,300 eQTLs were identified with 18%
mapping cis to the gene (cis eQTL) and the majority mapping trans to the gene (trans eQTL).
We then performed correlation analyses to identify the genes whose expression was best
associated with lesion severity in the male F
2
cohort. Through these analyses we have identified
a candidate atherosclerosis modifier gene that resides at 33 Mb on chromosome 17, precisely
at the Ath26 locus, whose expression is associated with 21% of the lesion variation in the F
2
cohort, and whose expression is controlled by a strong cis eQTL (LOD score 11.6). This
poorly annotated gene can now be tested to see if modulation of its expression can modify
lesion development or progression in a mouse model, and whether human genetic variation in
the orthologous gene is associated with atherosclerosis.
P168
Loss of BMPRII in Endothelium Induces Inflammation in Vitro and
Correlates with Atherosclerosis Progression in Human Coronary Arteries
Hannah Song, Georgia Institute of Technology, Atlanta, GA; Daiana Weiss, J D Vega, W R
Taylor, Emory Univ, Atlanta, GA; Hanjoong Jo; Georgia Institute of Technology and Emory
Univ, Atlanta, GA
Atherosclerosis is an inflammatory disease, occurring preferentially in arterial regions
associated with disturbed flow while sparing the undisturbed flow regions. We have shown that
exposure of endothelial cells (ECs) to disturbed flow stimulates production of bone morphogenic
protein 4 (BMP4), which leads to inflammatory responses - intercellular adhesion molecule-1
(ICAM-1) expression and subsequent monocyte adhesion. However, the underlying mechanism
by which BMP4 induces inflammation is unclear. Here, we examined which BMP receptors
(BMPR) mediate BMP4 action in ECs. Studies using mouse aortic ECs (MAEC), human umbilical
vein ECs (HUVEC), mouse thoracic aortas and human coronary arteries revealed that BMPRI
(ALK2 and 6) and BMPRII were expressed in ECs. Interestingly, immunostaining studies showed
that BMPRII was located in the cell-cell junction, colocalizing with VE-cadherin, in confluent
MAEC but not in ECs that were sub-confluent or at wounded edges. MAEC treated with
mouse-specific siRNA lost BMPRII expression in the cell-cell junction. The BMPRII knockdown
prevented phosphorylation of smad1/5/8 and stimulation of monocyte adhesion in response to
BMP4, suggesting a role for BMPRII as the BMP4 receptor. Unexpectedly, BMPRII knockdown
in ECs unleashed the inflammatory response in the absence of BMP4 as determined by
increased ICAM-1 expression and monocyte adhesion, which was blocked by an ICAM-1
neutralizing antibody (YN1). Moreover, BMPRII levels in ECs were progressively decreased in
more advanced atherosclerotic lesions in human coronary arteries. These findings suggest that
a decrease in BMPRII expression in ECs induces inflammation and is associated with more
advanced atherosclerotic disease states.
P169
Arsenic-induced Acceleration of Atherogenesis in Apoe-knockout Mice:
Role of Oxidative Stress and Inflammation
Sanjay Srivastava, Stanley E DSouza, J C States; Univ of Louisville, Louisville, KY
Chronic arsenic (As) ingestion causes inflammation and vascular dysfunction in humans. In the
present study, we examined the effect of As-feeding on atherogenesis in apoE-knockout mice.
Three week old mice were maintained on normal chow diet and received either tap water
(n14) or water containing 1 (n12), 4.9 (n12) or 49 (n16) ppm As. The mice were
sacrificed at age 16 weeks of age and atherosclerotic lesion formation, plasma lipids and
indices of oxidative stress and inflammation were examined. Plasma cholesterol and
phospholipids of the As-fed mice were comparable to the controls. Plasma triglycerides of the
As-exposed mice were 1025% lower as compared to the controls. As exposure induced a
dose dependent increase (1.53.0 fold) in the atherosclerotic lesion formation in the aortic
sinus and the aortic arch, whereas, little or no lesions were visible in the abdominal aorta. We
also performed time dependent studies with mice exposed to 49 ppm As. These studies
showed significant increase in the lesion area in the aortic sinus and aortic arch as early as
10 weeks of age (n12; P0.05) and throughout the aortic tree at 36 weeks of age (n12;
P0.05). Inflammatory cytokines and chemokines (TNF-, IL-1, MCP-1, VEGF) and lipid
peroxidation products-malondialdehyde (MDA) and 4,hydroxy-trans-2-nonenal (HNE) were
significantly increased (P0.05) in the plasma of these mice at 16 and 36 weeks of age.
Immunohistochemical analysis in these mice showed a 2-fold increase in the macrophage
accumulation in the aortic valve, whereas very little if any T-cells or smooth muscle cells were
detected in these lesions. These mice also showed increased staining for the markers of
inflammation (MCP-1) and oxidative stress (protein-HNE and protein-MDA) in the aortic valve.
Collectively, our data suggest that exposure to As enhances oxidative stress and inflammation
and accelerates atherosclerosis in apoE-knockout mice.
P170
The Potential of Locked Nucleic Acid Oligonucleotides in Treatment of
Hyperlipidemia
Ellen Marie Straarup, Niels F Nielsen, Jens Bo Hansen, Troels Koch, Henrik Oerum, Henrik F
Hansen, Christoph Rosenbohm; Santaris Pharma A/S, Hoersholm, Denmark
Hyperlipidemia with increased plasma levels of VLDL and LDL is typically associated with
diabetes and metabolic disorders. Apolipoprotein B-100 (apoB) is important for assembly of
VLDL in liver and the clearance of VLDL and LDL particles from plasma. Reduction of apoB
mRNA expression is known to lower the secretion of VLDL from the liver and reduce plasma
cholesterol and thereby the risk of development arteriosclerosis. Here we report on the
properties of Locked Nucleic Acid (LNA) oligonucleotides targeting the apoB mRNA. LNA is a
nucleic acid modification that increases affinity and improves stability of oligonucleotides.
These characteristics are of major importance in the development of therapeutic oligonucle-
otides. Several LNA oligonucleotides targeting the apoB mRNA were screened in vitro for
activity (measured as down regulation for the apoB mRNA level) and compounds with low
nanomolar IC50s were selected for in vivo studies. The result showed dose dependent down
regulation of apoB mRNA in mouse liver and jejunum. As a result of ApoB down regulation the
level of plasma cholesterol was reduced up to 70% compared to the saline control. Correlating
with apoB down regulation we observed dose dependent hepatic lipid accumulation but no
significant induction of the levels of transaminases. This study shows that LNA oligonucleotides
can be potent for specific down regulation of target mRNA in vivo in relation to treatment of
hyperlipidemia.
P171
Narrowing a Region Containing an HDL Gene on Mouse Chromosome 14
Zhiguang Su, Beverly Paigen; The Jackson Laboratory, Bar Harbor, ME
The inverse relationship between HDL levels and coronary artery disease has generated interest
in genetic factors contributing to variations in HDL levels. Because of the inherent difficulties
of carrying out linkage analysis for HDL in humans, many mammalian geneticists have turned
to mouse model. A mouse model is relevant because most quantitative trait loci (QTL) for HDL
map to homologous regions in mice and humans, indicating the same set of genes may
underlie variation in both strains. A set of four overlapping congenic strains for chromosome
14 has been constructed, in which a region from CAST has been introgressed by a series of
back-crosses onto the background of B6. Plasma HDL levels were examined at N10-generation
mice fed on chow. One congenic strain showed significant increases in HDL in both sexes: HDL
was 84.8 2.8 mg/dl (n9) in congenic males vs. 70.2 1.4 mg/dl (n14) in B6 males,
p0.00001 and 66.1 2.1 mg/dl (n10) in congenic females vs. 55.4 1.3 mg/dl (n12)
in B6 females, p0.0001. Genetic markers in all congenic strains showed that the region
containing the CAST allele responsible for the increase in HDL mapped between 69.25 to 71.59
Mb (build 36). This region contains 17 genes. The gene responsible for HDL variation must have
a difference in concentration or in function due to amino acid change. To help identify the gene
affecting HDL, we performed expression array analysis on multiple liver samples from CAST
and B6 mice on chow. Within this region, only one gene, nudt18 encoding nucleoside
diphosphate linked moiety X-type 18, has significant difference in expression; compared with
B6, CAST mice were 1.5 and 1.4 folds higher in males and females, respectively. We also
investigated the coding region polymorphisms between CAST and B6. Four genes had
2007 ATVB Poster Presentations e-67
by on March 25, 2011 atvb.ahajournals.org Downloaded from
polymorphisms that changed amino acid. These changes are Leu166Phe in gene Epb4.9
encoding erythrocyte protein band 4.9, Asn245Ser in Xpo7 encoding exportin 7, Thr310Met in
Dok2 encoding docking protein 2, and Thr619Ala in gene Fndc3a encoding fibronectin type III
domain containing 3a. None of these changes alter the acidity of amino acid, but two do change
the amino acid from polar to non-polar (Thr:Ala in Fndc3a and Thr:Met in Dok2). Further
evidence is needed to determine which of these is the gene affecting HDL.
P172
The Hemodynamic Environment Promotes Atherosclerosis-prone
Phenotypes in Endothelial and Smooth Muscle Cells via a
Mechanical-Transcription Coupling Mechanism
Nicole Hastings, Michael Simmers, Brian Wamhoff, Brett Blackman; Univ of Virginia,
Charlottesville, VA
Introduction Atherosclerosis is an inflammatory disease of the vasculature, developing as a
result of regional differences in arterial hemodynamics, where atheroprone flow leads to
increased susceptibility to atherogenesis. The internal carotid sinus (ICS) is distinguished by its
low disturbed flow profile (atheroprone) and is thus a common site of atherosclerotic lesion
formation, whereas the common carotid artery (CCA) is exposed to pulsatile laminar flow
(atheroprotective). Hypothesis A novel in vitro co-culture model has been developed to analyze
the mechanical-transcription coupling that occurs in human endothelial cells (EC) in contact
with human smooth muscle cells (SMC) during atheroprone hemodynamics. Here, we tested
the hypothesis that atheroprone flow applied to ECs induces SMC and EC phenotypes
characteristic of early atherogenesis. Methods Human ECs/SMCs were plated on the top and
bottom surface of porous transwell membranes (75mm) and grown to confluence. Hemody-
namic flow patterns derived from human MRI images of the ICS and CCA were applied to the
EC surface for 24 hours using a cone and plate flow device. Results/Conclusions ECs display
decreased alignment and elongation in response to atheroprone compared to protective flow,
while SMCs were oriented perpendicular to flow only during atheroprotective flow; both
reminiscent of in vivo architectures. Gene expression analysis confirms the presence of an SMC
atheroprone phenotype defined by reduction of SMC markers (smooth muscle -actin,
myocardin) and increased levels of transcription factor KLF4 and inflammatory gene VCAM-1,
resulting from the flow regime applied to ECs. Also, atheroprone-exposed ECs exhibited a
reduction in Tie2, eNOS and KLF2 gene expression and discontinuous staining pattern of
VE-cadherin, suggestive of a pro-remodeling state, loss of protective effects, and a more
porous permeability barrier. Interestingly, the chemokine interleukin-8 is upregulated in ECs
exposed to atheroprone flow, which may potentiate part of the SMC atheroprone phenotype.
This model indicates that SMCs undergo differential phenotypic changes in atheroprone flow
dependent upon EC mechano-coupling, leading to a priming effect reflecting early atherogen-
esis of the arteries.
P173
Degree of Acute Phase Response Predicts Extent of HDL Remodeling in
Human Inflammation
Sean P Heffron, Nehal N Mehta, Univ of Pennsylvania, Philadelphia, PA; Margarita de la
Llera-Moya, Childrens Hosp of Philadelphia, Philadelphia, PA; Leticia Pruscino, Jennifer
Tabita-Martinez, Meghan Wolfe, Karen Badelino, Daniel J Rader, Muredach P Reilly; Univ of
Pennsylvania, Philadelphia, PA
Introduction: The metabolic syndrome and atherosclerosis are chronic inflammatory disorders
associated with reduced HDL-cholesterol. The pathogenesis of HDL changes in these settings
in humans is poorly characterized. Hypothesis: We hypothesized that cytokine response would
predict the degree of HDL remodeling during inflammation, possibly through activation of
inflammatory lipases and induction of HDL-associated acute phase proteins. Methods: A
human endotoxemia model was used to examine whether baseline level or change in TNF,
CRP or IL-6 were associated with changes in HDL parameters (cholesterol, phospholipids (PL),
apoA-I, apoA-II, HDL-associated serum amyloid A (SAA)) and plasma endothelial lipase (EL)
mass. Serial plasma and serum samples were taken for 24 hours prior to and 24 hours
following intravenous administration of 3 ng/kg endotoxin in 20 healthy human volunteers.
Results: Baseline levels of TNF, CRP and IL-6 did not correlate with changes in HDL
parameters following endotoxin administration. However, increases in TNF levels correlated
with changes in HDL-PL (r -0.46, p 0.039) and EL mass (r 0.66, p 0.002), while
induction of IL-6 was associated with changes in several HDL parameters (PL, r -0.53, p
0.015; apoA-I, r -0.54, p 0.014; apoA-II, r -0.60, p 0.005) and increase in EL mass
(r 0.43, p 0.05). Notably, increases in EL mass correlated with decreses in HDL-PL (r
-0.44, p 0.05). Increases in CRP were strongly related to change in EL mass (r 0.71, p
0.001) and multiple HDL parameters (PL, r -0.74, p 0.001; apoA-I, r 0.49, p 0.029;
apoA-II, r -0.54, p 0.015; SAA, r 0.6, p 0.005). Conclusions: Acute inflammation
in humans induces atherogenic remodeling of HDL particles. While baseline levels of TNF,
CRP and IL-6 did not predict the extent of HDL alteration, the degree of inflammatory response
to experimental endotoxemia was highly correlated with induction of EL, acute phase
HDL-associated proteins and the extent of HDL remodeling. These findings provide strong
support for inflammatory cytokine-driven HDL remodeling in humans in vivo. This may be
mediated in part by cytokine induction of inflammatory lipases and increases in HDL-associated
SAA.
P174
VLDL Lipolysis Products Increase Monocyte Adhesion to Endothelial Cells
by Upregulation of CD11b and CD49d Integrins
Laura J Higgins, Limin Wang, Harold J Ting, Scott I Simon, John C Rutledge; Univ of
California, Davis, CA
A key step in atherogenesis is the recruitment of activated monocytes to damaged endothelium.
The postprandial state is characterized as a transient increase in circulating triglyceride-rich
lipoproteins such as very low-density lipoproteins (VLDL). Lipolysis of VLDL by lipoprotein lipase
(LpL) can result in activation of vascular cells such as monocytes and endothelial cells. Little
is known about the role lipolysis of postprandial VLDL plays in the activation of monocytes. We
hypothesized that postprandial VLDL lipolysis products increase adhesive interactions between
monocytes and endothelial cells by increasing expression of key integrins on monocytes. Blood
was collected from human volunteers after consumption of a moderately high fat meal and
VLDL was isolated by ultracentrifugation. Lipid classes were then separated from VLDL and
VLDL lipolysis products by solid phase extraction. THP-1 monocytes were incubated with VLDL,
VLDL LpL, or the separated lipid classes from each for 4 hours, and CD11b and CD49d gene
expression was measured using quantitative real-time PCR. To measure monocyte adhesion,
fresh human monocytes were treated with VLDL or VLDL LpL for 3 hours, while Human
Aortic Endothelial Cells (HAECs) were pre-treated with TNF to induce inflammatory activation.
Adhesion of monocytes under shear flow (2 dyn/cm
2
) over HAECs was recorded and rolling,
arresting, or transmigratory events were counted. THP-1 monocytes incubated with VLDL
lipolysis products showed a 3- and 6-fold increase in CD11b and CD49d expression,
respectively, compared to whole VLDL. In addition, the monoglyceride, diacylglyceride,
triglyceride, cholesterol and free fatty acid fractions separated from the VLDL lipolysis reaction
increased both CD11b and CD49d expression from monocytes to varying degrees. Treatment
of fresh monocytes with VLDL LpL increased all adhesive events to HAEC monolayers under
shear flow. These results show that VLDL lipolysis products increase the expression of integrins
necessary for adhesion of monocytes to endothelial monolayers, providing a possible
correlation between postprandial lipemia and inflammation associated with atherosclerosis.
P175
Combined Deficiency of (1,3)-fucosyltransferase-IV and -VII Is Required
to Limit Atherosclerosis in Mice Lacking the Low-density Lipoprotein
Receptor
Jonathon Homeister, Maria Morales-Levy; Univ of North Carolina, Chapel Hill, NC
Atherosclerosis is due to an inflammatory/immune response that requires leukocyte recruit-
ment to the vessel wall, mediated by E- and/or P-selectin and other leukocyte adhesion
molecules. (1,3)-fucosyltransferase (FucT)-IV and FucT-VII synthesize active fucosylated
selectin ligands. To determine a role for these enzymes in the atherosclerotic disease process,
this study tests the hypothesis that deficiency of FucT-IV and/or FucT-VII will decreased aortic
root lesion size in the low density lipoprotein receptor (LDLR) deficient mouse model of
atherosclerosis. Mice deficient in FucT-IV and/or FucT-VII were crossed onto the LDLR(-/-)
strain and fed a normal chow diet for six months. Aortic root lesion size was determined by
planimetry, plasma cholesterol and triglyceride concentrations by colorimetric assay, and blood
cell counts by automated blood analyzer. Cholesterol and triglyceride lipoprotein distribution
profiles were determined by FPLC. Lesion size [mm
2
; mean sem; (n)] in male mice was
decreased in FucT-IV(-/-)/FucT-VII(-/-)/LDLR(-/-) animals [0.02 0.003; (5); p0.05], but was
not decreased in FucT-IV(-/-)/LDLR(-/-) [0.07 0.001; (14)] or FucT-VII(-/-)/LDLR(-/-) [0.09
0.005; (14)] mice, compared to control LDLR(-/-) mice [0.10 0.02; (13)]. Lesion size in
female mice was not significantly different in FucT-IV(-/-)/LDLR(-/-) [0.22 0.03; (13)] or
FucT-VII(-/-)/LDLR(-/-) mice [0.13 0.07; (12)], compared to control LDLR(-/-) mice [0.17
0.03; (12)]. Cholesterol or triglyceride distribution among lipoprotein fractions was not
genotype-dependent. Significant changes in total plasma cholesterol or trigylceride, when
present, did not correlate with the observed genotype-dependent changes in lesion size.
FucT-VII(-/-)/LDLR(-/-) and FucT-IV(-/-)/FucT-VII(-/-)/LDLR(-/-) mice had a monocytosis (2.7
4.7 fold), a lymphocytosis (1.52.7 fold), and a granulocytosis (4.011.0 fold ), compared to
LDLR(-/-) mice. These results show that loss of selectin ligand activity achieved by deletion of
both FucT-IV and FucT-VII results in decreased aortic root lesion size in male LDLR(-/-) mice,
despite the presence of a significant leukocytosis.
P176
Does Elevated Macrophage uPA Expression Cause Plaque Rupture in
Advanced Atherosclerotic Lesions?
Jie H Hu, Nagadhara Dronadula, Goro Otsuka, David A Dichek; Univ of Washington, Seattle,
WA
Background: The cellular and molecular mechanisms of atherosclerotic plaque rupture are
poorly understood. Increased proteolytic activity of lesion macrophages is often proposed as a
cause of plaque rupture. Urokinase-type plasminogen activator (uPA), a protease that activates
plasminogen, is expressed in human atherosclerotic lesions, primarily by macrophages.
uPA/plasminogen can activate matrix metalloproteases, which have been implicated as causes
of plaque rupture. Hypothesis: We hypothesized that overexpression of uPA by macrophages
in advanced lesions of apolipoprotein E null (Apoe
-/-
) mice will cause plaque rupture. Methods:
Bone marrow from transgenic mice with macrophage-targeted uPA overexpression (SR-uPA
/
0Apoe
-/-
mice) or nontransgenic donors (SR-uPA
0/0
Apoe
-/-
mice) was transplanted into irradiated
35-wk-old SR-uPA
0/0
Apoe
-/-
recipients. Some of the donor mice were also transgenic for GFP.
Recipients BM reconstitution was examined at 7 wks by FACS analysis of peripheral blood.
Innominate lesions were examined at 12 wks by GFP immunostaining to determine whether
donor-derived cells were present, and by qRT-PCR at 810 wks to measure uPA expression.
Lesion histology will be examined at 10 wks to detect features indicative of plaque rupture.
Results: FACS analysis demonstrated adequate BM reconstitution of irradiated recipients:
7489% of peripheral blood leukocytes were GFP

. GFP and Mac-3 immunostaining of serial


e-68 Arterioscler Thromb Vasc Biol June 2007
by on March 25, 2011 atvb.ahajournals.org Downloaded from
sections showed that numerous donor-derived macrophages were present in innominate artery
lesions. TaqMan qRT-PCR showed significantly higher expression of uPA mRNA in innominates
from recipients of SR-uPA
/0
BM than in innominates from recipients of SR-uPA
0/0
BM (relative
uPA expression: 2.1 0.31 vs 0.10 0.036 arbitrary units; P0.001; n8). Conclusion: We
introduced uPA-overexpressing macrophages into advanced innominate artery lesions of
Apoe
-/-
mice. These macrophages express high levels of uPA after entry into the lesions,
resulting in a large (20-fold) increase in lesion uPA expression. This experimental setting will
allow us to test whether elevated macrophage-targeted uPA expression in innominate lesions
precipitates plaque rupture.
P178
Antiatherosclerotic and Anti-inflammatory Effects of Polyphenol-enriched
Fraction from Taraxacum coreanum Nakai in LDL ReceptorDeficient Mice
Jong-Min Han, Yong-Dae Park, Sojin An, Min-Jung Kim, Yue-Jan Jin, Woo S Lee, Tea-Sook
Jeong; KRIBB, Daejeon, Republic of Korea
There have been numerous reports demonstrating anti-atherosclerotic activity of polyphenols.
Natural polyphenols have a wide range of biological activities. The polyphenol-enriched fraction
(PEF) isolated from dried whole plants of Taraxacum coreanum Nakai decreased nuclear
factor-B (NF-B) activation. The PEF contains many biologically active polyphenols, luteolin
(1.28%), luteolin-7-O-glucoside (0.73%), caffeic acid (0.33%), apigenin (0.15%), and diosmetin
(0.09%), being the major constituents. In this study, we investigated early stages anti-
atherosclerotic effects of the PEF in a high-cholesterol diet-fed LDL receptor deficient (LDLR
-/-
)
mice. At 10 weeks of age, 20 male LDLR
-/-
mice were randomly divided into two groups (n
10) and fed a high-cholesterol diet (0.15% wt/wt diet, control group) or a high-cholesterol diet
supplemented with PEF (0.2% wt/wt diet) for 8 weeks. There were no differences in total
cholesterol and body weight between the control group and PEF-supplemented group during
the study period. However, the triglyceride level was decreased in the PEF-supplemented
group. Cross-sectioning of distal aortic roots also significantly revealed the difference in lesion
areas. The mean lesion areas of 10 consecutive sections stained with oil red O were
56.229.7 m
2
x10
3
in the PEF-supplemented group versus 93.838.1 m
2
x10
3
in control
group (P0.05). Also the protein levels which in turn regulated by NF-B, such as VCAM-1,
ICAM-1, TNF- and COX-2, were suppressed in aorta. In addition, the PEF significantly
suppressed the production of nitric oxide (NO) and the accumulation of intracellular reactive
oxygen species (ROS) in a dose-dependent manner by inhibiting NF-B activation in
LPS-induced RAW 264.7 cells. In conclusion, our results suggest that the PEF containing
luteolin, luteolin-7-O-glucoside, caffeic acid, apigenin, and diosmetin may be a promising
product for treatment of atherosclerosis by inhibiting activation of NF-B and provides the
additional rationale for application of anti-inflammatory therapeutic approaches for atheroscle-
rosis prevention.
P179
Downregulation of Tissue Inhibitor of Metalloproteinases Increases
Invasion, Proliferation, and Death of Macrophage-derived Foam Cells
Jason L Johnson, Andrew C Newby; Univ of Bristol, Bristol, United Kingdom
Foam cell macrophages secrete higher levels of matrix metalloproteinases (MMPs) than
non-foamy macrophages which contributes to plaque development and rupture. We investi-
gated the expression of the endogenous tissue inhibitor of metalloproteinases (TIMP)-3 during
foam cell formation, and its effects on macrophage and foam cell behaviour. Foam cells derived
from cholesterol-fed rabbits and by Ox-LDL loading of human macrophages demonstrated a
significant decrease in TIMP-3 mRNA (55%; p0.01 and 49%; p0.05, respectively) and
protein expression (84%; p0.0001) compared to control macrophages. Adding back TIMP-3
to foam cells significantly inhibited migration (51%; p0.05), proliferation (70%; p0.05) and
apoptosis (36%; p0.05), but had no effect on non-foamy macrophages. Immunocytochem-
istry for TIMP-3 on foam cells revealed a subset of these cells (28%) that were TIMP-3
negative. Furthermore, only cells negative for TIMP-3 invaded a synthetic basement membrane
using an in vitro invasion assay. In early rabbit atherosclerotic plaques, TIMP-3 expression was
associated with macrophages. However in advanced plaques, foam cells with little or no
TIMP-3 protein expression were found in the deeper layers of the plaque. These results
demonstrate that TIMP-3 is down-regulated when macrophages become lipid-loaded, and this
causes increased migration, proliferation and apoptosis. Furthermore, a distinct highly invasive
subset of macrophage-foam cells exists with very low TIMP-3 expression. We conclude that
loss of TIMP-3 from foam cells dramatically alters their potential to destabilise atherosclerotic
plaques.
P181
Liver X Receptors Enhance Apolipoprotein A-1 and Paraoxonase-1
Expression in Mouse Hepatocytes and Prevent the NF-kB Induced
Alterations in apoA-1, PON-1, and SAA in Inflammatory States
Atil Y Kargi, Chang Yeop Han, Mohamed A Omer, Alan Chait; Univ of Washington, Seattle,
WA
Atherosclerosis is a disorder of lipid metabolism and an inflammatory disease. Liver X receptors
(LXR) reciprocally regulate inflammation and lipid metabolism in macrophages. LXRs increase
expression of several genes involved in reverse cholesterol transport including ABCA1, ABCG1,
ABCG5, ABCG8, CETP, apoE, and CYP7A1. However, little is known about the role of LXRs in
regulating the hepatic expression of apoA-1, a key acceptor in reverse cholesterol transport.
LXR regulation of inflammatory gene expression involves the antagonism of NF-B signaling.
In hepatocytes, we have shown that NF-B activation upregulates SAA expression and
decreases paraoxonase-1 (PON-1) and apoA-1 production, changes which might decrease the
atheroprotective effects of HDL. Therefore, we hypothesized that LXR activation in hepatocytes
would increase apoA-1 and PON-1 expression while inhibiting the effects of inflammatory
cytokines on SAA, apoA-1 and PON-1. To test this hypothesis, we measured the effect of the
endogenous LXR ligand 22(R)- hydroxycholesterol (22HC) and TO901317, a potent and specific
synthetic LXR ligand, on SAA, apoA-1 and PON-1 expression in cultured mouse AML12
hepatocytes. Both 22HC and TO901317 increased hepatic apoA-1 and PON-1 expression as
much as 4 fold (p0.05) while SAA expression remained very low. Activation of LXR 4 h prior
to treatment with individual cytokines (10 nM of either IL-1 or TNF-) or other activators of
NF-B prevented the cytokine-mediated increase in SAA and decreases in apoA-1 and PON-1.
These LXR effects occurred at concentrations as low as 100 nM and peaked at 10 M for
TO901317 and between 10 M and 50 M for 22HC. Thus we have shown that LXRs, in
addition to their well known effects on multiple genes regulating cholesterol efflux, upregulate
hepatic expression of apoA-1, the major acceptor protein in reverse cholesterol transport.
Moreover, by inhibiting the effects of inflammatory cytokines on apoA-I, PON-1 and SAA, LXRs
also have an anti-inflammatory effect on HDL apolipoproteins. This reciprocal regulation of
lipoprotein metabolism and inflammation at the level of the hepatocyte could result in a more
atheroprotective HDL particle, which suggests a novel mechanism by which LXRs might
prevent atherosclerosis.
P182
Generation and Characterization of a C-Reactive Protein Knockout Mouse
Steven W Kerr, Huiping Jiang, Rosemarie Sellati, Hong Wang, Adedayo Hanidu, Jeffrey B
Madwed, Boehringer Ingelheim Pharmaceuticals Inc, Ridgefield, CT; Alexander J Szalai,
Mark A McCrory; The Univ of Alabama at Birmingham, Birmingham, AL
C-reactive protein (CRP) is well recognized as a marker of inflammation, but its role as an active
participant in an inflammatory response and whether it exacerbates cardiovascular and other
diseases remains controversial. Much of the difficulty associating a causal role to CRP in human
health or disease stems from the lack of a relevant rodent model because CRP has different basal
and stimulated blood levels in rodents compared to humans. Also, no case of human CRP deficiency
has been identified. In order to determine the role of CRP in inflammatory processes akin to those
experienced by humans, and to determine its impact on normal physiology, we engineered a mouse
strain that does not express the endogenous CRP gene. In these CRP
-/-
mice, the CRP gene was
floxed and then deleted by Cre-recombinase. CRP
-/-
were viable and fertile and showed no overt
physical or behavioral abnormalities despite the absence of CRP mRNA and protein (qRT-PCR and
Western blot). CRP
-/-
mice were put through a battery of inflammatory and immunological tests to
identify a potential functional role of CRP deficiency. We observed the following effects in the CRP
-/-
mice compared to wild type mice: (1) inhibition of cytokine production after LPS challenge (TNF-
and IL-10 blood levels were 57% and 74% lower, respectively, p 0.05), (2) inhibition in T-cell
cytokine production after -CD3 stimulation (INF- and IL-2 were present at levels 35% and 51%
lower, respectively, p 0.05), (3) we observed a 50% increase in T-cell independent IgM antibody
production after immunization with TNP-ficoll (p0.05). These data demonstrate that genetic
deficiency of CRP is not a lethal mutation in rodents and that CRP plays a significant role in both the
innate immune response to inflammation as well as the humoral response to immunization.
Importantly, these findings suggest that CRP plays an active role in orchestrating the inflammatory
and immune responses, and the changes seen due to deletion of the CRP gene indicate that
inhibition of CRP may have beneficial effects in inflammatory diseases such as atherosclerosis.
P183
Is Coronary Calcification Protective of ST-elevation Myocardial Infarction?
Shazib Khawaja, Nadeem Husain, Malik Ali, Hinan Ahmed, Asmir Syed, Frank Pettyjohn;
Univ of South Alabama, Mobile, AL
Background The association between coronary calcification and acute coronary syndromes
(ACS) especially ST elevation myocardial infarction (STEMI) has not been extensively studied.
It has been suggested that plaques containing calcium may be less vulnerable to rupture. We
studied the extent of coronary calcification as assessed by coronary angiography in the infarct
related artery among patients with STEMI in comparison with patients with chronic ischemic
heart disease requiring revascularization. We hypothesize that the presence of coronary
calcification may be protective for STEMI. Methods Coronary calcification, as assessed by
angiography, was graded as none, mild, moderate or severe at the site of lesion extending
5mm proximal and distal to the culprit lesion in patients with STEMI and chronic angina group.
Results 50 patients with STEMI as well as 50 consecutives patients with stable angina in a non
ACS setting presenting for outpatient coronary angiography and receiving PCI or CABG for
severe (69%) stenosis were studied. 72% of the patients with STEMI had no angiographic
calcification in comparison with only 10% of the patients with chronic angina requiring
revascularization. Overall there were more patients with moderate and severe calcification in
the chronic angina arm than the patients with STEMI. Conclusion In conclusion coronary
calcification is noted to be lower in culprit lesion among patients with STEMI as opposed to
patients with chronic angina. The low incidence of coronary calcification with STEMI supports
the possibility of calcium as protective in preventing plaque rupture.
PATIENT CHARACTERISTICS
STEMI (n50) Chronic Angina (n50)
Age(years) 49.6 60.4
Sex(M/FM) 37/13 34/16
Hypertension 35(70%) 43(86%)
Diabetes 8(16%) 16(32%)
Dyslipedemia 39(78%) 40(90%)
Tobacco Use 38(76%) 28(56%)
Results
STEMI Chronic Angina
No Calcification 36(72%) 5(10%)
Mild Calcification 9(18%) 21(42%)
Moderate Calcification 4(8%) 15(30%)
Severe Calcification 1(2%) 9(18%)
2007 ATVB Poster Presentations e-69
by on March 25, 2011 atvb.ahajournals.org Downloaded from
P184
High-density Lipoprotein Cholesterol Response to Statin Therapy in Korean
Hypercholesterolemic Patients
Min-Kyung Kim, Yeon-Yee Yoon, Seoul National Univ Hosp, Seoul, Republic of Korea;
Sang-Hyun Kim, Joo-Hee Jo, Myung-A Kim, Seoul Metropolitan Boramae Hosp, Seoul,
Republic of Korea; Dong-Ju Choi, Seoul National Univ Bundang Hosp, Sungnam, Republic of
Korea; Hyo-soo Kim, Dae-Won Sohn, Byung-Hee Oh, Young-Bae Park, Yun-Sik Choi; Seoul
National Univ Hosp, Seoul, Republic of Korea
Background and aims Statins generally increase high-density lipoprotein cholesterol (HDL-C),
but not in all patients. We designed a retrospective study 1) to assess the characteristics and
the different response patterns of lipoprotein level after statin therapy, comparing the patients
with decreased HDL-C level after statin therapy (poor response group) and the patients with
preserved or increased HDL-C level after statin therapy (favorable response group), 2) to
investigate the problems of statin-only-strategies in hypercholesterolemia especially in the
patients with high risk. Methods 516 patients, who were newly diagnosed as hypercholes-
terolemia, were enrolled. Their clinical characteristics, baseline and follow-up laboratory data
were analyzed. All patients had received one kind of statins for at least two months. Results
The poor response group showed more decrease of TC (p0.001) and LDL-C (p0.001) and
increase of TG (p0.001) than the favorable response group. Patients with initial low HDL-C
level showed poor responses to statins in terms of TC (p0.011) but better responses in terms
of HDL-C than those with initial normal HDL-C level (HDL-C increased by 11.4% vs. 2.2%
p0.001). Conclusions Serum HDL-C and TG level increased with statin therapy in some
patients, but decreased in the others. The risk of HDL-C decrease with statin therapy coexists
with large reduction of LDL-C level, so HDL-C raising therapy can be considered with statin
therapy, especially in the patients of high risk group.
P185
Endogenous Estrogenic Effects on Blood Pressure and Lipids in Normal
Women
Jei Kim, Jee Yeon Kim, Jong Sung Kim, Chungnam National Univ Hosp, Taejon, Republic of
Korea; Suk Hoon Lee, Chungnam National Univ, Taejon, Republic of Korea; Jae-Hoon
Chung; Korean Advanced Institute of Science and Technology, Taejon, Republic of Korea
Background: The present study evaluated the relationship between endogenous estrogen
levels and blood pressures or lipids in normal men and women. Methods: Experimental and
laboratory data was collected from normotensive subjects (123 men and 166 women) in their
thirties, forties and fifties who had normal laboratory profiles and no prior history of
cardiovascular risk. Differences of LDL-cholesterol (LDL), triglyceride (TG), and HDL-cholesterol
(HDL), and estradiol were compared by gender and age-groups in men and women. The
relationships between estrogen and blood pressure or lipid levels were evaluated using
correlation and stepwise multiple regression analysis in men and women. Results: Estradiol
levels decreased with advancing age in women only (p0.05). Overall, lower triglyceride (TG),
higher HDL, and lower blood pressure were observed in women compared to men (p0.05).
In women, LDL, TG, and systolic (SBP) and diastolic blood pressures (DBP) increased with
advancing age (p0.05). TG, SBP and DBP also increased with decreasing estradiol levels
(p0.05), independent of increasing age. Levels of HDL correlated positively with increased
estrogen only in women (p0.05). In multivariate analysis, age variable did not show any
relationship to the changes of blood pressures and lipids. SBP and DBP only had negative
relationship with estradiol levels. (p0.05) Only triglyceride among the lipid variables showed
negative relationship to estrogen increase. (p0.05) These correlations were not observed in
men. Conclusion: The present study observed the beneficial effects of higher endogenous
estrogen on the blood pressures and lipids in normal women. The estrogen-related benefits
observed were lower TG and SBP and DBP in young women than in older women or in men.
P186
Newly Developed PPAR- Agonist (R)-K-13675 Inhibits the Secretion of
Inflammatory Markers from Human Coronary Artery Endothelial Cells
Ken Kitajima, Shin-Ichiro Miura, Yoshinari Uehara, Keijiro Saku; Fukuoka Univ Sch of
Medicine, Fukuoka, Japan
Peroxisome proliferator-activated receptor-alpha (PPAR-) is a key regulator of lipid and
glucose metabolism and implicated in inflammation. (R)-K-13675 is a novel potent PPAR-
agonist with high subtype selectivity. Monocyte chemoattractant protein-1 (MCP-1) plays a key
role in the early stage of atherosclerosis. Therefore, we analyzed whether (R)-K-13675
decreases MCP-1 secretion and other inflammatory markers such as interleukin-6 (IL-6) and
interferon-gamma (INF-) in human coronary endothelial cells (HCECs). HCECs were main-
tained in different doses of (R)-K-13675 under serum starvation. After 20 hours, the levels of
MCP-1, IL-6 and INF- secretion in the medium were analyzed using ELISA. Treatment with
KPA-73979 at 0, 10, 50 and 100 nM, MCP-1 levels were significantly suppressed in dose
dependent manner (3.50.4, 2.80.1, 1.90.1 and 1.30.2 ng/ml, respectively). IL-6 and
INF- levels also significantly suppressed (IL-6: 1958, 1945, 1575 and 941 pg/ml,
respectively; INF-: 372, 333, 213 and 5.31.0 pg/ml, respectively). In addition,
(R)-K-13675 did not affect HCECs proliferation and tube formation in all doses. Thus,
(R)-K-13675 was associated with inhibition of inflammatory responses without affecting of
proliferation and angiogenesis, and may induce anti-atherosclerosis.
P188
Markers of Endothelial Dysfunction and Inflammation in Asymptomatic
Subjects at Increased Oxidation Stress
Pavel J Kraml, Petr Syrovatka, 3rd Faculty of Med, Charles Univ, Prague, Czech Republic;
Lenka Fialova, 1st Faculty of Med, Charles Univ, Prague, Czech Republic; Jana Potockova,
3rd Faculty of Med, Charles Univ, Prague, Czech Republic; Martin Vejrazka, 1st Faculty of
Med, Charles Univ, Prague, Czech Republic; Michal Andel; 3rd Faculty of Med, Charles Univ,
Prague , Czech Republic
Aim: Assessment of endothelial dysfunction and inflammatory markers in healthy assymptom-
atic subjects at increased oxidant stress. Methods: 75 healthy primary prevention men (aged
3060 yrs.) were ordered to tertiles according to plasma levels of advanced oxidation protein
products (AOPP). Subsequently markers of endothelial dysfunction:endothelium dependent
vasodilatoin (EDV), VCAM-1, ICAM-1, von Willebrand factor, nitrites/nitrates; intima media
thickness (IMT); markers of inflammation: IL-2, IL-6, IL-8, IL-10, IL-18, CRP US, TNF-alpha,
immunoglobulines (IgG, IgA, IgM), circulating IC; other markers of oxidant stress: total
antioxidant capacity of plasma (TAP), lipid hydroperoxides (HPL), body iron stores - ratio of
circulatory transferrin receptors and plasma ferritin (BIS); plasma lipids (total cholestero, HDL
cholestero, LDL cholesterol, triglycerides, Lp(a), ApoA1, ApoB) and markers of insulin resistance
(HOMA IR calculated from fasting glycemia and insulinemia, ) were compared between the 1
st
and the 3
rd
tertiles. Results: Men in the highest tertile showed increased plasma levels of
ICAM-1 (P0.05) and IMT (P0.05). There was no significant difference in all measured
inflammatory cytokines, or immunoglobulines. Howeveer, higher HOMA IR (P0.01), diabetic
dyslipidemia, hypercholesterolemia and hyperlipoproteinemia Lp(a) were more frequent in the
3
rd
tertile. Conclusions: Elevated oxidant stress is associated with endothelial dysfunction,
insulin resistance, hyperlipidemia but not markers of inflammation in early stage of
atherosclerosis.
P189
Elevated Expression of the Interleukin-8 Receptors CXCR1 and CXCR2 in
Peripheral Blood Cells in Obstructive Coronary Artery Disease
Deborah A Leonard, Niagara Univ, Niagara Falls, NY; Michael E Merhige, The Heart Cntr of
Niagara, Niagara Falls, NY; Robert S Greene; Niagara Univ, Niagara Falls, NY
Inflammation plays an important role in the initiation and progression of coronary artery disease
(CAD). We hypothesize that expression patterns of inflammatory cytokines and their receptors
can be correlated with severity and progression of CAD as monitored by positron emission
tomography/myocardial perfusion imaging (PET-MPI). Participants were recruited from sequen-
tial patients referred to the Heart Center of Niagara for PET-MPI. In preliminary experiments,
expression arrays were used to measure 84 cytokine and cytokine receptor mRNAs in
peripheral blood from patients matched for age, race, gender and traditional risk factors for
CAD. Interleukin-8 and its receptors CXCR1 and CXCR2 showed elevated expression in three
patients with persistent stress-perfusion defects compared to three patients with improved
perfusion and two with mild diffuse disease. In a larger case-control study, twenty five patients
with stress-induced perfusion defects were matched to patients with mild diffuse disease and
no previous history of CAD. These results confirmed that CXCR1 mRNA is elevated in males
with obstructive CAD (p0.02), but not in females (p0.53). A similar male-specific increase
in CXCR2 mRNA was seen (p0.03). A decrease in the coordinate expression of CXCR1, CXCR2
and IL-8 was seen in patients with stress-induced perfusion defects although it is unclear
whether this reflects a dysregulation of gene expression. The increase in mRNA levels for the
interleukin-8 receptors CXCR1 and CXCR2 in blood from males with obstructive CAD combined
with decreased expression in patients with improved perfusion suggests that these genes may
be useful as markers of both clinically overt CAD as well as patient response to aggressive
clinical management.
P190
Apolipoprotein E Suppresses Monocytic Cell Invasiveness
Yan Liu, SUNY at Stony Brook, Stony Brook, NY; Vasanthy Narayanaswami, Childrens Hosp
Oakland Rsch Institute, Oakland, CA; Craig C Malbon, Fayanne E Thorngate; SUNY at Stony
Brook, Stony Brook, NY
Apolipoprotein (Apo) E plays an important role in lipid metabolism as a major ligand in
receptor-specific lipoprotein uptake. Previous work in our laboratory showed that atheroscle-
rosis is reduced in two transgenic lines expressing too little ApoE (1% to 2% of wild type)
to correct their hypercholesterolemia. A possible mechanism by which low level ApoE reduces
atherosclerosis is suppression of the action of cytokines that promote monocyte invasion and
differentiation. Mitogen-activated protein (MAP) kinases mediate cellular responses to external
stress signals. We hypothesized that ApoE inhibits monocyte invasiveness through MAP kinase
signaling pathways. We analyzed invasion in THP-1 cells treated with either TNF-alpha
(50ng/ml) or IL1-alpha (2ng/ml). Cytokines stimulated a 13 fold increase in invasiveness in a
Matrigel invasion chamber compared to control. We tested if ApoE and MAP kinase inhibitors
regulate this response. THP-1 cells were treated with TNF-alpha in the absence or presence
of increasing amounts (210 ug/ml) of lipidated full-length ApoE3, or of LDL receptor binding
(amino acids 141149) ApoE peptide (10 ug/ml), or the MEK inhibitor PD98059 (50 uM) for 5
hours. ApoE3-lipid complex inhibits cytokine-induced invasion as well as suppresses basal
THP-1 cell invasion activity. ApoE peptide or the MEK inhibitor PD98059 that suppresses
activation of downstream Erk1,2 partially suppress invasive activity in response to cytokines as
well as basal invasion activity. These and other experiments suggest ApoE3 drives multiple
signaling pathways to suppress cell invasion. Expression of very low levels of ApoE3 appears
to curtail atherosclerosis at early stages, in part, by suppressing cell invasiveness thus
preventing monocytic cells from crossing the endothelial layer.
e-70 Arterioscler Thromb Vasc Biol June 2007
by on March 25, 2011 atvb.ahajournals.org Downloaded from
P191
Exposure to Gasoline Engine Emissions Increases Vascular Reactive
Oxygen Species and Activates Molecular Pathways Involved in Progression
of Atherosclerosis
Amie K Lund, Travis L Knuckles, JoAnn Lucero, JeanClare Seagrave, Jacob D McDonald,
Matthew J Campen; Lovelace Respiratory Rsch Institute, Albuquerque, NM
Cardiovascular disease is currently the leading cause of death in the US, with atherosclerosis
being responsible for approximately half of all CVD-related deaths. Epidemiological studies
have reported an association between environmental air pollution and increased rates of
cardiovascular morbidity and mortality. We have previously reported that subchronic exposure
to gasoline engine emissions results in increased aortic expression of several matrix
metalloproteinases (MMPs), which are markers of vascular remodeling and inflammation, in
atherosclerotic-prone ApoE
-/-
mice; however the underlying molecular mechanisms have not
yet been elucidated. We investigated the hypothesis that acute exposure to gasoline engine
emissions results in increased levels of vascular reactive oxygen species (ROS), which in turn
drive the subsequent induction of MMPs and related peptides such as endogenous tissue
inhibitors of MMPs, TIMPs, and endothelin-1 (ET-1). 10-wk old male ApoE
-/-
mice (n6 per
group), on a high fat diet, were treated orally with either 10 mmol/L of the SOD mimetic,
Tempol (avg. 41 mg/kg/day, beginning 24 h prior to exposure), or vehicle, and exposed via
inhalation for 6 h/d for a period of 1, 3, 7 d to either filtered air or gasoline exhaust (a 1:12
dilution with ambient air; 60 g PM/m
3
). TBARs analysis showed increased levels of vascular
oxidative stress by d7 of exposure to gasoline emission, which was ameliorated with
Tempol-treatment. Concurrent significant elevations in aorta MMP-2 and -9 (d7) and TIMP2
(d1) mRNA expression were also observed, each of which was attenuated by Tempol-
treatment. Interestingly, aorta ET-1 mRNA was significantly elevated by exposure d3, however
Tempol had no obvious effects on expression. Such findings suggest that exposure to gasoline
emissions results in increased vascular oxidative stress which mediates, at least in part,
expression of markers involved in vascular remodeling, inflammation, and the progression of
atherosclerosis.
P192
Selective Activation of Liver X Receptor Perturbs Hepatic Insulin
Signaling and Stimulates Production of Apolipoprotein B-containing
Lipoproteins
Abigale Miller, Heather Basciano, Mark Naples, Elaine Xu, Joanna Nelken, Khosrow Adeli;
Hosp for Sick Children, Toronto, Canada
Liver X receptor alpha (LXR alpha) is known to act as a master regulator of hepatic lipid
metabolism; however, the role of LXR alpha in regulation of hepatic assembly and secretion of
apolipoprotein B (apoB)-containing lipoproteins is not fully understood. Here, we examined the
link between LXR alpha activation and hepatic insulin signalling and apoB lipoprotein
production. In vivo activation of LXR alpha using a specific LXR agonist, TO901317, in Syrian
golden hamsters led to increased plasma cholesterol (28%, p0.001), triglyceride (TG) (72%,
p0.005), and apolipoprotein B (apoB) protein mass (54%, p0.0001), as well as very low
density lipoprotein (VLDL)-TG (239%), relative to vehicle-treated controls. LXR activation was
also found to activate sterol response element binding protein-1c (SREBP1c) and the SREBP1c
target genes stearoyl Co-A desaturase (SCD) and fatty acid synthase (FAS). Metabolic
pulse-chase labelling experiments with primary hamster hepatocytes showed an increased
number of newly synthesized apoB-containing lipoprotein particles secreted following LXR
activation. After stimulation with the LXR agonist, reductions in insulin receptor (IR) beta subunit
mass (35%, p0.02), insulin receptor substrate (IRS)-1 mass (12%, p0.03) and tyrosine
phosphorylation (36%, p0.003), as well as increases in phosphatase and tensin homologue
(PTEN) (18%, p0.03) and protein tyrosine phosphatase (PTP)-1B (30%, p0.001) protein
mass were observed. In contrast, there was a two-fold increase in IRS-2 mass (p0.05, and
an increasing trend in phosphorylation, p0.08) as well as reductions in plasma glucose levels
(19%, p0.001) following LXR stimulation. In conclusion, activation of LXR in the hamster led
to the induction of a mixed state of hepatic insulin resistance at the level of IRS-1 and
hypersensitivity at the level of IRS-2. These changes were accompanied by a considerable
increase in the number of circulating TG-rich VLDL-apoB particles likely due to enhanced
hepatic assembly and secretion of apoB-containing lipoproteins.
P193
Intermediate-conductance Calcium-activated Potassium Channel, KCa3.1,
Plays an Important role in Macrophage Migration in Atherosclerosis
Hiroto Miura, Kazuyoshi Toyama, Med College of Wisconsin, Milwaukee, WI; Yoshimasa
Fujiwara, Fujiwara Memorial Hosp, Katagami, Japan; Takashi Saito, Akita General Hosp,
Akita, Japan; Ossama Hatoum, Israel Institute of Technology, Haifa, Israel; David Gutterman;
Med College of Wisconsin, Milwaukee, WI
Infiltration of atherosclerotic plaques by macrophages is an essential step for the development
of atherosclerosis. We previously reported that long-term blockade of KCa3.1 reduces
development of atherosclerosis in apolipoprotein E knockout mice (EKO). Since ion channel
remodeling has been reported in activated macrophages, we hypothesized that increased
KCa3.1 activity is necessary for macrophage migratory function in atherosclerosis. Methods:
KCa3.1 expression and monocyte chemoattractant protein-1 (MCP-1, 10
-9
M)-induced migra-
tion were examined in peritoneal macrophages of EKO by immunocytochemistry and trans-well
migration assay. Macrophage accumulation and KCa3.1 expression were immunohistochemi-
cally examined in atherosclerotic lesions of aortic roots from EKO treated with vehicle or
TRAM-34 (120mg/kg/day), a specific KCa3.1 blocker for 12 weeks. Results: KCa3.1 expression
was markedly increased in peritoneal macrophages compared to peripheral blood or peritoneal
monocytes. TRAM-34 (10
-7
10
-5
M) dose-dependently suppressed MCP-1-induced macro-
phage migration (1.50.4-fold of control, p0.05 vs vehicle 2.40.3-fold, n10). Macro-
phages accumulated in atherosclerotic lesions of EKO were also positively immuno-stained for
KCa3.1. Chronic KCa3.1 blockade significantly prevented development of atherosclerosis in
EKO by 40% in conjunction with 60-% reduction of macrophage accumulation in the lesions
(macrophage-positive area; 8112 x 10
3
m
3
, p0.05 vs control 21920, n9). Migratory
response to MCP-1 was impaired also in macrophages collected from EKO chronically-treated
with TRAM-34 (1.50.1-fold of control, p0.05 vs vehicle 2.80.4-fold, n6). Long-term
treatment of EKO with TRAM-34 caused no obvious pathological or clinical signs of toxicity,
while plasma TRAM-34 concentrations were maintained at ranges specific to KCa3.1
(86648x10
-9
M, n6). In conclusion: KCa3.1 activity is associated with macrophage
migration in atherosclerosis, suggesting a pathophysiological role for KCa3.1 up-regulation in
atherogenesis. KCa3.1 blockade is a new therapeutic strategy for diseases involving activated
macrophages such as atherosclerosis.
P194
Atypical Antigen Presenting Cells in Grossly Normal Human Aorta
Mihail Moisenovich, Lomonosov Moscow State Univ, Moscow, Russian Federation; Olga
Pustovalova, Institute of General Pathology and Pathophysiology Russian Academy of Med
Sciences, Moscow, Russian Federation; Shamil Khapchaev, Lomonosov Moscow State Univ,
Moscow, Russian Federation; Irina Andrianova, Institute of Experimental Cardiology,
Cardiology Rsch and Production Cntr of Russian Federation Health Ministry, Moscow,
Russian Federation; Alexander Orekhov; Institute for Atherosclerosis Rsch, Russian Academy
of Natural Sciences, Moscow, Russian Federation
Histocompatibility class II molecules (HLA-DR) positive cells represented mostly by macro-
phages, dendritic cells and B-lymphocytes can orchestrate and affect inflammatory responses
in atherosclerotic lesions. We have recently demonstrated the HLA-DR cells in normal
(grossly uninvolved) human aortic intima. In present study we investigate phenotypical and
morphological characteristics of subendothelial intimacytes expressing HLA-DR. Intimal en face
preparations of grossly normal aorta were used for investigation. Visualization of antigens was
carried out using confocal laser scan microscopy and immunohistochemical methods. For the
quantitative measurements isolated subendothelial intimacytes were analyzed using a flow
cytofluorimeter. Four main morphotypes of HLA-DR cells have been identified: 1) large
stellate cells (more than 20 m) possessing two or more rounded processes; 2) round and
irregular-shaped cells over 10 m in size; 3) small round-shaped cells less than 10 m in size;
4) minor subpopulation was presented by stellate cells with polygonal body and sharped
processes that were situated parallel to endothelium. Over 90% of first population and almost
all cells of second and third subpopulations expressed CD45 indicating their monocyte origin.
Cells of fourth subpopulation were negative for CD45 but some contain alpha actin. The
processes of these cells interact with other cells of a similar shape that expressed alpha actin
but not HLA-DR.Thus intimal antigen-presenting cells have different origin. Immune response
in subendothelial intima may result from differentiation and activation of resident non-blood
intimacytes. Antigen-presenting function of resident intimacytes suggests unknown mecha-
nisms of adaptive immune response.
P195
Reinfusing Stem Cell Grafts with Increased Vascular Progenitors Reduces
Tissue Injury Caused by High-dose Chemotherapy in Hematopoietic
Transplantation
Tariq Iqbal, Univ of Ottawa, Ottawa, Canada; Thivisha Rajagopal, Ottawa Health Rsch
Institute, Ottawa, Canada; Michael Halpenny, Lin Yang, Lisa Martin, Antonio Giulivi,
Canadian Blood Services, Ottawa, Canada; Sheryl McDiarmid, Lothar Huebsch, The Ottawa
Hosp, Ottawa, Canada; David S Allan; Ottawa Health Rsch Institute, Ottawa, Canada
Background: Endothelial-like vascular progenitors (VPCs) can be collected in peripheral blood
stem cell (PBSC) grafts used in hematopoietic stem cell transplantation (HSCT). HSCT involves
toxic chemotherapy and/or radiation that can induce widespread tissue damage. The impact of
graft VPC content on transplant-related toxicity was assessed in autologous HSCT patients to
address the vascular regenerative capacity of hematopoietic grafts. Methods & Results: 17
patients (mean age 46 y) undergoing autologous hematopoietic transplantation at The Ottawa
Hospital were included. PBSC grafts were analyzed using a cell culture assay for VPC content
following adherence depletion of mononuclear cells on fibronectin-coated plastic dishes in
serum-rich conditions. Transplant toxicity was estimated using total length of hospital stay
(LOS) following graft infusion and the Seattle criteria for transplant-related organ toxicity in 8
organ systems each graded 0 - 3. Results: LOS following graft reinfusion was lower (16.4 vs
22.5 d, p0.03) and number of organs with grade 2 or 3 toxicity was reduced (0.6 vs 2.6
organs, p0.05) in patients with higher graft VPC content (n8, 1.0 x 10
2
VPCs/kg)
compared with reduced VPC content (n9, 1.0 x 10
2
VPCs/kg). Only 3 patients avoided any
significant organ toxicity (grade 0 for systems) and all 3 had increased graft VPC content.
Moreover, 5 of 8 patients with high graft VPC levels had minimal toxicity (grades 0 or 1 in all
organs) compared with only 1 of 9 patients in the low graft VPC group (p0.05). Importantly,
severe oral mucositis was reduced in patients with high compared with low graft VPC content
(0/8 pts with grade 2 or 3 mucositis vs 5/9 pts, p0.025). Graft VPC levels were independent
of graft CD34 counts, peripheral blood monocytes and Hb levels, age and disease (pNS).
Conclusion: Collection and reinfusion of autologous peripheral blood stem cell grafts with
higher VPC content appears to reduce tissue injury in hematopoietic transplantation. Identifying
factors that contribute to high graft VPC levels is needed. Regenerative cell therapy using
blood-derived VPCs warrants further investigation and may facilitate repair of tissue injury in
diverse organ systems such as myocardial or cerebral ischemia.
2007 ATVB Poster Presentations e-71
by on March 25, 2011 atvb.ahajournals.org Downloaded from
P196
Contrast-Enhanced Ultrasound Imaging of Atherosclerotic Carotid Plaque
Neovascularization: A New Surrogate Marker of Atherosclerosis?
Prakash Balan, Falak Shah, Matthew Weinberg, Vijaya Reddy, Matthew Feinstein, Marshall
Goldin, Steven Feinstein; Rush Univ Med Cntr, Chicago, IL
Objectives: To demonstrate that contrast enhanced carotid ultrasound (CECU) constitutes an
effective method for the detection of carotid intra-plaque neovascularization. Background: An
atherosclerotic plaque requires a nutrient blood supply, which is predominantly derived from
arterial vasa vasorum. A variety of factors (environmental and genetic) contribute to the
initiation and growth of atherosclerosis within vessel walls. Chemotactic factors, such as tissue
ischemic and hypoxic factors, stimulate the release of vascular endothelial growth factor (VEGF)
proteins, resulting in vessel wall angiogenesis. These developments often precede the
formation of the luminal plaque. In this report, we describe the use of contrast enhanced carotid
ultrasound (CECU) imaging for the detection and quantification of intra-plaque neovasculariza-
tion. The efficacy of CECU was measured against the neovascular density observed within the
tissue specimens obtained at the time of carotid endarterectomy surgery. Methods: Fifteen
patients with significant atherosclerotic carotid artery disease received a contrast enhanced
carotid artery ultrasound (CECU) examination prior to undergoing a carotid endarterectomy
(CEA). Two patients received bilateral endarterectomies, resulting in a total of 17 cases. At the
time of surgery, carotid plaque samples were surgically removed and stained with specific
vascular markers (CD31, CD34, von Willebrand factor, and hemosiderin) designed to identify
the presence and degree of neovascularization. The intra-plaque neovascularization recorded
on pre-operative CECU was correlated with the degree of neovascularization noted in the tissue
specimens. Results: The contrast-enhanced carotid ultrasound (CECU) neovascularization was
correlated to CD-31 stained tissue specimens. This correlation value was 0.68 using
Spearmans rank method. Conclusions: The ability of CECU to detect neovascularization
appeared to correlate with the presence and degree of intra-plaque neovascularization
observed by histologic comparison.
P197
Cyclic Strain Regulates the Notch/CBF-1 Signaling Pathway in Endothelial
Cells: Role in Angiogenesis
David Morrow, John P Cullen, Univ of Rochester, Rochester, NY; Paul A Cahill, Vascular
Health Rsch Cntr, Dublin, Ireland; Eileen M Redmond; Univ of Rochester, Rochester, NY
The role of hemodynamic forces, such as pressure-induced cyclic stretch, in the control of
angiogenesis remains poorly understood. The Notch signaling pathway regulates cell fate in a
variety of cell types. Notch receptors and target genes are upregulated following vascular injury
and are reportedly expressed on vascular endothelial cells (EC). We assessed the hypothesis
that Notch signaling mediates the cyclic strain-induced angiogenic response of EC. A Flexercell
system was used to expose human umbilical vein endothelial cells (p3-p7) to physiological
levels of cyclic strain (010% strain, 60 cycles/min, 024 h). Notch receptor and Notch target
gene mRNA and protein levels were determined by QRT-PCR and Western blot analysis,
respectively. Network formation on Matrigel was measured as an index of angiogenesis.
Exposure of EC to cyclic strain (10%) resulted in temporal regulation of Notch receptors 1 and
4 and target genes (hrt 1,2,3) at the mRNA and protein level; maximal increase at 4h, 21;8
and 23;9 fold increase, respectively for Notch 1 and 4 mRNA (n3). Moreover, cyclic strain
(10%, 24h) significantly increased EC angiogenesis; network length (AU) 775;127 vs
3928;400 for static and strained EC, respectively (n3, p0.05), while concomitantly
increasing mRNA expression of the growth factor Angiopoietin-1 (Ang-1) and its receptor
tyrosine kinase Tie- 2 by 7 and 3 fold, respectively. Knockdown of Notch 1 and 4 by siRNA, or
inhibition of Notch mediated CBF-1/RBP-Jk regulated gene expression by RPMS-1, resulted in
a significant decrease in cyclic strain-induced network formation and in Tie2 mRNA expression.
Notch 1 or Notch 4 siRNA, but not RPMS1, inhibited cyclic strain-induced Ang1. Constitutive
over expression of Notch 4 IC resulted in increased network formation, and Ang1 and Tie2
mRNA expression, under both static and strain conditions. These data demonstrate that (i) the
Notch signaling pathway in EC is biomechanically sensitive and (ii) that cyclic strain stimulated
angiogenesis is mediated, in part, through a Notch-dependent, Ang-1/Tie2 signaling pathway.
Mechanosensitive Notch signaling may thus represent a novel therapeutic target for diseases
involving angiogenesis.
P198
Androgen Increases Expression of Aortic AT1a Receptors and Restores
Angiotensin II-induced Abdominal Aortic Aneurysms in Castrated Male
ApoE-/- Mice
Xuan Zhang, Tracy Henriques, Alan Daugherty, Victoria English, Eva Langlois, Lisa Cassis;
Univ of Kentucky, Lexington, KY
Objective: Previous studies demonstrate that castration of male apoE-/- mice reduces
angiotensin II (AngII)-induced abdominal aortic aneurysm (AAA) formation to the level observed
in females, suggesting a primary role for androgen in promoting AAA formation. Recent studies
demonstrate a critical role for AT1a receptors (AT1aR) in AngII-induced AAA formation. The
purpose of this study was to determine if exogenous androgen administration to castrated male
apoE-/- mice would regulate abdominal aortic AT1aR expression and restore AngII-induced AAA
formation. Methods and Results: Male apoE-/- mice were castrated, and 1 week later
implanted with slow release pellets containing placebo (P) or dihydrotestosterone (DHT) (10 mg
pellet of each androgen/60 day sustained release). Values were compared to a group of
non-castrated (intact) male and female mice. Five weeks later, AT1aR mRNA abundance was
determined in the thoracic versus abdominal aorta. In intact male and female mice, AT1aR
mRNA abundance was greater in the abdominal than thoracic aorta. However, the fold
difference in AT1aR mRNA abundance between abdominal and thoracic aorta was greater in
male (4.57-fold) compared to female (2.18-fold) mice. Castration of male mice decreased
AT1aR mRNA abundance by 61% in abdominal, but not thoracic aortas, resulting in similar
AT1aR mRNA abundance between these aortic regions. DHT administration partially restored
AT1aR mRNA abundance in castrated male mice (AT1aR mRNA/18SRNA: intact, 1.74 0.54;
orchiectomy/DHT, 1.14 0.08). To determine if this dose of DHT would restore AAA
susceptibility in castrated male mice, we infused saline or AngII to castrated male mice
administered placebo or DHT. Administration of DHT to castrated male mice increased AAA
incidence from 27% to 75%, but had no effect on AngII-induced atherosclerosis. Conclusions:
These results suggest that androgen positively regulates the expression of the AT1aR in
abdominal aorta to increase AAA susceptibility in male apoE-/- mice.
P199
A Novel eNOS-independent Protective Action of Statin Against Angiotensin
II-induced Cardiovascular Remodeling and Renal Injury
Shusuke Yagi, Ken-ichi Aihara, Masashi Akaike, Yasumasa Ikeda, Yuka Sumitomo, Takashi
Iwase, Toshio Matsumoto; Tokushima Univ, Tokushima, Japan
Background: Statins are known to have pleiotropic actions mainly through activation and
up-regulation of the eNOS system. However, the effect of statins on cardio-renal insufficiency
caused by renin-angiotensin activation and the mechanisms of their actions are not fully
understood. Methods and Results: We used pitavastatin because this statin is not metabolized
by hepatic cytochrome P450, implying that pitavastatin has direct action on the cardiovascular
and renal systems. C57BL6/J mice at 10 weeks of age were infused with angiotensin II (Ang
II) (2.0 mg/kg/day) by an osmotic mini-pump for 2 weeks and were simultaneously
administered pitavastatin (0.2 mg/kg/day) or a vehicle. Echocardiography showed that
pitavastatin treatment improved AngII-induced left ventricular concentric hypertrophy and
diastolic dysfunction. Enhancement of perivascular fibrosis and medial thickening of the
coronary artery, of cardiac fibrosis, and of cardiomyocyte hypertrophy by AngII infusion were
significantly attenuated in pitavastatin-treated mice. Pitavastatin treatment also reduced
cardiac mRNA expression of p67phox and urinary excretion of 8-OHdG, an oxidative stress
marker. Ang II-induced phosphorylation of MAPKs (ERK1/2, JNK, p38), TGF-1 expression and
Smad 2/3 phosphorylation were all attenuated by pitavastatin treatment. Even in eNOS KO
mice, pitavastatin improved Ang II-induced cardiovascular remodeling and left ventricular
diastolic dysfunction. In addition, low glomerular filtration rate and enhancement of albuminuria
in Ang II-treated eNOS KO mice were improved by pitavastatin treatment. Pathological findings
of glomeruli showed that Ang II-induced PAS-positive deposition, TGF-1 expression and
oxidative stress evaluated by dihydroethidium staining were enhanced in eNOS KO mice, which
were attenuated by pitavastatin treatment. Survival rate of eNOS KO mice was decreased by
Ang II treatment; however, pitavastatin significantly improved the survival rate of Ang II-treated
eNOS KO mice. Conclusion: Pitavastatin has eNOS-independent protective actions against Ang
II-induced cardio-renal insufficiency through decreasing oxidative stress and suppressing
MAPKs and the TGF-1/Smad pathway.
P200
Triglyceride-rich Lipoprotein Lipolysis Products Induce Endothelial Cell
Inflammation That Is Mediated by Oxidized Lipids
Limin Wang, Rajan Gill, Univ of California, Davis, Davis, CA; John Newman, Univ of
California, Davis, and USDA, ARS, Western Human Nutrition Rsch Cntr, Davis, CA; John C
Rutledge; Univ of California, Davis, Davis, CA
Objective: Triglyceride-rich lipoprotein (TGRL) lipolysis products provide a pro-inflammatory
stimulus to the endothelium. We investigated the mechanism by which TGRL lipolysis products
induce endothelial cell dysfunction by examining individual lipid fractions. Methods and
Results: Lipid classes from human postprandial TGRL with or without lipoprotein lipase (LpL)
were extracted and separated using aminopropyl solid phase extraction columns. The amount
of free fatty acids (FFA) released from TGRL by lipoprotein lipase was 10-fold greater than that
found in untreated TGRL. The FFA extracted from TGRL lipolysis significantly increased the
expression of supernatant TNF in human aortic endothelial cells. Reactive oxygen species
(ROS) were measured by determining dichlorofluorescein diacetate fluorescence. Treatment
with both the FFA and cholesterol fractions caused ROS production; however, the FFA fraction
released from TGRL lipolysis doubled the production of ROS compared with that released from
TGRL only. The FFA-mediated increase in ROS was blocked by the cytochrome P450 2C9
inhibitor sulfaphenazole. Further, the levels of oxidized lipids within the FA fraction were
determined by HPLC/MS/MS. Linoleic acid metabolites 13-HODE (1.68-fold) and 9-HODE
(1.51-fold) were found in TGRL lipolysis group. Compared with linoleic acid, 13-HODE
significantly induced ROS production in human endothelial cells (1.56-fold). Summary and
Conclusions: The pro-inflammatory effect of TGRL lipolysis products is mediated, in part, by
the free fatty acid fraction. Further more, the linoleic acid derived alcohols, 13-HODE and
9-HODE are released from TGRL by LpL and may facilitate endothelial cell injury in vivo by
stimulating intracellular ROS production.
P201
WITHDRAWN
e-72 Arterioscler Thromb Vasc Biol June 2007
by on March 25, 2011 atvb.ahajournals.org Downloaded from
P202
Pulmonary HypertensionRelated Shear Stress Induces Pulmonary
Endothelial Dysfunction
Min Li, Robin Shandas, Wei Tan; Univ of Colorado at Denver Health Sciences Cntr, Denver,
CO
Introduction: Recent clinical measurements and subsequent computational modeling studies
have shown that patients with pulmonary hypertension have higher peak flow shear stress
(FSS) compared to normotensive subjects. Thus, we assessed the hypothesis that pulmonary
hypertension-related FSS induces pulmonary arterial endothelial cell (PAEC) dysfunction, which
may result in smooth muscle vasoconstriction and hypertrophy. Methods: Bovine PAECs were
subject to a laminar FSS profile covering a range of conditions from low-pathological (FSS: 0
dyne/cm
2
), normal physiological (FSS: 5, 20, 60 dyne/cm
2
), and high-pathological (FSS: 90, 120
dyne/cm
2
). Endothelial function was evaluated by examining vasodilator protein expression
(eNOS, PGIS, COX-1 and COX-2), vasoconstrictor expression (ET-1), and growth factor release
(VEGF). Cell signal studies were performed using flow-conditioned endothelial media to culture
smooth muscle cells (SMC). Subcellular mechanical structures (F-actin and VE-cadherin) were
examined to provide mechanotransduction evidence. Results: Compared to normal physiolog-
ical FSS exposure (20 dynes/cm
2
), low-pathological (0 dynes/cm
2
) and high-pathological FSS
(90 or 120 dynes/cm
2
) significantly downregulated eNOS expression by 1.49 0.20, 1.31
0.16 and 1.41 0.16, respectively. In the physiological FSS range (5, 20 and 60 dyne/cm
2
),
eNOS expression significantly increased with increasing FSS. Expressions of PGIS, COX-1 and
COX-2 did not increase significantly for FSS 60 dynes/cm
2
. These results suggest that
increased FSS favors the expression of eNOS, PGIS, COX-1 and COX-2 only in the physiological
range. Compared to 0 dynes/cm
2
, FSS of 90 and 120 dynes/cm
2
significantly increased VEGF
expression. SMC expression of SM a-actin and PCNA suggests hypertension-related FSS
increases SMC hypertrophy and proliferation. Conclusions: We conclude that pulmonary
hypertension related high FSS may play an important role in endothelial-mediated vasocon-
striction and vascular remodeling.
P203
Palmitate, but not Oleate, Reduces 3 Integrin Expression in Vascular
Smooth Muscle Cells
Kara N Standley, Jochen G Schneider, Washington Univ, St. Louis, MO; David R Clemmons,
Univ of North Carolina, Chapel Hill, NC; Clay F Semenkovich; Washington Univ, St. Louis,
MO
A relationship between fatty acid metabolism and several human diseases may be mediated by
changes in integrin expression patterns. Beta3 integrin, part of the promiscuous alphavbeta3
receptor expressed on smooth muscle cells, appears to mediate inflammation induced by high
fat diets containing palmitate in mouse models. Specifically, beta3 integrin null mice have
increased inflammatory markers and are more susceptible to atherosclerosis when challenged
with a high fat diet. To test the hypothesis that fatty acids affect beta3 integrin expression,
primary porcine aortic smooth muscle cells were treated with oleate- or palmitate-
supplemented cell culture media. Palmitate treatment did not induce apoptosis as detectable
by propidium iodide staining or fluorescent DNA end-labeling. Palmitate treatment (500
microM) reduced beta3 integrin mRNA by half compared to BSA treatment alone. This response
was dose-dependent and maximal at 24 hours of palmitate exposure. A lower concentration of
palmitate (250 microM) also caused a significant decrease in the beta3 integrin message level.
Beta3 integrin protein mass was also reduced after palmitate treatment as shown by Western
blot analysis. Unlike palmitate, 500 microM oleate treatment did not reduce beta3 integrin
expression. Palmitate did not affect beta3 integrin transcription in beta3 integrin promoter/
reporter gene construct experiments. Transcription inhibition followed by analysis of decay of
the beta3 integrin mRNA revealed that downregulation of the beta3 integrin message occurs,
at least in part, through mRNA destabilization. These results show that palmitate, the saturated
fatty acid most commonly consumed in the diets of industrialized countries, down-regulates the
expression of the beta3 integrin in smooth muscle cells through effects on mRNA stability, and
suggest a potentially novel mechanism underlying the induction of inflammation in vascular
cells by high fat diets.
P204
Hyperbaric Oxygen Induces bFGF and HGF Expression and Enhances Blood
Perfusion as well as Muscle Regeneration in Mouse Ischemic Hind Limbs
Tetsuichi Asano, Shouhei Shinozaki, Masaharu Shibayama, Toru Nakayama, Yoshihiro Mano,
Kentaro Shimokado; Tokyo Med and Dental Univ, Tokyo, Japan
Background: It is not clear how hyperbaric oxygen (HBO) affects ischemia-induced patho-
physiological responses such as angiogenesis and skeletal muscle regeneration. We studied
the effects of HBO on the functional and morphological recovery of ischemic hindlimbs, blood
perfusion and the local production of angiogenic growth factors in a mouse model. Method and
results: Mice were placed in pure oxygen under 3 atm one hour a day for 14 days after the
removal of a segment of left femoral artery. HBO-treated mice showed better functional
recovery and greater blood flow in the ischemic hindlimb than untreated mice. Histological
examination revealed the unatrophic muscle fibers with islands of small regenerating muscle
cells and angiogenesis only in HBO-treated mice. Regeneration of muscle was confirmed by the
increase in myf5 mRNA. The amount of mRNA for VEGF, HGF and bFGF was slightly increased
in the ischemic hindlimbs. HBO eliminated the increase in VEGF mRNA. In contrast, the amount
of mRNA for bFGF and HGF was further increased by HBO treatment. HBO transiently increased
Egr-1 in ischemic hindlimbs. Conclusions: HBO accelerates the recovery of ischemic hindlimbs
by increasing the production of bFGF and HGF and by promoting muscle regeneration in mice.
P205
Statin Treatment of Vascular Endothelial Cells Disrupts Caveolae via
Cholesterol Depletion and Redistribution of Caveolin-1 Expression
Richard A Bundey; Univ of California, San Diego, La Jolla, CA
Statins inhibit a rate-limiting enzyme in the biosynthesis of cholesterol, HMG-CoA reductase,
and are widely used to treat atherosclerosis. In addition to its role in atherosclerotic plaque
development, cholesterol is a requirement for the formation of caveolae, important signaling
microdomains of many cell types including vascular endothelial cells. Therefore the current
study tests the hypothesis that statin will have a direct effect on vascular endothelial cells, in
particular affecting the relationship between cholesterol and caveolae abundance and
localization. Experiments used human umbilical vein endothelial cells and bovine aortic
endothelial cells with lovastatin or simvastatin treatments. Confocal imaging of caveolin-1-
eGFP and fluorescently-tagged cholesterol analogues in live cells identified a concentration-
and time-dependent reduction of caveolin-1 and cholesterol at the plasma membrane in
response to statin, respectively. Immunoblot experiments determined that statin reduced the
caveolin-1 expression in buoyant sucrose-gradient fractions of Triton-X100-solubilized lysates
without a significant change in total cell caveolin-1 expression. However, high concentrations
(1uM, 48hr) of statin did reduce the global caveolin-1 expression but this correlated with
an increase in expression of the p17 fragment of caspase 3 indicating this may be a cytotoxic
effect of statin at high doses. Since caveolae are a site of endothelial nitric oxide synthase
(eNOS) and interaction with caveolin-1 has been shown to negatively regulate eNOS activity,
we measured nitric oxide production (using an electrochemical sensor) in real-time from
statin-treated cells; responses elicited by bradykinin (10nM) treatment of statin-treated cells
were 2-fold those of control-treated cells. In conclusion, the results indicate that statins, by
acting directly on endothelial cells, can produce a cascade of events initially reducing
cholesterol content of plasma membrane; a subsequent reduction in caveolin-1 expression at
the membrane; followed by an increase in eNOS activity. In addition to the cholesterol-lowering
effects of statins these direct actions of statin on endothelial cells may underlie some of their
therapeutic benefit in vivo
P206
Low Endothelial Shear Stress (ESS) Leads to Excessive Expansive
Remodeling of Coronary Atherosclerotic Subsegments
Yiannis S Chatzizisis, Brigham and Womens Hosp, Harvard Med Sch, Boston, MA; Michael
Jonas, Massachusetts Institute of Technology, Boston, MA; Ahmet U Coskun, Northeastern
Univ, Boston, MA; Roy Beigel, Benjamin V Stone, Massachusetts Institute of Technology,
Cambridge, MA; Charles Maynard, Univ of Washington, Washington, DC; Ross G Gerrity,
Med College of Georgia, Augusta, GA; William Daley, Novartis Pharmaceuticals Corp, New
Jersey, NJ; Campbell Rogers, Cordis Inc, Warren, NJ; Elazer Edelman, Peter H Stone,
Charles L Feldman; Brigham and Womens Hosp, Harvard Med Sch, Boston, MA
Background: The role of excessive expansive remodeling in the natural history of atheroscle-
rosis has not been studied. We investigated the hypothesis that low ESS leads to the excessive
expansive remodeling of coronary atherosclerotic subsegments. Methods: In 11 diabetic
hyperlipidemic swine, IVUS based 3D reconstruction of the coronary arteries was performed at
baseline (wk 23) and follow up (wk 30). Local ESS was assesed using computational fluid
dynamics, and plaque-free subsegments (n102) of 3 mm length were identified. The
coronary arteries (n31) were harvested at follow up, cryosectioned at the subsegments of
interest, stained histologically and intima/media ratio, lipid deposition (oil red O), inflammation
(CD45) and collagen content (picrosirius red) were quantified. Local remodeling behavior was
assesed by IVUS based on the external elastic membrane area of the subsegment of interest
and the remodeling characteristics of the artery as a whole and classified into excessive
expansive (EER), compensatory expansive (CER) and inadequate remodeling (IR). Results:
Subsegments with EER had larger plaques with more lipid deposition, inflammation and
collagen content than subsegments with CER or IR (Fig a). Baseline ESS was significantly lower
in subsegments with EER as compared to those with IR (Fig b). Follow up ESS remained low
in subsegments with EER or CER, and slightly increased in subsegments with IR (Fig b).
Conclusion: Low ESS leads to EER in atherosclerotic subsegments, and in this setting, the
adverse low ESS environment persists, thereby fostering continued lipid accumulation,
inflammation, matrix degradation, and development of a high risk plaque.
2007 ATVB Poster Presentations e-73
by on March 25, 2011 atvb.ahajournals.org Downloaded from
P207
Therapeutic Approach of Human Embryonic Stem CellDerived Endothelial
Cells in Mouse Ischemic Heart and Hind Limb
Yu Chen, Memorial Sloan-Kettering Cancer Cntr, New York, NY; Gabsang Lee, Jae-Hung
Shieh, Memorial Sloan-Kettering Cancer Ctr, New York, NY; Malcolm A S Moore; Memorial
Sloan-Kettering Cancer Cntr, New York, NY
Human embryonic stem cells (HESCs) have been established as a potential resource for cell
replacement therapy. Here, we determined the engraftment and reconstitution of functional
endothelial-like cells differentiated from HESCs (HES-ECs) in NODSCID mice. HES-ECs were
obtained from blast colonies and displayed endothelial characteristics by expression of KDR,
VE-Cadherin, vWF, Tie-2, and uptake of LDL. To monitor engraftment of HES-ECs, HESCs were
transfected with lentivirus-GFP/luciferase. HES-ECs (3e10
5
) were injected into either myocar-
dial infracted (MI) mouse heart or ischemia hind limb (IHL). Medium-only injections served as
a control (n6). Cell engraftment was determined at 2w and 4w (weeks) of cell transplantation
in both MI hearts and IHL by luciferase imaging. Doppler perfusion imaging was also performed
to evaluate limb blood flow by comparison of the ratio of blood flow in IHL to that in non-IHL.
Mice were sacrificed at 4w of cell transplantation to determine myocytes regeneration and new
vessel formation derived from the HES-ECs in both MI hearts and IHL. A positive Luciferase
image was obtained in both MI hearts and IHL at 2w and 4w of cell transplantation. Doppler
imaging indicated that HES-EC transplantation significantly improved blood flow as early as 1w
with a blood flow ratio (IHL/non-IHL) of 0.60.2 in the HES-EC group versus the control group
0.30.1 (blood flow ratio at day 3 of ischemia ligation was 0.20.1). The blood flow in HES-EC
mice was greatly improved thereafter in comparison to control mice. At 4w, cells positively
stained with cardiac specific protein cTnI or NKx2.5 were also positive for GFP in MI hearts,
which indicated regeneration of myocytes from transplanted HES-ECs. New vessel formation
was demonstrated by human specific vWF staining in both MI hearts and IHL with vascular-like
structure. Our data suggest that HESC-derived endothelial precursors could be used thera-
peutically for the treatment of ischemia heart and limb.
P208
The Frequency of Cyclic Stretch Determines the Proliferative and Apoptotic
Capacity of Vascular Smooth Muscle Cells in Vitro
Alberto Colombo, Paul Cahill, Caitriona Lally; Dublin City Univ, Dublin, Ireland
Cyclic stretch is a key factor in determining the proliferative and apoptotic capacity of vascular
smooth muscle cells (SMC). Changes in SMC growth are critical to vascular remodeling and
restenosis following injury. The aim of this work is to investigate the effect of cyclic stretch
applied at different frequencies on the proliferation and apoptosis of SMC. Bovine aortic SMC
were subjected to 5% cyclic stretch at different frequencies (0, 0.5 and 1 Hz) for up to 96 h
using a Flexercell Tension Plus FX-4000T

system with an applied equibiaxial heartbeat


waveform before cell proliferation and apoptosis was evaluated. The Vybrant

CFDA-SE dye
and Vybrant

Alexa Fluor 488

Annexin V and propidium iodide were utilized to determine cell


proliferation and apoptosis, respectively, using FACS analysis. Cell counts were also performed
using a hemocytometer to confirm changes in cell growth after a 24, 48 and 96 h period of the
applied stretch. Using both FACS analysis and cell counting it was found that cyclic stretch
decreased SMC proliferation in a temporal manner. Moreover, when cells were exposed to the
lower frequency, SMC proliferation was inhibited to a greater extent. (Fig 1). In parallel cultures
using FACS analysis, the level of SMC apoptosis following exposure to cyclic stretch was also
frequency dependent. There was a reduced level of apoptosis after 96 h at the higher
frequency. We conclude that cyclic stretch has a temporal antiproliferative effect on SMC in
vitro. When cells are subjected close to the physiological frequency condition, the growth and
apoptotic activity of these cells is reduced. Fig 1: Temporal effect of 5% cyclic stretch on
SMCs under varying frequencies after 96 hours.
P209
A Role for Formin Homology Proteins in Shear Stressinduced Endothelial
Cytoskeleton Remodeling
Fatima Maksoud, Bethany M Gaudet, John W Copeland; Univ of Ottawa, Ottawa, Canada
The frictional drag caused by the laminar flow of fluid over the surface of cells causes a shear
stress to the underlying cell layer. Endothelial cells shifted from static to flow culture conditions
undergo a unique and stereotypical response to this stimulus. They respond as a population to
re-pattern their cell-cell junctions and cytoskeletal networks to change from a cobblestone
appearance to a more elongated morphology that is polarized in the direction of flow. This
shear-stress induced morphology is also observed in vivo and is thought to be atheroprotective.
Formins are cytoskeleton remodeling proteins that regulate polymerization of actin filaments.
The cytoskeletal structures that are induced by activation of formin proteins are highly
homologous to the structures that are formed in endothelial cells in response to shear stress.
The function of formin proteins in endothelial cells in any context has not been addressed.
Therefore we wished to test the hypothesis that Formin proteins are required for endothelial
cytoskeleton remodeling in response to flow-induced shear stress. Using RT-PCR we have
shown that the same nine (of the 15 known) vertebrate formins are expressed in arterial
(HAEC), venous (HUVEC) and microvascular (HDMEC) endothelial cells. Expression of a dominant
negative formin derivative abolishes adherens junction formation in a Ca2 switch assay in
static HUVEC cultures. Expression of this same dominant negative construct also diminishes
flow-induced shear stress remodeling of the actin cytoskeleton in HUVECs in vitro. The effect
of our dominant negative formin derivative suggests that the activity of at least one formin is
required in endothelial cells for adherens junction formation. In addition our results are
consistent with a role for formins in mediating the cytoskeletal changes induced by
flow-induced shear stress. These results will be confirmed using RNAi to knockdown the
expression of individual formins in endothelial cells.
P211
The effect of Rosiglitazone and Pioglitazone on Myocardial Apoptosis
Aycan Erkan, Ufuk Univ, Ankara, Turkey; C imen Karasu, Rdvan Yalc n; Gazi Univ, Ankara,
Turkey
Background: The role of apoptosis in heart failure and reperfusion injury has been etablised
by previous studies. PPAR-gamma agonists rosiglitazone and pioglitazones are widely used for
the treatment of diabetes mellitus and insulin resistance and alsa have favorable effects on lipid
profile. Glitazones, on the other hand, are associated with worsening of heart failure. Thus, we
aimed to investigate the effect of glitazones on myocardial apoptosis. Methods: H9c2 rat
myocardial cell cultures were used as a model of apoptosis. Doxorubicin was used to induce
apoptosis. MTT assayed was performed to measure cellular cytotoxicity. Reactive oxygen
species (ROS) was taken as a surrogate of cytotoxicity. Intracellular ROS measurements were
assessed by a fluorescent reader using 5(6)-Chloromethyl-2,7-dichlorodihydrofluorescein
diacetate-acetyl ester (CM-H2DCFDA) in living cells. Caspase-3 and caspase-9 levels were
determined to assess myocardial apoptosis. Results and Conclusion: Although glitazones are
clinically associated with aggrevation of heart failure, the addition of rosiglitazone (10 M)
resulted in attenuation of cellular toxicity caused by doxorubicin in cardiomyocytes .
Additionally, rosiglitazone treatment decreased doxorubicin-induced ROS generation signifi-
cantly (p0.01) in living myocardial cells. Doxorubicin treated cells displayed higher caspase-3
and caspase-9 levels compared with controls, which are markers associated with apoptosis.
Rosiglitazone attenuated the elevation of both caspase activities at significant levels (p0.05).
Rosiglitazone decreased doxorubicin-induced caspase 3 activity by nearly a half from
231.98 9.01 (% caspase activity) to 127.1 15.91. Caspase 9 activity went down from
225.74 15.38 to 172.37 2.75. These findings were comparable to those of the well known
antioxidant, N-acethyl L-cystein. We also found similar antioxidative and anti-apoptotic effects
for pioglitazone (data not shown). As a conclusion, both rosiglitazone and pioglitazone
attenuate cellular cytotoxicity and apoptosis in doxorubicin treated myocardial cell cultures in
an in vitro setting, although glitazones are known to be associated with aggrevation of heart
failure.
P212
PTEN Depletion Enhances Vascular Smooth Muscle Cell Proliferation and
Accelerates Neointima Formation
Seth B Furgeson, Peter A Simpson, Nihal Kaplan-Albuquerque, James Cooper, Raphael
Nemenoff, Mary C M Weiser-Evans; Univ of Colorado Health Science Cntr, Denver, CO
A change in proliferative phenotype of vascular smooth muscle cells (SMC) from a highly
quiescent to a rapidly proliferating phenotype is a key component of neointima formation. The
PI3K/Akt pathway is a principal pathway involved in SMC proliferation and phenotypic
modulation. PTEN is a tumor suppressor gene that negatively regulates the PI3K pathway; our
previous research has shown that early inactivation of PTEN in response to vascular injury is
a critical event involved in neointima formation. The goal of this study was to determine the role
of PTEN in SMC function and neointima formation using both in vitro and in vivo techniques.
Rat aortic SMC were transfected with control or PTEN-specific siRNA oligonucleotides. Western
blotting was used to determine levels of total PTEN and phospho-Akt and SMC proliferation was
determined by BrdU immunocytochemistry. To study the effect of PTEN depletion in vivo, we
performed carotid artery ligation experiments on wild type mice (WT), global PTEN heterozy-
gous mice (PTEN), and SMC-targeted PTEN heterozygous mice (SM22-Cre;PTEN;
generated by crossing PTENfl/fl mice to transgenic mice expressing Cre recombinase (Cre)
under the control of the SM22 promoter). At day 13 following injury, mice were injected
with BrdU and tissues harvested on day 14. Uninjured and injured carotid arterial sections were
examined for neointima size and were stained for SM--actin and BrdU using immunoflu-
orescence/histochemistry. Compared to controls, SMCs transfected with PTEN siRNA exhibited
significantly decreased PTEN levels with corresponding increases in phospho-Akt and
enhanced proliferation under both basal and serum-stimulated conditions. Injured carotid
e-74 Arterioscler Thromb Vasc Biol June 2007
by on March 25, 2011 atvb.ahajournals.org Downloaded from
arteries from PTEN and SM22-Cre;PTEN mice showed markedly larger neointimas
compared to WT mice. Immunohistochemistry showed that the neointimas were strongly
SM--actin positive and the number of BrdU positive cells was significantly greater in
PTEN and SM22-Cre;PTEN mice versus WT mice. These results suggest that PTEN is
a potent suppressor of SMC proliferation and that SMC-expressed PTEN is an important
regulator of neointima formation in vivo.
P213
Hypercholesterolemia in the Metabolic Syndrome Increases the Functional
Expression of TRP Channels in Coronary Myocytes from Ossabaw Miniature
Swine
Zachary P Neeb, Jason M Edwards, Gregory M Dick, Michael Sturek; Indiana Univ,
Indianapolis, IN
Ca
2
is a ubiquitous second messenger and controls many cellular processes in vascular
smooth muscle, including proliferation. Activating Ca
2
-permeable channels of the transient
receptor potential (TRP) family contributes to smooth muscle proliferation. Specific TRP channel
isoforms can be regulated by the sarcoplasmic reticulum (SR) Ca
2
stores, such that depleting
the SR of Ca
2
couples to influx of Ca
2
. We tested the hypothesis that Ossabaw pigs that
manifest the metabolic syndrome when fed excess high fat/cholesterol diet have greater TRP
channel-mediated Ca
2
influx upon SR Ca
2
store depletion. Groups of pigs used were fed the
following diets for 28 weeks: normal diet healthy controls (C; N 5), high fructose (20% of
kcal; F; N 3), and high fructose, fat (46% of kcal), and cholesterol (2% by weight;
hyperlipidemic; H; N 3). Coronary atherosclerosis was noted only in H vs. C, while F was
not different than C as shown by intravascular ultrasound measures in vivo. Enzymatically
dispersed smooth muscle cells from right coronary artery segments were loaded with fura-2
to measure intracellular Ca
2
. Influx of Ca
2
through TRP channels was determined by
analyzing the Mn
2
-induced quench of fura-2 fluorescence at the Ca
2
-insensitive wavelength
of 360 nm upon SR Ca
2
store depletion with caffeine (5 mM; to release SR Ca
2
) and
cyclopiazonic acid (10 M; to prevent the re-uptake of Ca
2
by the SR) in a Ca
2
-free solution.
TRP channel activation was verified by blockade of Mn
2
influx with Gd
3
(10 M), but not the
voltage-gated Ca
2
channel antagonist nifedipine (10 M). The rate of Gd
3
-sensitive Mn
2
influx ( pixel intensity/minute) was significantly higher in cells from H (-2.82 0.36)
compared to C (-0.89 0.11) and F (-0.82 0.46). In conclusion, our data indicate that
hypercholesterolemia in the metabolic syndrome increases TRP channel-mediated Ca
2
influx
in coronary smooth muscle cells associated with atherosclerosis. We suggest that activation of
Ca
2
-permeable TRP channels may contribute to smooth muscle cell proliferation.
P214
TP508 Peptide Restores VEGF-induced Activation of eNOS in Hypoxic
Human Endothelial Cells
Barbara Olszewska-Pazdrak, Darrell H Carney, UTMB, Galveston, TX; Theresa W Fossum,
Texas A&M Univ, College Station, TX; Gerald M Fuller, Univ of Alabama, Birmingham, AL;
Travis Hein; Texas A&M Univ Health Science Cntr, Tempe, TX
Endothelial dysfunction (ED), is characterized by impaired nitric oxide (NO) production in
endothelial cells that is associated with chronic hypoxia. ED may have contributed to the limited
benefit of VEGF in myocardial revascularization clinical trials since it has now been shown that
VEGF and other factors appear to require NO production to induce angiogenesis. TP508 is a 23
amino acid peptide derived from a portion of human thrombin that triggers tissue repair effects
subsequent to vascular injury. We previously demonstrated that TP508 increases activation of
eNOS and prevents hypoxia-induced downregulation of eNOS expression in endothelial cells,
indicating that it reverses specific aspects of ED. We investigated the effect of TP508 on
VEGF-stimulated eNOS activation in endothelial cells exposed to chronic hypoxia. VEGF
treatment of normoxic human coronary artery endothelial (HCAE) cells increased NO production
by 2.5-fold (23738 nM vs 10218 nM) through a mechanism that involves activation of
eNOS. HCAE cells exposed to hypoxia (1% O
2
) for 24 h showed a 3- to 5-fold decrease in basal
level of eNOS activation as determine by western blotting using antibody specific for eNOS
phosphorylated at S1177. In these hypoxic cells, the maximal level of VEGF-stimulated eNOS
phosphorylation is 4 fold decreased compared to normoxic cells. In addition, hypoxia
decreased (2 fold) the basal level of NO production (4813 nM vs 10218 nM)) and caused
unresponsiveness of hypoxic cells to produce NO after VEGF stimulation. Treatment of HCAE
cells with TP508 for 24 h restores the ability of VEGF to activate eNOS in hypoxic endothelial
cells to the level observed in normoxic cells. These results demonstrate that TP508 reverses
certain effects of hypoxia that lead to endothelial dysfunction and restores the ability of VEGF
to activate eNOS. This study suggests that combinatorial therapies with TP508 and VEGF may
be more beneficial for therapeutic angiogenesis of the heart or peripheral tissues than VEGF
alone.
P215
The Chemotactic Potential of Serum of Patients Undergoing Cardiac
Surgery: The Time Course of Chemotactic Potential from Different Times of
Surgery
Pavel P Osmancik, CardioCntr, 3rd Med Sch, Charles Univ in Prague, Prague, Czech
Republic; Joerg Schmidt, Jozsef Bocsi, Joerg Hambsch, Peter Schneider, Attila Tarnok;
Cardiac Cntr Leipzig, Univ of Leipzig, Leipzig, Germany
Background: Cardiac surgery with cardiopulmonary bypass (CPB) induces massive perturba-
tion of the immune system altering leukocyte composition in the peripheral blood. This immune
response contributes to the adverse outcome with migration of activated cells to sites of
inflammation that is driven by attractant and repellent chemokines acting in concert. Patients
and methods: Nine patients underwent cardiac surgery with CPB were studied. Serum
samples were obtained 1d preoperative, after anesthesia, at the onset and the and of the CPB,
4h, 1d, 2d after surgery and at discharge. Peripheral blood leukocytes (PBL) from healthy
donors were isolated (Ficoll-Hypaque centrifugation) and 250,000 cells were placed into a
migration chamber (Costar) separated from a second lower chamber filled with patient serum
(20% in RPMI) by a filter (pore width 3m). After incubation (1h, 5% CO
2
, 37C) cells from top
and bottom chamber were removed and analysed by flow cytometry. Results: Chemotactic
activity increased at onset of anaesthesia followed by a phase of low activity immediately after
surgery and a second phase of high activity at post-operative days 12. In the first phase
mainly monocytes and NK-cells migrated. The in vitro results correlated with results obtained
by immunophenotyping of circulating PBL of the same patients showing that at CPB onset
monocyte and NK-cell count increases. After surgery of T- and B-cell count decreased probably
due to homing into lymphatic tissues. From both chambers the total number of cells recovered
was 515% below that of the initial cell number due to attachment of migrating cells to the
filter, which was quantified by LSC. Discussion: During paediatric cardiac surgery the
chemotactic activity of the serum changes following characteristic patterns. Manipulation of the
chemokine pattern might prove beneficial to prevent extravasation of cells leading to tissue
damage.
P216
Progenitor Cells: A Role in Vascular Calcification?
Shripad N Pal, Mirko Karan, Jonathan Golledge; James Cook Univ, Townsville, QLD,
Australia
Vascular calcification is an important determinant of cardiovascular mortality. It has been
suggested that bone marrow derived progenitor cells may contribute to the development of
arterial mineralisation. In this study we investigate the contribution of the circulating progenitor
cells towards the development of vascular calcification using an Osteoprotegerin knockout
(OPG-/-) murine model system, previously shown to develop calcification. The degree of
vascular calcification in the aorta was evaluated using an alizarin stain and a bioassay system
for the quantification of calcium (n16). Tail bleeds (n 104) at regular intervals were
analysed for progenitor cells such as stem cell antigen (Sca-1) and C-kit (CD 117) using flow
cytometry. The mean degree of calcification was observed to be more in experimental (OPG-/-)
mice (0.450.03 mg) than the control (C57/BL6) mice (0.210.02 mg). Comparative studies
also showed that the percentage population of progenitor cells were significantly more
(P0.05) in the OPG-/- than the C57/BL6 mice (Table 1). The degree of calcification was
correlated to the percentage population of the progenitor cells. Bone markers such as
osteopontin (P0.001), osteocalcin (P0.001) and bone alkaline phosphatase (P0.01) were
also expressed in Sca-1, C-kit positive cells. In conclusion, our findings suggest that circulating
progenitor cells may contribute to the development of artery calcification. Our findings have
implications for bone marrow based cellular therapies being developed.
TABLE 1: -C57 VS OPG-/- : COMPARISION OF PERCENTAGES OF PROGENITOR CELL
POPULATION
Mice (n16) Tail bleeds (n104) Sca/CKit- Sca/Ckit Sca-/Ckit
C57 (control) 0.78(0.09) 0.28(0.03) 0.19(0.02)
OPG-/- (experimental) 1.47(0.1) 0.68(0.06) 0.43(0.03)
P217
Differential Contribution of Cycloooxygenase-Isozymes to the Generation of
Prostacyclin and Prostaglandin E
2
by Endothelial Cells in Response to
Steady Laminar Shear Stress
Paola Patrignani, Luigia Di Francesco, G dAnnunzio Univ, Chieti, Italy; Antonio Piccoli, Mario
Negri Sud, Santa Maria Imbaro Chieti, Italy; Marta Capone, G dAnnunzio Univ, Chieti, Italy;
Virgilio Evangelista, Licia Totani; Mario Negri Sud, Santa Maria Imbaro Chieti, Italy
Prostacyclin (PGI
2
) and prostaglandin(PG)E
2
, the major prostanoids released from endothelial
cells, play different roles in cardiovascular(CV) homeostasis. PGI
2
is a general restraint on
endogenous stimuli to platelet activation, vascular proliferation and remodeling, hypertension,
atherogenesis, and cardiac function. Differently, PGE
2
accelerates atherogenesis and can
activate platelets. We explored the contribution of cyclooxygenase (COX)-isozymes and
down-stream specific synthases to the generation of PGI
2
and PGE
2
in endothelial cells in
response to steady laminar shear stress (LSS, 10 dyne/cm
2
) versus interleukin-1 (5 ng/ml).
Western blot analysis showed that primary human umbilical vein endothelial cells (HUVECs)
cultured in static conditions in the presence of foetal calf serum 5% expressed COX-1, PGI
2
synthase (PGIS), cytosolic PGE
2
synthase (cPGES) and microsomal PGES-2 (mPGES-2) but not
COX-2 and mPGES-1. They released 6-keto-PGF
1
(the hydrolysis product of PGI
2
) and PGE
2
(799340 and 559142 pg, respectively). After the loading of laminar shear stress for 6 h,
COX-2, mPGES-1 and mPGES-2 were induced. This was associated with a significant (P0.01)
increase of 6-keto-PGF
1
and PGE
2
generation (2372414 and 3600588 pg, respectively).
Under static conditions, IL-1 induced the expression of COX-2, mPGES-1 and mPGES-2. This
was associated with a significant increase in the generation of 6-keto-PGF
1
and PGE
2
generation (117446700 and 6376510 pg, respectively). Using NS-398 (a selective inhibitor
of COX-2) we showed that endothelial COX-2 is the dominant isozyme involved in PGI
2
biosynthesis in response to LSS and IL-1. In fact, it was suppressed by 60% and 90%,
respectively, by the compound. Interestingly, PGE
2
was produced principally by COX-1 in
response to LSS while only COX-2 contributed to the generation of the prostanoid in response
to IL-1 . In conclusion, our results show that in endothelial cells PGI
2
biosynthesis is coupled
preferentially with COX-2. Preservation of PGE
2
biosynthesis in front of profound suppression
of PGI
2
in endothelial cells, at physiological level of steady LSS, by selective inhibitors of COX-2
might contribute to the acceleration of the developing CV risk, in subjects at initial low risk.
2007 ATVB Poster Presentations e-75
by on March 25, 2011 atvb.ahajournals.org Downloaded from
P218
Activin A and Transforming Growth Factor-1 Have Distinct Functions in
Smooth Muscle Cell Migration
Bianca C Groenendijk, Germaine Benus, Mariska Vos, Thijs W Pols, Peter I Bonta, Joost O
Fledderus, Oscar L Volger, Anton J Horrevoets, Carlie J de Vries; AMC, Amsterdam, The
Netherlands
Activin A and Transforming Growth Factor- (TGF-) are members of the TGF- superfamily.
Both modulate vascular lesion formation beneficially, however, they involve distinct mecha-
nisms in this process. Activin A inhibits the formation of smooth muscle cell (SMC)-rich lesions,
whereas TGF-1 does not. In addition, TGF-1 induces production of extracellular matrix
components, resulting in a larger but more stable vascular lesion. Activin A and TFG-1 bind
unique cell-surface receptors, but both involve Smad-2, 3 and 4 in downstream signaling
pathways. To understand how TGF- and Activin A affect SMC-rich lesion formation differently,
we first investigated the presence of Activin receptor-like kinases (ALK) in SMCs. We
demonstrate that ALK-1, 2, 4, and 5 are all expressed in human SMCs. Western-blot analyses
showed that both Activin A and TGF-1 induce Smad-2 phosphorylation, however, also Smad-1
phosphorylation was observed in response to both factors. Since, to our knowledge, Activin A
has not been reported before to phosphorylate Smad-1, this was unexpected. Secondly, we
performed micro-array experiments followed by gene-set enrichment analyses on SMCs
stimulated with either Activin A or TGF-1. Several pathways were discovered to be
differentially regulated by these factors, including cellular migration. Functional assays
confirmed that Activin A does not affect SMC migration (323.622.7m vs. control:
330.117.2m, after 24 hours; n8, p0.05), whereas TGF-1 inhibits migration
(249.917.7m vs. control: 330.117.2m, after 24 hours; n8, p0.01), most likely
through increased expression of extracellular matrix proteins promoting firm attachment of the
cells. In conclusion, gene expression profiling experiments provide evidence that Activin A and
TGF-1, in spite of the activation of common intracellular signaling pathways, provoke
significant differences in cellular responses of SMCs, which may clarify their distinct effects on
the vessel wall.
P219
Calcium/Calmodulin-Dependent Protein Kinase II- Regulates Vascular
Smooth Muscle Cell Proliferation
Suzanne J House, Roman Ginnan, Albany Med College, Albany, NY; Shayn Armstrong,
Upstate/Chemicon Group, Temecula, CA; Harold A Singer; Albany Med College, Albany, NY
There is accumulating evidence that Ca
2
-dependent signaling pathways regulate proliferation
and migration of VSM cells contributing to the intimal accumulation of VSM observed in many
vascular diseases. In this study we investigated the role of the multifunctional serine/threonine
kinase, CaMKII, as a mediator of Ca
2
signals regulating VSM cell proliferation. Differentiated
VSM cells isolated from rat aortic media express primarily CaMKII gene products while
passaged primary cultures of de-differentiated VSM cells express primarily CaMKII
2
.
Experiments examining the time course of CaMKII isoform modulation revealed the process was
rapid following initial dispersion of aortic VSM with a significant increase in CaMKII
2
protein
and significant decrease in CaMKII protein within 30 hrs, coinciding with onset of DNA
synthesis and cell proliferation. Attenuating the initial upregulation of CaMKII
2
in primary
cultured cells using siRNA resulted in decreased serum-stimulated DNA synthesis and cell
proliferation. In passaged VSM cells, suppression of CaMKII
2
activity by overexpression of a
kinase-negative mutant, or suppression of endogenous CaMKII content using multiple siRNAs,
significantly attenuated serum-stimulated DNA synthesis and cell proliferation. Cell cycle
analysis following either inhibitory approach indicated decreased proportion of cells in G1, an
increase in proportion of cells in G2/M, and an increase in polyploidy, corresponding with
accumulation of multi-nucleated cells. In vivo we observed CaMKII isoform modulation in
response to vascular injury in the rat carotid artery balloon angioplasty model. Three days after
injury to the left carotid artery, there is a significant increase in CaMKII
2
and a significant
decrease in CaMKII. At 14 days after injury the neointima contained primarily the
2
isoform.
Incubation of the injured vessel with siRNA to the
2
isoform prevented the upregulation of the

2
isoform and inhibited neointimal formation by 80%. PCNA detection in the medial wall was
significantly attenuated at 3 and 7 days in siRNA-treated vessels. These results indicate that
CaMKII
2
is specifically induced during modulation of VSM cells to the synthetic phenotypic and
is a positive regulator of proliferation.
P220
c-Myc Expression Is Upregulated in HUVEC Subjected to Elevated Levels of
Cyclic Strain: Mechanism, Downstream Effects, and Modulation
Nicole E Hurley, Georgia Institute of Technology, Atlanta, GA; Tiffanie J Bialis, Univ of
Arizona, Tucson, AZ; Yumiko Sakurai, Georgia Institute of Technology and Emory Sch of
Medicine, Atlanta, GA; Suzanne G Eskin, Georgia Institute of Technology and Emory Univ
Sch of Medicine, Atlanta, GA; Laurence H Hurley, Univ of Arizona, Tucson, AZ; Larry V
McIntire; Georgia Institute of Technology, Atlanta, GA
Cyclic strain has been implicated in playing a pivotal role in the genetic regulation of vascular
physiology and pathology. We have investigated the hypothesis that in human umbilical vein
endothelial cells (HUVEC), pathogenic levels of cyclic strain activate the c-Myc promoter,
leading to c-Myc transcription and downstream gene induction. To determine expression and
time-dependency of c-Myc in HUVEC, mRNA and protein expression of c-Myc under
physiological (610% cyclic strain) and pathologic conditions (20% cyclic strain) were studied.
Both c-Myc mRNA and protein expression increased more than four-fold in HUVEC (P4-P5)
cyclically-strained at 20%. This expression occurred in a time-dependent manner, peaking in
the 1.52 hour range and falling to basal levels by 3 hours. Subsequently, the mechanism of
c-Myc transcription was investigated by using specific inhibitors to modulate c-Myc transcrip-
tional activation. These compounds, obtained from the University of Arizona Cancer Center,
attenuated cyclic-strain-induced c-Myc transcription by about 50%. Having established this
reduction in expression, we investigated how these effects modulate downstream genes that
are regulated by c-Myc. For example, we have examined the expression of Cyclin Dependent
Kinase 4, a c-Myc target gene, and preliminary results show that direct targeting of the c-Myc
promoter decreases stretch-induced gene expression. These findings may help in the
development of a novel therapeutic opportunity in vasculoproliferative diseases.
P221
Patterns of Protein Kinase C and Rho Activation During Cerebral
Vasospasm After Subarachnoid Hemorrhage
Sunil G Nair, Joseph F Clark, Gail J Pyne-Geithman; Univ of Cincinnati, Cincinnati, OH
Delayed Cerebral Vasospasm (CV), the phenomenon of sustained vascular contraction is seen
in about 40% of Subarachnoid Hemorrhage (SAH) cases that survive the initial hemorrhagic
event. This complex pathological vasospasm that occurs between 310 days after SAH results
in worsening of neurological deficits and increased morbidity. Despite intensive research
efforts, the etiology and molecular mechanism of CV still remains to be elucidated clearly. Our
laboratory has shown that Bilirubin Oxidation Products (BOXes) are present in greater
concentrations in the cerebrospinal fluid (CSF) of patients with SAH who suffer from vasospasm
than those who do not. Previous in vivo studies from our lab have shown that BOXes are
vasoactive and potentiate smooth muscle contraction similar to protein kinase C (PKC)
activators and phosphatase inhibitors. Our in vitro model of CV after SAH uses CSF from SAH
patients with (CSF
v
) and without (CSF
c
) CV. We compared the effects these with laboratory-
synthesized BOXes on porcine carotid arteries (PCA) and examined the activation of regulatory
proteins PKC , PKC and Rho. PCA rings are pre-stretched to optimum length and placed in
control solution (MOPS), CSF
c
, CSF
v
or BOXes (25 M) for 20 minutes or 3 hours and
translocation of PKC , and Rho from cytosol to membrane is analyzed using western blot.
Among different protein kinase C isoforms, and are present in appreciable amounts in
porcine carotid artery smooth muscle. Translocation of PKC , and Rho from the cytosol to
the membrane indicates activation. After 20 minutes there is greater translocation of PKC ,
and Rho in the presence of CSF
v
and BOXes as compared to CSF
c
and MOPS respectively.
After 3 hours there is still greater translocation of PKC and Rho and not PKC in the presence
of CSF
c
and BOXes when compared with CSF
c
and MOPS respectively. The temporal changes
in translocation of PKC-, and Rho are likely to be important for the initiation and
maintenance phases of vasospasm and will require further study to determine their relative
contributions to the pathology. BOXes are likely the major vasoactive compounds in CSF
v
that
causes the activation of these contractile proteins and results in increased calcium sensitization
of the vascular smooth muscles.
P222
Atorvastatin Attenuates Lrp5-mediated Aortic Valve Calcification and
Osteoporosis in LDLR-/- Hypercholesterolemic Mice
Nalini Rajamannan, Frank C Caira, Northwestern, Chicago, IL; Malayannan Subramaniam,
Mayo Clinic, Rochester, MN; Stuart Stock, Amy Flores, Northwestern, Chicago, IL; Thomas C
Spelsberg; Mayo Clinic, Rochester, MN
Cardiovascular calcification and osteoporosis are the leading causes of morbidity and mortality
in the aging population in the Unites States. Evidence indicates that hyperlipidemia plays a
paradoxical role in these disease processes (Parhami et al, ATVB, 1997 Apr;17(4):6807).
However, the hyperlipidemic mechanisms of atherosclerotic calcification and decrease bone
formation are not well understood and have not been defined in terms of aortic valve disease.
We have previously shown that aortic valve calcification expresses an osteoblast phenotype in
humans (Rajamannan et al,Circulation. 2003 May 6;107(17):21814). We hypothesize that
lipids play a role in aortic valve calcification and osteoporosis. We propose to test this
hypothesis in a hypercholesterolemia model. We also plan to test if atorvastatin plays a
protective role in this process by measuring the Lrp5 receptor expression in the heart and
bones. LDLR-/- mice (n60). Group I (n20) normal diet, Group II (n20) 1.25% chol diet
(w/w), and Group III (n20) 1.25% (w/w) chol dietatorv for the development of calcification.
The aortic valve and aortas (AVA) was examined for proliferation, calcification, and bone matrix
markers. Bone formation was assessed by micro Computed Tomography (microCT), calcein
injection, osteocalcin, cbfa-1 and osteopontin expression. Results: The cholesterol diet induced
complex bone formations in the calcified AVA with an increase in cellular proliferation,
osteopontin, osteocalcin, cbfa-1 expression and Lrp5 receptor (4 fold increase as compared to
control p0.05). Atorvastatin reduced bone formation, cellular proliferation, cbfa-1 and Lrp5
levels in the AVA (2 fold decrease as compared to chol (p0.05). Ex vivo analysis of calcein
label demonstrated an increase in calcein in the hypercholesterolemia AVA and bones with
attenuation of the label with atorvastatin in these tissues. The hypercholesterolemic femurs
demonstrated a decrease in Lrp5, Cbfa1 and OC and an increase in Lrp5, Cbfa1 and OC.
Conclusion: Hypercholesterolemic AV calcification and bone turnover is attenuated by
atorvastatin and is mediated in part by the Lrp5 osteoblast signaling pathway. This model may
have future implications in the treatment of this disease with statin therapy.
P224
Wnt3a Upregulates SM22 Gene Transcription in C3H10T
1
2 Mesenchymal
Cells via a Novel CAGAG Regulatory Element
Shawn L Shafer, Dwight A Towler; Washington Univ, St. Louis, MO
The regulation of -catenin-dependent gene transcription has been implicated in the
proliferation and phenotypic modulation of vascular smooth muscle cells (VSMC) during both
development and disease. In order to better understand the role for Wnt-dependent control of
the VSMC phenotype, we examined Wnt3as effects on C3H10T
1
2 (10T
1
2) cells, a multipotent
mesenchymal progenitor capable of adopting the mural VSMC phenotype in vivo. Following
Wnt3a treatment, 10T
1
2 cells developed a spindle-shaped, myofibroblast phenotype in culture.
Treatment with 5 ng/ml to 30 ng/ml of Wnt3a in 2% horse serum significantly upregulated the
expression of early VSMC markers such as SM22 (5-fold) and smooth muscle -actin (2-fold).
e-76 Arterioscler Thromb Vasc Biol June 2007
by on March 25, 2011 atvb.ahajournals.org Downloaded from
Wnt3a induction of SM22 was further enhanced by the use of media containing 10% FBS.
Additionally, Wnt3as increase in SM22 transcription was additive with the 4-fold induction
observed by 5 ng/ml TGF-
1
treatment (a 10-fold overall increase). BMP2 treatment had no
effect. In transiently transfected 10T
1
2 cells, 15 ng/ml of Wnt3a treatment for 2 days
upregulated the SM22 promoter-luciferase reporter activity 2- to 5-fold. A series of deletion
constructs mapped Wnt3a responsiveness to an area within the -213 to -190 region of the
promoter. We identified CCAAAG and CAGAG motifs within this area that closely resemble
cognates for TCF/LEF and Smad binding, respectively. Gel shift analyses using extracts from
Wnt3a-treated 10T
1
2 cells identified 4 specific DNA-protein complexes, two of which were
perturbed by treatment with antibodies to -catenin, TCF7, and Smad2/3, but not by antibodies
to Smad1 or Smad4. Cold competition with mutant oligos identified that the intact CAGAG motif
was required for all visualized binding complexes, but that the CCAAAG motif was dispensable.
Moreover, mutation of the CAGAG motif in the native -213 to 5 SM22 promoter decreased
overall transcriptional activity by 90% and abrogated Wnt3a induction. Thus, DNA-protein
interactions involving nuclear Smad2/3, TCF7, and -catenin at the -203 to -199 CAGAG motif
in the SM22 promoter can mediate Wnt3a induction of this early VSMC gene. These findings
expand our understanding of how paracrine Wnt signals may contribute to the phenotypic
modulation of vascular myofibroblasts.
P225
Cyclic Strain of Vascular Endothelial Cells Reduces Vascular Smooth
Muscle Cell Proliferation, Possibly via an MMP-2-dependent Mechanism
Maria T Killeen, Ronan P Murphy, Paul A Cahill, Philip M Cummins; Dublin City Univ, Dublin,
Ireland
Introduction: Hemodynamic forces, namely shear stress and cyclic strain play an important
role in physiological control of vascular tone, remodelling and associated pathologies.
Furthermore, these forces indirectly impact vascular smooth muscle cell fate decisions by
modulating vascular endothelial processes. MMPs (matrix metalloproteinases) play a vital role
in vascular cell fates via degradation of extracellular matrix substrates. Recent studies
demonstrate that these forces can modulate MMP expression and activity in different vascular
cell types. For these investigations, we examined how MMP-2 and MMP-9, induced in BAECs
in response to cyclic strain, putatively impact on BASMC proliferation. Methods: BAECs were
exposed to a defined level of equibiaxial cyclic strain using the Flexercell Tension Plus

FX4000

apparatus (10% strain, 60 cycles/min, 24 h, cardiac waveform). BAECs were


harvested for Real Time PCR to monitor MMP-2 mRNA levels whilst BASMCs were incubated
with BAEC strain conditioned media (BCM) and the cells were subsequently harvested and
analysed for proliferation by FACS analysis and cell counting. Results: Cyclic strain of BAECs
increased MMP-2 and MMP-9 mRNA levels by (1.20.1 and 1.90.1 fold respectively).
Subsequent incubation of BASMCs with BCM decreased proliferation to (0.860.1 and
0.820.1 fold) of control as monitored by FACS analysis and cell counts. This inhibitory effect
could be reversed by 10M GM6001, an MMP inhibitor and siRNA knockdown of MMP-2 (but
not MMP-9) ie. proliferation at (1.150.1 and 1.10.1 fold respectively) relative to control.
However, this event was not blocked by 20M Genistein (a protein tyrosine kinase inhibitor)
(0.770.2 fold) relative to control. Conclusion: Our findings indicate that cyclic strain of BAECs
reduces BASMC proliferation. Moreover blockade of MMP-2 induction but not MMP-9 appeared
to reverse this effect, suggesting a regulatory role for strain-induced endothelial-derived
MMP-2.
P226
Three-day High-flow Loaded Rabbit Carotid Artery Remodels Automatically
Ex Vivo
Masayo Komatsu, Yamamoto-Kumiai General Hosp, Noshiro, Japan; Masato Takahashi,
Kouich Kawamura, Mikio Kobayashi, Makoto Yoshida, Hiroshi Nanjo, Hirrotake Masuda;
Akita Univ Sch of Medicine, Akita, Japan
[Introduction] High blood flow induces endothelial cell proliferation and arterial dilatation.
Arterial dilatation starts as the tearing of internal elastic lamina (IEL gaps) at 4-day high flow
in the rabbit carotid arteries. As IEL gaps widen, artery dilates. At first we believed that high
flow was necessary during whole process. However, we wonder whether high-flow is
necessary in the whole process or transient high-flow is sufficient for the remodeling. Here, we
loaded 3-day high-flow to the rabbit carotid artery and then removed the artery for ex vivo to
observe whether internal elastic lamina might be torn without flow.[Method]In order to increase
blood flow, arterio-venous fistula (AVF) was created in the left common carotid artery and
jugular vein. 3 days after creation of AVF, left common carotid artery was removed and the
whole blood vessel was cultured in vitro (ex vivo) for 1 day. After 1-day, carotid arteries were
observed with light microscopy and laser scanning microscopy (LSM) (n4). Non-operated
animals (n4) and sham-operated animals (n4) were used for controls. [Result] IEL gaps
appeared in the ex vivo cultured artery. They were small and occasional. No IEL gaps were
observed in the controls. MIB-5 positive smooth muscle cells were observed in the media
beneath IEL gaps. [Discussion]We proved that 3-day high-flow loaded rabbit carotid artery gaps
internal elastic lamina after 1-day ex vivo. Therefore it is concluded that 3-day high-flow loaded
rabbit carotid artery remodels automatically ex vivo. We assume that the cause of remodeling
has already played and arranged within 3-day of high-flow, although no detectable
morphological initiation could be detected. We suspect that transient high-flow may incubate
smooth muscle progenitors into the arterial wall.
P227
High-density Lipoprotein Cholesterol Prevents Apoptosis in Endothelial
Progenitor Cells Through Activation of AMP-activated Protein Kinase
Bertrand Lapergue, Askar Mohammad, Ashfaq Shuaib; Univ of Alberta, Edmonton, Canada
Endothelial dysfunction is thought to play a critical role in the atherosclerosis. Emerging
evidence suggest pluripotent cells present in the peripheral blood called endothelial progenitor
cells (EPCs) help in maintaining the endothelial integrity during atherosclerotic vascular
disease. Therefore enhancing the number and function of EPCs should be beneficial in
preventing vascular disease. High-density lipoprotein (HDL) cholesterol has shown promising
results in protecting endothelial cell monolayer and reverse endothelial dysfunction. HDL might
exert at least in part its beneficial effect on vascular endothelium through EPCs mobilization.
However, the intra-cellular mechanism that is involved in the beneficial effect of HDL is not
completely known. We hypothesized that AMP-activated protein kinase (AMPK), an intracellular
kinase involved in cellular energy regulation, may constitute a key signalling enzyme in the
beneficial effect of HDL on EPCs. Methods and Results: We isolated EPCs from peripheral blood
in healthy subject using standard procedure. Apoptosis was induced in EPCs by camptothecin.
To assess protective effect of HDL on EPCs, we pre-treated EPCs with either HDL or
phenformin. Apoptotic cell population was quantified by flow cytometry. We found that HDL
reduced apoptosis in EPCs (HDLCamptothecin 19.6%0.91 versus Camptothecin
24.4%1.03 (p0.001)). Furthermore, we showed that phenformin, an AMPK agonist, also
reduced the apoptosis of EPCs (PhenforminCamptothecin 17%0.6 versus Camptothecin
24.4%1.03(p0.00001)). Finally, we determined that HDL-induced protective effect on EPCs
is mediated through the activation of AMPK, which was measured by Western Blot and
densitometry. Conclusions: Our results indicate that HDL has potent anti-apoptotic effects on
EPCs, which might be a contributory factor towards its anti-atherosclerotic. We found that HDL
can reduce camptothecin-induced apoptosis in EPCs. Furthermore, we also found that this
beneficial effect of HDL is mediated via activation of AMPK. Interestingly, activation of AMPK
by anti-diabetic drug phenformin also produces similar beneficial effects in EPC. Therefore our
study underlines a promising role of AMPK stimulation in endothelial homeostasis.
P228
Apolipoprotein(a) Stimulates Vascular Endothelial Cell Growth and
Migration and Signals Through Integrin
V

3
Lei Liu, Marlys L Koschinsky; Queens Univ, Kingston, Canada
Elevated plasma concentrations of lipoprotein(a) [Lp(a)] are an emerging risk factor for
atherothrombotic disease. Apolipoprotein(a) [apo(a)], the unique glycoprotein component of
Lp(a), contains tandem repeats of a plasminogen kringle (K) IV-like domain followed by
sequences that are highly homologous to the KV and protease domains. In light of recent
studies suggesting that apo(a)/Lp(a) affects endothelial function, we evaluated the effects of
apo(a) on growth and migration of cultured human umbilical vein endothelial cells (HUVECs). For
this purpose, we utilized a battery of recombinant apo(a) [r-apo(a)] variants representing
deletion and/or site-directed mutants of various kringle domains in the molecule in order to
determine the mechanism(s) of apo(a) action. Two full-length r-apo(a) variants, 12K and 17K,
were able to stimulate HUVEC growth and migration to a comparable extent; 17K also
decreased the levels of total and active transforming growth factor-. Using additional r-apo(a)
variants, we were able to determine that the observed effects of full-length r-apo(a) on HUVECs
were dependent on the presence of functional lysine binding site(s) present in kringle domains
in the apo(a) molecule. With respect to signaling events elicited by apo(a) in HUVECs, we found
that 17K treatment of the cells increased the phosphorylation level of focal adhesion kinase
(FAK) and mitogen-activated protein kinases (MAPKs) including ERK, p38 and JNK. Further to
this, we showed that LM609, a function-blocking antibody to integrin
V

3
, abrogated 17K
r-apo(a)-induced HUVEC growth and migration, as well as FAK and MAPK activation. Taken
together, our data suggest that apo(a) signals through integrin
V

3
to activate endothelial cells;
the stimulatory effects of apo(a) on growth and migration of these cells may reflect decreased
TGF- levels in the treated cells. Studies are ongoing to dissect the apo(a)-activated integrin
signaling pathway, and to determine the role of MAPKs in apo(a)-mediated effects on HUVEC
growth and migration.
P229
Molecular Mechanisms of Cell Cycle Gene Expression in Vascular Smooth
Muscle Cells
Rohit Malhotra, Coleen McNamara; Univ of Virginia, Charlottesville, VA
Vascular smooth muscle cell (VSMC) proliferation is a key component of vascular lesion
formation and restenosis in response to injury. The helix-loop-helix factor, Id3 has recently
emerged as a key regulator of VSMC growth. Phosphorylation of Id3 at serine 5 occurs in S and
G2/M phase of the cell cycle and modulates Id3 effects on VSMC growth and the expression
of the cyclin dependent kinase inhibitor p21. Id3 is a dominant negative inhibitor of the
functions of basic HLH (bHLH) factors such as ITF-2. We hypothesize that the interaction of Id3
with ITF-2 and the effects on p21 gene activation change in a cell-cycle and phosphorylation
dependent manner. To determine the effects of phosphorylation and cell cycle stage on Id3
partner interactions, the mammalian two hybrid system was used. VSMC were co-transfected
with Id3 or Id3S5A in the bait vector and ITF-2 in the activation domain vector. Cells were then
arrested in G0 using serum starvation, S phase with thymidine, and G2/M with nocodazole.
Samples were assayed for CAT reporter activity. To determine the effects of cell cycle and
phosphorylation on ITF-2 regulation of p21 gene activation, VSMC were transfected with pGL3
p21 promoter and pXS ITF-2 and again arrested at each cell cycle stage and then assayed for
luciferase activity. Results demonstrated that phospho-competent Id3 interacts with ITF-2 more
strongly than our phosphoablating mutant, Id3S5A. This interaction varies with cell cycle stage
and is highest at G2/M. Our promoter-reporter data demonstrate that p21 promoter activity
increases as the VSMC progress through the cell cycle. ITF-2 augments p21 promoter-reporter
activity at each cell cycle stage, but the increase in reporter activity above unstimulated is
reduced at G2/M. Taken together, results confirm a key cell cycle dependent role for Id3
phosphorylation in regulating cell cycle genes.
2007 ATVB Poster Presentations e-77
by on March 25, 2011 atvb.ahajournals.org Downloaded from
P230
Synergetic Interaction Between Aldosterone and Angiotensin II: Differential
Effects on RhoA, Akt, and MAP Kinase Signaling in Vascular Smooth
Muscle Cells
Augusto C Montezano, Glaucia E Callera, Alvaro Yogi, Rita C Tostes, Ottawa Health Rsch
Institute, Ottawa, Canada; Ernesto L Schiffrin, Lady Davis InstituteMcGill Univ, Montreal,
Canada; Rhian M Touyz; Ottawa Health Rsch Institute, Ottawa, Canada
Aldosterone (Aldo) exerts a synergistic mitogenic effect with angiotensin II (Ang II) through
ERK1/2 activation in vascular smooth muscle cells (VSMC). Whether similar interactions
influence contractile and migratory signaling pathways remain unclear. Here we investigated
cross-talk of c-Src, MAP kinases (ERK 5, p38 and JNK), RhoA, Akt and [Ca
2
]
i
signaling
between Aldo and Ang II. Cultured rat mesenteric vascular smooth muscle cells were studied.
Activation of MAP kinases and Akt was determined by immunoblotting using phospho-specific
antibodies. RhoA activity was assessed using the G-LISA RhoA activation assay kit. [Ca
2
]
i
was
measured by fura-2 methodology and fluorescence digital imaging. Whereas high concentra-
tions (10
-8
mol/L) of Aldo and Ang II significantly increased activation of c-Src (2-fold), low
concentrations (10
-10
mol/L) of Aldo and Ang II were without significant effect. Co-stimulation
of VSMCs with combined low dose Aldo and Ang II significantly increased c-Src phosphorylation
(0.5-fold above basal, p0.05), RhoA activity (1-fold, p0.05) and [Ca
2
]
i
transients (12-fold)
and decreased Akt phosphorylation (0.5 fold, p0.05). ERK 5, p38 and JNK were unaffected
by low dose Aldo/Ang stimulation. Effects were inhibited by a selective AT
1
receptor (AT
1
R)
antagonist, Ibersartan (10
-6
mol/L), or a mineralocorticoid receptor (MR) antagonist, eplerenone
(10
-6
mol/L). Our results suggest that Aldo/Ang II, through MR/AT
1
R, synergistically exerts a
positive effect on c-Src, RhoA and [Ca
2
]
i
and a negative effect on Akt. However, signaling
events involving ERK5, JNK and p38 are independent of Aldo/Ang II interactions. These findings
suggest that Aldo and Ang II act in a synergistic fashion to regulate defined signaling pathways,
particularly those related to VSMC migration and cell survival.
P231
Air Pollutant Chemicals and Oxidized Lipids Exhibit Genome-wide
Synergistic Effects on Endothelial Cells
Ke Wei Gong, UCLA Sch of Medicine, Los Angeles, CA; Wei Zhao, UCLA Sch of Public
Health, Los Angeles, CA; Ning Li, Berenice Barajas, UCLA Sch of Medicine, Los Angeles,
CA; Michael Kleinman, Univ of California, Irvine, CA; Constantinos Sioutas, Univ of Southern
California, Los Angeles, CA; Steve Horvath, UCLA Sch of Public Health, Los Angeles, CA;
Aldons J Lusis, Andre Nel, Jesus A Araujo; UCLA Sch of Medicine, Los Angeles, CA
Background - Ambient air pollution is associated with increased cardiovascular morbidity and
mortality. We have recently found that exposure to ambient ultrafine particulate matter, highly
enriched in redox cycling organic chemicals, promotes atherosclerosis in mice. We hypothesize
that these pro-oxidative chemicals could synergize with oxidized lipid components generated
in low-density lipoprotein (LDL) particles to enhance vascular inflammation and atherosclerosis.
Methods and Results - We have used human microvascular endothelial cells (HMEC) to study
the combined effects of a model air pollutant, diesel exhaust particles (DEP), and oxidized
1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine (ox-PAPC) on genome-wide gene
expression. We treated HMEC in triplicate wells with an organic DEP extract (5 g/ml), ox-PAPC
(10, 20 and 40 g/ml) or combination of both compounds for 4 hours. Gene expression profiles
were assessed by Illumina microarray technology and validated by qPCR. Both the DEP extract
and ox-PAPC co-regulated a large number of genes. All together, 1555 genes were significantly
upregulated (1.5 fold, p0.05) by the three DEP and ox-PAPC combinatory treatments. We
used weighted gene co-expression network analysis on the 3600 most varying genes to identify
co-expressed gene modules. We found three modules that were most highly enriched in genes
differentially regulated by the stimuli. These modules exhibited a pattern of additive/synergistic
interaction and concentrated 83% of the genes that were synergistically upregulated by both
DEP and ox-PAPC. These modules were also enriched in pathways relevant to vascular
inflammation such as antioxidant, apoptotic, inflammatory and unfolded protein response. We
validated this synergy in vivo by demonstrating that liver gene expression of apoE null mice
exposed to ambient ultrafine particles and fed a high fat diet exhibited significant upregulation
of the module genes. Conclusions - Diesel exhaust particles and oxidized phospholipids
synergistically affect the expression profile of several gene modules, that correspond to
pathways relevant to vascular inflammatory processes such as atherosclerosis.
P232
Increases in Plasma Cytokine Levels due to Inhalation of Diesel Exhaust in
Older Apolipoprotein E-deficient Mice
Lisa M Corey, Coralie Baker, Haley D Neff-LaFord, Jasmine H Wilkerson, Daniel L Luchtel,
Joel D Kaufman, Terrance J Kavanagh, Michael E Rosenfeld; Univ of Washington, Seattle,
WA
The cardiovascular (CV) system is distinctively sensitive to adverse effects following increases
in ambient particulate matter (PM), as exemplified by increases in morbidity and mortality
following periods of greater air pollution. Diesel accounts for a large percentage of PM in an
urban setting. Inflammation is associated with coronary events such as atherosclerotic plaque
rupture, thrombogenesis and embolization. Plasma and pulmonary cytokines play a critical role
as both mediators and markers of the inflammatory response to PM. We hypothesize a change
in both pulmonary and plasma cytokine levels following exposure to diesel exhaust (DE) in a
model of CV disease, the apolipoprotein E null (apoE
-/-
) mouse, that will suggest the
mechanisms involved in PM mediated CV events. To test this, male apoE
-/-
mice 2535 weeks
old (N 15/group) were exposed to 200 g/m
3
DE or filtered air (FA) for 1 day, 3 days, 1 week,
4 weeks or 8 weeks. At sacrifice, bronchoalveolar lavage fluid and plasma were analyzed for
interleukin (IL)-1, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, granulocyte-macrophage colony
stimulating factor (GM-CSF), interferon (IFN) and tumor necrosis factor (TNF) using the
BioRad BioPlex cytokine kit. All of the cytokines measured had the greatest increases following
1 day of DE exposure with a return to FA exposed levels with longer DE exposures. Interleukin
(IL)-1, IL-5, IL-6, IL-12, IFN and TNF were not detected in lung lavage fluid at any time
point. While IL-2, IL-4 and GM-CSF levels were unaltered, exposure to DE suppressed
pulmonary IL-10 production. In plasma, there was no change in IL-2, IL-10, IL-12 or GM-CSF
levels and IL-5 was not detected following DE exposure. In contrast, exposure to DE increased
plasma levels of IL-1, IL-4, IL-6, IFN and TNF. Plasma cytokine levels are normally low and
the increases demonstrate significant responses to DE. Based on the cytokines modulated,
there appears to be both a cell mediated and antibody mediated response to DE. This change
in circulating cytokines may directly affect the vasculature, cardiac muscle or atherogenesis
with a resultant adverse effect. We conclude that the complex cardiopulmonary inflammatory
response that characterizes CV effects due to DE exposure is mediated by cytokines.
P233
Diesel Particulate Instillation Dysregulates the Endothelial Transcriptome
John Maresh, Ralph V Shohet, Ria Nag; Univ of Hawaii, JABSOM, Honolulu, HI
Air pollution accelerates atherogenesis and cardiovascular disease. We examined the effect of
diesel exhaust on endothelial gene expression in vivo, utilizing hypercholesterolemic transgenic
mice. Tie2-GFP mice expressing green fluorescent protein regulated by the endothelial-specific
promoter Tie2 were crossed with apolipoprotein E knockout mice to generate Tie2-GFP/ApoE
null mice. Tie2-GFP and Tie2-GFP/ApoE mice were exposed to NIST diesel exhaust particulate
by intratracheal instillation of 100 ug. Within 24 hours of exposure, aortae (pooled from 4
animals) were dissected from the valve to the iliac bifurcation and rapidly processed by
proteolytic dissociation followed by fluorescence activated cell sorting to yield 100,000
endothelial cells of 95% purity. Changes in the abundance of endothelial transcripts were
then examined comprehensively by microarray techniques. Amplified RNA representing
25,000 cells was subjected to fluorescent labeling and hybridization to arrays created from the
Operon V3 long oligo set. Within the endothelium of both ApoE- null or wild-type mice, an
overlapping subset of 33 transcripts were consistently dysregulated by 1.5-fold by 24 hours.
These included inhibitors of bone morphogenetic signaling, chordin and Smad6, whose
dysregulation may relate to roles in arterial calcification. Transcripts relating to endothelial
growth regulation include Mapk7 (Mitogen activated protein kinase 7). Gmeb2 (glucocorticoid
modulatory element binding protein 2) may relate to regulation of inflammation. By uncovering
particulate-dysregulated endothelial transcripts we ultimately hope to allow a more complete
picture of pollution-related atherogenesis to emerge.
P234
Glutathione Biosynthesis Genes Protect Macrophages from Diesel
ExhaustMediated Toxicity
Haley Neff-LaFord, Pamela J Roque, Jim Stewart, Joel D Kaufman, Michael E Rosenfeld,
Terrance J Kavanagh; Univ of Washington, Seattle, WA
There are a growing number of epidemiological studies linking cardiorespiratory morbidity and
mortality to exposure to particulate matter, a major component of air pollution. These events
are likely mediated by several mechanisms, including inflammation and oxidative stress.
Oxidative stress can be attenuated by many antioxidants, including the tripeptide thiol
glutathione (GSH). GSH biosynthesis is rate-limited by glutamate-cysteine ligase (GCL), which
is composed of catalytic (GCLC) and modifier (GCLM) subunits. Macrophages play an important
role in the pulmonary immune response to particulate matter and are also involved in the
development of atherosclerotic plaques. Therefore, in order to study the protective role of GSH
in the development of oxidative stress-related diseases, our laboratory utilized a RAW264.7
macrophage cell model in which GCLC and/or GCLM are over expressed. Cells were exposed
to 0, 50 or 200 g/ml of diesel exhaust particles (DEP) for 18 hours and viability and GSH levels
were measured using flow cytometry. There was a 23 fold increase in GSH levels in GCLC over
expressing RAW cells. Exposure to 50 g/ml DEP decreased survival of normal macrophages
by over 75%. Elevated GCLC expression rescued cells from DEP-mediated cell death,
evidenced by a 2-fold increase in survival, while over expression of GCLM was not protective.
These preliminary data demonstrate the importance of GSH and GCL in protecting macrophages
from DEP-mediated toxicity.
P235
Oxidative Stress Modification of Apolipoprotein E in Environmental Tobacco
Smoke
Shiori Tamamizu-Kato, Olayemi Akintunde, Jason Wong, Childrens Hosp Oakland Rsch
Institute, Oakland, CA; Kent Pinkerton, Cntr for Health and the Environment, Univ of
California, Davis, CA; Koji Uchida, Graduate Sch of Agricultural Sciences, Nagoya Univ,
Japan; Hiroyuki Kato, Vasanthy Narayanaswami; Childrens Hosp Oakland Rsch Institute,
Oakland, CA
Apolipoprotein E (apoE), an anti-atherogenic protein, plays a crucial role in cardiovascular
diseases by regulating plasma cholesterol and lipoprotein metabolism. By assisting in the
transportation of very low-density lipoproteins and high density lipoproteins, apoE is able to
remove excess amounts of cholesterol and triglyceride from the blood and into the liver by
acting as a ligand for cell surface localized lipoprotein receptors and proteoglycans. Acrolein,
a chemical found in cigarette smoke, heated oils and as a natural by-product of age-related
oxidative stress, is highly reactive with the lysines, cysteines and histidines on proteins.
Previously, we demonstrated that acrolein modified recombinant human apoE3 in vitro leading
to functional impairment of the protein. The objective of the present study is to determine
whether acrolein in tobacco smoke modifies plasma apoE. Ten-week old male rats were
exposed to low doses of environmental tobacco smoke (ETS; 1 mg/m3 total suspended
particulate matter) for 6 h/ day for 3 days. This level of exposure is considered to be within a
range people may experience who are exposed to second-hand smoke. The control group was
exposed to filtered air (FA). Plasma from 4 rats was pooled and the lipoprotein fractions
separated by density gradient ultracentrifuge. Western blot analysis showed that the ETS group
e-78 Arterioscler Thromb Vasc Biol June 2007
by on March 25, 2011 atvb.ahajournals.org Downloaded from
had lower levels of apoE in their lipoprotein fractions compared to rats in the FA group. Using
immunoprecipitation assay, we determined that acrolein-modified apoE was present in both FA
and ETS groups. Compared to the FA group, the ETS group had higher levels of acrolein-
modified apoE in their lipid-free fractions. Based on these results, we propose that acrolein
from tobacco smoke modifies plasma apoE in rats exposed to ETS leading to a decreased
affinity of apoE for lipoproteins. Lipid binding is an essential requirement for apoE to serve as
a ligand for the low density lipoprotein receptor family of proteins and mediate effective
clearance of plasma triglyceride-rich lipoprotein particles. This has potential implications in
predisposing second-hand smokers to developing a pro-atherogenic profile and cardiovascular
disease.
P236
Pulmonary Diesel Pollution Exposure Dysregulates Key Cardiac and
Pulmonary Transcripts
John G Maresh, Ria Nag, Ralph V Shohet; Univ of Hawaii, JABSOM, Honolulu, HI
Air pollution accelerates atherosclerosis and cardiovascular disease. We examined the effect of
diesel exhaust particles on cardiac and pulmonary gene expression in vivo, utilizing transgenic
mice. Tie2-GFP mice express green fluorescent protein regulated by the endothelial-specific
promoter Tie2. Tie2-GFP mice were subjected to intratracheal instillation of a single dose of
100 ug of Diesel exhaust particles (NIST). Within 1 and 5 day of exposure, the heart and lungs
(pooled from 4 animals) were dissected and the total RNA was extracted. Changes in
abundance of transcripts were then examined comprehensively by microarray techniques. RNA
was subjected to amplification prior to labeling and hybridization to arrays created from the
Operon V3 long oligo set. Within both 1 and 5 days after exposure to diesel exhaust particles,
a subset of 11 transcripts detected by microarray were consistently dysregulated by more than
2 fold within heart. Within 1 and 5 days after exposure of diesel exhaust particles, an
overlapping subset of 18 transcripts was consistently dysregulated by greater than 2 fold within
lung. Within both heart and lung, expression of Abc1 (transporter with known roles in reverse
cholesterol transport and interleukin secretion) was dysregulated at 5 day. We speculate that
cardiac and pulmonary transcriptional dysregulation due to chronic diesel exhaust exposure
contributes to accelerated atherosclerosis.
P237
The Environmental Pollutant, Polychlorinated Biphenyl 77, Augments
Angiotensin II-induced Abdominal Aortic Aneurysm Formation in
Apolipoprotein E-deficient Mice
Violeta Arsenescu, Sara Police, Eva Langlois, Victoria English, Alan Daugherty, Lisa A
Cassis; Univ of Kentucky, Lexington, KY
Objectives: Polychlorinated biphenyls (PCBs) are omnipresent industrial pollutants that have
been linked to increased cardiovascular disease. Previous studies demonstrated that co-planar
PCBs (PCB77) increase proinflammatory gene expression in endothelial cells, macrophages and
adipocytes. Infusion of angiotensin II (AngII) to hyperlipidemic mice results in the formation of
abdominal aortic aneurysms (AAAs). AngII-induced AAAs are associated with pronounced
inflammation. The purpose of this study was to determine if PCB77 would augment
AngII-induced AAAs. Methods and results: Male ApoE-/- mice (3 months of age) were injected
twice (i.p.) with either vehicle (safflower oil) or PCB77 (170 M/kg) at 1 week prior to and 1
week after implantation of osmotic minipumps for infusion of saline or AngII (1,000 ng/kg/min)
for 28 days. Administration of PCB77 increased body weight gain in saline- and AngII-infused
mice (vehicle, 1.4 0.3, saline/PCB77, 3.6 0.3; AngII/PCB77, 2.2 0.4 g P 0.0036).
Elevations in body weight were associated with adipocyte hypertrophy in mesenteric adipose
tissue and liver steatosis from PCB77-injected mice. PCB77 increased systolic blood pressure
in saline-infused mice (vehicle, 102 3; PCB77, 117 4 mmHg). Infusion of AngII increased
blood pressure to a similar extent in vehicle (146 6 mmHg) and PCB77-injected mice (140
4 mmHg). Both AAA incidence (AngII/vehicle, 45%; AngII/PCB, 90%) and aortic diameter
measured by ultrasound (day 28: vehicle, 0.92 0.01; AngII/vehicle, 1.66 0.21;
AngII/PCB77, 2.32 0.15 mm, P0.05) were increased by PCB77. PCB77 also increased the
severity of AAAs, including mortality from ruptured AAAs. Unexpectedly, PCB77 resulted in
striking ectopic lipid deposition manifested in skeletal muscle (diaphragm), visceral peritoneum
and liver. Conclusions: PCB77 promoted AngII-induced AAA formation and severity. Elevations
in blood pressure, body weight and adiposity, and enhanced inflammation may have
contributed to detrimental effects of PCB77.
P238
Diesel Exhaust Enhances Vascular Oxidative Stress, Vasoconstriction, and
Venous Congestion in a Cardiomyopathic Hamster Model
Travis Knuckles, Amie Lund, Selita Lucas, Lovelace Respiratory Rsch Institute, Albuquerque,
NM; Sathish Babu, Univ of New Mexico Sch of Medicine, Albuquerque, NM; Matthew
Campen; Lovelace Respiratory Rsch Institute, Albuquerque, NM
Environmental air pollution has been associated with increased hospital admissions and death
due to heart failure. However, the exact mechanism(s) by which environmental air pollution
affects the heart and vasculature is currently unknown. Recent studies have found that
exposure to environmental air pollution enhances vasoconstriction in humans. We hypothesized
that diesel exhaust (DE), a major component of ambient urban air, could enhance vasocon-
striction and produce venous congestion in the presence of a failing heart. To test this
hypothesis, we exposed older cardiomyopathic hamsters (HCM; fractional shortening 0.15
0.20, normal 0.60) to freshly derived DE. HCM were exposed to 300g/m
3
of DE for 4 hr/day
for 2days and venous pressures (Pven) were obtained via radiotelemetry. Pven was significantly
increased to 13 mmHg compared to control animals (Pven 5 mmHg). Pven was did not
increase in similarly-exposed non-cardiomyopathic hamsters, suggesting that pre-existing
cardiac disease is prerequisite for the development of venous congestion. DE also enhanced
vascular oxidant generation in exposed animals as measured by lucigenin-enhanced chemi-
luminescence. Interestingly, HCM animals exposed to gasoline exhaust at 60g/m
3
, a 1:12
dilution with filtered-air, for 3hr for 1day did not have enhanced oxidant generation unlike DE
exposed animals. In a related series of studies, the vasoconstrictive effects of the volatile
organic components of DE were determined ex vivo using mouse mesenteric arteries and veins.
DE-exposed saline enhanced the vasoconstrictive effects of ET-1 in mesenteric arteries and
veins. Furthermore, mesenteric vessels exposed to a combined acetaldehyde, formaldehyde,
acetone, hexadecane and octadecane saline solution at concentrations found in DE-exposed
saline also had enhanced ET-1 vasoconstriction. However, the individual compounds did not
appear to replicate these effects. These data suggest that exposure to the complex components
of DE may induce venous congestion in susceptible subjects through enhanced vasoconstric-
tion due to vascular oxidative stress.
P239
Downregulation of Activated Factor XIII by Polymorphonuclear Granulocyte
Proteases Within Fibrin Clot
Zsuzsa Bagoly, Gizella Haramura, Laszlo Muszbek; Univ of Debrecen, Med and Health
Science Cntr, Debrecen, Hungary
Activated clotting factors are down-regulated by two major mechanisms involving protease
inhibitors or proteolytic degradation. Activated factor XIII (FXIIIa) cross-links fibrin chains and
other plasma proteins to fibrin; prolonged exposure of fibrin to FXIIIa results in highly
cross-linked clot resistant to degradation. No down-regulating mechanism for activated factor
XIII (FXIIIa) has been demonstrated, so far. As the haemostatic plug contains polymorphonuclear
granulocytes (PMNs) rich in proteolytic enzymes, we tested if these proteases are released in
fibrin clots, and could be responsible for the down-regulation of FXIIIa. As demonstrated by SDS
PAGE and activity measurements the supernatant of fMLP-stimulated granulocytes degraded
and inactivated purified FXIIIa. The activation of PMNs and the release of their proteolytic
enzymes in the fibrin clot formed from fibrinogen solution were verified by specific elastase,
cathepsin G and matrix metalloprotease-9 (MMP-9) substrates and by the demonstration of
their fibrinolytic effect. Western blotting experiments showed that PMN proteases released in
fibrin clot containing FXIII almost completely degraded both FXIII subunits. The elastase
inhibitor, ONO5046 partially inhibited the degradation of FXIII in fibrin clots supplemented with
PMNs. Cathepsin G and MMP-9 inhibitors provided less protection against the degradation of
the parent molecule, in these cases intermediate split products accumulated. The degradation
of FXIII by PMNs was also significant when the clot was made from whole plasma. Consistent
with this finding, 1-antitrypsin, the main plasma protease inhibitor provided only partial
protection of FXIII against degradation by PMN proteases in fibrin clots. The results suggest that
the degradation of FXIII subunits by the concerted action of PMN proteases released within the
clot represents a novel mechanism for the down-regulation of FXIIIa and protects fibrin from
being over-cross-linked and difficult to eliminate when it is not needed any more.
P240
Increased Bleeding Tendency in Patients with Hematological Malignancies
Giorgio Corinaldesi, Medicina Generale ASL 7, Ancona, Italy; Christian Corinaldesi; Universita`
Politecnica Delle Marche, Ancona, Italy
Patients with hematological malignancies (AML, ALL, multiple myeloma, B and T cells
lymphoma) may often develop thrombocytopenia attributed to chemotherapy, to cancer, and to
infection; they also have an increased risk of bleeding. The degree of bleeding severity includes
mostly multiple sites and a low platelet count; in particular, in older patients the pathogenesis
of this phenomenon is complex and it is related to platelet production, to the increased platelet
destruction (i.e. hypersplenism), to abnormalities in platelet function, to circulating inhibitors or
anticoagulants, and it may finally cause disseminated intravascular coagulation (DIC);
hyperfibrinolysis and proteolysis were frequently associated. Clinically significant bleeding
occurs in approximately 8%12% of patients and are often associated with cytogenetic
abnormalities involving chromosomes 7q (7q 22, 7q3234), 5q (5q11, 5q13-q21, 5q31), and
trisomy 8. Translocation AMLI/ETO from t(8;21), PML/RARalfa fusion transcript arising from the
t(15:17), and CBFB/MYH11 from inversion 16, t(4:11)(q21:q23), t(11:19)(q23:p13). Cytogenetic
markers are not innocent bystanders, and they should be monitored by cytogenetic analysis
because the mutation requires early identification to permit a useful tool for predicting which
patients are at risk of bleeding or relapse; for all these reasons, these markers have a great
prognostic significance. Patients with visible multiple petecchias, or echymosis, or multiple
teleangectases of the skin, gengival bleeding, epistaxis, hematuria, or gastrointestinal
hemorrage have been studied with particular attention (platelet count, PT, PTT, fibrinogen, FDP,
D-Dimer, TAT, AT-III, TM, antiplasmin activity). The management of bleeding disorders is based
upon two different options; recently two recombinant TPO (rHuTPO) and pegylated recombinant
megakaryocyte growth and development factor (PEG-rHuMGDF) increased the nadir platelet
count after about 5 days; otherways a platelet trasfusion may be needed when platelet count
lowers below 20.000/mmc.
P242
Increased Blood-borne Tissue Factor Activity Following Total Knee
Arthroplasty: A Contributor to Venous Thrombosis?
Gerhard J Johnson, VA Med Cntr and Univ of Minnesota, Minneapolis, MN; Linda A Leis, VA
Med Cntr, Minneapolis, MN; Ronald R Bach; VA Med Cntr and Univ of Minnesota,
Minneapolis, MN
Introduction Blood-borne tissue factor (TF) is increased in several disorders associated with
thromboembolism, and cell-associated TF appears to be important in thrombogenesis. Elevated
blood TF has been demonstrated primarily in patients with increased risk of arterial thrombosis,
but evidence for an association of elevated blood TF with risk of venous thromboembolism
(VTE) is lacking. Total knee arthroplasty (TKA) is associated with a risk of venous thrombosis
which is 4080% without thromboprophylaxis. Hypothesis We hypothesized that elevated
2007 ATVB Poster Presentations e-79
by on March 25, 2011 atvb.ahajournals.org Downloaded from
blood TF is a contributing factor to venous thrombosis following TKA. Methods Fifteen male
subjects who had unilateral TKA gave written, informed consent. Venous blood samples were
obtained before surgery, 5 min following tourniquet release and daily until hospital discharge
at day 47. All patients received prophylactic dalteparin and none developed VTE. Platelets and
mononuclear cells were isolated from EDTA anticoagulated blood by differential centrifugation.
A modified prothrombin time (PT) assay was used to quantify TF procoagulant activity. Innovin
was employed as the PT calibration standard. The encrypted TF procoagulant activity was fully
expressed by pretreatment with 20 M ionomycin in the presence of 12.5 mM CaCl
2
and 5 nM
activated factor VII. Results Blood mononuclear TF activity increased following TKA, peaked at
Day 4 and returned to near baseline by Day 7 (Table). Platelet TF activity did not change
significantly. The peak rise in TF activity preceded the median time to diagnosis of venous
thrombosis following TKA (7 days) observed in previously reported studies. Conclusion
Increased mononuclear cell-derived blood TF activity is a delayed response to TKA which is
predominantly independent of acute tissue injury. The close temporal relationship of the rise in
TF to the occurrence of VTE strongly suggests that blood-borne TF contributes to VTE following
TKA.
TF ACTIVITY (PG TF/ML BLOOD; MEAN SEM) *P<0.05 COMPARED TO PRE SURGERY
Pre
Surgery
Tourniquet
Removal Day 1 Day 2 Day 3 Day 4 Day 6 Day 7
Mononuclear
Cells
11.10.5 17.50.9 25.41.0* 25.91.1* 30.41.8* 39.87.7* 31.17.5* 19.91.4
Platelets 7.20.3 12.60.6 9.10.3 6.91.8 11.76.5 10.03.9 9.42.9 13.53.6
Total 18.20.6 30.01.1* 34.51.0* 33.21.2* 44.61.3* 49.88.2* 40.66.5* 33.42.2*
(n) (14) (9) (13) (9) (10) (4) (3) (3)
P243
Changes in Activated Factor XII Type A from Admission to Day 4 Predict
Recurrent Troponin T Positive Cardiac Events Following Admission for
Myocardial Infarction
Volker Poenitz, Trygve Brugger-Andersen, Stavanger Univ Hosp, Stavanger, Norway; David
Pritchard, Axis-Shield, Dundee, United Kingdom; Heidi Grundt, Dennis W Nilsen; Stavanger
Univ Hosp, Stavanger, Norway
Background: Recent research has demonstrated that in-vivo XIIa exists in several different
forms, and that activated Factor XII type A (XIIaA) concentrations are strongly predictive of
mortality. The studys aim was to assess the ability of changes in concentration of XIIaA from
admission to day 4 in predicting outcome in patients admitted with MI. Methods: Quartiles
were established based on the change in XIIaA between admission and day 4. New TnT positive
events and all cause mortality were correlated to the change in XIIaA in blood samples taken
at admission and 4 days post admission and compared at 30 days, 6, 12, and 24 months
follow-up in 319 patients admitted with MI. Analyses of XIIaA were performed on citrated
plasma using ELISA-methodology. Results: Changes in XIIaA concentration followed a normal
distribution, with the mode and median corresponding to no change. Those patients where
XIIaA decreased by 25 pM were in Q1 whilst those where XIIaA increased by 25 pM were
in Q4. Changes in XIIaA between admission and day 4 post-MI were associated with risk for
recurrent TnT positive events at all time points, with odds ratios as high as 8.78 (p0.021) at
30 days, comparing patients in Q4 with those in Q1. Patients in whom XIIaA increased had
higher mortality rates than those where XIIaA decreased, with OR ranging from 3.11 at 30 days
to 2.02 at 24 months (p0.05) Conclusion: Changes in XIIaA concentration from admission to
day 4 predict recurrent TnT positive events and all cause mortality.
P244
Serum Adiponectin Levels Associate with Tissue-type Plasminogen
Activator Activity in Patients with Advanced Carotid Atherosclerosis
Jani Saksi, Petra Ija s, Lauri Soinne, Riitta Lassila, Helsinki Univ Central Hosp, Helsinki,
Finland; Eija Saimanen, South Karelia Central Hosp, Lappeenranta, Finland; Oili Salonen,
Helsinki Univ Central Hosp, Helsinki, Finland; Petri T Kovanen, Wihuri Rsch Institute,
Helsinki, Finland; Markku Kaste, Perttu J Lindsberg; Helsinki Univ Central Hosp, Helsinki,
Finland
INTRODUCTION Adiponectin is an adipocyte-derived bioactive substance with multiple
functions including antiatherogenic properties. Recently, hypoadiponectinemia has been
associated with platelet activation in carotid atherosclerosis. OBJECTIVE The objective of this
study was to investigate the relation of serum adiponectin levels with advanced unstable
carotid atherosclerosis and detailed clinical data including variables of fibrinolytic activity.
METHODS The Helsinki Carotid Endarterectomy Study included 92 consecutive patients with
severe operable symptomatic (stroke or TIA) or asymptomatic carotid stenosis ( 70%,
NASCET). Blood samples were collected before surgery and processed immediately for the
determination of coagulation and fibrinolysis-associated variables. Tissue-type plasminogen
activator (tPA) antigen, -activity and plasminogen activator inhibitor-1 (PAI-1) antigen and
activity were measured from plasma samples collected according to the Leiden fibrinolysis
working party. Serum adiponectin levels were measured using a commercial enzyme-linked
immunosorbent assay. RESULTS Univariate correlations confirmed the previous positive
correlations of adiponectin with female gender, HDL, age and negative correlations with BMI,
triglycerides, PAI-1 antigen and activity. In addition adiponectin levels showed consistent
association with tPA antigen (r -0.380) and even more strongly with tPA activity (r 0.550)
(backward regression model). Patients with symptomatic and asymptomatic carotid stenosis
did not show significant differences in adiponectin levels although patients with no previous
history of cerebrovascular symptoms tended to have higher adiponectin levels (P 0.10).
CONCLUSIONS Adiponectin does not seem to be a risk factor for unstable carotid atheroscle-
rosis but it showed strong association with tPA activity, the surrogate marker of fibrinolytic
activity, in patients with advanced carotid atherosclerosis.
P245
Platelet Nitric Oxide Signaling Is Impaired in Essential Hypertension
Eugenia Gkaliagkousi, Ashish Shah, Patricia De Winter, Kings College London, London,
United Kingdom; Chrysanthos Zamboulis, Aristotle Univ of Thessaloniki, Thessaloniki,
Greece; James Ritter, Albert Ferro; Kings College London, London, United Kingdom
Essential hypertension (EH) carries an increased risk of thrombosis. Platelet-derived nitric oxide
(NO) inhibits platelet activation. We have previously shown that platelet NO synthase type 3
(NOS3) activation is impaired in patients with EH. We wished firstly to determine whether
platelet cyclic GMP (cGMP), an index of bioactive NO, is suppressed in patients with recently
diagnosed untreated mild EH, and secondly to investigate if the observed changes in NO
signalling are associated with alterations in the expression of proteins involved in this signalling
23 consecutively recruited untreated hypertensives (UH; blood pressure [BP]151.61.8/
92.71.7mmHg), and 23 normotensive controls of similar age and sex (NC; BP118.12.2/
74.61.7mmHg) were studied. cGMP was measured by radioimmunoassay in gel-filtered
platelets, in response to albuterol, 10
-5
mol/l, collagen 8g/ml or vehiclea NOS inhibitor
N
G
-monomethyl-L-arginine (L-NMMA,10
-4
M). The difference between platelet cGMP in the
absence and presence of L-NMMA was taken as NOS-attributable cGMP. Expression of total
platelet NOS3, phosphoserine-1177-modified NOS3 (pSer
1177
-NOS3), phosphoserine-633-
modified NOS3 (pSer
633
-NOS3), and sGC were determined by Western blotting of platelet
lysates, in 10 UH (BP:163.24.6/102.92.4mmHg) and 10 NC (BP122.53.3/
78.52.6mmHg). In platelets from NC, NOS-attributable cGMP was increased by albuterol or
by collagen, from 31.112.8 (basal) to 81.614.2 and 79.616.5 fmol cGMP/10
8
platelets
respectively (each p0.05). By contrast, basal NOS-attributable cGMP was undetectable in
platelets from UH and no increase was observed in response to either agonist. NOS-attributable
cGMP was lower in platelets from UH as compared to NT, both under basal conditions and on
stimulation with albuterol or collagen (p0.05 for basal, p0.01 for albuterol, and p0.001
for collagen stimulation). There was no significant difference in the platelet expression of total
NOS3, pSer
1177
-NOS3, pSer
633
-NOS3 or sGC, between the two groups. Basal and stimulated NO
signalling is impaired in platelets from UH. This is not explained by decreased expression of
NOS3 or sGC, or decreased phosphorylation of NOS3 at two serine residues known to modulate
NOS3 activity.
P246
DG-041, a Novel Antiplatelet Agent, Is a Potent and Selective Antagonist of
the EP3 Receptor for Prostaglandin E2
Jasbir Singh, deCODE chemistry, Woodridge, IL; Thorkell Andresson, Jose Ramirez, deCODE
genetics, Reykjavik, Iceland; Wayne Zeller, deCODE chemistry, Woodridge, IL; Mark Gurney;
deCODE genetics, Woodridge, IL
Background. Genetic studies in Iceland find that DNA variants in the EP3 receptor for
prostaglandin E2 are associated with increased risk for peripheral arterial disease (PAD),
myocardial infarction and stroke. The EP3 receptor is expressed on platelets, but not in the
vessel wall suggesting that aberrant signaling through the platelet PGE2/EP3 pathway may play
a role in atherothrombotic disease. PGE2 signaling through EP3 amplifies platelet responses to
platelet co-aggregants but has no effect in the absence of a co-aggregant. Methods. DG-041
is 2,3-Dichlorothiophene-5-sulfonic acid, 3-(-1-(2,4-dichlorobenzyl)-5-fluoro-3-methyl-1H-
indol-7-yl)acryloylamide, a novel, selective and potent EP3 antagonist. DG-041 was tested in
binding assays using appropriate [3H]-labeled ligands using receptors expressed in insect cells.
Platelet rich plasma was collected from humans and rats, then stimulated with collagen, ADP
or U446619 in the presence of sulprostone, an EP3 agonist. Results. DG-041 is a
noncompetitive antagonist of PGE2 binding to EP3 with an IC50 of 7 nM which is similar to the
natural ligand, PGE2 (IC50 4 nM). The compound is highly selective for EP3 showing 900
fold selectivity over the EP1, EP2, EP4, IP and FP receptors. The EP3 receptor is a member of
the G-protein receptor family regulating cAMP level in cells through activation of inhibitory Gi
proteins. When tested in CHO-K1 cells expressing EP3, DG-041 behaves as a full antagonist.
It is a potent antagonist of EP3 potention of platelet aggregation in ex vivo assays. DG-041 also
protects mice from pulmonary thromboembolism induced by either arachidonic acid or a
mixture of sulprostone and collagen. The compound has no effect on bleeding time in mice
after surgical incision. Conclusions. DG-041 is a novel anti-platelet that does not increase
bleeding time. It represents a novel therapeutic strategy for the treatment of PAD, MI & stroke.
DG-041 currently is in human clinical PhIIa trials.
P247
No Effect of Lipid Lowering by Simvastatin or Simvastatin and Ezetimibe
on Platelet Function in Patients with Type 2 Diabetes or Impaired Glucose
Tolerance and Coronary Artery Disease
Rickard E Malmstro m, Magnus Settergren, Felix Bo hm, Lars Ryde n, John Pernow, Paul
Hjemdahl; Karolinska Institutet, Stockholm, Sweden
Objective: In addition to lowering cholesterol levels, statin therapy may exert beneficial
non-lipid-related (pleiotropic) effects. We studied the effect of equal lipid lowering by high dose
simvastatin or the combination of the cholesterol absorption inhibitor ezetimibe and low dose
simvastatin on platelet function in patients with dysglycemia and stable coronary artery
disease. Methods: Thirty-nine patients with type 2 diabetes or impaired glucose intolerance
and stable coronary artery disease were randomized to either simvastatin 80 mg daily (S80;
n20) or ezetimibe 10 mg and simvastatin 10 mg daily (E10/S10; n19) for 6 weeks. Serum
lipids, inflammatory markers and platelet function were assessed before and after 6 weeks of
treatment. Platelet function was evaluated by whole blood flow cytometry and turbidimetric
aggregometry in platelet rich plasma with agonist stimulation ex vivo. Results: The two groups
were well balanced regarding age, BMI, blood pressure, medications and smoking habits.
Decreases in total and LDL cholesterol and CRP were similar in the two groups. LDL (mmol/l)
decreased from 3.00.2 to 1.70.2 with E10/S10 and from 3.00.2 to 1.50.2 with S80
treatment. CRP (mg/l) decreased from 5.21.0 to 4.10.9 (p0.01) with E10/S10 and from
e-80 Arterioscler Thromb Vasc Biol June 2007
by on March 25, 2011 atvb.ahajournals.org Downloaded from
4.21.0 to 3.20.7 (p0.09) with S80 treatment. All platelet parameters studied were similar
in the two treatment groups at baseline. Neither treatment affected ADP- or thrombin-induced
platelet P-selectin expression and fibrinogen binding. The maximum platelet P-selectin
expression (% positive cells) evoked by ADP (10 M) was 7912% and 7716% before, and
818% and 7715% after treatment with S80 and E10/S10, respectively. Neither treatment
affected ADP-induced platelet aggregation. The maximum platelet aggregation evoked by ADP
(10 M) was 7413% and 6913% before and 748% and 6814% after S80 and
E10/S10, respectively. Conclusion: Our results indicate that neither statin treatment per se nor
lipid-lowering by combined treatment exerts substantial inhibitory effects on circulating
platelets in patients with stable coronary artery disease and dysglycemia. Platelet stabilization
may not contribute as much as generally believed to the beneficial effects of lipid lowering.
P248
The VerifyNow

P2Y
12
Assay Does Not Eliminate the Need for a Baseline
Sample in Evaluating Inhibition of Platelet Aggregation by Clopidogrel
Chantal Pharand, Marie Lordkipanidze , Donald A Palisaitis, Erick Schampaert, Jean G
Diodati; Hopital Sacre -Coeur, Montreal, Canada
Background: The latest ACC/AHA guidelines recommend platelet aggregation studies to
determine platelet response to clopidogrel in selected patients. Therefore, emergence of
point-of-care assays enabling bedside testing, such as the VerifyNow

P2Y
12
system, might
prove useful in clinical settings. As off-drug baseline values are seldom available, TRAP
(thrombin receptor activating peptide) is used in the assay to approximate the inhibition
achieved by clopidogrel. Hypothesis: We assessed the hypothesis that the VerifyNow

P2Y
12
point-of-care assay is inadequate for quantifying inhibition of platelet aggregation by
clopidogrel. Methods: Sixty-eight patients scheduled for elective percutaneous coronary
intervention were enrolled. Platelet aggregation was measured by VerifyNow

P2Y
12
at baseline
and following clopidogrel administration. The inhibition reported by the VerifyNow

cartridge
[(1-(ADP post-clopidogrel/TRAP post-clopidogrel))*100] was compared to that calculated with
the actual baseline off-drug value obtained with the same methodology [(1-(ADP post-
clopidogrel/ADP pre-clopidogrel))*100]. Results: The stability of the TRAP baseline obtained
before and after clopidogrel ingestion was found to be modulated by the drug; only moderate
correlation was achieved between the two sets of values (r0.626, p0.0001), thus
suggesting that aggregation in response to TRAP may not serve as an optimal baseline. As a
result, the inhibition reported by the assay overestimated that calculated from off-drug data by
an average of 8% (95% confidence interval 1 to 15%, p0.03, paired T-test). Notwithstanding,
results showed moderate correlation (r0.715, p0.0001). Conclusion: The VerifyNow

P2Y
12
fails to accurately quantify inhibition achieved by clopidogrel compared to the standard
before and after testing. Although the technique is suboptimal, the results reported by the assay
correlate moderately with those obtained with the standard method. Thus, in the absence of
an off-drug blood sample, the VerifyNow

P2Y
12
assay might prove a viable compromise to
assess platelet inhibition provided by clopidogrel in patients.
P249
Platelet Activation and Effect of ApoE3 Are Altered in LRP8-deficient Mice
Jason O Robertson, Cleveland Clinic Lerner College of Medicine, Cleveland, OH; Eric J
Topol, Scripps Rsch Institute, La Jolla, CA; Jonathan D Smith; Cleveland Clinic Lerner
College of Medicine, Cleveland, OH
Myocardial infarction (MI) is understood to have a polygenic mode of inheritance and a complex
interaction with environmental factors. Our group has previously reported the results of a
genome wide association study and three confirmatory, large-scale, case-control studies
implicating polymorphisms in the low density lipoprotein receptor related protein 8 (LRP8 or
apoER2) gene in early onset MI. LRP8 has been identified on the platelet membrane and
indirect evidence suggests that lipidated apoE signals through LRP8 to inhibit human platelet
activation. We have used tail bleeding assays and flow cytometry with antibodies against
activated integrin IIb3 and P-selectin to examine platelet function in LRP8-deficient mice.
Bleeding times in 20% of LRP8-deficient mice reached the 10-min endpoint compared with 0%
of the wild type BL/6 mice (p0.0185). Moreover, integrin activation in response to both
thrombin (66.43 6.30% vs. 47.77 19.83%, p0.0394) and ADP (22.00 2.86% vs.
16.18 2.57%, p0.0157) was reduced in the LRP8-deficient mice compared to wild type
controls. Pre-incubation with DMPC:apoE3 complexes (3.75:1 w/w) significantly inhibited
integrin activation by thrombin in a dose-dependent fashion at physiological concentrations
(range: 1030 g/mL). At all doses of apoE3, inhibition was markedly attenuated in
LRP8-deficient mice (p0.05). P-selectin levels in response to platelet activation and apoE3
were not changed by LRP8-deficiency. Taken together, our results suggest that LRP8 is capable
of altering platelet activation by agonists through mechanisms both dependent on and
independent of apoE and begin to suggest a means whereby clot formation could be affected
in humans with a genetic alteration of this protein.
P250
Toll-like Receptor 4- and Toll-like Receptor 2-dependent Platelet Activation
Identify a Potential Route of Aspirin Resistance
Pavel N Shashkin, Manisha Sharma, Mark N Calabro, Gopal K Marathe, Thomas M
McIntyre; Cleveland Clinic, Cleveland, OH
Platelets contain cyclooxygenase-1 (COX-1) that generates prostaglandin H
2
that is metabolized
by thromboxane synthase to thromboxane A
2
(TxA
2
). TxA
2
is a potent vasoconstrictor and
mediator of platelet aggregation. Inhibition of COX-1, and consequently TxA
2
production, by
aspirin is an important tool in the treatment of inflammation and vascular diseases.
Hypo-responsiveness to aspirin treatment - aspirin resistance - is an important clinical problem
and not well defined. Recently platelets were shown to express TLR4 and TLR2, and
lipopolysaccharide (LPS), a TLR4 activator, is a potent agonist that induces reactive oxygen
species (ROS) production in platelets. Agonists of TLR4 and TLR2 (LPS, oxidized lipoproteins,
and other endogenous ligands) may increase thrombus formation and exacerbate the
development of atherosclerosis. We confirmed that TLR4 agonists activate platelets and
increase ROS formation. We also found that LPS, and the TLR2 agonist bacterial lipopeptide,
induced platelet P-selectin expression, and that ROS production was partially blocked by
pretreatment with antioxidant Trolox (water-soluble vitamin E analog). LPS also potentiated
platelet aggregation and made it less sensitive to aspirin treatment. This led us to test the
postulate that TLR4- and TLR2 agonists may have induced expression of aspirin-insensitive
cyclooxygenase-2 (COX-2). We detected COX-2 in agonist-treated platelets using real-time
RT-PCR, confocal microscopy, FACS, and Western blot. Trolox treatment significantly down-
regulated COX-2 protein expression in LPS-activated platelets. TxA
2
synthesis in LPS-treated
cells was less sensitive to aspirin, but sensitive to COX-2 inhibitor NS398. These observations
suggest that the induction of COX-2 in platelets by the agonists of TLR4 and TLR2 mediate
aspirin-insensitive production of TxA
2
, and so upregulation of COX-2 in platelets may be one
mechanism leading to aspirin resistance. Application of antioxidants together with COX-2
inhibitors may be useful in the treatment of acquired aspirin resistance.
P251
The Role of Akt in Glycoprotein Ib-IXMediated Platelet Activation
Hong Yin, Aleksandra Stojanovic, Nissam Hay, Xiaoping Du; Univ of Illinois at Chicago,
Chicago, IL
Under high shear rate flow conditions, initial platelet adhesion at the site of vascular injury is
mediated by the interaction between von Willibrand factor (VWF) and its platelet receptor, the
glycoprotein Ib-IX (GPIb-IX) complex. This interaction also initiates a signaling cascade, leading
to activation of the platelet integrin, IIb3. The signaling mechanism of GPIb-IX is not totally
clear. The cytoplasmic domain of GPIb-IX has been suggested to interact with signaling
molecules phosphoinositide 3-kinase (PI3-K), which has been shown to be involved in the
GPIb-IX signaling. An important signaling molecule that is activated by PI3-K is the protein
kinase, Akt. Thus, to understand the downstream signaling pathway of GPIb-IX signaling, we
investigated the role of protein kinase, Akt, in VWF-induced, GPIb-IX-mediated platelet
activation. We showed that GPIb-IX/VWF interaction-dependent platelet aggregation induced by
botrocetin or ristocetin was impaired in Akt-1 deficient mouse platelets or human platelets
treated with an Akt inhibitor, SH-6. In contrast, botrocetin-induced VWF binding to platelets or
platelet agglutination was not significantly affected. Similarly, GPIb-IX and integrin-dependent
platelet spreading on VWF was impaired in Akt-1-deficient or Akt inhibitor-treated platelets.
Under high shear rate flow conditions introduced by the cone-plate rheometer, GPIb-IX- and
integrin-dependent stable platelet adhesion on immobilized VWF was attenuated in Akt-1
deficient or SH-6 treated platelets. Thus, Akt is important in GPIb-IX and integrin-dependent
platelet adhesion, spreading and aggregation. The role of Akt-1 is independent of integrin
outside-in signaling, because Akt-inhibitor-treated or Akt-1 null platelets are not different from
control platelets in spreading on immobilized fibrinogen, which requires integrin outside-in
signaling but not integrin activation. Thus, Akt-1 plays an important role in GPIb-IX-mediated
platelet activation signaling pathway leading to integrin activation. Furthermore, ristocetin and
VWF induced the PI3-K-dependent Akt phosphorylation. Taken together, our data indicate an
important role for Akt in GPIb-IX-mediated platelet activation signaling.
P252
Blood and Hair Hg Levels Increase Blood Pressure but Do Not Affect
Vascular Reactivity
Leonelo E Bautista, James H Stein, Barbara J Morgan, Univ of Wisconsin, Madison, WI;
Noel Stanton, Wisconsin State Laboratory of Hygiene, Madison, WI; Terry Young, Javier
Nieto; Univ of Wisconsin, Madison, WI
Introduction: Some studies suggest that high levels of urine, hair or toenail Hg increase the
risk of atherothrombotic diseases, an effect that may be explained by oxidative damage to the
vascular endothelium. Hypothesis: We tested the hypothesis that high Hg levels impair the
vasodilating function of the vascular endothelium. Methods: We measured the association
between high blood and hair Hg and brachial artery flow mediated vasodilation (FMD%), middle
cerebral artery reactivity to CO
2
(MCAR%), heart rate variability (standard deviation of normal
RR intervals, SDNN), and hypertensive status in a consecutive sample of 101 subjects
participating in the Wisconsin Sleep Cohort study (mean age59.4 years; range: 4475;
52.5% males). Whole blood total Hg and hair total Hg were tested using inductively coupled
plasma mass spectrometry and cold vapor atomic fluorescence spectrometry, respectively.
Results: Geometric mean blood and hair Hg were 1.16 g/L (0.98, 1.38) and 270.1 ng/g
(226.1, 322.6). Blood and hair Hg were not significantly associated with FMD%, MCAR%, and
SDNN (Table). However, after adjusting for gender, age, body mass index, and fish intake,
people in the upper quartile of hair Hg were more than 4 times more likely to be hypertensive
(odds ratio: 4.19; 95% confidence interval: 1.28, 13.76). Similarly, hypertension was 1.4 times
more likely in those in the upper quartile of blood Hg, although this difference was not
statistically significant (odds ratio: 1.40; 95% confidence interval: 0.92, 2.12). Conclusion:
High hair and blood Hg levels do not seem to influence vascular reactivity, but may increase
the risk of hypertension. This could be explained by deleterious effects on kidney function.
DIFFERENCE IN VASCULAR FUNCTION PARAMETERS IN SUBJECTS IN THE UPPER AND
LOWER QUARTILES OF HG LEVELS
Blood Hg Difference p Hair Hg Difference p
FMD% 0.47 (-0.50, 1.42) 0.34 0.50 (-1.81, 2.81) 0.67
MCAR% 0.14 (-0.40, 0.67) 0.61 -0.85 (-2.21, 0.51) 0.22
SDNN 0.95 (-2.98, 4.88) 0.63 -1.45 (-12.4, 9.47) 0.79
Hypertension 1.40 (0.92, 2.12) 0.11 4.19 (1.28, 13.76) 0.02
2007 ATVB Poster Presentations e-81
by on March 25, 2011 atvb.ahajournals.org Downloaded from
P253
Nitric Oxide Release from Pulmonary and Coronary Vasculature with the
Use of Intravenous Iloprost and Nitroglycerin in Diabetic Patients
Undergoing Valvular Heart Surgery
Ayse Baysal, Dr Siyami Ersek Hosp, Istanbul, Turkey; Serpil Bilsel, Ozlem Gumustekin
Bulbul, Marmara Univ Med Sch, Istanbul, Turkey; Kemal Baysal; Tubitak Rsch Institute for
Genetic Engineering and Biotechnology, Istanbul, Turkey
Introduction: The aim of this study is to compare the effects of intravenous iloprost and
nitroglycerin in patients with type II diabetes mellitus undergoing valvular heart surgery.
Methods: Twenty five patients undergoing valvular replacement with pulmonary hyperten-
sion 25 mmHg were randomized to be given iloprost or nitroglycerin via a central pulmonary
catheter, and the levels of nitrite/nitrate were evaluated before incision (T1) and 20 minutes
after cardiopulmonary bypass. (T4). Aortic, coronary sinus and pulmonary mixed venous blood
samples were taken at the T1 and T4 time periods and the release of nitric oxide from the
coronary vasculature was determined by the difference between the aortic and coronary sinus
concentrations of nitrite and nitrate. Results: Before application of aortic cross clamp, at T1
period, the levels of nitrite/nitrate from the coronary vasculature were similar in both iloprost
(group 1)and nitroglycerin (group 2) groups ( 18.43 M/mL versus 13.8 M/mL (median), p
0.05).However, after the removal of the cross-clamp, a significant increase in nitric oxide is
observed in the group 2 at T4 comparing to group 1 ( 26.1 M/mL versus 34.2 M/mL, p
0.05). Conclusion: This study has shown that in patients with type II diabetes mellitus
undergoing valvular heart surgery the iloprost group did not show an increase in the release
of nitric oxide from the coronary vascular bed before aortic cross clamp and during reperfusion
period. This finding suggests that iloprost and nitroglycerin act via different mechanisms on
vascular smooth muscle cells during their use for pulmonary hypertension.
P254
Induction of Thrombotic Plaque Erosion in Balloon-injured Arteries of
Atherosclerotic Rabbits
Yu-guo Chen, Feng Xu, Rui-jian Li, Yi Sun, Chun-xi Liu, Yun Zhang; Qilu Hosp of Shandong
Univ, Jinan, China
Objective: Plaque erosion triggers thrombosis, serving as a vital pathologic basis of coronary
thrombotic events. This research sought to establish a model of thrombotic plaque erosion in
atherosclerotic rabbit aortas by staurosporine-triggered endothelial apoptosis. Methods:
Atherosclerotic plaques were established in 33 New Zealand rabbits by post-balloon-injury
high-cholesterol feeding for 3 months. The animals were randomized into two groups and their
plaque-rich abdominal aortas were induced with staurosporine (group A, n24) or saline
(group B, n9). Group A was randomized into group A
1
(n12) and group A
2
(n12); they
were triggered with 510
-5
or 110
-5
mol/L staurosporine, respectively. Three days after
induction, thrombotic score was performed according to angiography (angiographic obvious
thrombosis was defined as score 34). Serial sections of the induced segments were
conducted to count incidence of histological thrombosis. Results: Angiography showed that,
relative to group B, the incidence of obvious thrombosis was higher in groups A (P0.04), in
which it was higher for group A
1
(58.3%, P0.01) but not for A
2
(16.7%, P0.49). Serial
sections of the processed aortas revealed that there were more histological thrombosis in
groups A
1
and A
2
than in group B (P0.01, P0.02). Pathological analysis showed that,
non-ruptured and smooth-muscle-cell-rich fibrous caps at plaque/thrombi interfaces were
observed in serial sections stained with HE, Oil Red O and -actin, and endothelium integrity
according to CD31 staining were lower in groups A
1
and A
2
than in group B (P0.01,
respectively). Apoptosis scores according to TUNEL staining were higher in groups A
1
and A
2
than in group B (P0.01, respectively). Logistic analysis demonstrated that endothelial
apoptosis was an independent risk factor of histological thrombosis and angiographic obvious
thrombosis (OR15.22, P0.01; OR45.74, P0.01). Conclusion: a thrombotic plaque
erosion model can be successfully established by perfusing staurosporine into atherosclerotic
abdominal aortas; and 510
-5
mol/L staurosporine appears to be more suitable with a higher
angiographic obvious thrombosis. In addition, endothelial apoptosis may play a critical role in
thrombotic plaque erosion.
P255
An Orally Active Inhibitor of the TGF- Type I Receptor, ALK5, Adventitial
Myofibroblast Induction, and Vascular Remodeling in the Rat Carotid
Balloon Injury Model
Kai Fu, Michael J Corbley, Lihong Sun, Jessica Friedman, Feng Shan, James L Papadatos,
Donald Costa, Frank Lutterodt, Harry Sweigard, Jeannie Reis, Pamela Korsman, Scott
Bowes, Michael Choi, Ann Boriack-Sjodin, Robert Arduini, Dongyu Sun, Xiamei Zhang,
Jonathan Mead, Claudio Chuaqui, Kam Cheung, Xin Zhang, Mark Cornebise, Serene Josiah,
Juswinder Singh, Wen-Cherng Lee, Alan Gill, Leona E Ling; Biogen Idec, Cambridge, MA
Objective: TGF- antagonist antibodies, soluble receptor, antisense, decorin and gene therapy
with negative regulatory Smad7 have established the importance of TGF- in intimal thickening
and lumenal narrowing following vascular injury. This study evaluates the efficacy of a novel,
small molecule inhibitor of ALK5/ALK4 kinase, in the rat carotid injury model of vascular
fibrosis. Methods and Results: The small molecule, SM16, was shown to bind with high affinity
to ALK5 kinase ATP binding site using a competitive binding assay and biacore analysis. SM16
blocked Smad2 phosphorylation and inhibited TGF--induced PAI-luciferase activity in cells.
Good overall selective was demonstrated in a large panel of kinase assays, but SM16 also
showed nanomolar inhibition of ALK4 and weak (micromolar) inhibition of Raf and p38. In the
rat carotid injury model, SM16 dosed once daily orally at 15 or 30 mg/kg SM16 for 14 days
caused significant inhibition of neointimal thickening, lumenal narrowing and the induction of
adventitial smooth muscle -actin-positive myofibroblasts at 2, 4, 7, 10 or 14 days post-injury.
Conclusion: These results are the first to demonstrate the efficacy of an orally active,
small-molecule ALK5 inhibitor in a vascular fibrosis model and suggest the potential
therapeutic application of these inhibitors in vascular fibrosis.
P256
Reversal of Hypercholesterolemia Prevents Progression of Atherosclerosis
and Protects Against Arterial Thrombosis in Mice
Lorie Leo, Katina M Wilson, Jordan D Miller, The Univ of Iowa, Iowa City, IA; Stephen
Young, Univ of California, Los Angeles, Los Angeles, CA; Donald D Heistad, Steven R Lentz;
The Univ of Iowa, Iowa City, IA
Introduction: Hypercholesterolemia promotes atherosclerosis and increases thrombotic risk, but
little is known about the effects of cholesterol lowering on susceptibility to experimental
thrombosis. Hypothesis: We tested the hypothesis that reversal of hypercholesterolemia
induces regression of atherosclerosis and restores normal susceptibility to carotid artery
thrombosis in Reversa mice. Methods and Results: Male Reversa (Ldlr
/-
Apob
100/100
Mttp
fl/fl
Mx1-
Cre
/
) mice were fed a high fat diet and studied at 5 or 9 months of age (total cholesterol (TC)
76466 and 72665 mg/dL, respectively). To prevent hypercholesterolemia, Reversa mice
were injected with polyinosinic-polycytidylic acid (pI-pC) at one month of age to induce
Mx1-Cre and switch off hepatic Mttp gene expression, fed a control diet, and studied at 5 or
9 months of age (TC 6310 and 707 mg/dL, respectively). To reverse hypercholesterolemia,
Reversa mice were fed a high fat diet until 5 months of age, then injected with pI-pC and
switched to a control diet, and studied at 9 months of age (TC 7512 mg/dL). Atherosclerotic
lesion area was measured in aortic sinus cross-sections and en face preparations of the
thoracic aorta. At both sites, lesion area was greater in the hypercholesterolemia group
compared with the normocholesterolemia group (P0.05) and greater at 9 months compared
with 5 months (P0.05). In the reversed group, aortic sinus lesion area at 9 months was
similar to the hypercholesterolemia group at 5 months, and smaller than the hypercholester-
olemia group at 9 months (P0.05), indicating that atherosclerosis did not progress between
5 and 9 months. Susceptibility to carotid artery thrombosis was measured using a
photochemical injury model. Thrombotic occlusion occurred more rapidly in the hypercholes-
terolemia group than in the normocholesterolemia group at both 5 months (178 vs. 4612
minutes; P0.05) and 9 months (91 vs. 459 minutes; P0.05). Accelerated thrombosis
was corrected toward normal after reversal of hypercholesterolemia (3112 minutes; P0.05
vs. hypercholesterolemia group). Conclusions: Reversal of hypercholesterolemia normalizes
thrombotic susceptibility in atherosclerotic mice.
P258
Pentoxifylline Lowers Plasminogen Activator Inhibitor-1 Levels in Obese
Human Subjects
James A Muldowney, Judy L Francescon, Douglas E Vaughan; Vanderbilt Univ Sch of
Medicine, Nashville, TN
Obesity, an increasingly important risk factor for cardiovascular disease in men and women, is
associated with the premature development of atherosclerosis. A classical perspective of
cardiovascular risk does not adequately explain all of the cardiovascular events associated with
obesity. Elevations in plasma levels of PAI-1 (plasminogen activator inhibitor) and CRP
(c-reactive protein) are biochemical hallmarks of obesity and inflammation that likely contribute
to the increased risk of atherothrombotic events in patients with obesity. While PAI-1 and CRP
are known to be synthesized in vascular tissues, the liver and adipose tissue, the source and
mechanisms of increased PAI-1 and CRP in obesity are incompletely understood. There is
strong experimental evidence that TNF- (tumor necrosis factor-) plays an important role in
regulating PAI-1 production in obese mice, and inhibition of TNF- by pentoxifylline reduces
PAI-1 production in adipose tissue. We tested the hypothesis oral pentoxifylline will lower
plasma PAI-1 and plasma CRP levels in obese patients. We randomized 13 obese subjects
(mean age 38 2 years, 77% female) in a double-blind manner to 400 mg of pentoxifylline
three times daily (n5) or to placebo three times daily (n8) for 56 days. PAI-1, high sensitivity
CRP (hs-CRP) and TNF- were measured at days 0, 28 and 56. There was a strong correlation
between baseline PAI-1 and hs-CRP levels in all subjects ( r 0.766, P 0.002). PAI-1 levels
decreased 10 5 ng/ml in the pentoxifylline arm while the placebo arm saw an increase in
plasma PAI-1 levels of 54 over the course of the study (P 0.007). Pentoxifylline appeared
to reduce hs-CRP levels (1.81.0 mg/L) compared to placebo (0.20.3 mg/L), but this did not
achieve statistical signficance (P 0.08). TNF- levels were essentially unchanged throughout
the study. These data suggest that pentoxifylline may be an effective agent in lowering PAI-1
and CRP in obese subjects, which could portend a better cardiovascular risk profile in this
patient population.
P259
Bilirubin Oxidation Products Elicit Significantly Elevated Thromboxane
Production in Rat Brain
Gail J Pyne-Geithman, Danielle N Caudell, Amanda L Harm, Leigh A Chasse, Univ of
Cincinnati, Cincinnati, OH; Kenneth R Wagner, Univ of Cincinnati/VA Med Cntr, Cincinnati,
OH; Joseph F Clark; Univ of Cincinnati, Cincinnati, OH
Bilirubin Oxidation products (BOXes) have been found in the CSF of patients with cerebral
vasospasm after SAH, as well as in hematoma and perihematomal brain of animal models of
ICH. We have found that BOXes are vasoactive, both in vitro and in vivo, and so wanted to
investigate possible effects of Boxes on the inflammatory responses of the brain itself. Control:
animals were anaesthetized and perfused with 0.9% saline. The brains were removed and
flash-frozen. ICH: 100L autologous blood was infused into the right cerebral cortex. The
animal was allowed to recover for 24 hours, then the brain was harvested as for the control
animal. BOXes: a cranial window was opened over the right cerebral hemisphere and 25L of
23M BOXes was applied directly to the surface of the brain. The animal was allowed to
recover for 24 hours, then the brain was harvested as for the control animal. Contralateral
cortex (from ICH and BOXes brains) was homogenized and assayed for Thromboxane B
2
using
e-82 Arterioscler Thromb Vasc Biol June 2007
by on March 25, 2011 atvb.ahajournals.org Downloaded from
an EIA kit (Cayman Chemicals). Total protein was assayed using the BCA method (Pierce). The
results indicate that BOXes induce TX production in the brain, in the absence of thrombin and
other blood components normally present in hemorrhagic stroke, This suggests a role for
BOXes in other complications (than vasospasm) following hemorrhagic stroke such as edema,
inflammation and immune responses. It may also hint at a putative role for therapeutic use of
inhibitors of TX production, such as COX-2 inhibitors.
P261
5-Amino-4-Imidazole Carboxamide Riboside Inhibits Tissue Factor
Induction in Endothelial Cells and Monocytes
Weiyu Zhang, Yuqing Huo; Univ of Minnesota, Minneapolis, MN
AMP-activated protein kinase (AMPK) is tightly regulated by the ratio of intracellular AMP/ATP
and plays a central role in regulation of energy homeostasis under metabolic stress. Recent
studies link AMPK to an anti-inflammatory effect such as inhibition of cytokine production and
adhesion molecule expression. In this study, we used 5-amino-4-imidazole carboxamide
riboside (AICAR), one of AMPK activators to investigate whether AMPK plays a role in
anti-thrombotic effect. Tissue factor (TF), a critical initiator of physiologic and pathologic
coagulation, plays an important role in initiation and propagating thrombus formation in heart
attack and stoke. In an in vitro assay, the clotting activity of TF in human umbilical vein
endothelial cells (HUVEC) and peripheral blood mononuclear cells were induced by physiologic
agonist LPS, TNF-a and IL-1b. Pretreatment of cells with AICAR inhibited clotting activity by
90 %. Inhibition of TF clotting activity by AICAR appears dose and time-dependent.
Suppression of TF clotting activity correlated a decrease of TF expression at protein and mRNA
levels. ZM 241385, a specific adenosine A2a receptor antagonist, did not block the effect of
AICAR on TF suppression, indicating inhibition of TF by AICAR was not due to adenosine
production and A2A occupancy. We are using AMPK knockout or knockdown cells in our study.
The consequent results will provide information on the extent of AMPK dependency in
AICAR-mediated TF inhibition.
P262
Nocturnal Oxygen Desaturation and the Prevalence of Metabolic Syndrome
Imran H Iftikhar, Fairview Hosp, Cleveland Clinic, Cleveland, OH; Mansoor Ahmed,
Southwest Cleveland Sleep Cntr, Cleveland, OH; Robert P Blankfield; Case Sch of Medicine,
Cleveland, OH
Objective: Many studies in the past have identified an association between obstructive sleep
apnea (OSA) and the elements of MS. Recently some studies have found a direct association
between the severity of OSA and the prevalence of MS. In this study we sought to determine
an association between the severity of nocturnal oxygen desaturation and the prevalence of MS
in a community subpopulation diagnosed with obstructive sleep apnea. Methods: A cross
sectional retrospective analysis of the data of 419 patients was performed at a community
sleep disorders diagnostic center. MS was considered to be present in patients if three out of
the following criteria were met: hypertension, diabetes, hyperlipidemia and a body mass index
of greater than 30. Nocturnal oxygen desaturation was considered to be significant if the
oxygen concentration during the sleep study desaturated below 90 %. Results: A total of 22
patients were identified with MS. The NCSS and PASS statistical software was used for the
analysis. A correlation value of 0.93 and a p value of 0.000 was found. Although the correlation
value was not significant, the study found that in this subpopulation with OSA, in general, a
higher percentage of total sleep time spent below 90% was accordingly associated with a
higher number of cases with MS (graph 1). Conclusions: The study found a dose dependent
association between the severity of nocturnal oxygen desaturation and the prevalence of MS.
Whether or not the reversal of nocturnal oxygen desaturation through CPAP treatment corrects
the syndrome needs to be tested in a large multi center prospective study.
P263
Hyperglycemia-Induced Cell Growth and Gene Expression via Serum
Response Element Through Pkc, Rhoa, and Rho-Kinase in Vascular
Smooth Muscle Cells
Kai Ishiko, Tsuyoshi Sakoda, Takahumi Akagami, Takashi Doi, Toshio Naka, Takeshi
Tsujino, Tohru Masuyama, Mitsumasa Ohyanagi; Hyogo College of Medicine, Nshinomiya,
Japan
The impressive correlation between cardiovascular disease and alterations in glucose
metabolism has raised likelihood that atherosclerosis, heart failure and type 2 diabetes may
share common antecedents. Postprandial hyperglycemia has been shown the important role for
onset and development of heart failure and cerebral infarction by several large-scale clinical
trials. Recently, chronic hyperglycemia has been reported to enhance the vasoconstrictor
response by Rho-kinase and PKC. Furthermore, oral PKC selective inhibitor has been reported
to show the effective therapy for the cardiovascular complications of diabetes. We have
reported Phenylephrine showed enhancement of vasoconstrictor response in a spontaneous
diabetes mellitus model, OLETF ( Otsuka-Long-Evane-Tokushima fatty ) rat. However, the
mechanism of hyperglycemia on these reactions, especially the influence to signal transduction
pathway by hyperglycemia has not been well understood. Therefore, we examined the effect
of hyperglycemia on cell growth and gene expression in rat aorta smooth-muscle cells
(RASMCs). Hyperglycemia accelerated the growth of RASMCs with concentration dependent
manner. Furthermore, c-fos gene expression was also increased by hyperglycemia. Phenyl-
ephrine activated c-fos gene expression. Hyperglycemia augmented Phenylephrine-induced
c-fos gene expression synergistically with dose dependent manner. The deletion analysis
revealed c-fos serum response element (SRE) accounts for c-fos gene expression. PKC-,
RhoA, and Rho-kinase were involved in this signal transduction pathway. Furthermore,
PKC-activated c-fos SRE expression was inhibited by RhoA and Rho-kinase. These results
indicate RhoA and Rho-K are downstream molecules of proten kinase C ( PKC )-. A HMG-CoA
reductase inhibitor, Fluvastatin inhibited hyperglycemia-augmented these reactions by inhibi-
tion of RhoA. Furthermore, catalytic domain mutant of PKC- also inhibited these reactions.
Hyperglycemia itself increased the cell growth and gene expression. Furthermore, it also
modifies and augments the cell growth and gene expression by 1-AR-mediated stimulation.
Statin and PKC inhibitor might be effective for hyperglycemia-induced cardiovascular
dysfunction.
P264
Red Blood Cell Fatty Acid Composition and the Metabolic Syndrome: NHLBI
GOLDN Study
Edmond K Kabagambe, Univ of Alabama at Birmingham, Birmingham, AL; Michael Y Tsai,
Univ of Minnesota, Minneapolis, MN; Paul N Hopkins, Univ of Utah, Salt Lake City, UT; Jose
M Ordovas, Tufts Univ, Boston, MA; James Peacock, Univ of Minnesota, Minneapolis, MN;
Ingrid Borecki, Washington Univ, St. Louis, MO; Donna K Arnett; Univ of Alabama at
Birmingham, Birmingham, AL
Different fatty acids may vary in their effect on the metabolic syndrome (MetS). We tested
whether fatty acids measured in red blood cells (RBC) are associated with the MetS or its
components. Men (n 486, 49 16 y) and women (n 535, 49 16 y) from 187 families
from Utah and Minnesota were studied as part of the Genetics of Lipid Lowering Drugs and Diet
Network (GOLDN) study. Fatty acids in RBC were measured with gas chromatography while
data on confounders were obtained from interviewer-administered questionnaires. The
prevalence of the MetS as defined by the updated Adult Treatment Panel III criteria was 37.0%
in Utah and 39.8% in Minnesota (P0.05). In a multivariate model that included four fatty acid
classes, age, sex, physical activity, alcohol intake, smoking and pedigree (modeled as a
random effect), polyunsaturated and saturated fatty acids were significantly associated with the
MetS (Table 1). We observed significant (P0.05) correlations between fatty acid classes and
components of the MetS (data not shown). Saturated fat (r 0.10) , monounsaturated fat (r
-0.12) and polyunsaturated fat (r -0.11) were significantly correlated (P0.05) with fasting
insulin. In conclusion, polyunsaturated fatty acids were inversely associated with the MetS
while saturated fatty acids were positively associated with the MetS probably through their
effect on lipids, insulin and systolic blood pressure. These data suggest that RBC fatty acid
profiles could be a good marker for the metabolic syndrome.
TABLE 1. ODDS RATIOS (95% CI) FOR FATTY ACID CLASSES AND THE
METABOLIC SYNDROME
Variable
Quartile
1
Quartile
2
Quartile
3
Quartile
4
P for
trend
Polyunsaturated fatty acids 1.00 0.69 (0.461.06) 0.63 (0.400.99) 0.40 (0.240.65) 0.0003
Saturated fatty acids 1.00 1.23 (0.791.89) 1.44 (0.922.27) 1.62 (1.012.62) 0.04
Monounsaturated fatty acids 1.00 0.78 (0.511.20) 0.72 (0.461.13) 0.81 (0.501.32) 0.39
Trans fatty acids 1.00 1.25 (0.831.90) 1.26 (0.821.94) 0.96 (0.591.56) 0.89
2007 ATVB Poster Presentations e-83
by on March 25, 2011 atvb.ahajournals.org Downloaded from
P265
WITHDRAWN
P266
Elevated Triglyceride with Low HDL-C and Risk of Coronary Heart Disease
in Diabetics: The Strong Heart Study
Jennifer S Lee, Univ of California Davis Med Cntr, Sacramento, CA; Barbara V Howard,
MedStart Rsch Institute, Hyattsville, MD; Ying Zhang, Univ of Oklahoma Health Sciences
Cntr, Oklahoma City, OK; Richard R Fabsitz; National Heart, Lung, and Blood Institute,
Bethesda, MD
An elevated triglyceride (TG) and decreased high-density lipoprotein cholesterol (HDL-C) has
been shown to be a characteristic dyslipidemia in insulin resistance and type 2 diabetes. We
examined whether this dyslipidemia is a predictor of coronary heart disease (CHD) in type 2
diabetic patients. The Strong Heart Study is a large population-based prospective study of
cardiovascular disease (CVD) in 13 American Indian communities in the U.S. The baseline visit
(1989 to1992) consisted of a personal interview, exam, and lab tests, including serum fasting
lipids. Diabetes status was determined based on World Health Organization recommendations.
Incident CHD was identified through medical records or patient medical histories and confirmed
by a committee of blinded CVD experts. A total of 2,059 participants aged 4574 years had type
2 diabetes but no known CHD at baseline. Of these545 participants were diagnosed with CHD
over 14 years. Participants were categorized into four groups based on having comparable
incidence rates according to their TG and HDL-C tertile levels: Highest TG tertile and lowest
HDL-C tertile (High TG-low HDL-C); highest TG tertile and mid-to-highest HDL-C tertile (High
TG); lowest-or-mid TG tertile and lowest HDL-C tertile (Low HDL); and lowest-or-mid TG
tertile and mid-or-highest HDL-C tertile (Referent). Univariate and multivariate (Cox proportional
hazard models) methods were used to estimate the hazard ratio (HR) for incident CHD.
Age-adjusted incidence of CHD (in events/1000 person-years), was 20.3 in the referent group,
24.7 in Low HDL-C, 33.5 in High TG, and 44.8 in High TG-low HDL-C (Ptrend 0.001).
Compared to the referent group, the HR of CHD (95% confidence interval) were 1.24 (0.95,
1.62); 1.50 (1.19, 1.90); and 1.70 (1.38, 2.16) in the three groups, respectively, after
adjustment for age, gender, smoking, hypertension, body mass index, and albumin to
creatinine ratio. The HR for High TG-low HDL-C was attenuated but remained significant
(1.54 (1.20, 1.97)) after additional adjustment for LDL cholesterol. The combination of elevated
TG and decreased HDL-C is an independent predictor of CHD in type 2 diabetics. This
dyslipidemia might be a useful marker to identify diabetics who are at increased risk of CHD.
P267
Delineating the Relationship Among the Components of the Metabolic
Syndrome: Insight from the National Health and Nutrition Examination
Survey, 19992002
Jou-Wei Lin, Lian-Yu Lin; National Taiwan Univ Hosp Yun-Lin Branch, Dou-Liou City, Taiwan
Background: The pathogenesis of the metabolic syndrome (MS) involves complex interactions
among the main components of MS. To analyze all these components as a whole, we
constructed a structural equation model (SEM) to examine the hypothesis that obesity plays the
central role in the development of MS. Methods: A total of 3,596 Caucasians aged 19 years old
and above without current use of anti-hypertensive, oral hypoglycemic agents, or lipid-lowering
medication, who participated in the National Health and Nutrition Examination Survey (NHANES)
19992002, was included for analysis. Anthropometric measurements, biochemical profiles
(lipids, fasting glucose, and insulin), and high-sensitivity CRP were measured. A SEM was
constructed to elucidate a pathway in which obesity might be the most important early step in
the etiological cascade leading to full MS. Results: The results of SEM demonstrated that
obesity was positively associated with elevated CRP level (B0.05, p0.001). This higher
inflammatory state could result in insulin resistance (B0.32, p 0.001), which in turn lead
to dyslipidemia (B0.06, p0.001) after adjustment of potential confounding factors. Obesity
could also directly and positively affect blood pressure (B0.51, p0.001), without the
mediation of insulin resistance and/or inflammation. Conclusions: Our study has demonstrated
that obesity plays a central role in the development of insulin resistance and dyslipidemia
through the mediation of a proinflammatory state. Obesity also directly leads to higher blood
pressure. The SEM has provided a comprehensive view to illustrate the complex inter-play of
the main components in the development of the MS.
P268
Comparison Between Adiponectin and Leptin in Relation to Metabolic
Syndrome in Japanese Women
Kunihiro Matsushita, Hiroshi Yatsuya, Koji Tamakoshi, Hirotsugu Mitsuhashi, Takahisa
Kondo, Toyoaki Murohara, Hideaki Toyoshima; Nagoya Univ Graduate Sch of Medicine,
Nagoya, Japan
Background: Two representative adipokines, adiponectin and leptin, have been suggested to
play important roles in the pathogenesis of metabolic syndrome (MetS). However, little
information exists on their respective features in relation to MetS, particularly in women.
Objective: To investigate the respective associations of adiponectin and leptin with each MetS
component and its clustering. Methods: We studied 769 middle-aged Japanese women
without a history of cancer or cardiovascular disease. Since it was less likely that changes in
these adipokines cause an increase in adiposity, we focused on the association of adiponectin
or leptin with 4 MetS components other than central obesity. Thus, we computed odds ratios
(ORs) in favor of the presence of each MetS component or its clustering (2 components)
according to a 1-SD decrease in log-adiponectin and a 1-SD increase in log-leptin with
multivariate logistic regression analyses, in which both adipokines were simultaneously entered
as explanatory variables. Results: Interestingly, adiponectin had a closer association than leptin
with low high-density lipoprotein cholesterol (HDL-C) and hyperglycemia (Table). In contrast,
the association with elevated blood pressure was significant only for leptin. Hypertriglyceride-
mia was significantly associated with both adipokines. As a result, both adipokines were
independently and significantly associated with the clustering of MetS components. Conclu-
sion: Although both adiponectin and leptin were independently associated with MetS, their
associations with each MetS component were distinctive. Specifically, decreased adiponectin
seemed to play a greater role in the reduction of HDL-C and the elevation of glucose. So did
increased leptin in the elevation of blood pressure. These data may have important implications
both for inferring the etiological role of these adipokines in causing MetS and for introducing
them as targets for MetS treatment.
TABLE. COMPARISON OF ADIPONECTIN AND LEPTIN IN RELATION TO METS
Dependent variables Adiponectin Leptin OR (95% CI)* P
Low HDL-C 1.84 (1.402.42) 0.001 1.28 (0.941.73) 0.12
Hypertriglyceridemia 1.41 (1.111.79) 0.005 1.89 (1.432.50) 0.001
Hyperglycemia 1.31 (1.001.71) 0.051 1.02 (0.761.37) 0.91
Elevated blood pressure 1.08 (0.911.28) 0.41 1.41 (1.171.70) 0.001
Clustering of 2 components 1.52 (1.201.93) 0.001 1.67 (1.272.19) 0.001
*ORs (95% CIs) by a 1-SD decrease and increase of log adiponectin and log leptin,
respectively, adjusted for age and smoking.
P271
Thiazolidinediones Attenuate the Angiotensin II-mediated Enhanced
Vascular Responses in High Fat DietFed Rats by Altering the
Characteristics of L-type Calcium Channels
Bhoomi Viswanad, Poduri Ramarao; National Institute of Pharmaceutical Education and
Rsch, Mohali, India
Background/rationale:- Insulin resistance has emerged as a mechanism leading to diabetes
mellitus and hypertension. However, insulin sensitizers such as pioglitazone and rosiglitazone,
[peroxisome proliferator activated receptor- (PPAR) agonists] are reported to reduce blood
pressure (BP) in hypertensive models by altering L-type calcium channel functions. Objective/
Hypothesis: To establish the cause and effect relationship of insulin resistance and
vasculopathy and find the role of L-type calcium channels. Methods: Ang II-induced
contractions were studied isometrically in thoracic aortic rings isolated from control and high
fat diet (HFD) fed rats. To evaluate the involvement of L-type calcium channels in Ang II
mediated contraction, cumulative concentration responses curves (CRC) to Ang II was
constructed in the presence of various concentrations (0.01 nM -1M) of nimodipine
(dihydropyridine sensitive L-type calcium channel blocker)and the -log IC
50
was estimated.
Receptor radioligand binding studies for Ang II receptors and L-type calcium channels were
carried by [
3
H]-Ang II and [
3
H]-PN-200110, respectively. Results: The rats fed with HFD for four
weeks exhibited the conglomeration of characteristic features of insulin resistance syndrome,
such as obesity, hyperinsulinemia, mild hyperglycemia, hypertriglyceridemia, hypercholester-
olemia, glucose intolerance and hypertension. Maximal contractile response (E
max
) to Ang II was
increased in HFD fed rats as compared to control rats. In addition, B
max
values and affinity of
Ang II receptors and L-type calcium channels are increased, respectively. Nimodipine
dose-dependently blocked the Ang II-induced contractions in a noncompetitive manner and its
-log IC
50
was significantly lower in aortic rings from HFD fed rats (8.87 0.17) compared
(P0.05) to control (9.78 0.18) rats. Rosiglitazone and pioglitazone treatment for 14 days
restored -log IC
50
values (p0.05) comparable to that of control (9.88 0.28 & 9.82 0.34,
respectively). Conclusions: Thiazolidinediones attenuate the development of hypertension and
improves the vascular dysfunction induced by Ang II by altering the L-type calcium channel
functions in HFD fed rats.
P272
Repetitive Glucose Fluctuations Accelerate Macrophage Adhesion to
Endothelial Cells and Formation of Arteriosclerotic Lesions in
Apolipoprotein E-Deficient Mice
Tomoya Mita, Hirotaka Watada, Kosuke Azuma, Ryuzo Kawamori; Juntendo Univ, Tokyo,
Japan
Background Postprandial hyperglycemia is regarded as an independent risk factor for the
progression of atherosclerosis. Recently, we demonstrated that repetitive postprandial hyper-
glycemia per se induced monocyte adhesion to endothelial cells by using a new en face method
e-84 Arterioscler Thromb Vasc Biol June 2007
by on March 25, 2011 atvb.ahajournals.org Downloaded from
for optimal observation of endothelial surface in Goto-Kakizaki (GK) rats {Azuma K. et al. 2006
ATVB}. However, it is still unknown whether glucose fluctuations actually accelerate the
formation of atherosclerosis. Previously accelerated atherosclerotic changes were observed
both in streptozotocin injected Apolipoprotein (apo) E-dificient mice and apoE-difficient mice
clossed with db/db mice. However these mice showed further increase in cholesterol level
compared with apo-E deficient mice and profoundly higher glucose level. So, these model were
not suitable to evaluate the effect of glucose fluctuation per se on the progression of
atherosclerosis. Aim The aim of this study was to investigate the effect of glucose fluctuation
on the process of atherosclerosis. Methods We used apo E-deficient mice which maltose was
compulsorily given twice daily as the model with repetitive postprandial glucose spikes. We
investigated the number of macrophage adhesion to endothelial cells and the lesion size of
fibrotic arteriosclerosis with and without the administration of miglitol, an -glucosidase
inihibitor on this model. Macrophage adhesion to endothelial cells in thoracic aorta was
quantitated by a novel enface method for optimal observation of endothelial surface after
immunohistochemical staining for Mac-2. The lesion size of fibrotic arteriosclerosis in the
proximal aorta was measured by elastica-van Gieson staininig. Results Maltose administration
induced daily blood glucose fluctuation without further increase in cholesterol level in
ApoE-deficient mice. The number of macropahge adhesion was increased at 1 week after the
beginning of maltose administration and the lesion size was increased at 4 weeks after the
beginning of maltose administration. This increase was prevented by the simultaneous
administration of miglitol. Conclusion Our data demonstrated that glucose fluctuations
accelerate atherosclerosis independent of serum cholesterol level in vivo.
P273
Telmisartan Improves Metabolic Syndrome in Addition to Its Vascular
Protection in High-fat DietFed Insulin-Resistant Rats
Poduri Ramarao, Godugu Chandraiah, Bhoomi Viswanad; National Institute of Pharmaceutical
Education and Rsch, Mohali, India
Background/rationale:- Increased vascular contraction by angiotensin II and decreased
relaxation by impaired endothelial function causes the development of vascular dysfunction in
insulin resistance (IR). However, peroxisome proliferator activated receptor (PPAR )
activators have been documented for vascular protection by unclear mechanisms. Hence, the
inhibition of RAS and activation of PPAR simultaneously could have the beneficial effects in
IR. Methods:-Angiotensin II (Ang II) and acetylcholine (ACh) mediated vascular responses were
studied isometrically in thoracic aortic rings isolated from normal and high fat diet (HFD) fed
rats. Other biochemical parameters were analyzed by commercially available spectrophoto-
metric kits. Receptor radioligand binding studies for Ang II receptors were carried by [
3
H]-Ang
II. Telmisartan (AT1 blocker & partial PPAR agonist), pioglitazone and losartan were
administered orally for 14 days prior to all the experiments. Results:- HFD-fed rats exhibited
characteristic features of metabolic syndrome (MetS) viz., obesity, hyperinsulinemia, mild
hyperglycemia, hypertriglyceridimia, hypercholesterolemia, glucose intolerance and hyperten-
sion. Vascular dysfunction was evident from the increased maximal contractile response to Ang
II and decreased vascular relaxation to ACh in thoracic aortic rings. Angiotensin receptor
blocker (ARB), losartan improved the vascular dysfunction without altering metabolic param-
eters. Pioglitazone improved the metabolic parameters with a marginal improvement of
vascular parameters. However, the telmisartan produced similar kind of effects of combination
of pioglitazone and losartan including down regulated AT
1
receptors with an addition of weight
reduction potential. Conclusion:- Telmisartan improves abnormal metabolic and vascular
profile in addition of weight reduction potential in MetS. Due to coexistence of metabolic
syndrome and CVDs, the availability of the multifunctional dual pharmacophores like
telmisartan can treat more than just blood pressure or metabolic disturbances which could be
of considerable clinical value.
P274
Tannin Extracted from Sumac Inhibits Vascular Smooth Muscle Cell
Migration
Hanieh Zargham, Thao Trinh, Gaetan Thibault, Ramin Zargham; IRCM, Montreal, Canada
Vascular smooth muscle cell (VSMC) migration is an integral part of the pathogenesis of
atherosclerosis. Sumac (Rhus coriaria) leaves are believed to have cardio-protective effects.
Therefore, Sumac, which is a rich source of tannin antioxidants, was tested for its capacity to
inhibit VSMC migratory activity. Materials & Methods: Tannin was extracted and purified from
ground sumac. Cultured rat carotid VSMCs were treated with different concentrations of tannin.
Transmembrane migration assay was used to detect VSMC migratory activity as a response to
platelet derived growth factor (PDGF)-BB. After 10 days of tannin treatment, equal number of
VSMCs were loaded on to the top of the inserts as well as the bottom of the wells. Cells
migrated through the inserts and also cells seeded on the bottom of the wells were counted
after fixation and staining. Results: A significant reduction (62%) of VSMC migration was
observed in the tannin treated cells. To rule out any possible toxicity and cell death, only cells
on the bottom of the wells were counted. No difference between the tannin-treated group and
the controls was observed in the number of cells seeded on the bottom of the wells.
Conclusion: Our data suggests that tannin extracted from Sumac possesses potent antimi-
gratory activity. Sumac may have implications for the prevention or treatment of atherosclerosis
and its clinical manifestations. Further experimentations, especially in vivo, are required to
examine the cardioprotective effect of Sumac.
P275
Carboxyl Ester Lipase Deficiency Exacerbates Dietary Lipid Absorption
Abnormalities and Resistance to Diet-Induced Obesity in Pancreatic
Triglyceride Lipase Knockout Mice
Dean Gilham, Eric D Labonte , Juan C Rojas, Ronald J Jandacek, Philip N Howles, David Y
Hui; Univ of Cincinnati, Cincinnati, OH
Pancreatic triglyceride lipase (PTL) is generally accepted as the principal enzyme involved in the
hydrolysis of dietary fat, thereby mediating its absorption. However, triacylglycerol (TAG)
absorption was minimally altered in PTL-/- mice. This study tested the hypothesis that the
compensatory enzyme in TAG hydrolysis and absorption in PTL-/- mice is carboxyl ester lipase
(CEL). PTL/CEL-/- double knockout mice were generated via crossbreeding. The ability of wild
type, PTL-/-, CEL-/- and PTL/CEL-/- mice to take up dietary lipids and to attain body weight on
a high fat diet was assessed. Net TAG absorption was reduced from 91.50.7% in wild type
mice to 80.13.7% in PTL-/- mice (p0.05). Strikingly, this was reduced to 61.13.8% in
PTL/CEL-/- mice (p0.01). Defective free cholesterol absorption reported previously in PTL-/-
mice was confirmed in this study. Cholesterol absorption in PTL-/- mice was 41% less than
wild type (p0.05), but this was not exaggerated in PTL/CEL-/- mice (44% less than wild type;
p0.01). Additionally, absorption of retinyl palmitate from the intestinal tract to the plasma was
reduced by 45% and 60% in PTL-/- mice (p0.05) and PTL/CEL-/- mice (p0.01)
respectively. On a high fat diet, food intake was not different between mice with the various
genotypes. After 15 weeks on the high fat diet, body weight of CEL-/- mice increased as wild
type mice, but PTL-/- and PTL/CEL-/- mice weighed an average of 6.2 g and 8.6 g less
respectively (p0.01). Body composition analysis showed that both PTL-/- and PTL/CEL-/-
mice consuming the high fat diet carry less fat and more lean mass than wild type mice. In
summary, we show that PTL and CEL together are responsible for a major portion of dietary
fat and fat-soluble vitamin absorption. Our results support free cholesterol absorption requiring
efficient TAG hydrolysis in the proximal gut. Although the residual 5060% of dietary TAG
absorption in PTL/CEL-/- mice suggests an additional TAG lipase exists in the gut, deficiency
of both PTL and CEL confers protection against diet-induced obesity. Thus specific inhibition of
PTL and CEL may be a therapy to suppress diet-induced obesity without fat-soluble vitamin
deficiency or steatorrhea associated with total inhibition of lipolytic enzymes in the intestinal
tract.
P276
Intestinal Cholesterol Uptake in SR-B1 and Niemann-Pick C1 Like 1
(NPC1L1) Knockout Mice
Sean M Lally, Lizbeth M Hoos, Maureen Maguire, Glen T Tetzloff, Li-ji Zhu, Harry R Davis,
Scott W Altmann; Schering Plough Rsch Institute, Kenilworth, NJ
Niemann Pick C1 Like1 (NPC1L1) is a sterol transporter localized in jejunal enterocytes and is
critical for intestinal cholesterol uptake and absorption. Scavenger Receptor-B1 (SRB1) is also
localized in jejunal enterocytes and has been proposed to play a role in cholesterol absorption.
We determined if deficiency of SR-B1 and NPC1L1 results in additional effects on cholesterol
absorption and whole-body cholesterol homeostasis using knockout mouse models. Plasma
cholesterol levels (mg/dl) were measured in 4 animal groups: 133 in wild type (WT), 105 in
NPC1L1 (-/-), 223 in SR-B1 (-/-) and 213 in SR-B1/NPC1L1 (-/-) chow-fed mice. Increases in
plasma cholesterol levels in SRB1 (-/-) and SR-B1/NPC1L1 (-/-) groups occurred in HDL-C.
Hepatic cholesterol levels were not different among the groups. Cholesterol absorption (fecal
dual isotope) was 55% WT, 5.7% (-90%) NPC1L1 (-/-), 50.3% SR-B1 (-/-), and 4.2% (-92%)
SR-B1/NPC1L1 (-/-). Acute 2 hr cholesterol absorption was reduced 82% in both NPC1L1 (-/-)
and SR-B1/NPC1L1 (-/-) groups. Intestinal uptake was reduced approximately 50% in both
NPC1L1 (-/-) and in SR-B1/NPC1L1 (-/-) mice indicating that SR-B1 deficiency causes no
statistically significant additional effects on cholesterol uptake and absorption beyond the
reductions in NPC1L1 (-/-) mice. Mice fed a cholesterol/cholate diet were evaluated. Hepatic
cholesterol levels for SR-B1 (-/-) mice on diet showed a 3-fold increase compared to wild type
mice. There was a 2.5 fold reduction in cholesterol ester levels in the SR-B1/NPC1L1 (-/-)
compared to wild type animals, with no change when compared with the chow diet. NPC1L1
(-/-) mice showed no difference between chow and cholesterol fed diets. To better understand
the changes in cholesterol levels observed in response to diet, mRNA expression levels of
pertinent cholesterol metabolism genes were measured in the intestine and the liver. From the
results, it would appear that in mice, NPC1L1 seems to be the critical intestinal cholesterol
transporter, with no apparent role for SR-B1 in intestinal cholesterol absorption.
P277
12/15-Lipoxygenase Activity Reduces ABCG1-Mediated Cholesterol Efflux
Through Increased ABCG1 Phosphorylation in Macrophages
Melissa H Nagelin, Allison J Wojcik, Jeremy P Mauldin, Suseela Srinivasan, Jerry L Nadler,
Catherine C Hedrick; Univ of Virginia, Charlottesville, VA
A key step in early atherogenesis is the formation of lipid-laden foam cells. Oxygenation of
low-density lipoprotein (LDL) is a critical step in foam cell formation. There are multiple
mechanisms for in vivo LDL oxidation, and the enzyme 12/15-lipoxygenase (12/15LO)
contributes to this oxidation. 12/15LO incorporates molecular oxygen in a stereospecific
manner into arachidonic acid to form 12- and 15-S-hydroxyeicosatetraenoic acids (12SHETE/
15SHETE). Disruption of the 12/15LO gene protects mice from atherosclerotic lesion formation,
while transgenic mice that overexpress 12/15LO are more prone to lesion development. We
recently found that J774 macrophages that stably overexpress leukocyte 12/15LO (Plox-86)
have a 35% reduction in cholesterol efflux to HDL compared to mock-transfected cells (Mock
4). Treatment of control J774 cells or Mock 4 with the 12/15LO product 12SHETE (500nM, 24
hours) also reduced efflux to HDL (15% reduction in J77412SHETE and 34% reduction in
Mock 412SHETE). Concurrently with the reduction in efflux, we saw a 2-fold reduction in
protein expression of the ABC transporter ABCG1 following 24 hour treatment of macrophages
with 12SHETE or in Plox-86 12/15LO-expressing macrophages, indicating that the reduction in
2007 ATVB Poster Presentations e-85
by on March 25, 2011 atvb.ahajournals.org Downloaded from
cholesterol efflux is due to reduced ABCG1 expression. However, we found no changes in
ABCG1 mRNA expression in these macrophages by 12/15LO products, suggesting that the
decrease in ABCG1 protein observed by 12/15LO activity is not caused by transcriptional
changes in ABCG1 mRNA. Thus, we hypothesized that ABCG1 is regulated post-transcriptionally
by 12/5LO products. We examined ABCG1 phosphorylation by immunoprecipitation (IP). IP
studies revealed increased threonine and tyrosine phosphorylation of ABCG1 following
treatment with 12SHETE (500nM, 24 hours) or in Plox-86 12/15LO-expressing cells. This
protein phosphorylation may lead to degradation of ABCG1, thereby increasing macrophage
foam cell formation via reducing cholesterol efflux from the macrophage. Thus, these data
provide evidence for a novel role of 12/15LO in promoting foam cell formation. Understanding
the role of ABCG1 in macrophages will aid in developing beneficial therapies that target genes
involved in cholesterol metabolism.
P278
The Role of ES-4 as a Neutral Cholesteryl Ester Hydrolase in Hepatic Cells
Saj Parathath, Snjezana Dogan, NYU Med Sch, New York, NY; Earl H Harrison, Ohio State
Univ, Columbus, OH; Edward A Fisher; NYU Med Sch, New York, NY
The ability of cells to control cholesterol levels is a balance between the de novo synthesis and
uptake of cholesterol versus secretion and efflux of cholesterol. Excess cholesterol stored as
can be toxic and is stored as cholesteryl esters (CE). CE can supply free cholesterol (FC) to be
utilized for synthesis of steroids, bile acids or for efflux from the cell, through the action of
non-lysosomal CE hydrolases. These hydrolase(s) have been given the general name neutral
cholesteryl ester hydrolase (NCEH). Our data indicate that the protein esterase 4 (ES-4) is a
strong candidate as a rat hepatic NCEH. We have determined in rat hepatic cells the effects of
modulating ES-4 levels using siRNA and overexpression, on total cholesterol, CE, and FC levels
and FC efflux. Rat hepatoma McA cells overexpressing ES-4 showed decreased CE levels
compared to control cells indicating that ES-4 can hydrolyze CE in intact cells. Also in cells
where ES-4 levels were suppressed by 75% by siRNA, there was increased (by 50%) CE levels.
To determine whether the change in CE was from ES-4 mediated hydrolysis, we used
radiolabeled H
3
-CE in McA cells with varying ES-4 levels. The results showed that in
ES-4-siRNA treated cells there was a 40% reduction in CE hydrolysis and an increase of 60%
hydrolysis in cells overexpressing ES-4 compared to control cells. In cholesterol efflux studies,
cells with suppressed ES-4 levels showed reduced efflux of FC to 10% serum; cells
overexpressing ES-4 had increased efflux. These results strongly indicate that ES-4 is a NCEH
in hepatic cells. Currently we are evaluating the role of ES-4 in cholesterol metabolism using
primary rat hepatocytes.
P279
Identification of a Novel Lipid Efflux Defect That Is Not Due to Mutations in
the ABCA1 Gene but to Regulation of ABCA1 Protein
Shirya Rashid, Michel Marcil, Isabelle Ruel, Jacques Genest; McGill Univ, Montreal, Canada
Currently, HDL deficiency due to genetic causes is attributed to mutations in three genes -
LCAT, Apo A1 and ABCA1. These genes, however, do not account for the majority of cases of
low HDL. We have identified 42 French-Canadian subjects with severe HDL-c deficiency (5
th
percentile of the population) in whom mutations in LCAT, Apo A1 and ABCA1 have been
excluded (via candidate gene, haplotyping and linkage analysis approaches). To further identify
individuals in whom the low HDL phenotype is due to defective HDL synthesis, cellular lipid
efflux assays were performed in skin fibroblasts from the subjects. The fibroblasts were loaded
with free cholesterol and incubated with lipid-free apoA 1 (24 h for each) to stimulate cellular
lipid efflux. The assay results indicated two probands in whom both cholesterol and
phospholipid efflux are defective (75% and 70% of normal controls) in response to cholesterol
stimulation (20ug/mL). However, stimulation with 22-hydroxycholesterol/9 cis retinoic acid
(22OH/RA) (2.5 ug/mL) resulted in near-normal cholesterol and phospholipids efflux. To
investigate the cause of the efflux defect in the probands, we utilized real time RT-PCR and
Western blot approaches. We investigated the expression of ABCA1 mRNA (using RTPCR) in
response to both cholesterol stimulation and 22OH/RA stimulation (dose-response and
kinetics). We observed no difference in ABCA1 mRNA expression between normal control and
patient fibroblasts in response to either cholesterol or 22OH/RA stimulation. Interestingly,
Western blot results showed that ABCA1 protein expression is upregulated in response to both
cholesterol and 22OH/RA stimulation in normal control cells but in patient cells only 22OH/RA
stimulation resulted in increased ABCA protein expression. This identifies a new lipid efflux
defect in humans that is not due to mutations in the ABCA1 gene but rather in ABCA1 protein
regulation. Also, binding studies were carried out showing that apoA-I binding to ABCA1 in
patient cells is significantly increased above normal levels with 22OH/RA stimulation, indicating
a possible compensatory effect in lipid binding and efflux with 22OH/RA. Thus, overall, we have
identified a new mechanism leading to impaired ABCA1 mediated lipid efflux in humans.
P280
Human StarD4: Localization and Sterol-transport Characteristics of a
StAR-related Lipid Transfer Protein
Daniel Rodriguez-Agudo, Shunlin Ren, VA Med Cntr/VCU, Richmond, VA; Phillip B Hylemon,
VCU, Richmond, VA; Kaye Redford, VA Med Cntr, Richmond, VA; William M Pandak; VA Med
Cntr/VCU, Richmond, VA
StarD4 mRNA has been detected in heart, lung, liver, and kidney, and is a presumed
intracellular sterol transport protein based upon its START domain. Objective: Characterize this
novel START domain protein by determining its localization and its ability to transport sterols.
Methods: Human StarD4 was His-tag purified and a StarD4 polyclonal antibody generated for
Western analysis and immunocytochemistry. Results: Binding assays showed recombinant
StarD4 was selective in its binding of cholesterol in a molar ratio of 1:1, but was unable to bind
any of the other tested sterols. As we have previously observed with StarD1 and StarD5,
Western analysis detected high levels of StarD4 protein in human monocytes/macrophages.
Despite prior detection of high mRNA levels within liver tissue, StarD4, like StarD5, was not
found in freshly isolated or cultured human hepatocytes. Immunocytochemistry localized
StarD4 intracellularly to the macrophage cytosol. Cellular fractionation confirmed cytosolic
localization of full length (24kDa) StarD4 with a smaller (18kDa) StarD4 degradation band also
detected in the mitochondrial fraction. To corroborate cholesterol binding observed with
recombinant StarD4, evidence for intracellular cholesterol binding/transport/metabolism was
looked for following StarD4 expression in hepatocytes, i.e. cells which do not express
detectable StarD4. Interestingly, StarD4 overexpression led to a 5-fold increase in bile acid
synthesis via the CYP27A1 initiated mitochondrial pathway and an increase in cholesterol ester
formation. Increased cholesterol ester formation was also observed following StarD4 overex-
pression in macrophages. Conclusion: The presence of StarD4, previously described as
ubiquitous to sterol metabolizing tissues, can at this time be ascribed to its localization within
tissue macrophages. Furthermore, its selective binding of cholesterol and its ability to
mobilize/transport cholesterol suggest StarD4 to be an important intracellular cholesterol
transporter in macrophages. Accumulating evidence suggests that StarD1, StaD5, and StarD4
transport and target cholesterol in a selective fashion important to the maintenance of
macrophage cholesterol homeostasis.
P281
A Novel Endoplasmic Reticulum-Derived Organelle Abundantly Present in
Cholesterol-Rich Human Macrophages and Expresses Elevated Acyl-CoA:
Cholesterol Acyltransferase 1 Enzyme Activity
Naomi Sakashita, Kumamoto Univ, Kumamoto, Japan; Catherine C Chang, Dartmouth Med
Sch, Hanover, NH; Xaofeng Lei, Motohiro Takeya, Kumamoto Univ, Kumamoto, Japan;
Ta-Yuan Chang; Dartmouth Med Sch, Hanover, NH
Under hyperlipidemic condition, aortic macrophages continue to internalize modified low-
density lipoprotein (LDL), and are transformed into cholesteryl ester-rich foam cells. The
modified LDL liberates free cholesterol, which is converted to cholesteryl esters by the enzyme
acyl coenzyme A:cholesterol acyltransferase 1 (ACAT1). These events occur during early stages
of atherogenesis. Using immunoelectron microscopy, we had previously shown that, in human
monocyte-derived macrophages grown under normal lipidemic condition, ACAT1 is mainly
located in the endoplasmic reticulum (ER); however, when these cells are overloaded with
modified LDL, a significant portion of the ACAT1 signal is present in small, ER derived vesicles.
To further pursue this finding, in the current work, we prepared postnuclear cell homogenates
from the THP-1 macrophages, and subjected them to subcellular fractionation using Opiti-prep
ultracentrifugation. Each subcellular fraction was analyzed for ACAT1 protein content and for
ACAT enzyme acitivity in vitro. The results show that, under normolipidemic condition, the
ACAT1 protein is mainly distributed among the middle density fractions characteristic of the ER;
the ACAT enzyme activity in vitro in each fraction is low. After treating the cells with aggregated
LDL, a significant portion of the total ACAT1 protein emerge in a low buoyant density fraction;
the ACAT enzyme activity in this fraction is much higher than those present in the normal ER
fractions. Further purifications by using differential centrifugation and immunoisolation
procedures disclosed that the low-density, ACAT1 activity rich fraction possess markers for
both the ER and for the trans-Golgi network, but are devoid of the marker for the plasma
membrane. Confocal microscopic analysis suggested that, in cells treated with aggregated
LDL, the ACAT1 signal significantly colocalizes with the trans-Golgi network signal. Overall,
these data suggest that cholesterol loading of macrophages induces the formation of a novel
cellular compartment derived from ER, and is rich in cholesterol content. ACAT1 residing in
such compartment can efficiently esterifies cholesterol, thus preventing excessive built up of
free cholesterol in the ER.
P282
12/15-lipoxygenase Deficiency Decreases Neointimal Formation in Male
ApoE-knockout Mice in Response to Endothelial Denudation
Hamid Deliri, Rohit Malhotra, James W Chumley, Stephanie N Oldham, John M Sanders,
Nahum Meller, Coleen McNamara; Univ of Virginia Health System, Charlottesville, VA
Background: Vascular smooth muscle cell (VSMC) proliferation is a key component of the
response to injury in vascular disease. The hyperlipemic apolipoprotein E deficient mouse
(ApoE
-/-
) is widely used as a model of neointimal formation in response to carotid wire injury.
12/15-lipoxygenase enzyme (12/15-LO), homologous to human 15-lipoxygenase, has been
implicated in the pathogenesis of VSMC proliferation in vitro and in response to endothelial
denudation in the normolipemic rat carotid endothelial denudation model. The in vivo role of
12/15-LO in response to injury in a hyperlipemic state is unknown. Hypothesis: Mice deficient
in both ApoE and 12/15-LO (DK) will have reduced neointimal formation following endothelial
denudation as compared to ApoE
-/-
mice. Materials/Methods: DK and ApoE
-/-
mice were
placed on Western diet at 1012 weeks of age. After one week, wire endothelial denudation
of the left common carotid artery (LCCA) was performed. Mice were euthanized 28 days
following injury and the LCCAs were removed and paraffin embedded. Beginning at a point
240m proximal to the carotid bifurcation, eight consecutive sections (each 120m apart)
were collected and stained using Russells modified Movat method. The neointimal area was
then measured using Image-Pro software. Mann-Whitney two-tailed U-test was used for data
analysis. Data is presented as meanSEM. Results: Nineteen DK (9 m, 10 f), and 24 ApoE
-/-
mice (13 m, 11 f) were entered in the study. There was no statistically significant difference
between genotypes when comparing cholesterol, triglyceride, LDL and glucose levels.
Neointimal area was reduced by 54.6% in DK compared to ApoE
-/-
males (26461 8843m
2
vs. 58223 8602m
2
, p0.033) but not in females (40818 7262m
2
vs. 33479
7176m
2
, p0.55). Conclusion: Neointimal formation following endothelial denudation is
reduced in male DK mice, providing evidence that 12/15-LO is a key regulator of neointimal
growth in response to vascular injury in the hyperlipemic ApoE
-/-
mouse. Interestingly, there is
no difference in the neointimal response to injury in female DK mice, suggesting a gender
specific effect of 12/15-LO.
e-86 Arterioscler Thromb Vasc Biol June 2007
by on March 25, 2011 atvb.ahajournals.org Downloaded from
P283
ACE2 Expression in Adipose Tissue Is Regulated by High-fat Diets but Not
by Angiotensin II
Manisha N Gupte; Univ of Kentucky, Lexington, KY
Objectives: Angiotensin converting enzyme type-2 (ACE2) is a monocarboxypeptidase which
cleaves both angiotensin (Ang) I and II to produce Ang17. Recent data suggest that ACE2
functionally controls the renin-angiotensin system (RAS), metabolizing AngII and regulating
blood pressure. ACE2 mRNA is abundantly expressed in mouse adipose tissue, and increases
markedly during differentiation of 3T3-L1 cells. In kidney and aorta, ACE2 is regulated by AngII,
ACE inhibitors or AT1 receptor (AT1R) antagonists. The purpose of this study to define
mechanisms for regulation of adipocyte ACE2, focusing on components of the RAS, or high fat
(HF) feeding. Methods and Results: We examined the effect of AngII, losartan, or an AT2
receptor antagonist on ACE2 mRNA abundance in differentiating 3T3-L1 adipocytes. While
ACE2 mRNA increased markedly during adipocyte differentiation, there was no effect of
treatments. In adipose tissue from female AT1aR deficient mice, ACE2 mRNA abundance was
not altered. To determine the effect of high fat (HF) feeding on adipose ACE2 expression,
C57BL/6 female mice were fed either normal laboratory diet (chow) or a high fat diet (HF, 45%
Kcal as fat) acutely (1 week) or chronically (20 weeks). Female AT1aR -/- mice were also fed
normal or HF diets for 20 weeks. ACE2 mRNA expression and activity in adipose tissue were
increased within 1 week of HF feeding in C57BL/6 mice. At 20 weeks, body weight (BW) was
increased in C57BL/6 mice, but not in AT1aR-/- mice fed a HF diet. Surprisingly, systolic blood
pressure (SBP) was decreased by HF feeding in C57BL/6 (by 17 mmHg) and AT1aR-/- mice (by
13 mmHg). ACE2 mRNA abundance was markedly increased in adipose tissue from 20 week
C57BL/6 mice fed a HF diet (ACE2/18s rRNA: chow: 0.19 0.01, HF: 0.51 0.05; P0.05),
but was not altered in the kidney. Similar effects of HF feeding on adipose ACE2 expression
were observed in AT1aR-/- mice. Conclusions: These results demonstrate that adipose ACE2
expression is not regulated the RAS. In contrast, HF feeding markedly increased adipose ACE2.
Elevations in adipose expression of ACE2 by HF feeding were accompanied by a reduction in
blood pressure. Regulation of ACE2 by specific types of fatty acids may serve as a protective
mechanism against obesity-induced cardiovascular disease.
P284
An Insertion Mutation in the Promoter Increased Myosin Light Chain
Kinase Expression in Hypertension
Yoo-Jeong Han, Wen-Yang Hu, Mariann Piano, Primal de Lanerolle; Univ of Illinois at
Chicago, Chicago, IL
Introduction- Blood pressure is a strong genetic trait that is determined by quantitative trait
loci of an individual. Many candidate genes responsible for regulating blood pressure have been
identified. However, relatively few studies have focused on the genes for smooth muscle
contractile proteins despite the fact that smooth muscle contractility and growth are key
contributors to vascular function and resistance. Smooth muscle contraction is regulated by the
actin-myosin II interaction, which in turn is regulated by phosphorylation and dephosphorylation
of regulatory myosin light chain (RLC). The RLC phosphorylation is catalyzed primarily by
smooth muscle myosin light chain kinase (smMLCK). Therefore, changes in the expression of
smMLCK could elicit changes in blood pressure. Hypothesis- We hypothesized that a genetic
mutation in the smMLCK promoter could alter smMLCK expression and contribute to the
development of high blood pressure. Methods and Results- We investigated the regulation of
smMLCK gene expression using spontaneously hypertensive rats (SHR) as an experimental
model. Expression of smMLCK in arteries increases during the development of high blood
pressure and is always greater in blood vessels from SHR compared to normotensive rats.
Analysis of the DNA sequences of the promoters isolated from SHR and normotensive rats
revealed that SHR contain a 12 bp insertion. This insertion consists of 6 pairs of CT repeats and
adopts a non-B DNA structure, an intramolecular triplex DNA structure in supercoiling DNA. This
structure positively regulates the promoter activity in SHR partly by increasing histone dynamics
on the region. In vivo, inhibiting smMLCK activity decreases blood pressure in SHR. Conclusion
and Prospective- These data provide novel insights into the genetic factors that increase blood
pressure and demonstrate the importance of smMLCK expression in the development of
hypertension in SHR. These animal studies lay the foundation for studies on human
hypertension and possibly provide new insights into diagnosis and treatment of hypertension
in humans.
P285
Apolipoprotein B Gene Mutations and Fatty Liver Disease in Japanese
Hypobetalipoproteinemia
Shoji Katsuda, Masa-aki Kawashiri, Akihiro Inazu, Hayato Tada, Masayuki Tsuchida,
Yoshibumi Kaneko, Atsushi Nohara, Toshihide Okada, Junji Kobayashi, Hiroshi Mabuchi,
Masakazu Yamagishi, Kanazawa Univ, Kanazawa, Japan; Tsuyoshi Nozue, Ichiro Michishita;
Yokohama Sakae Kyosai Hosp, Yokohama, Japan
Background: Familial hypobetalipoproteinemia (FHBL) is an autosomal dominant hereditary
disorder characterized by decreased plasma levels of low-density lipoprotein cholesterol
(LDL-C). FHBL is considered as genetically heterogeneous, and the best-characterized cases
are due to mutations of the apolipoprotein B (apoB) gene, which produce truncated forms of
apoB. FHBL are thought to be related to nonalcoholic fatty liver disease (NAFLD), which may
progress to nonalcoholic steatohepatitis (NASH) or liver cirrhosis. However, few data exist
regarding the relationship between FHBL and NAFLD. Methods: To screen truncated apoB
variants, we performed immunoprecipitation of apoB protein in 14 subjects with primary
hypobetalipoproteinemia (LDL-C70 mg/dl). For those without truncated apoB in plasma, we
performed PCR single-strand conformational polymorphism analysis of the apoB gene to detect
truncated apoB shorter than apoB-30, which is usually absent from plasma. Results: We
identified two individuals with truncated apoB: an apoB-82 homozygote and a heterozygote for
a novel truncation termed apoB-13.7. The apoB-82 homozygote is asymptomatic despite her
extremely low levels of LDL-C (13 mg/dl). Moreover, fat-soluble vitamin levels are normal,
possibly due to spared secretion of apoB-48 and increased levels of high-density lipoprotein
cholesterol (89 mg/dl). NAFLD was observed in 7 of the 14 subjects, including both patients
with apoB-82 and apoB-13.7. The parents of the apoB-82 homozygote, also, had fatty liver. We
performed liver biopsy in the apoB-13.7 patient, who was diagnosed as severe fatty liver by
computed tomography. The liver showed simple steatosis but no diagnostic evidence of NASH.
Conclusions: Our results demonstrate that apoB gene mutations might not be rare and that
NAFLD might be frequent in Japanese hypobetalipoproteinemia.
P286
Insulin Inhibits Inflammation in Endotoxemic Mice in a Phosphatidylinositol
3-Kinase-dependent Manner
Linda Kidd, Gernot A Schabbauer, James P Luyendyk, Rachel E Tilley, Michael Tencati,
Todd Holscher, Nigel Mackman; The Scripps Rsch Institute, La Jolla, CA
Abstract Insulin reduces inflammation and morbidity in critically ill patients when used to
control pro-inflammatory hyperglycemia. Recent studies indicate that insulin may have
glucose-independent anti-inflammatory effects in endotoxemia models. To date, the mecha-
nism by which insulin reduces inflammation has not been elucidated. Insulin is a well known
activator of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway. We
hypothesized that insulin activation of the PI3K/Akt pathway mediates its protective effects
during endotoxemia. We have shown that activation of this pathway reduces LPS induction of
pro-inflammatory cytokines and mortality in endotoxemia. The objectives of this study were; 1)
to establish a model that tests if insulin decreases inflammation, morbidity, and mortality during
endotoxemia and 2) to determine if these protective effects are mediated by activation of the
PI3K/Akt pathway. Using a non-hyperglycemic mouse model of endotoxemia, we continuously
administered a very low dose of insulin that did not alter blood glucose levels. We found that
insulin decreased plasma levels of IL-6, TNF, and sICAM-1, and decreased morbidity and
mortality. Furthermore, the effects of insulin were enhanced in p85
-/-
mice, which have
increased insulin-mediated PI3K/Akt signaling. Finally, treatment of endotoxemic C57BL6/J
mice with a PI3K inhibitor (wortmannin) abolished the anti-inflammatory effects of insulin. We
conclude that insulin reduces LPS-induced inflammation, morbidity, and mortality through
activation of the PI3K/Akt pathway. These findings may ultimately have important implications
regarding the use of insulin for treating normoglycemic critically ill patents.
P288
Tissue Factor mRNA Isoforms Expression and Modulation in
Lymphomonocytes and Platelets of Patients with Acute Coronary
Syndromes
Marta Brambilla, Univ of Milan, Milano, Italy; Deborah Colnago, Monica Demetrio, GianCarlo
Marenzi, Karl Gertow, Paolo Biglioli, Elena Tremoli, Marina Camera; Cntr Cardiologico
Monzino, Milano, Italy
Tissue Factor (TF), the key initiator of the coagulation cascade, is responsible for the
thrombogenicity of the atherosclerotic plaque. Studies on patients with acute coronary
syndrome (ACS) showed that TF plasma levels, monocyte- and platelet-associated TF are
higher than in stable angina (SA) patients. Recently, an alternative spliced form of TF (asTF) has
been discovered, which is soluble, circulates in the blood and exhibits procoagulant activity.
Purpose. To examine TF and asTF mRNA expression in lymphomonocytes and platelets of
patients with ACS, SA and in control subjects. Methods. We studied 16 patients with ACS, 14
patients with SA and 12 healthy subjects. The three groups were matched for age, gender and
other clinical variables. Total RNA was extracted from peripheral lymphomonocytes and from
washed platelets free of leukocyte contamination and full length TF as well as asTF mRNA
levels were assessed by RT-PCR and real time PCR. Results. TF mRNA expression in resting
lymphomonocytes was barely detectable in all subjects. Conversely, a consistent expression of
asTF mRNA levels was observed in ACS (rel. exp: 0.38 0.06, p0.05) compared to in SA
patients (rel. exp: 0.19 0.04) and controls (rel. exp: 0.12 0.05). In vitro lipopolysaccharide
stimulation of lymphomonocytes upregulated TF mRNA expression in all samples with the
highest induction observed in ACS patients (TF rel. exp. vs unstimulated sample: 151.4 24.3
in ACS, 57.8 5.2 in SA and 26.8 3.4 in control subjects; p0.05 vs control). By contrast,
the asTF induction by lipopolysaccharide was similar in ACS and SA patients and in control
subjects, (asTF rel. exp. vs unstimulated samples: 38.3 13 in ACS, 54.8 6.7 in SA and
56.7 9.4 in control subjects). Platelet associated TF mRNA levels were significantly higher
in ACS patients (rel. exp: 3.11 0.51, p0.05) compared to SA (rel. exp: 2.5 0.87) and
control subjects (rel. exp: 0.7 0.1). No asTF mRNA was detectable in any platelet sample.
Conclusions. These data provide evidence for the first time that different TF mRNA isoforms
present in linfomonocytes and platelets of ACS patients can contribute to the hypercoagulability
associated with the disease.
P289
Acute Aortic Dissection as an Inflammatory Disease: Acute Phase Protein
and Cytokine Changes and Prognostic Implications
Wei-Tien Chang, Chien-Hua Huang, Matthew H Ma, Min-Shan Tsai, Chiung-Yuan Hsu,
Yuan-Teh Lee, Fang-Yue Lin, Wen-Jone Chen; National Taiwan Univ Hosp, Taipei, Taiwan
Introduction: Inflammation has been implicated in the pathogenesis of cardiovascular
diseases. The role of inflammation in acute aortic dissection, however, has not been thoroughly
investigated. Hypothesis: We hypothesize that inflammatory response occurs in acute aortic
dissection. The level of inflammation is associated with different pathogenic processes and is
of prognostic implication. Methods: From Jan. 2001 to July 2003, 32 patients presenting with
acute aortic dissection to a tertiary medical center were included. Eight patients with
comparable blood pressure and no aortic and inflammatory diseases served as the control. The
serum was sampled on arrival of the emergency department for measuring high-sensitivity C
2007 ATVB Poster Presentations e-87
by on March 25, 2011 atvb.ahajournals.org Downloaded from
reactive protein (hs-CRP), interleukin (IL)-1, IL-6, and tumor necrosis factor- (TNF-).
Comparisons were made between aortic dissection and the control groups, patients with
Marfan syndrome and those without, and patients with and without leakage/rupture. The
correlations with the survival outcome were also evaluated. Results: Compared to the control,
the patients with aortic dissection had significantly higher hs-CRP (1.98 2.66 vs. 0.17
0.11 mg/dl, p0.001) and IL-6 (25.86 16.82 vs. 1.32 0.76 pg/ml, p0.001). The levels
in patients without Marfan syndrome were even higher (hs-CRP: 2.26 2.89 mg/dl, IL-6:
30.40 15.36 pg/ml, P0.05 and 0.001 vs. control). For Marfan syndrome, only IL-6 was
significant higher than control (12.23 13.94 pg/ml, p0.05) and the extent was smaller. The
hs-CRP is not significantly different from the control. In patients with evidences of
leakage/rupture, the IL-6 was higher than those without (33.04 10.62 vs. 21.55 18.55
pg/ml, p0.05). No significant differences were noted in IL-1 and TNF-. For prognosis, the
patients who failed to survive to discharge had significantly lower hs-CRP than those who
survived (0.72 1.19 vs. 2.33 2.86 mg/dl, p0.05). The level was indeed not significantly
different from the control. Conclusion: Acute aortic dissection is associated with increased IL-6
and hs-CRP. IL-6 may implicate different pathogenic processes and the chance of leakage/
rupture. Poor initial hs-CRP response, on the other hand, is correlated with poor survival
prognosis.
P290
PKD, a Novel Component in Lysophosphatidylcholine-triggered Signaling
Pathway, Controls Egr-1 Expression in Monocytic Cells
Mingqi Tan, Feng Hao, Xuemin Xu, Univ of Tennessee, Knoxville, TN; Guy M Chisolm,
Lerner Rsch Institute, Cleveland, OH; Mei-Zhen Cui; Univ of Tennessee, Knoxville, TN
Monocyte activation is an important early event in the development of atherosclerosis and other
inflammatory diseases. The nature of monocyte activation is not completely understood. We
report here that lysophosphatidylcholine (lysoPC), a prominent component of oxidized low
density lipoprotein and present in arterial lesions, induces rapid and marked protein kinase D
(PKD) activation in monocytic THP-1 cells. Our data also reveal that PKD activation is required
for the activation of both ERK and p38 MAPK. Activation of ERK MAPK, but not p38 MAPK,
controls the expression of the early growth response gene (Egr-1), which has been reported to
be a key transcription factor regulating many cellular functions in atherosclerotic lesions. Our
results have also identified that, of the three components of PKD, namely PKD1, PKD2 and
PKD3, only PKD2 is expressed in these cells. PKD2 controls Egr-1 expression through ERK
MAPK after lysoPC activation. Our results reveal that PKD is a novel intracellular component in
lysoPC signaling pathways and in monocytic cell activation. Furthermore, our data point to a
novel function for PKD in the regulation of Egr-1 expression. Using multiple approaches,
including siRNA silencing and dominant negative mutants, we conclude that lysoPC-induced
PKD2 activation is required for Egr-1 expression in monocytic THP-1 cells. Our results suggest
a role for PKD in the development of atherosclerosis.
P291
MCP-1/CCR2 Involves AT1 Receptor Antagonism Induced Anti-inflammation
via PI3K/Akt in SHR
Qiuyan Dai, Mengdan Xu, Shanghai First Peoples Hosp, Shanghai, China; Min Yao,
Massachusetts General Hosp, Harvard Med Sch, Boston, MA; Baogui Sun; Shanghai First
Peoples Hosp, Shanghai, China
To understand what is the mechanism involve in the AT1A-induced anti-inflammation, we
investigate whether the MCP-1 and its receptor involves in this process and how to
modulated.Methods: We investigated whether AT1A has the effect on anti-inflammation of
blood vessel and what relationship with systolic pressure decreased by using several AT1A in
different doses or without treatment in SHR. Twenty-four male SHRs were treated with vehicle
(HC), or Losartan including low and high doses (LL, HL), or Telmisartan (T) for 4 wk. Six WKYs
with same age as SHR were used as a control(NC). The macrophages in aorta vessel wall were
measured by anti-ED-1 antibody immunolabelling. The left ventricular mass of heart/ body
weight , aorta intimal-midial thickness / Diameter, and aorta perivascular fibrosis ration be
detected after AT1A treatments; The gene expression and protein level of MCP-1/CCR2 were
determined in peripheral circulation by RT-PCR and ELISA;Signaling pathway involved in the
AT1A inhibited-MCP-1 secretion were performed by pre-treating the mouse monocytes with
Ang II, AT1A, or Ang II plus PI3K inhibitor, and then determining the MCP-1and the
phosphorylation of Akt were detected by western blotting.Results: Compared with NC Group,
ED-1 positive cells of the aorta wall in the HL, LL and T groups were significantly increased
(P0.01). Different doses of AT1As effectively causes an effect of anti-inflammation on blood
vessel wall of SHR, which is independent of systolic pressure decreased. The gene expressions
of MCP-1 and CCR2 in heart, kidney, aorta and mononuclear cells in peripheral circulation and
the protein level of MCP-1 in serum were significantly increased after treatments (P0.01).
AT1A improved the cardiovascular remodeling in SHR, especially in high-dose. The phosphor-
ylation of PI3K/Akt kinase invovles in the AT1A-inhibited MCP-1 secretion in mouse
monocytes.Conclusions: AT1A has an effect of anti-inflammation on blood vessel wall in SHR,
which is independent of systolic pressure decreased; and this function might useful for
improving the cardiovascular remodeling, especially in high-doses. MCP-1/CCR2 modulates the
AT1A-induced anti-inflammation in SHR through PI3K/Akt signaling pathway.
P292
Histone H2B as a Functionally Important Plasminogen Receptor on
Macrophages
Riku Das, Tim Burke, Thomas Herren, Edward F Plow; Cleveland Clinic Foundation,
Cleveland, OH
Active inflammation has been identified as a defining feature of vulnerable atherosclerosis
plaques which rupture and lead to arterial thrombosis and cardiovascular events, such as
myocardial infarction, stroke and peripheral occlusive disease. Plasminogen (Plg) facilitates
macrophage migration to sites of inflammation and injury. The cellular functions of Plg depend
upon its binding to receptors (Plg-Rs), which facilitate its activation to plasmin (Plm), protect
Plm from inhibition and harness protease activity to the cell surface. However, the particular
Plg receptors (Plg-Rs) that are involved in Plg-mediated macrophage migration during
inflammation are not well defined. We have investigated the expression of three previously
characterized Plg-Rs, -enolase, annexin II and p11, on the surface of two mouse macrophage
cell lines (RAW264.7 and J774A.1) and on thioglycollate (TG) induced mouse peritoneal
macrophages. In addition, we have also characterized surface expression and function of
histone H2B (H2B), a newly identified Plg-R on these cells. Using Fab fragments of anti-H2B,
anti--enolase, anti-annexinII, and p11, that each specifically blocks Plg binding to their target
proteins, we have shown that all of these receptors were contributed to Plg binding to the
macrophage cell lines, with H2B playing a particularly prominent role. On TG-induced
macrophages, H2B contributed 4550% of the Plg binding capacity, whereas -enolase,
annexin II and p11 contributed 25%. In an in-vitro matrigel invasion assay, a function of H2B
in the cellular response could also be demonstrated. Interestingly, treatment of mice with
anti-H2B Fab led to a marked reduction in macrophage recruitment (50%) towards TG,
whereas Fab to another Plg-R or nonimmune Fab had limited effects. Taken together, these
data suggest that multiple Plg-R contribute to Plg binding to macrophages, and among these,
H2B plays a very prominent role.
P293
Preferential Accumulation of CX3CR1bright Dendritic-like Cells in
Atherosclerotic Mouse Aortas
Tracy L Deem, John M Sanders, Anthony C Bruce, Klaus Ley; Univ of Virginia,
Charlottesville, VA
Mouse and human blood contain both inflammatory and resident-type monocytes. In this study,
we tracked these monocyte subsets using CX3CR1/GFP knockin mice. In blood, both subsets
express equal amounts of CD115 and F4/80. Inflammatory, CX3CR1dim monocytes express
more dectin-1, Ly-6G, 7/4, CD62L, CD11a, CD49b, CCR2, and I-Ab than resident-type,
CX3CR1bright, which express more CD80. This expression pattern remained unchanged when
CX3CR1/GFP mice were crossed into the ApoE-/- background, except for a 10-fold decrease
in Ly-6c expression in CX3CR1dim cells. GFP bone marrow cells from CX3CR1/GFP mice
express low levels of GFP and no CD11c when differentiated into macrophages by M-CSF, but
high levels of GFP and CD11c when differentiated to dendritic cells (DCs) by GM-CSF and IL-4.
Brightly fluorescent cells with a dendritic cell shape were present in the wall of the aorta and
the femoral artery of CX3CR1/GFPApoE-/- mice, and their number was tripled by feeding
these mice with western diet. Immunofluorescence showed GFP DC-like cells under the
endothelial layer and in the media. Both CX3CR1dim and CX3CR1bright cells in the aortic wall
express CD11c and CD86 at similar levels. CX3CR1dim cells express more CD31, CCR2, CD80,
F4/80, 7/4, CD31, CD62L, CCR7, and I-Ab from CX3CR1bright cells, consistent with a more
mature or activated DC phenotype. CX3CR1bright cells are at least three fold overrepresented
in CX3CR1/GFPApoE-/- mice on western diet, accounting for up to 62% of all GFP cells.
Adoptively transferred bone marrow cells in atherosclerotic aortas of ApoE-/- recipients were
80% CX3CR1bright after 48h. Our data suggest that CX3CR1bright monocytes preferentially
home to atherosclerotic aortas and assume a dendritic cell-like phenotype. Since atheroscle-
rotic aortas also contain large numbers of lymphocytes, it is plausible that these DCs may
participate in an active immune response in the vessel wall.
P294
Development of Bead-based Multiplexed Immunoassays for Simultaneous
Quantification of Circulating Cardiovascular Biomarkers in Rat Serum and
Plasma
Jiaxin Dong, Terry Whitehead, Xiao Qiang, Hank Hwang, Jehangir Mistry, Shaoquan Ji;
LINCO (now part of Millipore), Saint Charles, MO
Rat is one of the common models for biomedical research, including cardiovascular diseases
in elucidating mechanism of action, new drug screening and preclinical trials. Levels of soluble
biomarkers such as BNP, Troponin T, TIMP-1, sICAM-1, sE-Selectin, VEGF, MPO, vWF and
Fibrinogen are associated closely with cardiovascular diseases (e.g. atherosclerosis, throm-
bosis, and hypertension). These are important indicators for disease model establishment, drug
in vivo testing and efficacy evaluation. In order to provide useful tools for researchers, we
developed three multiplexed immunoassay panels based on Luminex

xMAP technology. Panel


A can detect and quantify simultaneously 11 biomarkers (BNP, TIMP-1, MPO, vWF, Troponin I,
Troponin T, IL-6, TNF, MCP-1, VEGF, PAI-1) from as little as 15ul of samples. Panel 2 (soluble
E-Selectin and soluble ICAM-1) and Panel 3 (Fibrinogen and Adiponectin) use less than 1ul of
samples. All three panels require a simple overnight assay protocol. Briefly, diluted samples are
incubated overnight at 4 C in a 96-well microtiter filter plate with a mixed population of
polystyrene beads, which are immobilized with specific capture antibodies. Each bead set
contains two internal fluorophores for bead identification. After washing, the captured analytes
on beads were incubated for 2 hr at RT with a cocktail of biotinylated detection antibodies.
Following subsequent incubation with streptavidin-phycoerythrin, the fluorescent signals on
beads are quantified through a Luminex

Reader. Within each panel, there is no significant


cross-reactivity among different antibody pairs. According to individual needs, the panel can be
customized to detect selective analytes. The assays are analytically robust (sample dilution
linearity, 100 25%; spike recovery, 90 20%) and reproducible (intra-assay CV%, 10%;
inter-assay CV%, 15%). The multiplexed assay panels also may be used for tissue culture
supernatant and tissue/cell lysate samples. These new immunoassay panels provide conve-
nient, flexible and economic tools for simultaneously quantifying multiple CVD biomarkers in
small volume rat samples.
e-88 Arterioscler Thromb Vasc Biol June 2007
by on March 25, 2011 atvb.ahajournals.org Downloaded from
P295
Cloning of Human Antibodies to Oxidation-specific Epitopes from Both
Innate and Adaptive Immunity
Lotte F Hansen, Karsten Hartvigsen, Douglas A Woelkers, Yury I Miller, Univ of California,
San Diego, La Jolla, CA; Sohvi Horkko, Univ of Oulu, Oulu, Finland; Joseph L Witztum; Univ
of California, San Diego, La Jolla, CA
Oxidation-specific epitopes are present on oxidized LDL (OxLDL), on cells undergoing
apoptosis, and in atherosclerotic lesions. Mice have natural germline IgM antibodies (Ab) that
recognize oxidation-specific epitopes. These Abs are part of innate immunity and exhibit
important biological functions in atherogenesis such as ability to bind to apoptotic cells, inhibit
uptake of OxLDL and apoptotic cells by macrophages, and confer atheroprotection in mice. Ab
titers to oxidation-specific epitopes are found in human adult and umbilical cord blood (UCB)
plasma and in atherosclerotic lesions, but it is not known if these Abs are germline. Also, the
nature and role of Abs in general to oxidation-specific epitopes in human atherosclerosis is
poorly understood. Human UCB contains significant IgM Ab titers to oxidation-specific epitopes.
These IgM titers represent a nave immune repertoire without exposure to exogenous antigens
and without clonal expansion of hypermutated Abs. We hypothesize that humans, similar to
mice, have germline Abs to oxidation-specific epitopes that play important roles in atheroscle-
rosis. We have generated human Fab (Ab fragment) phage display libraries from five patients
with familial hypercholesterolemia and seven UCB samples and screened these libraries
against oxidation-specific antigens. The clones from both libraries have been sequenced and
compared to obtain information on gene usage. The clones from the adult libraries are of
adaptive origin whereas several clones from the UCB libraries are near germ-line and one clone
is a full germ-line natural Ab originating from innate immunity. Antigen specificity was
examined for single Fab clones and revealed numerous clones highly specific to certain
oxidation-specific epitopes. Selected Fabs from both the adult and UCB libraries have been
shown by deconvolution microscopy and flow cytometry to bind apoptotic cells and also stain
atherosclerotic lesions.These results show that both adaptive and innate immunity generate
Abs against oxidation-specific epitopes and these Abs bind to apoptotic cells and atheroscle-
rotic lesions. Further studies may elucidate the role of these Abs in atherosclerosis and provide
new insights to diagnosis and treatment of patients.
P296
Comparison of Fenofibrate Alone or Atorvastatin Alone on Plasma
Inflammation, Adhesion, and Oxidation Markers in Type 2 Diabetic
Subjects with Moderate Hypertriglyceridemia
Jean-Charles Hogue, CRML, CHUL du CHUQ, Que bec, Canada; Benot Lamarche, INAF, Laval
Univ, Que bec, Canada; Andre J Tremblay, Jean Bergeron, Claude Gagne , Patrick Couture;
CRML, CHUL du CHUQ, Que bec, Canada
Type 2 diabetes mellitus is associated with elevated plasma triglyceride (TG) levels, low
HDL-cholesterol (C) and high incidence of cardiovascular disease. HMG-CoA reductase
inhibitors or fibrates are frequently used in the treatment of diabetic dyslipidemia but their
specific impact on the inflammatory process involved in atherosclerosis remains to be fully
characterized. The objective of this two-group parallel study was to investigate the differential
effects of atorvastatin 20mg/d alone (N20) or micronized fenofibrate 200mg/d alone (N19)
on inflammation, adhesion and oxidation markers in type 2 diabetic subjects with moderate
hypertriglyceridemia. Atorvastatin decreased plasma-C (-38.2%, P0.0001), plasma-TG
(-38.3%, P0.0001), plasma apo B (-44.1%, P0.0001) and LDL-C (-43.4%, P0.0001), and
increased HDL-C (16.5%, P0.0007). Atorvastatin decreased plasma levels of CRP (-26.9%,
P0.004), sICAM-1 (-5.4%, P0.03), sVCAM-1 (-4.4%, P0.008), sE-selectin (-5.7%,
P0.02), MMP-9 (-39.6%, P0.04), sPLA
2
(-14.8%, P0.04) and oxLDL (-38.4%, P0.0001).
On the other hand, fenofibrate treatment decreased plasma C (-12.1%, P0.0001) and plasma
TG (-41.4%, P0.0002) and increased LDL-C (15.7%, P0.04) and HDL-C (10.0%,
P0.05). Fenofibrate decreased plasma levels of sE-selectin (-6.0%, P0.04) but increased
the plasma levels of sPLA
2
(22.5%, P0.004). Fenofibrate had no significant effect on CRP
levels. In conclusion, the results of the present study suggest that atorvastatin is potent to
reduce inflammation, oxidation and monocyte adhesion in type 2 diabetic subjects with
moderate hypertriglyceridemia while fenofibrate decreased sE-selectin levels only and had an
unwanted effect on sPLA
2
levels.
P297
Effects of Artemisia princeps Pampanini Cultivated Sajabal on LDL
Oxidation and Atherosclerosis in LDLR
-/-
Mice
Min-Jung Kim, Sojin An, Jong-Min Han, Yue-Jan Jin Jin, Tea-Sook Jeong; KRIBB, Daejeon,
Republic of Korea
Oxidatively modified LDL (oxLDL) plays a key role in the development of atherosclerotic lesions.
Many animal and clinical studies have shown the beneficial results of various antioxidants
treatment on reducing atherosclerotic lesions and overall cardiovascular risks. The objective of
this study was to determine the anti-atherosclerotic effects of Artemisia princeps Pampanini
cultivated Sajabal. Jaceosidin isolated from A. princeps Pamp. cv. Sajabal has an LDL-
antioxidant activity. The ethanolic extracts of A. princeps Pamp. cv. Sajabal containing 9.7%
(w/w) jaceosidin reduced atherosclerotic lesions in LDLR
-/-
mice. We explored the effect of
jaceosidin on Cu
2
-mediated human LDL oxidation. Jaceosidin showed the potent LDL-
antioxidant activity with IC
50
values of 3.9 M in the thiobarbituric acid-reactive substances
(TBARS) assay. Jaceosidin inhibited the formation of conjugated diene during Cu
2
-induced
LDL oxidation as well as the human macrophage-mediated LDL oxidation. In in vivo study, the
effect of the extracts was investigated on the atherosclerotic lesion formation in LDLR
-/-
mice.
LDLR
-/-
mice (10 weeks old) were randomly divided into two groups (n 10 per group). The
mice were fed a western-type atherogenic diet alone (control group) or an atherogenic diet
supplemented with the extracts (1% wt/wt diet, SJ group) for 8 weeks. The extracts reduced
the aortic lesion formation and macrophage accumulation at 37% and 43%, respectively,
compared to controls. In aorta, the extracts decreased the transcriptional levels of CD36,
LOX-1, ABCA1, ICAM-1, VCAM-1, TNF-, IL-1, COX-2 and iNOS and increased the levels of
CYP7 family. The extracts not only reduced epididymal fat accumulation (28%) and plasma lipid
peroxidation (16%), but also corrected associated hyperlipidemia. These findings indicate that
the extracts from A. princeps Pamp. cv. Sajabal effectively ameliorates atherosclerotic lesion
formation by inhibiting lipid peroxidation, scavenger receptors (CD36 and LOX-1) and adhesion
molecules (ICAM-1 and VCAM-1) expression, which are crucial for the development of a fatty
streak lesion.
P298
Phenotypic Switching in Macrophages by Oxidized Phospholipids Involves
Toll-like Receptor 2
Alexandra Kadl, Norbert Leitinger; Univ of Virginia, Charlottesville, VA
Functional heterogeneity is a hallmark of cells of the mononuclear phagocyte system.
Macrophages (Ms) have been ascribed both pro- and anti-inflammatory properties. Classical
activation by INF and LPS leads to M1-Ms, while alternative activation by IL4 leads to
M2-Ms, that trigger either a Th1 or a Th2 response, respectively. Recently it has been
hypothesized that Toll like receptors (TLR) recognize endogenous danger signals, such as
oxidized phospholipids (OxPL), and their involvement in the development of atherosclerosis has
been suggested. Here we show that oxidized 1-palmitoyl-2-arachidonoyl-sn-3-glycero-
phosphorylcholine (OxPAPC), polarizes Ms towards a unique phenotype, different from the M1
and M2-types. Phenotypic switching of Ms by OxPL is reflected by upregulation of KC, Mip2,
IL1, COX-2 and HO-1, while the expression of the typical M1 markers TNF and IL12, as well
as the M2 marker arginase 1 are downregulated. This macrophage phenotype exhibits
increased survival, reduced migratory capability and facilitated lipoprotein uptake, and is
present in atherosclerotic lesions. In order to address the question of involved receptors, we
used Ms from mice lacking TLR2. We show that OxPAPC-induced expression of the
chemokines Mip2 and KC is dependent on TLR2, while the expression of IL1 and HO1 is
independent of TLR2. To identify molecular structures in OxPL that determine M polarization
via TLR2 we fractionated OxPAPC into long chain (m/z 782) and short chain fractions (m/z
782). We could show that TLR2-dependent COX-2 is induced by the long chain fraction of
OxPAPC, while TLR2-dependent KC is induced by the short chain fraction of OxPAPC, that
contains 1-palmitoyl-2-oxovaleroyl-sn-glycero-3-PC (POVPC), 1-palmitoyl-2-glutaroyl-sn-
glycero-3-PC (PGPC), and Lyso-PC. Together, we show that OxPAPC via TLR2 induces a unique
M-phenotype.
P299
Giant-cell Arteritis Is a Not So Rare and Not So Easy to Handle Form of
Vascular Inflammation: Analysis of 5 Years of Experience
Endre Kolossvary, Katalin Farkas, Istvan Kiss; Szent Imre Teaching Hosp, Budapest, Hungary
Introduction: GCA is a rare systemic vasculitis of uncertain, probably T cell driven immuno-
logical origin. Early diagnosis is essential in prevention of increased cardiovascular mortality
and impaired quality of life. Objective: identification of patients affected by GCA and treated in
our hospital in the last 5 years. Analysis of their clinical data in order to improve diagnosis and
therapy. Method: analysis of clinical history, diagnostic algorithm, efficacy of therapy especially
coritcosteroid regimen with a follow up period of 5 years. Results: We analysed the data of 24
identified patients (779 years old), which is a population wider than the expected prevalence.
18 patients showed typical cranial, while 6 patients showed large vessel manifestation which
are two distinct entities characterized by a different cytokine pattern in the vascular wall. The
period of time between the first symptoms and the diagnosis was more than one year that
represents the unawareness of this condition. In case of large vessel manifestation vascular
ultrasound examination proved to be essential. Despite of a long steroid therapy (mean 19
months) 2 relapses occurred on the avarage, which is indicative of the limitation of
corticosteroid therapy. Complications resulting from corticosteroid therapy, like diabetes,
fracture due to osteoporosis, myopathy, infections were prevelant in the examined population.
Conclusion: GCA is probably much more frequent than generally considered. Diagnostic
procedures include clinical observation, vascular ultrasound and histology. Corticosteroid
therapy is effective to diminish the symptoms, but in the long run it can be regarded
suboptimal, therefore new immunological therapy is needed.
P300
C-Reactive Protein Is Associated with Prompt Development of Severe
Cardiac Allograft Vasculopathy and Increased Need for Posttransplant
Coronary Interventions
Carlos A Labarrere, Gonzalo Campana, George Boguslawski, Miguel A Ortiz, Marcelo J Sosa,
Colin Terry, Methodist Rsch Inst, Indianapolis, IN; Douglas E Pitts, Clarian Health Transplant
Cntr, Indianapolis, IN; Jacqueline A ODonnell, Krannert Institute of Cardiology, Indianapolis,
IN; David A Hormuth; Clarian Health Transplant Cntr, Indianapolis, IN
Background: Elevated levels of C-reactive protein (CRP) are associated with development of
cardiac allograft vasculopathy (CAV). We asked if the increase in CRP level soon after
transplantation would be correlated with early development of CAV, early progression of CAV
to severity, and need for coronary interventions in heart transplant recipients. Methods: We
used ELISA to measure CRP levels in sera obtained from 161 patients (median: 7
samples/patient) during the first 3 months after transplantation; a median CRP value was
calculated for each patient. Patients were grouped using AHA criteria as having CRP 1 mg/L,
13 mg/L and 3 mg/L. The CAV, CAV severity and CAV progression were evaluated with serial
annual coronary angiograms (median: 4/patient). Coronary interventions were defined as
coronary artery bypass or percutaneous transluminal coronary angioplasty/stents. Results:
Early post-transplantation CRP levels (1 mg/L, n40; 13 mg/L, n39; 3 mg/L, n82)
were significantly related to early onset of CAV (p0.001) and to the rapid progression to
2007 ATVB Poster Presentations e-89
by on March 25, 2011 atvb.ahajournals.org Downloaded from
severe CAV (p0.001). A strong association was found between the need for coronary
interventions and the CRP concentration (p0.017). Conclusions: The data suggest that a
proinflammatory environment rich in CRP favors early development of severe CAV and
increases the need for coronary interventions in heart transplant recipients.
P302
Lipoprotein Accumulation and Antigen Presentation in Grossly Normal
Human Aorta
Alexander Orekhov, Institute for Atherosclerosis Rsch, Russian Academy of Natural
Sciences, Moscow, Russian Federation; Shamil Khapchaev, Lomonosov Moscow State Univ,
Moscow, Russian Federation; Olga Pustovalova, Institute of General Pathology and
Pathophysiology, Russian Academy of Med Sciences, Moscow, Russian Federation; Irina
Andrianova, Institute of Experimental Cardiology, Cardiology Rsch and Production Cntr of
Russian Federation Health Ministry, Moscow, Russian Federation; Mihail Moisenovich;
Lomonosov Moscow State Univ, Moscow, Russian Federation
Earlier we have found that subendothelial intimacytes in grossly normal human aorta express
HLA-DR and CD1a molecules that are involved in peptide and lipid/carbohydrate antigen
presentation, respectively. We hypothesize that low density lipoprotein (LDL) stimulates
antigen-presenting function in subendothelial cells. To investigate LDL distribution in grossly
normal subendothelial intima antibodies against apoB were used. Antigen was visualized using
confocal laser scan microscopy and immunohistochemical methods. We localized apoB both
inside the cells and associates with extracellular matrix. Intracellular apoB was found both in
CD1a and HLA-DR cells. CD1a and HLA-DR were expressed not only by typical
antigen-presenting monocyte-derived cells but also by resident intimacytes (peryicytes and
smooth muscle cells). HLA-DR and CD1a molecules were associated with membrane
antigen-presenting structures and intracellular structures transporting antigen from endoplas-
mic reticulum to cell surface through antigen-loading vesicles. The size of HLA-DR and
CD1a vesicles were similar and ranged from 0.2 to 1 m. Intracellular apoB was colocalized
with CD1a vesicles. In HLA-DR cells, the majority of vesicles containing apoB were closely
associated with HLA-DR structures but not colocalized. This suggests the difference in
antigen processing occurring in HLA-DR and CD1a compartments. CD1aapoB cells
were found in association with lymphocytes of various stages of differentiation that may reflect
the lymphocyte activation in subendothelial intima. High degree of colocalization of antigen-
presenting molecules and apoB supports the suggestion that LDL accumulation in subendo-
thelial intima stimulates activation and differentiation of intimal antigen-presenting cells.
P303
A Role for Galectin-3 as an Amplifier of Inflammation in Atherosclerotic
Plaque Progression
Marianna Papaspyridonos, Joe P de Bono, Keith M Channon, Univ of Oxford, Oxford, United
Kingdom; Alberto Smith, Kevin G Burnand, Kings College London, London, United Kingdom;
David R Greaves; Univ of Oxford, Oxford, United Kingdom
Galectins are a family of lectins that are involved in inflammation, cell adhesion, apoptosis and
chemotaxis, and can also function as scavenger receptors. We identified Galectin-3 (Gal-3) as
a highly abundant transcript in a whole- transcriptome scan of unstable atherosclerotic plaques
obtained from carotid endarterectomy specimens. Gal-3 was selected as a candidate
therapeutic target and potential biomarker of atherosclerotic plaque progression. We assessed
the hypothesis that increased levels of soluble Gal-3, found in advanced human and murine
atherosclerotic plaques, could exacerbate vascular inflammation by stimulating macrophages
to express chemokines and other pro-inflammatory molecules. Gal-3 was found to be
up-regulated in unstable plaque regions of carotid endarterectomy specimens compared to
stable regions from the same patient ( 2.5 -fold), at the mRNA (n12) and protein level
(n9), as determined by qRT-PCR and western blotting analysis. Gene expression analysis of
atherosclerotic plaques of ApoE -/- mice on a high-fat western-type diet showed that Gal-3
expression also increases with age and lesion size (3.1-fold increase from 8 weeks, n6, to
16 weeks n6). In vitro, Gal-3 mediates monocyte chemoattraction and causes an up to
10-fold increase in human macrophage expression of pro-inflammatory mediators, such as
TNFalpha and RANTES in a dose-dependent manner, as revealed by microarray (Illumina
Sentrix arrays) and qRT-PCR analysis. Finally, conditioned media from Gal-3 treated human
macrophages (after Gal-3 blocking) also mediates chemoattraction of monocytes through a
pertussis toxin-sensitive, dose-dependent fashion. In conclusion, Gal-3 may represent a new
class of inflammatory mediators that contributes to atherosclerotic plaque progression both
through monocyte chemoattraction and macrophage activation. Gal-3 could serve as a novel
therapeutic target in anti-inflammatory strategies for the treatment of atherosclerosis.
P305
C-Reactive Protein Produced by U937 Monocyte-like Cells Produces Strong
Proinflammatory Effects on U937 Cells Themselves
Katsuya Tashiro, Eiichi Ishii, Yuhei Hamasaki; Saga Med Sch, Saga, Japan
(Background) Accumulation of monocytes and/or macrophages is observed in inflammatory
lesions. Therefore, it is generally accepted that monocytes and macrophages should play
important roles in the progression of various inflammatory diseases such as infectious
diseases, auto-immune diseases and atherosclerosis. In previous our studies, we reported that
U937 monocyte-like cells produce C-reactive protein (CRP) by themselves and stimulation with
interferon (INF)-gamma could upregulate production of CRP in U937 cells (6
th
ATVB meeting,
2005). These results reveal that monocytes and macrophages might be one of important
resources of CRP in focal inflammatory lesions. Further, we demonstrated that the composition
of CRP produced by U937 was changed by stimulation with INF-gamma. This change was
enhanced by additional stimulation with oxidized LDL (XIV International symposium on
atherosclerosis 2006). However, effects of this CRP is still unknown at all. (Results) U937 cells
regularly expressed m-RNA of CRP and the level of m-RNA were increased after stimulation
with INF-gamma. Production of CRP has been already observed with Immunocytochemical
staining, Westernblotting and Immunoprecipitation. In this study, we examined proinflammatory
effects of CRP produced by U937 cells on themselves using the lysate of cells. Cell lysate was
prepared by sonication and centrifuge. Cell lysate of U937 cells clearly upregulated expressions
of inflammation related proteins, such as MCP-1, iNOS, COX2, IL-6 and Oxidized LDL receptor.
These effects of lysate were enhanced in cells stimulated with IFN-gamma with or without
oxidized LDL. On contrary, after only CRP was removed by immunoprecipitation, these
proinflammatory effects were weakened significantly. (Conclusion) U937 cells are one of
precious resources of CRP and stimulation with INF-gamma and LDL can upregulate its
production. Co-stimulation with INF-gamma and oxidized LDL are synergistic. These results
suggest that monocytes and macrophages in atherosclerotic lesions might accelerate vascular
wall inflammation by local production of both native and modified CRP.
P306
WITHDRAWN
P307
Intracellular Stress Activates BZIP Transcription Factors to Induce the Liver
Inflammatory Response
Kezhong Zhang, Jun Wu, Sung H Back, Kazuya Yamano, Hongzhi Miao, Randal J Kaufman;
The Univ of Michigan, Ann Arbor, MI
Atherosclerosis, the major cause of heart diseases, is an inflammatory disease in which
immune mechanisms interact with metabolic risk factors to initiate and propagate disease
lesions. Endoplasmic reticulum (ER) stress is known to induce the unfolded protein response
(UPR), an intracellular signaling pathway from the endoplasmic reticulum (ER) to nucleus to
protect cells from stress caused by accumulation of unfolded or misfolded proteins. Here we
found that ER stress, oxidative stress and pro-inflammatory signaling can interact and merge
into each other to activate the liver inflammatory response that contributes to atherogenesis.
XBP1, ATF6 and CREBH are ER stress-inducible basic leucine Zipper (bZIP) transcription factors
of CREB/ATF family. ER stress and oxidative stress induced by high-homocysteine diet,
inflammatory cytokines TNFa, IL6, IL1b or Lipopolysaccharide (LPS) can induce processing of
XBP1, ATF6 and CREBH to be activated in mouse liver. Under cellular stress or inflammatory
conditions, activated XBP1, ATF6 and CREBH can interact with each other to activate
transcription of the major inflammatory genes encoding C-reactive protein (CRP), Serum
Amyloid P-component (SAP) and Serum Amyloid A (SAA). In engineered mice defective in ER
stress-transducer molecules IRE1a, XBP1, CREBH and/or ATF6, transcription of the CRP, SAP
and SAA3 mRNAs and production of secreted CRP, SAP and SAA3 proteins were significantly
reduced in response to LPS that induces ER stress, oxidative stress and inflammation,
compared to those of control mice. Our studies suggest that cellular stress and inflammatory
stimuli activate bZIP transcription factors XBP1, CREBH and ATF6 to induce the inflammatory
response. Those studies provide molecular links by which intracellular stress activates liver
inflammation, and will be informative to developing novel methods to control inflammatory
diseases, particularly atherosclerosis.
P308
Increases in apoB-lipoprotein Secretion During Differentiation of Caco-2
Cells due to Enhanced Transcription of MTP
Kezhi Dai, M Mahmood Hussain; SUNY Downstate Med Cntr, Brooklyn, NY
Microsomal triglyceride transfer protein (MTP) and apolipoprotein B (apoB) are essential for
lipoprotein assembly. In the intestine, the differentiated enterocytes produce apoB-lipoproteins.
Caco-2 cells produce apoB-lipoproteins after spontaneously differentiating into enterocyte-like
cells in culture. We analyzed changes in apoB secretion and MTP activity in Caco-2 cells
cultured on Transwells for 23 weeks. ApoB secretion and MTP activity were barely detectable
in non-differentiated cells but increased 5 to 6-fold during differentiation. Interestingly, MTP
mRNA increased more than 10-fold during differentiation while variance in apoB mRNA were
less than 1-fold indicating that induction in MTP, not apoB, is responsible for the increased
apoB secretion. We used this model system to investigate the molecular basis for the
transcriptional induction of MTP. Studying a series of 5 end truncated human MTP promoter
sequences revealed that a 204-bp sequence, conserved during evolution, was sufficient to
simulate differentiation-dependent MTP expression. Site-directed mutagenesis of putative
elements in 204-bp and co-transfection of candidate transcription factors revealed that HNF1
and DR1 elements and HNF4, HNF1 and HNF1 proteins are involved in the basal
expression. For differentiation-dependent expression proximal and distal DR1 elements were
important. However, differentiation-dependent induction of MTP was not correlated with
e-90 Arterioscler Thromb Vasc Biol June 2007
by on March 25, 2011 atvb.ahajournals.org Downloaded from
changes in HNF4, HNF1 and HNF1. Instead, profiling differentiation-dependent changes in
several candidate transcription factors associated with DR1 element unveiled a putative
repressor, NR2F1 (also called Ear3 or COUP-TFI), whose expression was reduced by more than
50% after Caco-2 cell differentiation. Knockdown of NR2F1 by siRNA in undifferentiated Caco-2
cells increased MTP promoter activity. In summary, our data indicate increased transcription of
MTP enhances apoB-lipoprotein production and reduced expression of NR2F1 during differ-
entiation induces MTP in Caco-2 cells. Tissue-specific regulation of NR2F1 may provide novel
therapeutic solution to control MTP expression in the intestine.
P309
Initiation of Apolipoprotein B-Containing Lipoprotein Assembly Is
Independent of Microsomal Triglyceride Transfer Protein Activity
Nassrin Dashti, Medha Manchekar, Zhihuan Sun, Jere P Segrest, Yanwen Liu; Univ of
Alabama at Birmingham, Birmingham, AL
We previously proposed that the initiation of apolipoprotein (apo) B particle assembly occurs
when
1
domain of apoB folds into a three-sided lipovitellin-like lipid binding cavity to form
the apoB lipid pocket. We demonstrated, based on experimentally-derived results and
molecular modeling, that the N-terminal 1000 amino acid residues (
1
domain) of apoB
(designated apoB:1000) are competent to complete the lipid pocket without a structural
requirement for microsomal triglyceride transfer protein (MTP) and that this primordial apoB
particle is phospholipid-rich. These results, however, did not rule out a MTP-mediated lipid
transfer to apoB:1000. In this study, we investigated the putative role of MTP in the initial
lipidation of apoB:1000 by employing metabolic labeling of stable transformants of McA-
RH7777 cells with [
35
S]methionine/cysteine, [
14
C]oleic acid, and [
3
H]glycerol in the presence or
absence of BMS-197636 and BMS-200150, two inhibitors of MTP lipid transfer activity.
BMS-197636 at 0.1 M and BMS-200150 at 5, 10 and 20 M had no detectable effect on the
synthesis, lipidation, and secretion of apoB:1000-containing particles. At 40 M BMS-200150,
the secretion of intact apoB:1000 particles was decreased by only 1520% and this reduction
was attributable to the effect of high concentration of this compound generally on hepatic
protein synthesis, as reflected in 2030% inhibition in albumin secretion. In addition, MTP
inhibitors had no effect on the lipid composition of secreted apoB:1000-containing particles.
Under these experimental conditions, the synthesis, lipidation, and secretion of apoB-100-
containing particles in HepG2 cells were inhibited by 9097% and secreted particles had a
lower content of triglycerides and a higher level of phospholipids. In conclusion, our studies
provide compelling evidence that the initial addition of phospholipids to apoB:1000 and the
initiation of apoB lipoprotein assembly occur independently of MTP lipid transfer activity. We
propose that a lipid transfer protein other than MTP mediates the formation of the
phospholipid-rich primordial apoB particle.
P312
Native 2D Gel Analysis of HDL Subpopulations: A Proteomics Approach
Lita A Freeman, Marcelo J Amar, Angel Aponte, Wells Wu, Rong-Fong Shen, Robert T
Shamburek, Alan T Remaley, Silvia Santamarina-Fojo; NIH-Natl Heart, Lung, and Blood Inst,
Bethesda, MD
HDL is a heterogeneous mixture of lipoproteins with a density of 1.0631.21 g/ml. A powerful
method of analyzing HDL subpopulations is the native 2D gel electrophoresis technique of
Asztalos and colleagues, which fractionates HDL into its native components by charge in the
first dimension and then by size in the second dimension. Traditionally the composition of these
particles has been analyzed by Western blotting. However, Western analysis is limited by the
need for probing with different antibodies, and unanticipated proteins will not be detected. Here
we analyze the composition of HDL subpopulations using a proteomics approach. HDL was
isolated from three normolipidemic individuals by sequential density gradient centrifugation and
separated by native 2D gel electrophoresis. Gels were stained with Coomassie Blue to visualize
protein-containing particles in the HDL fraction. Individual spots were manually picked and
trypsinized and the protein composition of each spot was analyzed by LC-MS/MS. As expected,
apoA-I was the predominant apolipoprotein in alpha-migrating particles. Alpha-migrating
particles also contained apoA-II, alpha-1-antitrypsin, SAA4, and PON1; apoE, apoD, and apoM
were also present in alpha-migrating particles of higher molecular weight. Additional previously
identified low-abundance HDL components, such as haptoglobin and the ryanodine receptor,
were also localized to alpha-migrating particles. apoA-I was confirmed as the predominant
apolipoprotein in pre-alpha-migrating particles. apoA-II was also present in particles migrating
with pre-alpha mobility. Our results, in conjunction with those of others, are suggestive of
considerable heterogeneity in HDL subpopulations. In conclusion, native 2D gel electrophoresis
of HDL followed by proteomics analysis is a novel method to characterize high density
lipoprotein particle composition. This method may help to elucidate the functional roles of HDL
subpopulations.
P313
Diet Enriched with Oxidized Fatty Acids Lowers Triglyceride Levels in Mice
Mahdi O Garelnabi, Priscilla Jaichander, Ohio State Univ Sch of Medicine, Columbus, OH;
Nadya Khan-Merchant, InVasc Therapeutics, Atlanta, GA; Sampath Parthasarathy; Ohio State
Univ Sch of Medicine, Columbus, OH
We have previously shown through cell culture and animal studies that the intestine efficiently
absorbs oxidized linoleic acid and atherosclerosis is increased in animals fed a high cholesterol
diet in the presence of oxidized linoleic acid. Animals fed non-atherogenic diet did not develop
atherosclerosis even in the presence of oxidized fatty acids. Oxidized fatty acids have also been
shown to be ligands for PPARs and could induce genes for antioxidant enzymes such as
catalase. In the present study, we fed C57BL6 mice normal mouse diet in the presence of oleic
or oxidized linoleic acid (13-hydroxyoctadecadienoic acid, 13-HODE) at 50 mg per animal per
day for two weeks. There was no major changes in the lipid or lipoprotein classes in 13-HODE
fed animals as compared to oleic acid fed animals, except that the triglyceride values were
significantly lower (40.37% of controls) in 13-HODE treated animals (Table). Paraoxonase
(PON1) activity did not change between the two groups. We observed a lowering of plasma
triglycerides in human subjects who consumed oxidized fatty acids as compared to linoleic
acid. The result suggests that oxidized fatty acids may also have anti-atherogenic properties
properties in the absence of dietary cholesterol. However, 13-HODE also appeared to have
pro-inflammatory properties which might counteract any potentially beneficial effects. It is
possible that 13-HODE might affect the synthesis and metabolism of triglycerides or triglyceride
containing lipoproteins. Further studies are in progress.
Group CHOL mg/dL TG mg/dL HDLc mg/dL PON1 (OD)
Control 56.45 72.575 47.435 0.609
Oxidized fatty 54.05 29.31 42.405 0.589
P314
Independent Distributions of Apos A-II, E, and A-I Among Intracellular and
Newly Secreted High-Density Lipoproteins of HepG2 Cells
Baiba K Gillard, Hu-Yu A Lin, John W Gaubatz, John B Massey, Henry J Pownall; Baylor
College of Medicine, Houston, TX
The human hepatoma cell line, HepG2, secretes apo A-I as free and lipidated (HDL) species.
Apo A-I is lipidated intracellularly (20%) and in 1-hour media (30%; Chisholm et al. 2002).
The corresponding biogenic itineraries of apos A-II and E3, cysteine-containing apos that
dimerize with other cysteine-containing proteins, are less clear. Apo A-II in human plasma and
in HepG2 media is homodimeric; plasma apo E3 occurs as 45% monomer, 26% homodimer,
and 29% A-II-E heterodimer. Given that lipid surfaces catalyze dimerization of human apo A-II
(Gillard et al 2005), we hypothesized that intracellular lipidation of apo A-II promotes
dimerization, and because of its higher lipophilicity, intracellular and newly secreted apo A-II,
and perhaps apo E, would be more heavily lipidated than apo A-I. We compared the lipidation
of intracellular and newly secreted apos A-II, E, and A-I. According to density gradient
centrifugation and size exclusion chromatography (SEC) apos A-II and E are lipidated
intracellularly. Apos A-II and E have distinctive dimerization kinetics apo A-II dimerizes
intracellularly; secreted apo E is monomeric but dimerizes in media. Western blots of SEC
fractions revealed that apos occur with VLDL, LDL, HDL, and as lipid-poor and lipid-free
proteins; 20% of intracellular apo A-I co-elutes with nascent HDL; 80 % of intracellular apo
A-II is in the nascent HDL fraction; 90% of intracellular apo E co-elutes as VLDL. In 2 hour
HepG2 media, 50% of apo A-I is in an HDL fraction; the rest is lipid-poor or lipid-free. Most
apo A-II is in the nascent HDL fraction; while most of the apo E is on larger HDL particles as
well as on LDL particles. Newly secreted apo A-II is 100% homodimer, while apo E is
100% monomer, but forms homodimers and apo A-II-E heterodimers over 24 hr. Conclusion:
Apo A-I, A-II and E are processed differently in HepG2 cells with respect to lipidation and
dimerization, and heterodimerization of apos A-II and E requires association of apos on a
common particle.
P315
In Vitro Production of Active ApoJ/Clusterin
Eddy S Konaniah, Scott E Street, Joshua E Basford, David Y Hui, Norman A Granholm; Univ
of Cincinnati, Cincinnati, OH
The traditional source for human apoJ / clusterin is pooled human plasma. The purification
protocol is tedious, potentially hazardous, and prone to bacterial contamination. Moreover,
there is no potential to produce a modified form of apoJ / clusterin. We have utilized an in vitro
approach to address these issues. HEK293 cells were stably transfected with an expression
construct that contains cDNA for human apoJ / clusterin. We used western blots to identify
apoJ / clusterin as a protein that is prominent in the culture supernatant. In a tritiated thymidine
uptake assay with A7r5 cells, the same culture supernatant significantly suppresses growth
factor stimulated uptake of tritiated thymidine in A7r5 cells, as expected. Exogenous apoE
evokes a similar suppressive effect on thymidine uptake in growth factor stimulated A7r5 cells
and we used this response as a positive control for responsiveness of the A7r5 cells. We know
that apoJ / clusterin possesses heparin binding domains and we propose to utilize affinity
chromatography to purify apoJ / clusterin from the culture supernatant. We anticipate that this
in vitro approach will provide a consistent source of apoJ / clusterin that is biologically active.
The potential is present to determine on vascular smooth muscle cells not only the receptor(s)
but also the domains of the apoJ / clusterin molecule that evoke the protective function of apoJ
/ clusterin in vascular biology.
P316
WITHDRAWN
2007 ATVB Poster Presentations e-91
by on March 25, 2011 atvb.ahajournals.org Downloaded from
P317
Delayed in Vivo Catabolism of Small Dense LDL: A Stable Isotope Study
Katsunori Ikewaki, Yoshinobu Nakada, Yae Inoue, Seibu Mochizuki; Jikei Univ Sch of
Medicine, Tokyo, Japan
Background and aims: Small dense LDL (sdLDL) is an emerging risk factor for coronary heart
disease. However, detailed in vivo metabolism of sdLDL has been poorly understood. In order
to assess sdLDL metabolism, we performed in vivo kinetic studies utilizing stable isotopically
labeled leucine in 10 hypercholesterolemic patients and 5 healthy controls. Effects of statin on
sdLDL metabolism were also investigated. Methods: Deuterated leucine was injected and blood
samples were collected up to 48hours. sdLDL was isolated by heparin-magnesium (JLR
44:2193, 2003), followed by ultracentrifugation (d 1.019 - 1.063g/dL). VLDL, IDL, and LDL
were also isolated by sequential ultracentrifugation. ApoB was precipitated by isopropanol
method, then hydrolyzed and derivatized to determine tracer/tracee ratios of apoB by
gas-chromatography mass spectrometry. Results: Fractional catabolic rate (FCR) was esti-
mated by SAAMII software. The FCR of sdLDL apoB was 0.23 0.06 pools/day, which was
35% lower than that of LDL apoB of 0.35 0.12 pools/day (p0.001). Furthermore, statin
therapy significantly increased sdLDL apoB FCR by 67% (p0.01), in addition to 94% increase
in LDL apoB FCR. Conclusions: This is the first in vivo kinetic evidence of the delayed
catabolism of sdLDL which was improved by statin therapy. Therefore, the impaired catabolism
is translated into the longer residence time, thus supporting the proatherogenic nature of
sdLDL.
P318
IRE1 Restricts Chylomicron Production B by Selectively Degrading MTP
mRNA
Jahangir Iqbal, Kezhi Dai, SUNY Downstate Med Cntr, Brooklyn, NY; Tracie DeVries-Seimon,
Columbia Univ, New York, NY; Rivka Jungreis, Miho Oyadomari, New York Univ Med Cntr,
New York, NY; George Kuriakose, Columbia Univ, New York, NY; David Ron, New York Univ
Med Cntr, New York, NY; Ira Tabas, Columbia Univ, New York, NY; M Mahmood Hussain;
SUNY Downstate Med Cntr, Brooklyn, NY
Microsomal triglyceride transfer protein (MTP) is obligatory for the production of intestinal
chylomicrons to absorb dietary fat and fat-soluble vitamins as well as dietary and biliary
cholesterol. Inositol-requiring enzyme 1 (IRE1), a membrane-anchored kinase/ribonuclease,
plays a key role in relieving the stress induced by the buildup of misfolded proteins in the
endoplasmic reticulum. Although seemingly unrelated, we provide evidence for a cross talk
between these two processes to avoid hyperlipidemia during postprandial state. IRE1 is
expressed ubiquitously, but the intestinal epithelium expresses an additional protein IRE1.
Plasma cholesterol and triglyceride were significantly increased in cholesterol-fed Ire1b
-/-
mice
due to increased chylomicron production. Moreover, the activity and mRNA of MTP were
markedly enhanced in the intestine, but not the liver, of these mice. In human hepatoma Huh7
cells, cholesterol enhanced MTP levels but not when IRE1 was expressed. IRE1 specifically
decreased MTP mRNA in these cells. IRE1 had no effect on the MTP promoter, but reduced
MTP mRNA levels expressed under a heterologous promoter indicating for post-transcriptional
degradation. In IRE1 expressing cells, the degradation of the 3 end of the MTP mRNA was
prevented by siRNAs targeting 5 to 3 exonucleases, XRN1 and XRN2. These studies indicate
that IRE1 regulates MTP mRNA levels involving post-transcriptional degradation. Thus, IRE1
deficiency leads to intestine-specific induction of MTP and increased lipid absorption in
response to a high-cholesterol diet. These findings have potentially important mechanistic and
therapeutic implications related to the role of ER stress in the production of intestinally derived
atherogenic lipoproteins.
P319
ACAT2 and Human Hepatic Cholesterol Metabolism: Identification of
Important Gender-related Differences
Zhao-Yan Jiang, Curt Einarsson, Go sta Eggertsen, Karolinska Institute, Stockholm, Sweden;
Matthew A Davis, Wake Forest Univ, Winston-Salem, NC; Lei Wang, Sheng-Dao Zhang,
Ruijin Hosp, Shanghai Jiaotong Univ Sch of Medicine, Shanghai, China; Mats Eriksson,
Karolinska Institute, Stockholm, Sweden; Tian-Quan Han, Ruijin Hosp, Shanghai Jiaotong
Univ Sch of Medicine, Shanghai, China; Lawrence L Rudel, Wake Forest Univ,
Winston-Salem, NC; Paolo Parini; Karolinska Institute, Stockholm, Sweden
Objective ACAT2 is specifically expressed in hepatocytes and plays a role in hepatic cholesterol
esterification in human. To further elucidate its physiologic role in human cholesterol
metabolism, liver biopsies from 19 gallstone patients (female/male, 10/9) and 12 gallstone-free
patients (female/male, 8/4) were collected, and analyzed for ACAT2 activity and expression.
Results Age and BMI were matched between groups and genders, as well as plasma total
cholesterol and triglycerides. As expected, HDL cholesterol and Apo AI were significantly higher
in females than in males. Hepatic ACAT2 activity did not differ between gallstone and
gallstone-free patients and no correlation was observed between the hepatic ACAT2 activity
and the biliary cholesterol content. Interestingly in females, hepatic ACAT2 activity was 1/4 of
what observed in males (7.2 1.2 vs. 29 9.1 pmol/min/mg protein, P0.01). This
gender-related difference was also seen at protein level, but was not found at mRNA level.
Furthermore, the hepatic activity of ACAT2 correlated negatively with serum HDL cholesterol
(r -0.68, P 0.05) and with Apo AI (r -0.61, P 0.05). Conclusion A strong
gender-related different in hepatic ACAT2 activity is present in human liver, and alterations in
hepatic ACAT2 activity seems not to underlay the genesis of gallstone disease. Furthermore, a
new role for ACAT2 in the regulation of HDL cholesterol levels may be hypothesized: when
hepatic ACAT2 activity is low, free cholesterol may be preferentially secreted into HDL particles
rather than into bile.
P321
Human Macrophage ABCG1 Expression and Function Is Decreased in
Patients with Type 2 Diabetes
Jeremy Mauldin, Melissa Hatley, Allison Wojcik, Suseela Srinivasan, Carlos Ayers, Coleen
McNamara, Catherine Hedrick; Univ of Virginia, Charlottesville, VA
Coronary artery disease is the most common cause of death for people with Type 2 diabetes,
with diabetic patients being up to four times more likely to develop coronary artery disease than
their non-diabetic counterparts. A key early event in the development of atherosclerosis is
macrophage foam cell formation. The ABC transporter, ABCG1, plays an important role in
macrophage reverse cholesterol transport. Previous work by our lab in mouse models of Type
2 diabetes show a decrease in macrophage ABCG1 protein expression, leading to a decrease
in cholesterol efflux to HDL and increased macrophage lipid accumulation. Here, we show
similar data in human patients with Type 2 diabetes. Human blood was obtained from
consenting patients with and without Type 2 diabetes. Monocytes were isolated and
differentiated into macrophages in vitro. Western blot analysis of the human macrophages
revealed that ABCG1 protein expression was decreased by approximately 40% in diabetic
patients compared to controls, without changes in ABCA1 protein levels. Cholesterol efflux
experiments revealed a 30% decrease in cholesterol efflux to HDL by diabetic macrophages
compared to controls. Further, we saw no change in efflux to lipid free Apo-A1 by diabetic
macrophages, indicating no loss of function of ABCA1. Additionally, macrophages from diabetic
patients had significantly more lipid accumulation than non-diabetic patients when challenged
with oxidized LDL, as measured by oil red O staining and gas chromatography. In conclusion,
Type 2 diabetes appears to decrease human macrophage ABCG1 expression, resulting in
decreased cholesterol efflux to HDL and increased lipid accumulation. Thus, therapies to
upregulate ABCG1 expression in macrophages may be important for preventing atherosclerosis
development in diabetes.
P322
The Effect of Simvastatin on Surrogate Markers of Vascular Health in
Youth
Catherine J McNeal, Donnie Wilson, Clifford Buckley, Jose Santiago, Juhee Song, Scott and
White Hosp, Temple, TX; Ronald Macfarlane, Texas A&M Univ, College Station, TX; Guoyao
Wu; Texas A&M Univ, Temple, TX
Failure to diagnose preclinical CVD in youth misses a major opportunity to prevent the
long-term consequences of this disease. We have conducted a pilot study to evaluate surrogate
vascular markers (SVMs) that are associated with early arterial injury including flow-mediated
vasodilatation, carotid intima media thickness, arterial stiffness, and biomarkers including cell
adhesion molecules (ICAM-1, VCAM-1), methylarginines (asymmetric dimethylarginine (ADMA),
symmetric dimethylarginine (SDMA)), and C-reactive protein (C-RP). We hypothesized that one
or more of these SVMs which are linked to early pathological vascular changes will identify
high-risk youth with early vascular injury compared to a healthy group and that these markers
will tend to normalize with risk factor reduction. We further hypothesized that one or more of
the markers will correlate with the Pathological Determinants of Atherosclerosis in Youth (PDAY)
risk score. Ten subjects without any known risk factors and 22 hypercholesterolemic (HC)
youth, aged 1020 yrs. were recruited from the pediatric clinic. The majority of the HC group
was also obese. The HC group was randomized to diet 20 mg of simvastatin vs. placebo for
24 weeks followed by a forced titration to 40 mg vs. placebo for 24 weeks ending with a final
evaluation after a 12 week washout period of diet alone. The markers that best distinguished
the HC from the control subjects were C-RP (p0.03), VCAM-1 (p0.05) and SDMA (p0.04).
With the exception of ICAM-1 and C-RP, the marker values improved in the treatment group
more than the placebo group and the relative changes were the largest for the methylarginines
- a marker closely tied to insulin resistance. The markers that demonstrated the highest
correlation with the number of risk factors were VCAM-1 (r 0.45, p0.055) and SDMA
(r0.48, p0.04). PDAY risk scores were calculated for each subject and SDMA (r0.50,
p0.03) and Arg/SDMA (r-0.50, p0.04) again emerged as the most highly correlated
SVMs. Consistent with the relatively low PDAY risk scores and a minimally thickened IMT, the
data suggest that the majority of subjects do not have advanced atherosclerotic changes. We
conclude that SVMs are a useful index of vascular injury in high-risk youth.
P323
Dietary CholesterolMediated Stimulation of Chronic LXR Activation,
Dyslipidemia, Obesity, and Insulin-resistant Diabetes: Evidence for
Synergistic Interactions with Dietary Fat and Fructose
Abigale Miller, Heather Basciano, Mark Naples, Elaine Xu, Khosrow Adeli; Hosp for Sick
Children, Toronto, Canada
Evidence is presented for an important link between dietary cholesterol, chronic LXR
activation, and development of severe dyslipidemia and insulin resistance. Previously, our
e-92 Arterioscler Thromb Vasc Biol June 2007
by on March 25, 2011 atvb.ahajournals.org Downloaded from
laboratory has shown that chronic high fructose feeding readily induces insulin resistance and
a metabolic dyslipidemia state. However, the animals did not show evidence of obesity or
progression to type 2 diabetes. When hamsters were fed a diet rich in fructose (40%), fat
(30%), and cholesterol (0.25%) (the FFC diet), rapid induction of weight gain, insulin resistance
and dyslipidemia was observed within 12 weeks. Prolonged feeding (622 weeks) led to rapid
progression to hyperglycemia (suggesting development of overt type 2 diabetes) and hepatic
steatosis (largely due to liver accumulation of cholesterol ester). The development of these
metabolic disturbances was cholesterol concentration-dependent. Supplementation of chow
diet with cholesterol alone or fat alone did not induce these metabolic defects, suggesting that
dietary cholesterol acts synergistically with dietary fructose and fat to induce a state of severe
dyslipidemia, steatosis and type 2 diabetes. Importantly, the FFC diet was found to induce
strong activation of the nuclear receptor LXR in the liver. We postulated that dietary cholesterol
feeding may act as a direct chronic activator of LXR, and this may underlie the more severe
metabolic abnormalities observed. Parallel feeding studies were then performed in hamsters
with high fructose and high fat diets supplemented with either varying amounts of cholesterol
(0, 0.05% or 0.25%) or the specific LXR agonist TO901317 (25 mg/kg) for 14 days.
Interestingly, the lipid and lipoprotein profile of hamsters fed diets supplemented with high
cholesterol (0.25%) were closely similar to those in hamsters treated chronically (2 weeks) with
an LXR agonist, indicating that LXR activation may be at least partially responsible for the
chronic effects of cholesterol. The data clearly implicates dietary cholesterol, synergistically
acting with dietary fat and fructose, as a major determinant of the severity of insulin resistance
and dyslipidemia, an affect possibly mediated via chronic LXR activation.
P324
Vascular Function of LOX-1 Overexpressing Mice on High-fat Diet
Gregor Muller, Birgit Eichhorn, Ursula Ravens, Evelyn Schrock, Univ of Technology Dresden,
Dresden, Germany; Tatsuya Sawamura, National Cardiovascular Cntr Rsch Institute, Suita,
Osaka, Japan; Henning Morawietz; Univ of Technology Dresden, Dresden, Germany
Background: The major receptor for endothelial uptake of oxidized low-density lipoprotein
(oxLDL) is lectin-like oxLDL receptor-1 (LOX-1). In this study, we analyzed the impact of
high-fat diet on vascular function in transgenic mice overexpressing LOX-1. Methods and
Results: In LOX-1 overexpressing mice, bovine LOX-1 transgene was mapped by fluorescence
in situ hybridization to chromosome 3 (region 3H34) of the mouse genome and found to be
inserted in multiple repeats. Because LOX-1 overexpressing mice fed normal chow showed no
effect on vascular function, we tested whether LOX-1 overexpression affects contractile
function in different vascular beds of male mice (8 weeks old) fed a high-fat Western-type diet
for 10 weeks. Similarly aged and fed C57BL/6 wildtype (WT) animals were studied for
comparison. Acetylcholine (ACh)-induced relaxation was determined in phenylephrine (PE)-
preconstricted aorta, saphenous artery, and mesenteric artery.
Parameter Aorta Aorta
Saphenous
artery
Saphenous
artery
Mesenteric
artery
Mesenteric
artery
WT LOX-1 WT LOX-1 WT LOX-1
ACh [% PE] 15.81.5 18.81.2 36.23.9 46.93.0
P0.0001
36.24.1 54.45.3
P0.035
logEC50 [M] -6.90.2 -7.10.3 -4.90.01 -4.70.01
P0.0001
-5.10.01 -4.70.01
P0.0001
ACh/L-NAME [% PE] 80.718.2 94.77.1 48.83.9 69.15.5
P0.024
42.84.3 72.05.3
P0.005
NO fraction [% PE] 17.02.6 18.34.9 2.30.6 0.70.3 0.50.3 0.30.3
The concentration response curves of the NO donor nitroprusside did not differ between LOX-1
and WT mice. The membrane potential in the saphenous artery was -75.81.4 mV in LOX-1
mice and -73.30.5 mV in WT. Conclusion: We conclude that LOX-1 receptor overexpression
on high-fat diet can affect vascular function.
P325
The Citrus Flavonoid Naringenin Inhibits Apob100 Secretion and Improves
Dyslipidemia and Insulin Resistance in High-fatFed LDLR-/- Mice
Erin E Mulvihill, Emma M Allister, Brian G Sutherland, Dawn E Telford, Cynthia G Sawyez,
Jane Y Edwards, Murray W Huff; Robarts Rsch Institute, London, Canada
Naringenin, a citrus flavonoid, potently inhibits the assembly and secretion of apoB100
containing lipoproteins (apoB) from HepG2 and primary mouse hepatocytes. In the present
study, we determined if addition of naringenin to a high fat (Western) diet could inhibit hepatic
apoB100 overproduction and improve the dyslipidemia, insulin resistance and glucose
tolerance, in LDL-receptor deficient (ldlr-/-) mice. Four groups, chow, Western (42% calories
from fat and 0.05% cholesterol) and Western 1% or 3% w/w naringenin, were fed ad libitum
for 4 weeks (n12 per group). The Western diet significantly elevated plasma cholesterol (C)
and triglyceride (TG) approximately 3-fold, which were dose-dependently decreased by up to
40% by naringenin. Secretion of TG and apoB100 into plasma, as assessed by the tyloxapol
technique were increased 2- and 4-fold in Western-fed mice. Both parameters were
significantly decreased (-100% and -50% respectively) by 3% naringenin, demonstrating that
naringenin inhibits hepatic apoB100 secretion in vivo. The Western-fed mice had significantly
elevated TG accretion in liver (2-fold), intestine (4-fold) and skeletal muscle (1.3-fold) when
compared to chow, which were corrected by 3% naringenin (-34%, -80% and -40%
respectively, all P0.05). Naringenin inhibited the Western-diet induced weight gain,
independent of changes in caloric intake or intestinal TG absorption, suggesting decreased TG
synthesis and increased fatty acid (FA) oxidation. The significant increases in hepatic mRNA of
srebf-1c (5-fold) and rates of TG synthesis (1.4-fold) in Western-fed mice were completely
normalized by 3% naringenin. The expression of hepatic mRNA for acox and cpt1, genes
important in FA oxidation were significantly increased by 32% and 17% respectively, as
compared to Western-fed mice suggesting that naringenin could decrease TG availability for
apoB100 secretion through stimulation of FA oxidation. Naringenin also prevented the glucose
intolerance and insulin resistance induced by the Western diet. In summary, naringenin
improves dyslipidemia, insulin resistance and tissue TG content in high fat-fed ldlr (-/-) mice,
suggesting a novel approach for treatment of the metabolic syndrome.
P326
Inhibiting Proteasomal Degradation of Microsomal Triglyceride Transfer
Protein (MTP) Prevents Carbon TetrachlorideInduced Steatosis
Xiaoyue Pan, Farah N Hussain, Jahangir Iqbal, Miriam H Feuerman, M Mahmood Hussain;
SUNY Downstate Med Cntr, Brooklyn, NY
Carbon tetrachloride (CCl
4
) interferes with triglyceride secretion by unknown mechanisms and
causes steatosis. CCl
4
decreased plasma lipids, apoB-lipoproteins, MTP activity and protein,
and increased intestinal as well as hepatic lipids in mice in a concentration and time dependent
manner. Moreover, CCl
4
decreased apoB-lipoprotein production, MTP activity and protein, had
no effect on MTP mRNA, and increased cellular retention of triglycerides in primary enterocytes,
colon carcinoma and hepatoma cells. Metabolic labeling revealed that CCl
4
had no effect on
MTP synthesis, but induced its post-translational degradation involving ubiquitinylation and
proteasomes. In contrast, a MTP antagonist inhibited lipid transfer activity without reducing
protein levels. We hypothesized that CCl
4
might covalently modify MTP and signal for its
destruction. Incubation of cells with
14
CCl
4
resulted in its incorporation into MTP and the
amounts of modified MTP increased when proteasomal degradation was inhibited. In contrast,
inhibition of mixed function oxygenases prevented MTP modification. These studies indicate
that CCl
3
generated by cytochrome P450 oxygenases covalently attaches to MTP and this
modification targets MTP for proteasomal degradation. To determine if inhibition of proteolysis
could prevent CCl
4
toxicity, mice were fed with CCl
4
with or without lactacystin. MTP protein
and activity were decreased in the intestine and liver of CCl
4
fed mice and lactacystin partially
protected these losses. Furthermore, accumulation of triglyceride and cholesterol in these
tissues was prevented when mice were fed with CCl
4
and lactacystin. Since inhibition of
proteasomal degradation increases accumulation of modified MTP, these data indicate that
non-degraded, modified MTP is able to prevent steatosis. Thus, CCl
4
induces post-translational
degradation without affecting its lipid transfer activity, whereas MTP antagonist inhibits lipid
transfer activity without causing its destruction. These studies identify MTP as a major target
of CCl
4
and its degradation as a novel mechanism involved in the onset of steatosis, and
suggest that inhibition of proteolysis may prevent some forms of steatosis.
P327
Investigation of Levels of Serum Lipids in Patients with Periodontitis
Compared with Healthy Individuals
Hosein H Shahsavarani, Mohammad taghi M Shahsavarani, Jalaleddin H Hamissi; Qazvin
Univ of Med Science, Qazvin, Iran (Islamic Republic of)
Objective: Periodontal disease is a bacterial infection, which has been classified as a local
chronic inflammation. The literature suggested that a link exists between the presence of
severe periodontitis and several systematic health changes including an altered lipid
metabolism. The aim of this study was to investigate the relationship between chronic
periodontitis and serum lipid levels. Material and Methods: The level of serum lipids(CHL, TG,
HDL and LDL) of total 30 patients with Chronic periodontitis (CPITN score III & IV) ranging in age
between 30 to 40 years were examined and compared with data obtained from 30 healthy
individuals(control group). The relationship of serum lipids and periodontal disease and CPITN
index was tested by means of SPSS software (Ver 13.0). Results: The present of periodontal
disease was significantly related with higher total cholesterol in case group (P0.05).
Triglycerides and HDL were higher in patients but no statistically significant differences were
observed with control group. LDL did not show any difference between case and control
groups. Conclusion: Analysis of these data revealed that Chronic periodontitis enhances the
chance of occurrence of Hyperlipidemia in healthy people. The findings of this research support
the reports linking increased prevalence of changing serum lipids among patients with
periodontal disease.
P328
Lipoprotein Subfraction Responses Differentially Predict Changes in
Lipoprotein-associated Phospholipase A2 During Prescription Omega-3
Therapy
Robert A Shalwitz, Reliant Pharmaceuticals, Liberty Corner, NJ; Kevin C Maki, Provident
Clinical Rsch and Consulting, Inc, Bloomington, IN; Ralph T Doyle, Reliant Pharmaceuticals,
Liberty Corner, NJ; Christie M Ballantyne; Cntr for Cardiovascular Disease Prevention,
Methodist DeBakey Heart Cntr, Houston, TX
Lipoprotein-associated phospholipase A
2
(Lp-PLA2), a secretory product of macrophages,
circulates bound to low- (LDL) and high-density lipoprotein (HDL) particles, and is an
independent predictor of cardiovascular event risk. The present investigation assessed the
influence of triglyceride lowering with prescription omega-3-acid ethyl esters (P-OM3) on
Lp-PLA2 concentration and lipoprotein subfraction levels (assessed by nuclear magnetic
resonance spectroscopy) in men and women with triglycerides 200 to 499 mg/dL while on
statin therapy. After 8 weeks on simvastatin 40 mg/d, 256 subjects were randomly assigned
to 4 g/d of P-OM3 (Omacor

, 465 mg/g EPA and 375 mg/g DHA) or placebo for an additional
8 weeks. P-OM3 treatment lowered Lp-PLA2 concentration (231 to 200 ng/mL, p0.002 vs
placebo). The Lp-PLA2 response was not significantly related to changes in LDL or HDL
cholesterol levels, but was associated with changes in LDL (r0.30, p0.002) and HDL
(r0.25, p0.01) particle concentrations. The change in small LDL particle concentration
(20.5 nm) was associated with the Lp-PLA2 response (r0.24, p0.02), but the change in
large LDL particle concentration was not (r-0.02, p0.84). The change in small HDL particle
concentration (r0.01, p0.94) was not associated with the Lp-PLA2. Changes in medium
(r0.24, p 0.02) and large (r0.25, p0.01) HDL particle concentrations were both
associated with the Lp-PLA2 response, but in opposite directions. Thus, P-OM3-induced
2007 ATVB Poster Presentations e-93
by on March 25, 2011 atvb.ahajournals.org Downloaded from
changes in Lp-PLA2 are associated with changes in LDL and HDL particle concentrations, and
differentially related to changes in subfractions of these lipoproteins. These findings are
consistent with those of prior studies showing that Lp-PLA2 is enriched in small, dense,
electronegative LDL particles and may have implications for understanding how lipid therapies
influence Lp-PLA2 and associated cardiovascular risk. Funding provided by Reliant Pharma-
ceuticals.
P329
The Lipid Peroxidation Product Malondialdehyde Impairs ABCA1 Cholesterol
Export from Cells Through Site-specific Crosslinking of Apolipoprotein A-I
Baohai Shao, John F Oram, Jay W Heinecke; Univ of Washington, Seattle, WA
Objective - LDL extensively modified by malondialdehyde (MDA) is a classic ligand for
scavenger receptors that promote macrophage foam cell formation in vitro. Protein-bound MDA
has been detected in atherosclerotic lesions by immunohistochemistry, implicating lipid
peroxidation in the pathogenesis of atherosclerotic vascular disease. However, most studies of
MDA have focused on LDL and relied on antibodies that react with unknown epitopes.
Remarkably little is known about the specific sites and chemical nature of MDA adducts in
proteins. Moreover, the possibility that MDA-modified HDL plays a role in atherogenesis has
received little attention. We have investigated the possibility that one important target for MDA
might be apolipoprotein A-I (apoA-I), the major HDL protein. Results - Lipid-poor apoA-I
promotes efflux of cellular cholesterol from macrophage foam cells by the ABCA1 pathway.
ApoA-I exposed to increasing concentrations of MDA progressively and dramatically lost its
ability to remove cholesterol from cultured cells. Tandem mass spectrometric analysis
demonstrated that specific lysine residues (K118, K133, K195, and K226) as well as the
N-terminal amino group were modified by MDA in high yield. Importantly, cross-linked lysine
residues were identified as the major product of apoA-I exposed to MDA. We observed
significantly faster movement on non-denaturing PAGE of MDA-modified apoA-I, suggesting
that cross-linking between lysine residues condenses the molecular structure of the
apolipoprotein. One major cross-linking site in apoA-I involved K226, which is located in helix
10 of the protein. This region plays a critical role in triggering apoA-I lipid association and sterol
efflux by the ABCA1 pathway. Conclusions - These observations suggest that the high reactivity
of K226 promotes its selective cross-linking with other lysine residues. The reaction of K226
with MDA disrupts a key structural element of apoA-I, thereby inhibiting cholesterol efflux by
ABCA1. Our observations indicate that MDA may interfere with normal HDL function by
site-specifically cross-linking lysine residues in apoA-I, which might play a critical role in
atherogenesis by impairing cholesterol removal from artery wall cells.
P330
VLDL from Obese Zucker Rats Contain Elevated Eicosanoids and
Octadecanoids (Oxylipins) That Are Released by Lipoprotein Lipase
Gregory Shearer, Sanford Rsch/USD, Sioux Falls, SD; John Newman; USDA, ARS, Davis, CA
Objective: Oxidized fatty acid metabolites (oxylipins) such as eicosanoids and octadecanoids
are found in plasma and their levels are closely related to inflammatory processes. The role of
lipoproteins in transport of oxylipins is poorly understood. We investigated the effect of
hypertriglyceridemia on their distribution in VLDL, their esterification into lipids and their
availability for release by lipoprotein lipase (LpL). Methods and Results: Plasma and VLDL
from 4 obese and 4 lean Zucker females was obtained. Oxylipins with and without base
hydrolysis were extracted followed by HPLC/MS/MS allowing quantitation of 31 oxylipin
species. There was no difference in the relative abundance of plasma oxylipins. VLDL
composition was different than plasma (p0.001). Total molar abundance was increased in
obese VLDL (1003 127 vs. 277 56 pmol/mL, p0.002). Mid-chain alcohols (hydroxy-
eicosatetraenoic and hydroxyoctadecadienoic acids) were elevated on average 8.0 0.4 fold
over control levels. Epoxides were also increased (769 99 vs. 114 36 pmol/mL, p0.001)
driven largely by a 25 fold increase in 5,6 epoxyeicosatrienoic acid (5,6 EET) (515 73 vs.
21 12). The remaining epoxides were increased 3.9 2.2 fold and 9,10-
epoxyoctadecenoate was not increased (p0.259). Neither diols, triols nor ketones were
increased, and conspicuously the 5,6 EET breakdown product 5,6 dihydroxyeicosatetraenoic
was decreased (15 2 vs. 23 2, p0.017 uncorr). The oxylipins were esterified into lipids,
with 85 3% of appearing only after base hydrolysis. There was no difference in the levels
of unesterified oxylipins. Incubation with 5 g/mL lipoprotein lipase yielded 3.6 0.8 fold
more mid-chain alcohols and 2.2 0.4 fold more epoxides than heat inactivated LpL or vehicle
from obese VLDL, however we could not detect LpL mediated release from control VLDL.
Summary and Conclusions: While the total plasma content of oxylipin species is not
appreciably altered in plasma of obese Zucker rats, VLDL are appreciably altered by the
expansion of an LpL-labile compartment, presumably in triglycerides. With the exception of 5,6
EET, this expansion is characterized by pro-inflammatory oxylipins and could expose
TG-consuming tissues to increased inflammatory stress.
P331
A Novel Role for Apolipoprotein A-V: Association with Intracellular Lipid
Droplets
Xiao Shu, Trudy M Forte, Robert O Ryan; CHORI, Oakland, CA
Accumulating evidence indicates apolipoprotein A-V (apoA-V) is an important regulator of
triacylglycerol (TG) metabolism. Studies with mice and human have shown that a deficiency of
apoA-V is associated with increases in plasma TG levels. Given that plasma levels of apoA-V
are extremely low, we hypothesized that this protein functions intracellularly by affecting the
assembly and/or secretion of apoB-containing particles. Overexpression of apoA-V in Hep3B
cells cultured in medium supplemented with oleic acid altered neither the amount of apoB
secreted nor the density distribution of apoB-containing particles. Fluorescence microscopy
studies were carried out on oleic acid supplemented McArdle-RH7777 cells overexpressing
human apoB100 and apoA-V to determine whether these proteins traffic together in the
secretory pathway. Confocal fluorescence microscopy images revealed that apoA-V does not
interact with apoB intracellularly. Whereas apoB localized to the endoplasmic reticulum, apoA-V
was found in a distinct cellular compartment comprised of a cluster of spherical structures. Nile
Red fluorescence staining identified these structures as intracellular lipid droplets. ApoA-V-
green fluorescent protein fusion protein localized to the surface of the lipid droplets and
co-localized with adipophilin, a known hepatocyte lipid droplet surface protein. The data reveal
a unique association of apoA-V with intracellular neutral lipid droplets suggesting a function for
this protein in lipid storage and/or mobilization.
P332
The Influence of Apolipoprotein A-II Introduction on Apolipoprotein A-I
Conformation in High-density Lipoproteins
R A Gangani D Silva, W Sean Davidson; Univ of Cincinnati, Cincinnati, OH
The metabolism of high density lipoprotein (HDL) is likely influenced by the interaction and
conformation of its two major protein constituents; apolipoprotein (apo) A-I and apoA-II. To
understand the impact of apo A-II on apoA-I in HDL we have begun to monitor apoA-I
conformational changes on mixed apoA-I/A-II discoidal HDL particles (LpA-I/A-II) compared to
apoA-I only HDL particles (LpA-I). Homogeneous, 96 LpA-I/A-II was reconstituted with apoA-I:
apoA-II in a 2:1 ratio and a total of three protein molecules per particle. These mixed particles
were subjected to analysis by a cross-linking and mass spectrometry approach previously used
to study LpA-I and discoidal apoA-II HDL (Lp A-II) particles. After cross-linking, delipidation and
exhaustive tryptic digestion of LpA-I/A-II were specifically searched for the short and long range
apoA-I cross-links we previously identified in LpA-I particles. Almost all short-range cross-links
(7/8) previously identified in LpA-I particles were present in the LpA-I/A-II particles. However,
only 2/9 long range cross-links were present. Furthermore, all short range cross-links (5/5)
pertinent to LpA-II were present in LpA-I/A-II but none of the long range cross-links are found
(0/8). These observations strongly suggest that, when present together in a single HDL particle,
both apoA-I and apoA-II adopt different conformations than when present in isolation. Further
studies are currently underway to understand molecular details of LpA-I/A-II particles using
other complimentary mass spectrometric techniques as hydrogen deuterium exchange.
P333
Helix 10 Mutations in ApoA-I from Atherosclerosis-sensitive Mice Result in
Decreased Lipid Binding and Cholesterol Efflux from Macrophages
Timothy J Sontag, Catherine A Reardon, Godfrey S Getz; Univ of Chicago, Chicago, IL
The C57BL/6J (C57) mouse strain exhibits atherosclerotic lesions that are at least five-fold
larger than those of other strains and has significantly lower plasma HDL and apoA-I levels. The
apoA-I sequence of the C57 mouse is identical to the other strains except for a two amino acid
residue variation in alpha helix 10 (K228Q, A229V). Helix 10 along with helix 9 are important
for the initial association of lipid-free apoA-I with lipid, and helix 10 mutations in humans have
been reported to reduce cholesterol efflux from cells and lower plasma HDL levels. We
hypothesized that the strain-specific variations in helix 10 of mouse apoA-I alter the lipid
binding characteristics of the protein and subsequently its ability to efflux cholesterol/
phospholipid from cells, thereby altering the formation of nascent HDL and subsequently
mature HDL. This may account for the observed lower plasma HDL levels in C57 mice which
may contribute to the high susceptibility to atherosclerosis in this strain. The mRNA levels of
a variety of genes involved in HDL synthesis and catabolism were compared between C57 and
FVB mice, with apoA-I mRNA levels higher in FVB liver. Pulse-chase analysis revealed apoA-I
synthesis levels that were slightly greater in liver slices from FVB mice but the same in the
intestine of the two mice. Also similar were the in vivo rates of catabolism of nascent HDL,
mature HDL, and lipid free apoA-I from both strains, in both C57 apoA-I/apoE double knockout
mice that lack HDL and in wild type C57 and FVB mice. The two mouse apoA-I variants
demonstrated significant differences in their HDL binding affinity as well as in their ability to
mediate cholesterol efflux from macrophages, supporting the hypothesis that the initial lipid
binding affinities of the mouse apoA-I variants may regulate the resulting plasma HDL levels.
P334
Reduced Plasma Corticosterone Levels in LDLr-/- , ApoA-I-/- Mice: A
Model to Study the Role of HPA Axis in the Metabolic Syndrome
Manal Zabalawi, Manish S Bharadwaj, Lijun Tang, Mary G Sorci-Thomas; Wake Forest Univ
Med Cntr, Winston-Salem, NC
ApoA-I, the main structural protein found in HDL delivers the majority of cholesterol required
for adrenal steroid hormone synthesis. In our previous studies, LDLr-/-, apoA-I-/- mice have
been shown to have 10-fold less adrenal cholesterol compared to LDLr-/- mice. This decrease
in adrenal cholesterol is mainly due to the reduction in adrenal esterified cholesterol levels. The
goal of this study was to determine if the reduction in adrenal cholesterol ester would lead to
changes in adrenal function and alter plasma corticosterone and ACTH concentrations. Both
LDLr-/-,apoA-I-/- and LDLr-/- mice were fed chow or an atherogenic diet for 8 wks. At the end
of the study, adrenals were removed and the plasma corticosterone and ACTH levels were
measured. Adrenals from both chow and diet fed LDLr-/-, apoA-I-/- mice showed a 4-fold
upregulation in the expression of mRNA encoding HMG CoA reductase and the P450 side-chain
cleavage enzyme when compared to LDLr-/- mouse adrenals. This upregulation in adrenal
cholesterol synthesis apparently could not compensate for the lack of apoA-I derived
cholesterol since LDLr-/-,apoA-I-/- mice showed a 1.5-fold lower plasma corticosterone
concentration compared to LDLr-/- mice. When corticosterone was supplemented to the
drinking water of diet-fed LDLr-/-,apoA-I-/- and LDLr-/- mice, corticosterone levels were
increased in both genotypes, while LDLr-/-,apoA-I-/- mice showed a dramatic 10-fold
increase in ACTH levels accompanied by a redistribution of adipose tissue from peripheral to
central sites when compared to LDLr-/- mice. We hypothesize that diet-fed LDLr-/-, apoA-I-/-
mice may be a useful model to study the role of apoA-I in regulating the HPA axis and its
association with the metabolic syndrome.
e-94 Arterioscler Thromb Vasc Biol June 2007
by on March 25, 2011 atvb.ahajournals.org Downloaded from
P335
Profound Cardiac Metabolic Changes Caused by Coronary Disease
Anabelle Chase, Christopher Jackson, Gianni Angelini, Saadeh Suleiman; Bristol Heart
Institute, Bristol, United Kingdom
We have recently shown that apolipoprotein E knockout (apoE
-/-
) mice fed a high-fat,
Western-style diet for 6 months, develop occlusive coronary lesions and show evidence of
myocardial infarction. The same mice fed a normal rodent diet appear to remain healthy, and
therefore provide excellent littermate controls for the study of coronary disease. Thus, the aim
of this work was to investigate the direct myocardial effects of coronary disease. Male apoE
-/-
mice were weaned onto a normal rodent diet, and at approximately 8 weeks old, animals were
either switched onto a high-fat, Western-type diet (21% fat; 0.15% cholesterol) or were
maintained on normal rodent diet for 6 months. Isolated hearts from both groups were used to
measure cardiac metabolites and function. Isolated myocytes were used to measure
mitochondrial flux (NAD

/NADH), contractile function and calcium transients. Mice fed high-fat


diet had significantly increased levels of cardiac lactate (from 42 6 to 68 6 nmol/mg
protein), decreased glycogen content (from 0.077 0.008 to 0.036 0.005 mg/g wet weight)
and decreased levels of ATP (from 16 0.9 to 11 1 nmol/mg protein). Evidence of metabolic
stress in diseased hearts was confirmed in isolated perfused myocytes which showed
increased NAD

/NADH ratio (from 0.27 0.02 to 0.35 0.02). These metabolic differences
did not alter functional characteristics of isolated perfused hearts or myocytes. However both
isolated myocytes and intact perfused hearts from animals with coronary disease were
significantly more resistant to cardiac insults than control animals. In conclusion, coronary
disease induced by high-fat diet has profound metabolic effects on the myocardium. These
changes appear to alter vulnerability to cardiac insults, which may be due to ischemic
preconditioning.
P336
Prediction of the Localization of High-Risk Coronary Atherosclerotic
Plaques Based on Low Endothelial Shear Stress: A Serial IVUS and
Histopathology Natural History Study
Yiannis S Chatzizisis, Brigham and Womens Hosp, Harvard Med Sch, Boston, MA; Michael
Jonas, Massachusetts Institute of Technology, Cambridge, MA; Ahmet U Coskun,
Northeastern Univ, Boston, MA; Roy Beigel, Benjamin V Stone, Massachusetts Institute of
Technology, Cambridge, MA; Charles Maynard, Univ of Washington, Washington, DC; Ross G
Gerrity, Med College of Georgia, Augusta, GA; William Daley, Novartis Pharmaceuticals Corp,
New Jersey, NJ; Campbell Rogers, Cordis Inc, Warren, NJ; Elazer R Edelman, Charles L
Feldman, Peter H Stone; Brigham and Womens Hosp, Harvard Med Sch, Boston, MA
Background: The role of low ESS in the progression of atherosclerotic plaques and evolution
of these plaques to thin cap fibroatheromas (TCFA) has not been studied. We investigated the
effect of low ESS in the development of TCFA in swine coronary arteries. Methods: In 11
diabetic hyperlipidemic swine, IVUS-based 3D reconstruction of the coronary arteries was
performed at baseline (wk 23) and follow up (wk 30). Baseline ESS was calculated using
computational fluid dynamics, and plaque-free subsegments of interest of 3 mm length were
identified (n142). Coronary arteries (n31) were harvested at follow up, cryosectioned at the
subsegments of interest and stained histologically. Intima/media ratio, min cap thickness, lipid
deposition and inflammation were quantified and atherosclerotic lesions were histopathologi-
cally classified to minimal (MIN), intermediate (INT) and TCFA. Results: The magnitude of low
ESS was significantly associated with larger plaque size, increased lipid deposition, inflam-
mation and cap thinning (p0.001). Low ESS and hyperlipidemia were independent predictors
of the development of TCFA or INT vs. MIN, whereas the differentiation of early lesions to TCFA
vs. INT was associated with hyperlipidemia and hyperglycemia (Fig, Table). Conclusion: The
magnitude of low baseline ESS determines the severity and heterogeneity of atherosclerotic
lesions and, in combination with systemic risk factors, predicts the development of TCFA.
These findings provide a perspective for the identification of early stages of a high risk plaque,
thereby enabling highly selective pre-emptive coronary interventions to prevent adverse
coronary events.
P337
The Lysine Binding Site in Kringle IV Type 10 Is Required for
Apolipoprotein(a)-mediated Changes in Vascular Endothelial Cell Phenotype
Taewoo Cho, Marlys L Koschinsky; Queens Univ, Kingston, Canada
Substantial evidence indicates that endothelial dysfunction plays a critical role in atherogenesis.
We previously demonstrated that apolipoprotein(a) [apo(a); the distinguishing protein compo-
nent of the atherothrombotic risk factor lipoprotein(a)] elicits rearrangement of the actin
cytoskeleton in human umbilical vein endothelial cells (HUVECs), characterized by increased
central stress fiber formation and increased cell permeability. These effects are mediated by
increased myosin light chain (MLC) phosphorylation via a Rho/Rho kinase-dependent signaling
pathway. Apo(a) contains kringle (K)IV and KV domains similar to those in plasminogen: apo(a)
contains 10 types plasminogen KIV-like sequences, followed by sequences homologous to the
plasminogen KV and protease domains. Several of the apo(a) kringles contain lysine-binding
sites (LBS) that have been proposed to contribute to the pathogenicity of Lp(a). Here, we tested
recombinant apo(a) [r-apo(a)] variants containing both amino-and carboxyl-terminal truncations
of the molecule, and found that the effect of apo(a) on increases in MLC phosphorylation and
HUVEC permeability can be attributed to the kringle IV type 10 (KIV
10
) domain, within the
carboxyl-terminal half of the apo(a) molecule. Accordingly, 17KAsp(full length apo(a) species
with a mutation in the strong LBS in KIV
10
) does not elicit these effects, nor does 17K r-apo(a)
in the presence of the lysine analog epsilon-aminocaproic acid. In keeping with our previous
observations, the effects of apo(a) on MLC phosphorylation and EC permeability were abrogated
by Rho/Rho kinase inhibitors as well as the MLC kinase inhibitor ML-7. We have shown that
17K-r-apo(a) treatment of HUVECs enhances the Rho kinase-mediated phosphorylation of MLC
phosphatase at Thr 850, which inactivates the phosphatase activity and leads to an increase
in phosphorylation of MLC by MLC kinase; this effect was not observed using the 17KAsp
variant. Taken together, our findings indicate that the strong LBS in apo(a) KIV
10
mediates all
of our observed effects of apo(a) on HUVEC phenotype. Studies are ongoing to further dissect
the molecular basis of these findings.
P338
CCR7 Is Functionally Required for Atherosclerosis Regression and Is
Activated in Vivo by LXR
Jonathan E Feig, Jonathan R Hoffman, Ines Pineda Torra, Michael J Garabedian, Edward A
Fisher; New York Univ Sch of Medicine, New York, NY
We have developed a mouse model of atherosclerosis regression by changing, after plaque
formation, the plasma environment of the plaque from hyperlipidemia to normolipidemia by
transplanting aortic segments from apoE-/- mice to wild type recipients (regression environ-
ment). As a control, mice with hyperlipidemia were also transplant recipients to continue the
progression environment. In this model, we reported emigration of plaque foam cells to regional
and systemic lymph nodes after 3 days in the regression environment. During regression, the
foam cells had features of dendritic cells (DCs). Because DCs require the chemokine receptor
CCR7 for migration, we measured its mRNA and protein, and found foam cell expression of this
factor to be induced, but only during regression. Further experiments using blocking antibodies
to CCR7 demonstrated a functional requirement for it in regression. We have recently reported
that LXR mRNA increases in foam cells during regression and were interested in the present
studies whether LXR and CCR7 were linked. Using a murine model of immature DCs, we found
that CCR7 expression is increased 89 fold upon LXR activation by the agonist T0901317.
Importantly, this increase is dependent on CCR7 gene transcription, because pretreatment with
actinomycin D abolished the observed response. To extend the results in vivo, we treated
western-diet fed apoE-/- mice with LXR agonist. This also induced CCR7 expression in foam
cells. Foam cell content and lesion area decreased by 21% and 24%, respectively, consistent
with a report that a LXR agonist promoted regression of atherosclerosis in LDLR-/- mice. The
dependence of the agonists effects on CCR7 was demonstrated by treatment of apoE-/-
CCR7-/- with LXR agonist: foam cell content was only decreased by approximately 9%. In
conclusion, CCR7 is required in a mouse model for plaque regression with LXR being a factor
that can modulate its expression.
P339
Fibroblast and Aortic Smooth Muscle Cell Activation Is Regulated by
Niemann-Pick Type C2 Protein
Chad Csepeggi, David Y Hui, Andrey A Frolov; Univ of Cincinnati, Cincinnati, OH
Fibroblast and smooth muscle cell (SMC) phenotypic change represents a major underlying
cause in several pathologies, including maladaptive vessel wall remodeling induced by
hypertension, atherosclerosis, and angioplasty. Niemann-Pick type C2 (NPC2) gene has been
recently identified as the second gene in the autosomal recessive cholesterol storage disorder,
the NPC disease. It encodes a secretory, 151 amino acid protein with unknown function. This
report identifies NPC2 protein as a novel autocrine/paracrine factor that negatively regulates
fibroblast and SMC activation, thus playing an important role in regulation of aortic tissue
remodeling. This conclusion is supported by the following observations. First, loss of NPC2
protein in primary human dermal fibroblasts resulted in their activation. This was reflected by
a significant up-regulation of the SMC markers expression, growth factors and their receptors,
as well as by induction of the collagen and inflammatory cytokine genes. The latter correlated
strongly with the increased activity of NF-B ( 2.5-fold). Second, silencing of NPC2 gene in
aortic SMC through siRNA transfection resulted in stimulation of the cell migration toward PDGF
by 2.5-fold. Third, NPC2-deficient cells displayed constitutive activation of the EGF and FGF
receptor tyrosine kinase (RTK) signaling, which is consistent with the NPC2 primary function as
a suppressor of the RTK activation/transactivation. These findings could provide a solid basis
for the development of novel therapeutics to treat the coronary artery and cerebral vascular
diseases.
P340
The Receptor for Advanced Glycation Endproducts Induces Cellular
Migration via Binding of Its Intracellular Domain to Diaphanous-1:
Implications for Vascular Biology and Disease
Barry I Hudson, Anastasia Z Kalea, Maria Arriero, Evis Harja, Eric Boulanger, Jan Kitajewski,
Ann Marie Schmidt; Columbia Univ, New York, NY
Ligand binding to the Receptor for Advanced Glycation Endproducts (RAGE) activates a cascade
of intracellular signaling events which lead to vascular dysfunction. The short cytoplasmic
domain of RAGE is essential to activate these pathways, and hence the understanding of how
2007 ATVB Poster Presentations e-95
by on March 25, 2011 atvb.ahajournals.org Downloaded from
this domain functions in the intracellular environment is significant. In this study, we identified
using the yeast two-hybrid assay that the RAGE cytoplasmic tail directly binds Diaphanous-1
(Dia-1); a molecule that mediates intracellular signaling and cellular motility. Evidence
supporting this interaction is as follows: First, co-immunoprecipitation (IP) and binding of
epitope tagged RAGE tail and Dia-1 was confirmed in transfected cells. Second, RAGE/Dia-1
was co-IPed from RAGE expressing cells, but not from DN-RAGE (RAGE tail deleted) expressing
cells. Third, by confocal microscopy in intact cells, RAGE and Dia-1 co-localized after
RAGE-ligand stimulation, with significantly less co-localization observed with DN-RAGE and
Dia-1 expressing cells. To demonstrate the interaction between RAGE and Dia-1 was direct, in
vitro expressed GST-RAGEtail directly bound Dia-1, but not GST alone. Next, we prepared
distinct domains of Dia-1 to delineate the precise domain of Dia-1 that binds the RAGE tail.
Experiments revealed the RAGE tail interacted with the Formin Homology domain (FH) of Dia-1,
but not the Rho GTPase binding domain. The vascular relevance of this interaction was
identified by the expression of Dia-1 and its co-localization with RAGE in aortic ECs and SMC
and monocyte-derived cells. To test the biological relevance of the RAGE / Dia-1 interaction,
functional studies were performed using siRNA to knockdown Dia-1 expression or scramble
siRNA as control. Compared to siRNA scramble control, Dia-1 siRNA blocked RAGE ligand
stimulated cellular migration and invasion. In conclusion, we have identified a novel signal
transduction mechanism linking the intracellular function of RAGE to Diaphanous-1. We
propose that blockade of RAGE/Dia-1 interaction represent a novel target for therapeutic
intervention in vascular disease.
P341
Multiplexed Immunoassays for Simultaneous Quantification of Soluble
Cardiovascular and Metabolic Biomarkers in Mouse Blood
Terry Whitehead, Jiaxin Dong, Qiang Xiao, Hank Hwang, Jehangir Mistry, Shaoquan Ji;
LINCO, now part of Millipore, Saint Charles, MO
Mouse is a key model in cardiovascular research. Limited blood volume has increased the
difficulty of measuring multiple biomarkers necessary for understanding the biological
processes associated with cardiovascular disorders (e.g. atherosclerosis, inflammation and
atherothrombosis). Using the Luminex

xMAP technology, we developed multiplexed immu-


noassay panels requiring small sample volume for simultaneous quantification of multiple
circulating biomarkers (e.g. apolipoproteins, adhesion molecules, acute phase proteins, and
proinflammatory cytokines, etc) in mouse samples. Based on analyte compatibility, four
separate multiplexed immunoassay panels were developed (Panel 1: sICAM-1, sVCAM-1,
sE-Selectin, MMP-9, PAI-1; Panel 2: Adiponectin, Apolipoprotein A1, Apolipoprotein E, and
Fibrinogen; Panel 3: 22 cytokines and chemokines; and CRP single-plex assay). Samples were
incubated overnight at 4 C in a 96-well microtiter filter plate containing a mixed population of
polystyrene beads with unique fluroscent signature and covalently immobilized with various
specific capture antibodies. After washing, captured analytes on beads were incubated for 1 h
at RT with a cocktail of biotinylated detection antibodies. Following subsequent incubation with
streptavidin-phycoerythrin, fluorescent signals on beads were quantified using a Luminex

Reader. Each antibody pair targeting an individual analyte is highly specific, with no or
negligible cross-reactivity to other analytes within the panel. The assay robustness is
demonstrated by acceptable precisions (CV 15% for inter-assay variations; CV 10% for
intra-assay variations), and accuracy (100 30%) for serum or plasma samples. According to
analyte concentrations, serum or plasma samples require no dilutions (Panel 3), dilutions of
approximately 1:100 (Panel 1), 1:200 (CRP), or 1:5,000 (Panel 2). Thus, the total serum/plasma
volume required for measuring all analytes is less than 55 l for duplicate measurement. The
assays may be used for other sample types (e.g. cell culture supernatant, tissue/cell lysate).
These novel assay panels serve as accurate, reproducible and economic tools for simultaneous
quantification of multiple CVD and metabolic biomarkers in mouse samples.
P342
The Apolipoprotein(a) Present in the Carotid Artery Plaques of Subjects
with High Plasma Lipoprotein(a) Reacts Against an Antibody Specific for
Oxidized Phosphatidylcholine, Which Is Endowed with a Proinflammatory
Potential
Stephen T Joy, Priyesh N Patel, Ditta Pfaffinger, Celina Edelstein, Angelo M Scanu; Univ of
Chicago, Chicago, IL
Introduction-In previous studies in human macrophage cultures we showed that human
apolipoprotein(a), apo(a), has pro-inflammatory properties attributable to the presence of
oxidized phosphatidylcholine (ox-PC) chemically linked to lysine residues in apo(a), kringle V.
Hypothesis-Based on these observations and that circulating apo(a) is found in human carotid
artery plaques, we set out to determine whether plaque apo(a) also contains ox-PC adducts,
on the assumption that this post-translationally modified apo(a) may play a role in the formation
and progression of the atherosclerotic plaque. Methods-Subjects undergoing elective carotid
artery endarterectomy were divided into low and high Lp(a) according to their plasma Lp(a)
levels determined prior to surgery (low: a 3 mg/dL; high: a 20 mg/dL of protein).
Immediately after resection, the plaques were frozen in OCT and sections were immunostained
with CD68 to identify macrophage dense(M-d) and macrophage-poor(M-p) regions followed by
laser capture microdissection (LCM) of these regions. LCM-dissected tissues were extensively
extracted with Acetate buffer, pH 6.0, containing 6M GdHCl and Western blot analyses were
carried out using either an anti apo(a) specific antibody or a monoclonal antibody, T-15, specific
for ox-PC. Results-The results showed that: 1) in the plaques, apo(a) was present in the high
Lp(a) subjects ; 2) apo(a) was partially fragmented and reacted against T-15 indicating a
linkage to ox-PC; 3) in turn, in the low Lp(a) subjects no T-15 reacting material was detectable
in the absence of apo(a); 4) similar data were obtained in the M-d and M-p areas.
Conclusions-Our data indicate that there is a correlation between plasma and plaque
concentrations of apo(a). We show that plaque apo(a) can undergo cleavage, an expression of
the known proteolytic microenvironment and also post-translational modification via ox-PC
adducts shown previously in vitro to have pro-inflammatory properties. We speculate that in
subjects with high plasma Lp(a), the apo(a) complexed to ox-PC may contribute to the formation
and progression of the carotid plaques.
P343
Apolipoprotein E Receptor-2 Deficiency Alters Atherosclerotic Plaque
Composition in the Aortic Root and Exacerbates Abdominal Atherosclerosis
in LDL Receptor-Deficient Mice
Eddy S Konaniah, Joshua E Basford, David Y Hui; Univ of Cincinnati, Cincinnati, OH
Apolipoprotein E Receptor-2 (apoER2) is a member of the LDLR family of apoE-binding
receptors. ApoER2 is expressed abundantly in vascular endothelial cells and is also present in
vascular smooth muscle cells, but its role in vascular functions has yet to be described. This
study was initiated to determine if vascular apoER2 influences the development of atheroscle-
rotic lesion in a model of hypercholesterolemia by crossing apoER2-KO mice with LDLR-KO
mice and then feeding them a western-type diet for 24 weeks. Whole aortas of apoER2/LDLR-
dKO and LDLR-KO displayed similar amounts of plaque accretion in the aortic arch and
proximal regions of innominate, carotid, and subclavian arteries. However, analysis of aortic
root lesion morphology revealed variations of fat deposition, smooth muscle cell (SMC) and
collagen distribution between LDLR-KO mice with or without apoER2. Lipid deposits were
greater and distributed more evenly throughout aortic root lesions of LDLR-KO compared to
apoER2/LDLR-dKO mice. Lesions in the LDLR-KO mice also showed more SMC and less
collagen near the luminal surface whereas apoER2/LDLR-dKO mice have fewer SMCs and
increased collagen in the same region. These data suggest that the lack of apoER2 expression
in the aortic roots decreases lipid accumulation, reduces SMC activation, and increases
collagen production to yield a more stable fibrous plaque lesion compared to the unstable
lipid-rich lesions observed in apoER2 expressing LDLR-KO mice. Despite differences in aortic
root lesion composition, we were unable to detect significant difference in nitrosylated tyrosine
residues and VCAM expression in the aortic roots of LDLR knockout mice with or without
apoER2. Interestingly, absence of apoER2 in male LDLR-KO mice significantly increased plaque
formation in abdominal aortas compared to male LDLR-KO mice with apoER2 expression. The
exaggerated plaque size in abdominal aortas of the apoER2/LDLR-dKO male mice was
correlated to the presence of increased nitrosylated tyrosines compared to those observed in
LDLR-KO mice with apoER2. Thus, apoER2 expression in the abdominal aorta may function to
normalize endothelial function and protect against hypercholesterolemia-induced uncoupling of
endothelial nitric oxide synthase.
P344
Aging Induces a Decreased Vascular Remodeling Response in LDLr-/-
Mice: Identification of Quaking as a New Player in Vascular Homeostasis
Adriaan O Kraaijeveld, Ramon de Nooijer, LACDR Leiden Univ, Leiden, The Netherlands;
Ernst E van der Wall, Leiden Univ Med Cntr, Leiden, The Netherlands; Theo J van Berkel,
LACDR Leiden Univ, Leiden, The Netherlands; Wouter J Jukema, Leiden Univ Med Cntr,
Leiden, The Netherlands; Erik A Biessen; LACDR Leiden Univ, Leiden, The Netherlands
Objective: Aging is regarded as a major risk factor for atherosclerosis. It is still unclear if it
directly and/or indirectly impacts the process of atherogenesis, and by which genetic pathways
this is regulated specifically. Therefore, we not only evaluated the effect of aging in collar
induced atherosclerosis on lesion size, composition and vascular remodeling, but also tried to
identify differentially regulated genetic pathways in the context of atherosclerosis upon vascular
aging. Methods: Atherosclerotic lesions were elicited in carotid arteries of young (12 weeks,
n16) and aged (52 weeks, n15) LDLr-/- mice by bilateral perivascular collar placement
after six weeks of Western type diet. Six weeks later, left carotid arteries as well as aortic roots
were harvested and analyzed histologically. In addition, micro-array analysis was performed on
the right carotid arteries. Results: Carotid plaque (40,500 vs. 14,800 m
2
, P0.04) and media
areas (52,300 vs. 33,600 m
2
, P0.02) were larger in young vs. aged mice. In line with this
finding, aortic root plaque area was larger in young mice (7,38x10
5
vs. 5,06x10
5
m
2
, P0.03).
Intriguingly, due to a substantial outward remodeling in younger mice (total vessel area
179,400 vs. 113,500 m
2
, P0.04), carotid lumen areas were increased compared to aged
mice (86,572 vs. 65,122 m
2
, P0.04). No differences were seen in plaque composition,
suggesting that age per se is not a causal factor in plaque stability. Micro-array analysis
revealed a significant up-regulation of the vasculogenesis pathway in aged mice, with specific
up-regulation of the Quaking (Qk) gene. Qk protein was detected in carotid and aortic root
plaques and mainly observed in endothelial cells and macrophages. Qk function was
subsequently assessed in vitro; gene silencing with Qk siRNA caused an increased wound
healing response after scratching an endothelial cell monolayer (P0.02), indicative of an
important role of Qk in cell proliferation and/or migration. Conclusions: Decreased athero-
genesis and outward remodeling accompany increasing age in our collar induced atheroscle-
rosis model. Qk seems to be an important player in age-dependent vascular homeostasis.
P345
Protein Kinase C-delta Mediates PDGF-BBInduced Stabilization of MCP-1
mRNA in Smooth Muscle Cells
Bin Liu, Yi Liu, Latika Dhawan, Zhenggen Jin, Mark B Taubman; Univ of Rochester,
Rochester, NY
We have previously shown that PDGF-BB enhances monocyte chemoattractant protein-1
(MCP-1) mRNA stability in SMC by down-regulating a ribonuclease activity. To identify
downstream targets of the PDGF recepter, rat aortic SMC at 60% confluence were treated with
5A

M of actinomycin D (Act D) and PDGF-BB (5ng/ml) for 3 hrs in the presence of inhibitors
of PDGF-mediated signaling pathways, and MCP-1 mRNA levels were measured by RT-PCR.
1A

M of PP2 (Src inhibitor), PD98059 (MAP kinase kinase, MEK, inhibitor), wortmannin
(PI3-kinase inhibitor) or DPI (NADPH inhibitor) failed to block the effect of PDGF, whereas
U73122, a phospholipase C (PLC) inhibitor completely blocked the effect. We next examined
e-96 Arterioscler Thromb Vasc Biol June 2007
by on March 25, 2011 atvb.ahajournals.org Downloaded from
downstream targets of PLC, Go 6850 (an inhibitor of PKC,
2

, , , and I

) blocked
the effect of PDGF whereas Ro320432 (an inhibitor of PKC,
2

,
2
II,
3
, ) or
BAPTA (a Ca2 chelator) did not. To further elucidate the particular PKC isozyme(s) involved
in the mRNA stabilizing effect, siRNAs to PKC, , and
1
4 were transfected into SMC at
a ratio of 60 pmol siRNA/3 million cells. The efficacy of each siRNA was more than 70% as
determined by immuno-blotting. Reduction of PKC blocked the stabilizing effect of PDGF-BB
on MCP-1 mRNA, whereas reduction of PKC or
1
4 did not. The above studies suggest
a novel role for PKC in mediating the effect of PDGF on mRNA stability. Further elucidation
of the signaling pathway(s) through which PDGF-BB enhances MCP-1 mRNA stability may
provide new approaches for inhibiting vascular inflammation and atherosclerosis.
P346
Gene Expression and Pathway Analyses of Atherosclerotic Aorta Reveals
Significant Dysregulation of Calcium Signaling Pathway
Solida Mak, Ba-Bie Teng, Lawrence C Shimmin, James E Hixson; Univ of Texas, Houston,
Houston, TX
Atherosclerosis is a complex disease resulting from the interactions of genetic and environ-
mental risk factors leading to heart failure and stroke. We hypothesize that risk factors interact
to alter expression of genes and pathways in the aortic wall and hence render it susceptible
to atherosclerosis. Using an atherosclerotic mouse model (Ldlr
-/-
/Apobec1
-/-
designated as
LDb), we performed microarray gene expression analysis to investigate candidate genes and
pathways which are perturbed by the following risk factors: genetics (control C57BL/6 vs. LDb
mice), shear stress (lesion-prone vs. lesion-resistant regions in LDb mice), diet (chow vs. high
fat fed LDb mice) and age (2-month-old vs. 8-month old LDb mice). Male C57BL/6 and LDb
mice (n16/group) were fed on either a chow or a high fat diet, sacrificed at 2- and
8-months-old, and RNA was isolated from the aortic lesion-prone and -resistant segments.
Using Affymetrix Murine 430 2.0 chips (n64), we profiled differentially expressed genes with
the following criteria: p-value of 0.05, fold change 1.5 and FDR 0.05. The data were
normalized using two normalization methods the invariant probe sets (dChip) and the quantile
normalization (known as RMA), the statistical analysis was performed using t-test and ANOVA
and pathway analyses (Pathway Express by Wayne State) were performed. The results revealed
19 altered biological pathways. Both statistical analyses ranked calcium signaling as the most
perturbed pathway followed by focal adhesion, MAPK, complement and coagulation, cytokine-
cytokine receptor, apoptosis, circadian rhythm, PI3 signaling, Toll-like receptor, Wnt signaling
and so on. When we stratified the data by age, MAPK pathway was perturbed at 2-month of
age, whereas Calcium signaling pathway was most perturbed in the 8-month-old mice. In
summary, our analyses demonstrated that genes in MAPK pathway might play a significant role
in the onset of atherosclerotic disease, which is augmented to perturb Calcium signaling
pathway in the later stage. In conclusion, genes involved in MAPK and Calcium signaling
pathways may serve as important targets for future therapeutic intervention.
P347
Ultrasensitive Confocal Imaging of C-Reactive Protein Binding to
Fc-Receptors
Dimitar E Manolov, Kyrylo Tron, Carlheinz Ro cker, Martin Ka chele, Gu nter U Nienhaus, Jan
Torzewski; Univ of Ulm, Ulm, Germany
Background: C-reactive protein (CRP), the prototype human acute phase protein, is widely
regarded as a key player in cardiovascular disease. Fc-receptors have been controversially
discussed since 1999 to be receptors for CRP. Applying of FACS analysis and anti-CRP
antibodies or radioactive labeled CRP to resolve the CRP binding led to false positive results.
Aim of the studies: Definition of CRP-binding to Fc-receptors applying highly sensitive
technique overwhelming methodological problems leading to artifacts. Methods: Using
ultrasensitive confocal imaging analysis and gently labeled CRP we were able to overcome that
problems. The technique enables us to perform observations and incubations on single native
cells and precise association and equilibrium analysis. Results: Recently we showed that CRP
binds FcRIIa (CD32) on transfected COS-7 cells. The avidity we assessed was 100-fold
lower than the one estimated for the anti-CRP/CRP complex. In a work that followed up we
demonstrated a CRP binding to FcRI (CD64) on transfected COS-7 cells. The calculated avidity
was similar to that estimated for FcRIIa. In presence of -chain it increased up to 30-fold. It
is important to note that the dissociation of CRP from the cell surfaces could not be detected
over the time course of several hours and is thus extremely slow. Conclusion: Considering the
pentameric structure of CRP, we hypothesize that multivalent binding and receptor clustering
are crucially involved in the interaction of CRP with nucleated cells. Actual state: We have been
performing ongoing studies on monocytic cell lines and COS-7 cells transfected with FcRIII
(CD16) and -chain that could enrich the conception of CRP binding to receptors on cellular
membranes. We believe that the clear definition of CRP interaction with cellular receptors could
be of significant clinical importance for clarification of CRP pathological role in atherogenesis
and for the elaborating of efficient corresponding therapy.
P348
COX-2 Gene Deletion Increases Advanced Aortic Arch Atherosclerosis in
the ApoE-/- Mouse Model
Sarah E McClelland, Leslie R Ballou, Desmond J Fitzgerald, Orina A Belton; Conway
Institute, UCD, Dublin, Ireland
Cyclooxygenase-2 (COX-2) catalyses the formation of prostaglandins from arachidonic acid.
COX-2 expression is induced in the early phase of the inflammatory response, and again during
the late phase, when it is has a role in the resolution of inflammation. Atherosclerosis has a
chronic inflammatory component, associated with increased COX-2 expression and COX-2
derived prostaglandin production. The role of COX-2 in atherosclerosis is unclear. We have
shown that selective COX-2 inhibition does not affect early atherosclerosis in the ApoE-/-
(Belton, 2003). To date, however, the effect of COX-2 gene deletion has not been investigated.
We have, therefore, generated an ApoE-/- mouse model genetically deficient in COX-2 (DKO),
to address the role of COX-2 in early and late atherosclerosis. COX-2-/- mice were crossed with
ApoE-/- mice to generate ApoE-/-/COX-2-/- double knockout (DKO), ApoE-/-/COX-2, and
ApoE-/-/COX-2/ mice. Mice (n3 per group) were fed a 1% cholesterol diet for 8 or 20
weeks. Serum cholesterol levels did not differ between groups at either time point, nor did
serum thromboxane levels, indicating that platelet activation was equivalent in all groups. At
8 weeks, there was no difference in atherosclerotic lesion burden in the aortic arch or
descending aorta of ApoE-/-/COX-2-/-, ApoE-/-/COX-2 or ApoE-/-/COX-2/ mice. Follow-
ing 20 weeks cholesterol feeding, however, plaque burden was significantly increased in the
aortic arch of the ApoE-/-/COX-2-/- compared with the ApoE-/-/COX-2/ (61.92.8% v
33.85%, P0.01), and was increased to an intermediate level in the ApoE-/-/COX-2
(52.91.2%, P0.05). En face analysis of the descending aorta revealed that, although there
was a trend towards a modest increase in plaque burden in the DKO (21.85.3% v
15.62.4%), this did not reach statistical significance. The discrepancy between aortic arch
and descending aorta atherosclerosis may reflect differing rates of plaque formation at different
areas of the aortic tree, as has been reported elsewhere (Moore, 2005). In conclusion, these
data imply an anti-atherosclerotic function for COX-2 in late atherosclerosis, and its absence
may accelerate advanced atherosclerotic plaque development at some lesion prone sites of the
aortic tree.
P349
Passive Immunization with IK-17, a Human Oxidation-specific Antibody,
Reduces Progression of Atherosclerosis in LDLR
-/-
mice
Esther Merki, Peter X Shaw, Joseph L Witztum, Sotirios Tsimikas; Univ of California San
Diego, La Jolla, CA
Introduction Experimental evidence suggests that immunization strategies targeting oxidation-
specific epitopes may be atheroprotective. IK-17 is a fully human Fab antibody that binds both
malondialdehyde(MDA)-LDL and copper-oxidized LDL (OxLDL) and also inhibits uptake of
OxLDL by macrophages. We therefore hypothesized that passive immunization of LDLR-/- mice
with IK-17 will reduce the progression of atherosclerosis. Methods and Results 24 male
LDLR-/- mice (6 weeks old) were placed on a 1.25% cholesterol/21% milkfat diet for 2 weeks
to increase total cholesterol (TC) levels to 1500 mg/dl and to initiate atherosclerosis. They
were then switched to a 0.5% cholesterol diet and reached TC levels of 600 mg/dl. The mice
were then randomized to treatment with IK-17 (2.5 mg/kg) vs. PBS control intraperitoneally 3
times per week. After 14 weeks of treatment, atherosclerosis was quantitated as the % of the
aortic atherosclerotic area and as the aortic root area. There was significant reduction in %
aortic atherosclerosis surface area in the IK-17 group compared to the control group (4.9%
0.8 vs. 6.9%1.6, P0.001), whereas there was only a trend for reduction in the aortic root
area with IK-17 treatment (1.98 0.57 m
2
vs. 2.250.28 m
2
, p0.17). The diet led to
significant and progressive increases in autoantibody titers and titers of immune complexes to
OxLDL in both groups. There was a significant increase in anti-human antibodies in the IK-17
group but not in the control group. Conclusion Passive immunization with human oxidation-
specific antibody IK17 in LDLR
-/-
mice led to a reduction in atherosclerosis in the entire aorta
and a trend in the aortic root. This study suggests that human antibodies to OxLDL may prevent
progression of atherosclerosis.
P350
Analysis of Factors Influencing the Location of Coronary Artery Disease in
a Hospital-based Patient Population
Tayyab Mohyuddin, Asad Sheikh, Paula Engelking, Sarah Luongo, Susan Pachowitz, Jodie
Rady, Michael P Cinquegrani, Ulrich Broeckel; Med College of Wisconsin, Milwaukee, WI
Coronary artery disease (CAD) shows a complex disease pattern with regard to the extent and
location of disease. The location of developing coronary artery plaques is of considerable
importance as it significantly influences the myocardium at risk, prognosis as well as
therapeutic options. Recently, we described in a large collection of families with myocardial
infarction that genetic factors contribute substantially to the location of the disease. In
comparison to a disease manifestation in the distal segments, atherosclerotic disease at the
ostium and proximal segments show a particularly high heritable component. The aim of this
study was to characterize a hospital -based patient cohort with regard to disease and risk factor
characteristics in order to better identify patients with higher-risk disease. We analyzed 411
coronary angiograms (201 male, 210 female; mean age 62.2yrs) with regard to the location and
degree of stenosis. The prevalence of an ostial lesion was 6.7% while 40.0% of the patients
had lesions in the proximal segments and 3.4% exhibited lesions both in the proximal segments
and the ostium. There was no significant difference with regard to age, gender, and risk factors
such as hypertension, diabetes or hypercholesterolemia between patients with and without
ostium stenosis. Patients with a proximal disease pattern were significantly older (65.0 yrs vs.
61.4 yrs; P0.05) while there was no significant difference with regard to other risk factors.
In a logistic regression including age, gender and classical risk factors, only age was a
significant predictor of ostial disease (p0.008). Further analysis for the degree of ostial
stenosis confirms the significant influence of age (p0.05). In conclusion, in this cohort of
patients only a moderate higher age characterizes patients with ostium or proximal disease and
classical risk factors do not allow for the identification of this high-risk patient population. In
order to identify patients with ostial and proximal disease, novel disease markers would have
to be identified. Given the heritability of this disease manifestation, we are currently pursuing
genetic markers in addition to other non-traditional risk factors.
2007 ATVB Poster Presentations e-97
by on March 25, 2011 atvb.ahajournals.org Downloaded from
P351
Glycemia, Triglycerides, and Disease Severity Are Best Associated with
Higher Platelet Activity in Patients with Coronary Artery Disease
Pavel P Osmancik, Frantisek Bednar, Petr Stros, Leona Pavkova, Karel Jirasek, Petr
Widimsky; CardioCntr, 3rd Med Sch, Charles Univ in Prague, Prague, Czech Republic
Background: Although higher platelet activity has been described in patients with acute
coronary syndromes consistently by many authors, iconsistent findings are reported about the
relation of platelet activity to disease severity in stable patients with chronic coronary artery
disease. Nevertheless, most reports studied only very small groups of patients. The aim of our
study was to assess the relation of platelet activity to disease severity in sufficient number of
patients with chronic coronary artery disease. Methods: One hundred and sixty stable patients
with chronic coronary artery disease were studied (25 with single-, 63 with double- and 72 with
triple-vessel disease). 95% of them were on aspirin, 3% on clopidogrel medication. No patient
suffered from acute coronary syndrome. Platelet activity was determined as membrane
expression of antigens CD62P (P-selectin, as % of positive cells) and CD41 (part of GpIIb/IIIa
integrin, as mean fluorescence intensity) by flow cytometry. Platelet aggregability was
measured by optical ADP-optical aggregometry. Data sets were compared by Kruskal-Wallis
test, correlation by Spearman test. Data are shown as meanSD. Results: Membrane CD62P
expression correlated with vessel severity (p0.001, Kruskal-Wallis test). Patients with
triple-vessel disease had the highest CD62P expression (1.810.19) followed by patients with
double-vessel (1.640.22) and single-vessel (0.690.09) disease. Positive correlation was
found between CD62P expression with triglycerides (r0.49, p0.05) and CD41 with fasting
glucose (r0.48, p0.05). No differences in ADP aggregability were found between groups.
Conclusion: Higher platelet activity is present in patients with more severe coronary artery
disease. More aggressive anti-platelet treatment in these patients should be considered,
especially when metabolic syndrome is simultaneously present.
P352
Human Prostacyclin Receptor Polymorphisms and Atherogenesis
Paola Patrignani, Stefania Tacconelli, Concetta Di Febbo, G dAnnunzio Univ, Chieti, Italy;
Karen Douville, Dartmouth Med Sch, Hanover, NH; Maria D Guglielmi, Marta L Capone,
Ettore Porreca, G dAnnunzio Univ, Chieti, Italy; John Hwa; Dartmouth Med Sch, Hanover, NH
Selective inhibitors of cyclooxygenase 2 (COX-2) confer a small but absolute risk of myocardial
infarction and stroke presumably through inhibition of COX-2-dependent prostacyclin (PGI
2
).
PGI
2
acts as a general restraint on endogenous stimuli to platelet activation, vascular
proliferation and remodeling, hypertension, atherogenesis, and cardiac function risk. Develop-
ment of genetic biomarkers will be useful to identify the patients uniquely susceptible at
developing risk of cardiovascular complications by selective inhibition of COX-2. We aimed to
investigate the association between 31 distinct single nucleotide polymorphisms (SNPs) in the
human PGI
2
receptor (IP) and intima-media thickness(IMT) of the common carotid arteries, a
surrogate measure for systemic vessel disease, in 40 individuals with a previous objectively
confirmed deep vein thrombosis (DVT) and 21 controls, i.e. individuals without DVT but with
comparable cardiovascular risk factors (cigarette smoking, diabetes, hypertensive status, lipid
levels, body mass and systemic inflammatory status index, i.e. CRP, fibrinogen). We identified
5 SNPs which were not statistically significant different between the 2 groups (using Chi square
test). Three were synonymous (no alteration in the coding amino acid sequence),V53V(DVT,
40% versus controls, 13%), V196 (DVT, 2.5% versus controls, 0%), S328S (DVT, 60% versus
controls, 57%), and 2 were nonsynonymous (change in the coding amino acid sequence),
P226T (DVT, 2.5% versus controls, 0%), and R212C (DVT, 7.5% versus controls, 5%).
Interestingly, R212C polymorphism is associated with functional deficiencies. In DVT and
controls, IMT values were not significantly different (1.120.45 and 1.130.28 mm,
meanSD). However, we found that the 4 individuals carriers of R212C polymorphism (3 in
DVT and 1 in control group) were characterized by significant (P0.006) higher IMT values
versus all population (1.670.37 versus 1.080.37 mm). None of the 4 individuals were
carriers of factor V Leiden. In conclusion, our results suggest a possible contribution of IP in
the progression of atherosclerosis in humans and pave the way to perform larger studies
assessing a possible correlation between R212C polymorphism and vascular disease.
P353
Serum Total Bilirubin Level Is Inversely Associated with the Likelihood of
Peripheral Arterial Disease: National Health and Nutrition Examination
Survey, 19992004
Todd S Perlstein, Reena Pande, Joshua A Beckman, Mark A Creager; Brigham and
Womens Hosp, Boston, MA
Background: Endogenous protective mechanisms that lessen susceptibility to peripheral
arterial disease (PAD) are unknown. Bilirubin, a potent antioxidant and cytoprotectant, is a
strong candidate for atheroprotection. We hypothesized that higher bilirubin levels would
diminish the likelihood of developing PAD. Methods and Results: We examined the association
of serum total bilirubin level with PAD in the National Health and Nutrition Examination Survey,
19992004. PAD was defined as an ankle brachial index of 0.90. A total of 7074 adults had
complete data for analysis. The overall prevalence of PAD was 5.8%. PAD prevalence inversely
associated with bilirubin level (figure). A 1 mg/dL increase in bilirubin was associated with a
48% reduced likelihood of PAD (0.52 [0.33; 0.83]) after adjustment for age, gender,
race/ethnicity, BMI, smoking status, diabetes, hypertension, hypercholesterolemia, chronic
kidney disease, CRP, and homocysteine. Additional adjustment for liver disease and alcohol
intake did not modify this result. Categorizing subjects as having low ( 0.5 mg/dL),
intermediate (0.60.9 mg/dL), and high bilirubin ( 1.0 mg/dL) levels revealed that a bilirubin
level 1.0 mg/dL (14% of the population) was independently associated with a 51% reduced
likelihood of PAD (0.49 [0.32; 0.76]). Conclusions: Increased serum total bilirubin is associated
with a marked reduction in PAD prevalence. This association is independent of known PAD risk
factors and not confounded by liver disease or alcohol intake. These results are consistent with
the known antioxidant and cytoprotectant properties of bilirubin and suggest that bilirubin
lessens susceptibility to PAD.
P354
Association of A-290G Polymorphism of CYP3A4 Gene in Response to
Atorvastatin Therapy in Coronary Artery Disease
Aruna Poduri, Ajay Bahl, Yash P Sharma, Madhu Khullar; Postgraduate Institute of Med
Education and Rsch, Chandigarh, India
The P450 cytochromes are super family of hemeproteins involved in the metabolism of various
drugs and the isoenzymes show wide variation influencing both drug responses. Cytochrome
3A4 (CYP3A4) is the primary enzyme in the metabolism of Atorvastatin (lipid lowering drugs)
and activity varies 10-folds in different ethnic populations. In the present study, we have
examined the association of the functional variant in CYP3A4 gene in response to Atorvastatin
in coronary artery disease (CAD) patients in North Indian population. It was a case-control study
consisting of 101 CAD patients & 102 controls. We studied the single nucleotide polymorphism
in the promoter region (A-290G) of CYP3A4 gene by Polymerase chain reaction - restriction
fragment length polymorphism. The genotype frequencies of CYP3A4 gene were, AA: 33.67%,
AG: 39.60% and GG: 26.73% in patients group and in controls the frequencies were, AA:
59.60%, AG: 28.43 & GG: 11.78%. The frequency of homozygous mutant genotype GG was
significantly higher in patients as compared controls (p 2.76 respectively & a significant
reduction was seen in the levels after treatment with statin (p Standard error
P355
Chronic Intermittent Hypoxia Induces Atherosclerosis in C57BL/6J Mice
Vsevolod Y Polotsky, Vladimir Savransky, Ashika Nanayakkara, Jianguo Li, Shannon Bevans,
Annabelle Rodriguez, Philip L Smith; Johns Hopkins Univ, Baltimore, MD
Introduction: Obstructive sleep apnea (OSA), a condition leading to chronic intermittent
hypoxia (CIH), is associated with hyperlipidemia, atherosclerosis and a high cardiovascular risk.
A causal link between OSA and atherosclerosis has not been established. Hypothesis: CIH may
induces atherosclerosis in C57BL/6J mice. Methods: Forty male C57BL/6J mice, 68 weeks
of age, were fed either a high cholesterol diet (HCD) or a regular chow diet (RD) and subjected
either to CIH or intermittent air (IA) for 12 weeks. During exposure to CIH, FIO
2
was reduced
from 20.9 to 4.9 0.1 % over a 30 s period and then rapidly reoxygenated to room air levels
in the subsequent 30 s period. The CIH and IA states were induced during the light phase
alternating with 12 h of constant room air during the dark phase. After the exposure, animals
were sacrificed. The heart and proximal aorta were embedded in OCT and cross-sections were
examined by Oil Red O staining. The descending aorta was examined in en face preparation
stained with Sudan IV. Results: Nine out of ten mice simultaneously exposed to CIH and HCD
developed atherosclerotic lesions in the aortic origin and descending aorta. In contrast,
atherosclerosis was not observed in mice exposed to IA and HCD or in mice exposed to CIH and
RD. HCD resulted in significant increases in serum total and LDL cholesterol (LDL-C) levels and
a decrease in HDL cholesterol. Comparing to mice exposed to IA and HCD, combined exposure
to CIH and HCD resulted in marked progression of dyslipidemia with further increases in serum
total cholesterol (206 8 mg/dl vs. 172 12 mg/dl, respectively, p 0.05) and LDL-C
(124 4 mg/dl vs. 106 6 mg/dl, p 0.05), a 2-fold increase in serum lipid peroxidation
(malondialdehyde level of 1.33 0.11 M vs. 0.61 0.05 M, p 0.05), and up-regulation
of an important hepatic enzyme of lipoprotein secretion, stearoyl coenzyme A desaturase 1.
Conclusions: CIH causes atherosclerosis in the presence of diet-induced dyslipidemia.
P356
Deficiency of Herp, an ER Stress Protein, Suppresses Atherosclerosis in
apoE-deficient Mice
Shohei Shinozaki, Kaoru Hatori, Tokyo Med and Dental Univ, Tokyo, Japan; Koichi Kokame,
National Cardiovascular Cntr Rsch Institute, Osaka, Japan; Tuyoshi Chiba, Tokyo Med and
Dental Univ, Tokyo, Japan; Toshiyuki Miyata, National Cardiovascular Cntr Rsch Institute,
Osaka, Japan; Kentaro Shimokado; Tokyo Med and Dental Univ, Tokyo, Japan
Introduction: Herp is ER stress protein originally found in vascular endothelial cells. It is not
clear whether Herp is expressed in the artery or whether it affects the development of
atherosclerosis. Methods: Mice deficient both of Herp and apoE (Herp-/-;apoE-/-) were
generated by cross- breeding Herp-deficient and apoE deficient mice. Mice were fed normal
chow for 72 weeks and sacrificed. Atherosclerotic lesion formation, blood levels of cholesterol,
TG, glucose and insulin, and mRNA for Herp, inflammatory cytokines and VCAM-1 were
examined. Effects of Herp decificncy was also studied in cultured macrophages. Results: Herp
mRNA and protein were detected in the aorta. Atherosclerotic lesion was significantly smaller
e-98 Arterioscler Thromb Vasc Biol June 2007
by on March 25, 2011 atvb.ahajournals.org Downloaded from
in Herp-/-;apoE-/- mice than in apoE-/- mice, while there was no significant difference in
serum cholesterol, TG and FFA levels. ER stress induced IL-1b in cultured macrophages, and
Herp deficiency decreased the expression. Amounts of mRNA for IL-1b and VCAM1 were
significantly lower in Herp-/-;apoE-/- mice than in apoE-/- mice. Conclusions: Herp deficiency
suppresses atheroscrelosis in apoE-/- mice. It is partly attributed to decrease in ER-stress-
induced IL-1b production by acrophages and decreased VCAM-1 expression in the artery.
P357
Leptin Induces C-reactive Protein Expression in Vascular Endothelial Cells
Prachi Singh, Michal S Hoffmann, Robert Wolk, Abu S M Shamsuzzaman, Virend K Somers;
Mayo Clinic and Foundation, Rochester, MN
There is increasing evidence of an association between leptin and increased cardiovascular
risk. Several studies have shown an independent interaction between high leptin levels and
atherosclerosis, myocardial infarction, stroke, and coronary artery intima-media thickness,
suggesting that high levels of leptin imply increased cardiovascular risk. The molecular
mechanisms underlying the association of leptin with poor cardiovascular outcomes are not
well understood. C-reactive protein (CRP) elicits proatherogenic effects in the vascular
endothelium and positively correlates with leptin levels in human subjects. There have been no
studies investigating the role of leptin in regulating CRP expression in vascular endothelial cells.
We tested the hypothesis that leptin induces CRP expression in human coronary artery
endothelial cells (HCAEC) and sought to determine the signaling pathways involved. We
confirmed the presence of both long and short isoforms of the leptin receptor in HCAEC.
Incubation of HCAEC with leptin (0400 ng/ml) caused a dose dependent increase of CRP
mRNA and protein. This leptin-induced increased CRP expression was attenuated in the
presence of anti-leptin receptor antibodies and also by inhibition of extracellular signal-
regulated kinases1/2 (ERK1/2) by PD98059 (2040 M). Time (060 min) and leptin
concentration (0200 ng/ml) dependence of ERK1/2 phosphorylation were evident in response
to leptin treatment. Leptin (100 ng/ml) also elicited reactive oxygen species (ROS) generation.
Inhibition of ROS by catalase (400 g/ml) prevented ERK1/2 phosphorylation. Thus, leptin
induces CRP expression in HCAEC via activation of the leptin receptor, increased ROS
production and phosphorylation of ERK1/2. The local expression of CRP in HCAEC likely plays
an important role in the development and progression of atherosclerotic lesions. In conclusion,
these studies suggest a mechanism for the proatherogenic effects of leptin. Leptin may provide
a target for the development of preventive and therapeutic strategies against inflammatory
mechanisms predisposing to cardiovascular disease.
P358
Environmental and Lipid OxidationDerived Aldehydes Induce Unfolded
Protein Response in Endothelial Cells
Sanjay Srivastava, Elena N Vladykovskaya, Bradford G Hill, Yonis Ahmed, Aruni Bhatnagar;
Univ of Louisville, Louisville, KY
Accumulating evidence suggest that endoplasmic reticulum (ER) stress and unfolded protein
response (UPR) contribute to several disease process including atherosclerosis. Increased
expression of UPR target genes activating transcription factor 3(ATF3) and ATF4 has been
reported in human atherosclerotic lesions. Staining for these proteins co-localized with staining
with antibodies that recognize the aldehydic epitopes of oxidized LDL, suggesting that lipid
derived aldehydes could be involved in mediating ER stress and UPR. In the present study, we
examined the role of endogenous aldehydes generated during phospholipid oxidation [e.g.,
1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine (POVPC) and 4-hydroxy, trans-2-
nonenal (HNE) and acrolein, an environmental aldehyde in the induction of ER stress in human
umbical vein endothelial cells (HUVEC) and human aortic endothelial cells (HAEC). Our data
suggest, that these aldehydes (50 M) cause the activation of double stranded RNA-activated
protein kinase-like ER kinase (PERK) by 1.53.0 fold in endothelial cells. Moreover, HNE (1050
M) and POVPC (1025 M) also cause the activation of its downstream effectors eIF2
(eukaryotic initiation factor-2) by 1.55 fold, ATF4 by 1.53.0 fold and ATF3 by 210 fold in
these cells. POVPC (1025 M) also caused the activation of mitogen activated protein (MAP)
kinases-ERK, p38 and JNK by (36 fold) in endothelial cells. Reduction of POVPC to its
corresponding alcohol, 1-palmitoyl-2-(5-hydroxyvaleroyl)-sn-glycero-3-phosphocholine (PH-
VPC) significantly inhibited the activation of JNK and ERK but not of p38. To further examine
the ability of these aldehydes to promote ER stress responses, we examined the effect of
POVPC and HNE on UPR responsive protein, CHOP (C/EBP homologus protein). Our data show
that both HNE (25 M) and POVPC (25 M) induced the expression of CHOP in endothelial cells.
Collectively, our data suggest that aldehydes derived from intrinsic lipid peroxidation or the
environmental aldehydes induce ER stress and triggger unfolded protein response in
endothelial cells, which may be a significant feature of their atherogenic effects.
P359
Plaque Wall Stress Correlates Negatively with Atherosclerosis Progression:
Possible New Hypotheses for Plaque Progression
Dalin Tang, Worcester Polytechnic Institute, Worcester, MA; Chun Yang, Beijing Normal
Institute, Beijing, China; Joseph D Petruccelli, Worcester Polytechnic Institute, Worcester,
MA; Fei Liu, Tom Hatsukami, Univ of Washington, Seattle, WA; Sayan Mondal, Worcester
Polytechnic Institute, Worcester, MA; Chun Yuan; Univ of Washington, Seattle, WA
Introduction It has been well-accepted that atherosclerosis initiation and progression correlate
positively with low and oscillating flow wall shear stresses. However, this shear stress
mechanism cannot explain why advanced plaques continue to grow under elevated flow shear
stress conditions. Hypothsis There is a negative correlation between advanced plaque
progression and structural maximum principal stress (Stress-P
1
) in the plaque. Method Nine
multi-year in vivo MRI data sets were acquired from 9 patients and were segmented to identify
specific plaque features including the lumen, wall boundary, necrotic core, calcifications, and
other components using a custom made processing software CASCADE developed by the MRI
team in this group. Based on the segmented plaque data, 3D computational multi-component
models with fluid-structure interactions were constructed to obtain flow and stress/strain
distributions which were used to quantify correlation between mechanical stress/strain
conditions and plaque progression data. Results For each sample, 57 slices showing plaque
progression and 100 nodal points / per slice were selected to perform statistical analysis.
Negative correlations between plaque progression measured by wall thickness increase (WTI)
and Stress-P
1
/Strain-P
1
were found. The plaque growth function for one patient was found to
be: WTI 0.000107 - 0.000000414 Stress-P
1
(WTI unit: cm/day; stress unit: KPa). The
growth functions are highly individual-dependent. More complete correlation analysis including
other factors such as flow shear stress, plaque component size and plaque cap thickness will
be reported at the conference. We believe that this is the first time quantitative human plaque
growth functions have ever been quantified and reported. Conclusions Quantitative human
plaque growth functions were determined from our pilot study which supports our proposed
hypothesis and may be used to predict further plaque progression. Multi-year patient screening
with computational progression analysis may lead to improved prediction accuracy so that
proper procedures may be taken to prevent possible plaque rupture.
P360
Increased Meal-induced Oxidative Stress in HIV-infected Patients
Mary F Walter, Atlanta VA Med Cntr, Decatur, GA; Eric Folch, Aneesh Mehta, Carlos del Rio,
Emory Univ, Atlanta, GA; David Rimland, W V Brown, Atlanta VA Med Cntr, Decatur, GA;
N-Anh Le; Emory Univ, Atlanta, GA
HIV infection is a potential risk factor for cardiovascular disease. Impaired postprandial lipemia
and increased production of oxidatively modified lipoproteins have been demonstrated in
non-HIV patients with documented CAD. We assessed indices of lipoprotein-associated
oxidative stress (OS) in HIV patients (4 M and 10 F under HAART management with protease
inhibitors) during postprandial (PP) lipemia. Mean (SD) fasting lipids (mg/dL) were CHOL: 136
(26), TG: 159 (184), HDLc: 47 (15) and LDLc: 75 (23). Following a standardized liquid meal (600
kcal, 30% fat, 20,000USP vitamin A), impaired PP lipemia was noted in all subjects with
maximum increase in PP TG ranging 37 to 260% (mean maximum change of 140%; p0.007).
Analysis of retinyl ester (RE) kinetics indicated delayed clearance out of both the chylomicron
(Chylo) and remnant fractions with a mean increase in Chylo TG by 4-fold and in Chylo RE by
40-fold. Triglyceride-rich lipoproteins (TRL, d 1.020 gm/mL) isolated 4 hrs PP contained
higher levels of peroxides (TBARS, 2.10 1.1 nmoles/mg of TG) as compared to TRL isolated
from fasting plasma (0.67 0.32 nmoles/mg of TG), p 0.001. This 3-fold increase in TBARS
was not seen in non-HIV controls (data not shown). Of interest, there was no detectable
increase in preformed peroxides associated with postprandial LDL, either in HIV or in non-HIV
subjects. As with non-HIV patients with documented CAD, we also observed a transient
fat-induced reduction in the levels of circulating autoantibodies (AAb) against malondialdehyde-
modified LDL, mean normalized levels at 2, 4 and 8 hrs PP were 0.71 0.16 (p0.001),
0.85 0.2 and 0.91 0.12, respectively. Our data suggests that HIV patients have
increased oxidative stress as demonstrated by acute changes induced by a standardized test
meal enriched in PUFA. These meal-induced changes in oxidative stress may contribute to the
risk for CVD in these patients.
P361
Regulation of Homocysteine Transport in Vascular Cells
Xiaohua Jiang, Fan Yang, Eugen Brailoiu, Temple Univ Sch of Medicine, Philadelphia, PA;
Hieronim Jakubowski, UMDNJ-New Jersey Med Sch, Newark, NJ; Xiaofeng Yang, Temple
Univ Sch of Medicine, Philadelphia, PA; Andrew I Schafer, Univ of Pennsylvania Sch of
Medicine, Philadelphia, PA; William Durante, Univ of Missouri Sch of Medicine, Columbia,
MO; Hong Wang; Temple Univ Sch of Medicine, Philadelphia, PA
Increased levels of plasma homocysteine is an independent risk factor for cardiovascular
disease and has cell-type distinct proatherosclerotic effects on vascular cells. In this study, we
characterized L- homocysteine transport in cultured human aortic endothelial and aortic
smooth muscle cells. L-homocysteine was transported into vascular cells in a time-dependent
fashion. L-homocysteine transport activity was about 2-fold higher in aortic smooth muscle
cells. In addition, L-homocysteine transport in both cell types was mediated by sodium-
dependent and independent carrier systems. Competition studies revealed that the neutral
amino acids cysteine, glycine, serine, tyrosine, alanine, leucine, and methionine, and inhibitors
of the cysteine transport systems inhibited L-homocysteine uptake in both cell types, but the
inhibition was greater in endothelial cells. Eadie-Hofstee plots demonstrated that L-Hcy
transport in endothelial cells had a Michaelis constant (Km) of 79M and a maximum transport
velocity (Vmax) of 873 pmol/mg protein/min. In contrast, homocysteine transport in aortic
smooth muscle cells had a lower affinity (Km212M) but a higher transport capacity
(Vmax4192 pmol/mg protein/min). Interestingly, increases in pH (pH 6.58.2) only inhibited
L-homocysteine uptake in endothelial cells. Moreover, L-homocysteine transport in endothelial
cells was partially inhibited by lysosomal inhibitors. Our studies indicate that L-homocysteine
shares transporter systems with cysteine and can be inhibited for transport by multiple neutral
amino acids in vascular cells, and that L-homocysteine transport involves lysosomal transport
in endothelial cells. The specific lysosomic feature of L-homocystein transport in endothelial
cells may contribute to cell type specific growth inhibitory effects and therefore play a role in
homocysteine atherogenic potential.
P362
Activation of the Endothelial S1P1 Receptor Inhibits Erk1/2 Phosphorylation
in Type 1 Nonobese Diabetic Mice Through Induction of MAP Kinase
Phosphatase-3
Angela M Whetzel, David T Bolick, Catherine C Hedrick; Univ of Virginia, Charlottesville, VA
Endothelial activation is a key early event in vascular complications of Type 1 diabetes. The
non-obese diabetic (NOD) mouse is a well-characterized model of Type 1 diabetes. We recently
2007 ATVB Poster Presentations e-99
by on March 25, 2011 atvb.ahajournals.org Downloaded from
found that diabetic NOD mice have increased endothelial activation, with increased production
of MCP-1 and IL-6, and a 30% induction of surface VCAM-1 expression. Using freshly isolated
aorta in an ex vivo adhesion assay, we found that diabetic NOD mice have a 4-fold increase
in monocyte adhesion to aortic endothelium (153 monocytes bound in control NOD mouse
aorta versus 719 monocytes bound in diabetic NOD mouse aorta, p0.0001). Further, the
sphingolipid sphingosine-1-phosphate (S1P) prevents monocyte:endothelial interactions in
these diabetic NOD mice. Previously, we reported that S1P treatment of diabetic NOD
endothelial cells (EC) reduced Erk1/2 phosphorylation by 90%, with no significant changes in
total Erk1/2 protein. To further investigate the mechanism causing the dramatic downregulation
of Erk1/2 phosphorylation by S1P, we examined expression of mitogen-activated kinase
phosphatase-3 (MKP-3), a cytoplasmic phosphatase expressed in EC that dephosphorylates
Erk1/2 to prevent mobilization to the nucleus for gene transcription. S1P caused a significant
3-fold increase in MKP-3 expression in EC. To mimic the S1P induction of MKP-3 in diabetic
NOD EC, we overexpressed MKP-3 in EC. Overexpression of MKP-3 in EC decreased Erk1/2
phosphorylation. We next incubated EC with either 100nM S1P, 1uM SEW2871, a S1P1
receptor-specific agonist or with 10uM VPC 23019, a S1P1/3 receptor antagonist. S1P and
SEW2871 significantly increased expression of MKP-3 and reduced Erk1/2 phosphorylation, but
VPC 23019 decreased the expression of MKP-3, both results supporting a role for S1P1 in
MKP-3 regulation. We also found that U0126 (10uM), a novel MEK1/2 inhibitor, blocked the
anti-inflammatory action of S1P, resulting in an 80% increase in monocyte adhesion.
Correspondingly, there was a 4-fold decrease in MKP-3 levels suggesting that U0126 regulates
MKP-3 expression. Thus, a primary mechanism for the anti-inflammatory action of S1P in
diabetic NOD EC is inhibition of ERK1/2 phosphorylation through induction of MKP-3 expression
via the S1P1 receptor.
P363
Acute Changes in Shear Stress Induce Expression of Inflammatory Proteins
in a Mouse Coarctation Model
Nick J Willett, Georgia Institute of Technology/Emory Univ, Atlanta, GA; Dardo E Ferrara,
Emory Univ, Atlanta, GA; Jin Suo, Georgia Institute of Technology, Atlanta, GA; Daiana
Weiss, Emory Univ, Atlanta, GA; Robert E Guldberg, Don P Giddens, Georgia Institute of
Technology, Atlanta, GA; W Robert Taylor; Georgia Institute of Technology/Emory Univ,
Atlanta, GA
Blood flow has been identified as an important factor in the pathogenesis of atherosclerosis.
Under disturbed flow conditions, wall shear stress (WSS) is transduced by the endothelium into
a biological signal producing a localized inflammatory response. However, most of the
associated data have been drawn from cell culture studies in artificial hemodynamic
environments. Therefore, we developed a novel mouse aortic coarctation model to alter the
hemodynamic environment in vivo. This model utilizes shape memory polymer clips to provide
a high degree of control over aortic diameter and subsequently WSS. We employed this model
to test the hypothesis that acute changes in WSS in vivo, induces upregulation of inflammatory
proteins related to the pathogenesis of atherosclerosis. WSS was mapped through a
computational fluid dynamic model generated by reconstruction of the aortic morphology by
microCT imaging and determination of the velocity profile by MR phase contrast imaging. The
WSS map was then correlated to inflammatory marker expression by enface immunohisto-
chemical staining. The aortas were stained with quantum dots conjugated to anti-ICAM and
VCAM antibodies that were imaged via single photon confocal microscopy. The results of the
modeling showed that during systole, the WSS had a 2.5 fold increase in the neck of the
coarctation. WSS reached magnitudes on the order of 1,000 dynes/cm
2
, significantly larger
than the WSS in humans. Conversely, there was an 8 fold decrease in WSS downstream of the
coarctation with magnitudes of 50 dynes/cm
2
(n5). The staining showed that in regions with
acute changes in WSS and/or flow reversal there is a direct correlation to an increase in the
expression of ICAM and VCAM. We conclude that although WSS is higher in magnitude in mice
than in humans, areas of relatively lower WSS and/or flow reversal are associated with
increased expression of inflammatory proteins involved in the pathogenesis of atherosclerosis.
P364
Significance of Focal Coronary Calcification Adjoining Noncalcified Plaques
Evaluated by Multislice Computed Tomography
Nobusada Funabashi, Masae Uehara, Yoko Mikami, Yumi Shiina, Issei Komuro; Chiba Univ,
Chiba, Japan
Purpose To determine the significance of focal coronary calcified plaques (CP) adjoining non
calcified plaques (NCP) using multislice computed tomography (CT). Materials and Methods
We evaluated the coronary arteries of 348 subjects using ECG-gated multislice CT. We
classified subjects into the following four groups; (1) with focal CP adjoining NCP, (2) without
focal CP adjoining NCP, but with NCP alone or NCP separate from CP, (3) with CP without NCP,
and (4) neither NCP nor CP. Groups were compared as to age, sex, coronary risk factors (RFs)
(hypertension [HT], diabetes mellitus [DM], hyperlipidemia [HL] smoking history, and obesity).
Results The 4 groups had 81, 43, 147, and 77 subjects, respectively. Subjects in Group 4 were
significantly younger than the other groups, but there was no significant difference among
Groups 13. There were significantly more males in Group 1 (80%) than in Groups 2 (63%,
P0.05), 3(54%, P001) and 4(34%, P0.01). The incidence of HT and DM were higher in
Group 1 (69% and 36%, respectively) than in Group 4 (34% and 12%, respectively, P0.01),
and the incidence of HL was higher in Group1 (62%) than in Group2 (40%, P0.05) and Group
4 (25%, P0.01). The incidence of smoking was higher in Group 1 (64%) than in Group 3(41%,
P0.01) and Group 4(27%, P0.01). The incidence of obesity was higher in Group 1 (44%)
than in Group 3(28%, P0.05). There were no significant differences in the incidence of any
RFs between Groups 2 and 3. Comparing Groups 1 and 2, the incidence of RFs (P0.01),
number of males (P0.01), DM (P0.05), HL (P0.01), and smoking (P0.05) were
significantly higher in Group 1 than in Group2. Variables were used in logistic regression
models with the presence of focal CP adjoining NCPs as the dependent variable in subjects with
NCPs (sum of Groups 1 and 2). HL and obesity (relative risks 2.40 and 2.52 [95% CIs:
1.025.65 and 1.036.18, respectively]) were associated with increased incidence of focal CP
adjoining NCPs. Conclusion Coronary RFs were more frequent in subjects with focal CP
adjoining NCP than in other groups. Focal CP adjoining NCP may indicate the most advanced
coronary arteriosclerosis and may be important in the treatment of coronary artery disease.
P365
Association of On-treatment Lipid Levels and Carotid Plaque Progression
Assessed by Magnetic Resonance Imaging
Xue-Qiao Zhao, Hunter R Underhill, Chun Yuan, Univ of Washington, Seattle, WA; Joel S
Raichlen, AstraZeneca, Wilmington, DE; John Waterton, AstraZeneca, Macclesfield, United
Kingdom; Nayak L Pollisar, The Mountain-Whisper-Light Statistical Consulting, Seattle, WA;
Fei Liu, William S Kerwin, Univ of Washington, Seattle, WA; Tobias Saam,
Ludwig-Maximilians Univ, Munich, Germany; Baocheng Chu, Norihide Takaya, Wendy
Hamar, Thomas S Hatsukami; Univ of Washington, Seattle, WA
Background: Lowering of low-density lipoprotein cholesterol (LDL-C) via statin therapy has
been associated with regression in coronary atherosclerosis. We examined the association
between on-treatment lipid levels and changes to carotid plaques as identified by magnetic
resonance imaging (MRI) after 2 yrs of rosuvastatin (RSV) therapy. Methods: Forty-three
subjects (70% men; mean age 65 yrs) with LDL-C 100 and 250 mg/dL and 16%79%
carotid stenosis by ultrasound were administered daily RSV (low dose [5 mg] or high dose
[40/80 mg]) for 2 yrs. Multi-sequence, high-resolution carotid MRI at 1.5T was done at baseline
and 2 yrs. A custom-designed image analysis tool was used to collect wall area (WA), lumen
area (LA), normalized wall index (NWIWA/[WALA]) and lipid-rich necrotic core (LRNC) area
measurements of the index artery at both time points. Changes in arterial measurements and
lipid levels were compared using paired students t-test. Spearmans correlation coefficient (r)
was calculated for relationship between on-treatment lipid levels and changes in arterial
metrics. Results: In 33 subjects with matched baseline and 2-yr scans, RSV reduced total
cholesterol (TC) by 34% (p0.0001), LDL-C by 50% (p0.0001), triglycerides (TG) by 12%
(p0.01) and Apo B by 39% (p0.0001). The mean LA, WA and NWI for the population as a
whole did not change significantly after 2 yrs of RSV. Compared to baseline, the mean % of
LRNC decreased by 29.6% after 2 yrs of RSV (absolute change -1.6%; p0.03). Subjects with
carotid plaque progression (increased NWI; n19), compared to those without (decreased NWI;
n14), had significantly higher mean levels of TC (162 vs 128 mg/dL, p0.006), LDL-C (83
vs 58 mg/dL, p0.005), TG (143 vs 98 mg/dL, p0.006) and Apo B (99 vs 71 mg/dL,
p0.002). Change in NWI and on-treatment lipid levels were correlated for TC (r0.39,
p0.03), TG (r0.36, p0.04) and Apo B (r0.39, p0.03). Change in % LRNC correlated
with on-treatment LDL-C (r0.35, p0.05). Conclusion: Greater lipid response to RSV therapy
was associated with a reduction in plaque size. Individuals with less response to lipid-lowering
therapy continued to have plaque progression. These findings support the idea of very intensive
lipid lowering to achieve reduction in atherosclerosis.
P366
WITHDRAWN
P367
Ethanol Stimulates Endothelial Cell Angiogenic Activity via a
Notch/CBF-1/RBP-JkDependent Pathway
David Morrow, John P Cullen, Univ of Rochester, Rochester, NY; Paul A Cahill, Vascular
Health Rsch Cntr, Dublin, Ireland; Eileen M Redmond; Univ of Rochester, Rochester, NY
Notch signaling regulates cell fate in a variety of cell types and a role has been demonstrated
for this pathway in angiogenesis. Epidemiological studies have reported various associations
between alcohol consumption and several seemingly distinct diseases, including atheroscle-
rosis, rheumatoid arthritis and certain cancers, all of which, however, involve angiogenesis. We
assessed the hypothesis that ethanol (EtOH) modulates endothelial cell (EC) angiogenic activity
via a Notch/CBF-1/RBP-Jk dependent pathway. Human umbilical vein EC were treated with
EtOH and Notch receptor and Notch target gene mRNA and protein levels determined by
QRT-PCR and western blot analysis, respectively. Network formation on Matrigel was used as
an index of angiogenesis. Exposure of EC to EtOH (25mM, 24 h) significantly increased EC
network formation; network length (AU) 822;118 vs 1834;292 for control vs EtOH,
respectively (n4, p0.05), while concomitantly increasing Notch receptor 1,3 and 4 mRNA
and protein expression; 2.48;0.33, 1.73;0.2 and 3.77;1 fold increase, respectively, for
mRNA levels. In addition, Notch target gene (hrt 13) mRNA was significantly increased by
1.34;0.52, 2.24;0.34 and 2.78;0.87, respectively. Selective knockdown of Notch 1 and
4 receptors by siRNA resulted in a significant decrease in EtOH-induced network formation;
e-100 Arterioscler Thromb Vasc Biol June 2007
by on March 25, 2011 atvb.ahajournals.org Downloaded from
network length (AU) 4229;163 vs 2550;367 and 4229;163 vs 1830;489 for Notch
1 and 4, respectively. Moreover, inhibition of endogenous Notch mediated CBF-1/RBP-Jk
regulated gene expression using Epstein-Barr virus encoded RPMS-1 resulted in a significant
decrease in ethanol-induced network formation; network length (AU) 2200;380 vs
1090;265 for control EtOH vs RPMS-1 EtOH. These data demonstrate that ethanol, at a level
consistent with moderate consumption, regulates the expression of Notch receptors and
downstream target genes in endothelial cells. Moreover, ethanol-stimulated EC angiogenic
activity is mediated via a Notch/CBF-1/RBP-JK dependent pathway. These actions of ethanol
may be relevant to the effects of alcohol consumption and disease progression.
P368
Important Role of Erythropoietin Receptor in Promoting Vascular
Endothelial Growth Factor Expression and Angiogenesis in Peripheral
Ischemia in Mice
Makoto Nakano, Kimio Satoh, Yoshihiro Fukumoto, Yoshitaka Ito, Yutaka Kagaya, Naoto
Ishii, Kazuo Sugamura, Hiroaki Shimokawa; Tohoku Univ Graduate Sch of Medicine, Sendai,
Japan
Background: Prognosis of patients with severe peripheral artery disease (PAD) still remains
poor when there are no indications of revascularization therapies such as bypass surgery or
percutaneous transluminal angioplasty. It has been demonstrated that exogenous erythropoi-
etin (Epo) can stimulate angiogenesis in ischemic tissues. We have also recently demonstrated
that endogenous Epo/Epo receptor (EpoR) system plays an important protective role in
hypoxia-induced pulmonary hypertension. In this study, we examined the role of Epo/EpoR
system in ischemia-induced angiogenesis in EpoR
-/-
-rescued mice that lack EpoR in most
organs except erythroid-lineage cells. Methods and Results: Two weeks after femoral artery
ligation, blood flow recovery, activation of vascular endothelial growth factor (VEGF)/VEGF
receptor system, and mobilization of endothelial progenitor cells (EPCs) were all impaired in
EpoR
-/-
-rescued mice as compared with wild-type (WT) mice. Bone marrow (BM) transplanta-
tion with WT-BM cells in EpoR
-/-
-rescued mice partially but significantly improved blood flow
recovery after hindlimb ischemia. The extent of VEGF up-regulation and the number of
BM-derived cells in ischemic tissue were significantly less in EpoR
-/-
-rescued mice compared
with WT mice even after BM reconstitution with WT-BM cells. Similarly, the recovery of blood
flow was significantly impaired in recipient EpoR
-/-
-rescued mice that had been transplanted
with WT-BM or EpoR
-/-
-rescued-BM as compared with recipient WT mice. Furthermore, the
Matrigel implantation assay and aortic ring assay showed that microvessel growth in vitro was
significantly reduced in EpoR
-/-
-rescued mice as compared with WT mice. Conclusions: These
results indicate that vascular EpoR system, in addition to Epo, plays an important role in
angiogenesis in response to hindlimb ischemia through up-regulation of VEGF/VEGF receptor
system, both directly by enhancing neovascularization and indirectly by recruiting EPCs and/or
BM-derived proangiogenic cells. Therefore, vascular EpoR system may be a new therapeutic
target for the treatment of ischemic cardiovascular diseases, including PAD.
P369
Unfolded Protein Response Mediates Induction of VEGF in Endothelial Cells
by Oxidized Phospholipids
Olga V Oskolkova, Taras A Afonyushkin, Bernd R Binder, Valery N Bochkov; Med Univ of
Vienna, Vienna, Austria
Previous work in our group identified angiogenic activity of oxidized phospholipids (OxPLs) in
several in vitro and in vivo models. Furthermore, we have shown that effects of OxPLs are
mediated by upregulation of VEGF, IL-8 and COX-2-derived prostanoids, which stimulated
endothelial cells (ECs) in an autocrine manner. In this work, we asked a question about the
mechanisms mediating OxPL-induced upregulation of VEGF. We found that VEGF is induced by
a variety of OxPLs differing in polar head groups and oxidized sn-2 residues. This relaxed
structural specificity makes receptor-mediated mechanisms unlikely. Our findings suggest that
induction of VEGF by OxPLs is mediated by unfolded protein response (UPR). Several chemically
different UPR inducers upregulated levels of VEGF mRNA. Furthermore, OxPLs induced
elevation of protein levels of a major UPR effector, ATF4. Knocking down ATF4 by siRNA
dramatically decreased induction of VEGF by OxPLs. Furthermore, electrophoretic mobility shift
assay demonstrated that OxPL treatment induced DNA-binding activity in cell extracts, which
interacted with oligonucleotide corresponding to the ATF4-binding site from human VEGF
promoter. In summary, our data suggest that OxPLs induce endoplasmic reticulum stress
leading to unfolded protein response finally resulting in upregulation of VEGF gene transcription.
P370
Fractalkine Induces Angiogenesis by Stimulating Vascular Endothelial
Growth Factor-A Production by Human Vascular Endothelial Cells and
Improves Hind Limb Ischemia
Ki Hoon Han, Jewon Ryu, Jin-Ae Shin, Cheol Whan Lee; Asan Med Cntr, Seoul, Republic of
Korea
Background : Angiogenesis facilitates the process of inflammation or improves ischemic
condition. The present study investigated the detailed mechanism by which fractalkine (Fkn),
a CX3C chemokine, induces angiogenesis in vivo. Methods and Results : Fkn induced vessel
sprouting from excised rat aorta and angiogenesis on chicks chorioallantoic membrane (CAM)
through CX3CR1 activation. Immunoblotting analysis and EMSA showed that Fkn upregulated
hypoxia-inducible factor 1 alpha (HIF-1) by cultured human aortic endothelial cells (HAECs),
which in turn induced mRNA and protein productions of VEGF-A, a dominant isoform of vascular
endothelial growth factor (VEGF), through pathways involving p42/44 MAPK, not through RhoA.
Fkn potently induced angiogenesis on CAM and ears of C57/BL6 female mice in vivo, which
was inhibited by a specific Rho inhibitor and a VEGF receptor 2 (KDR) blocker. The condition
of hinlimb ischemia induced by obliterating rats left common femoral artery using a metal coil
through remoted access increased expression of Fkn and VEGF-A in the ischemic tissue, and
the blood flow to the ischemic hindlimb was improved by injection of rat-specific whole-length
Fkn protein. Conclusions: Fkn-induced angiogenesis involves two sequential steps; the
induction of HIF-1 and VEGF-A gene expression through CX3CR1 activation, and the
subsequent VEGF-A/VEGFR-2-induced angiogenesis through RhoA-dependent pathway. The
development of new microvessels by Fkn may contribute to the growth of plaques and
neoplasm during the process of atherosclerosis. On the other hand, the potent induction of
angiogenesis by Fkn can be used as therapeutic strategy for peripheral ischemia.
P371
Oxidized Low-Density Lipoprotein Induces Endothelial Progenitor Cell
Apoptosis by Inhibiting PI3 Kinase/Akt Pathway via Tyrosine Nitration
Guodong Tie, Jinglian Yan, Yagai Yang, Brian Park, Robert Raffai, Univ of California, San
Francisco, San Francisco, CA; Louis M Messina; Univ of Massachusetts, Worcester, MA
Objective: The mechanisms by which risk factors such as hypercholesterolemia reduce the
number of circulating endothelial progenitor cells (EPC) in patients with coronary artery disease
(CAD) are incompletely understood. We tested the hypothesis that oxidized low density
lipoprotein (oxLDL) induces EPC apoptosis by inactivating PI3 kinase/Akt cell survival pathway.
Methods and Results: In EPCs harvested from wiltype (wt) mice, oxLDL induced apoptosis and
inhibited Akt phosphorylation in a dose-dependent manner. Overexpression of constitutively
active Akt rescued oxLDL induced EPC apoptosis. Immunoprecipitation demonstrated that
oxLDL significantly increased tyrosine nitration on the p85 subunit of PI3 kinase, which resulted
in dissociation of p85 and p110 subunits of PI3 kinase. Treatments with SOD, L-NAME, as well
as epichetechin or FETTPs blocked tyrosine nitration on p85 subunit of PI3 kinase, restored Akt
phosphorylation and rescued apoptosis induced by oxLDL. Interestingly, oxLDL also increased
p38 phosphorylation in cultured EPC in a dose-dependent manner. Treatment with the
pharmacologic inhibitor of p38, SB203580, further increased oxLDL induced Akt inhibition and
EPC apoptosis, suggesting an interaction between Akt and p38 MAP kinase pathways. Lastly,
EPCs harvested from hypercholesterolemic hypomorphic apolipoprotein E mice deficient in LDL
receptor (Apoe
h/h
Ldlr
-/-
mice) showed a higher rate of apoptosis than EPCs from wt mice.
Conclusions: oxLDL inhibits the PI3 kinase pathway and induces apoptosis by causing tyrosine
nitration of PI3 kinase and inhibiting Akt mediated cell survival signalling in cultured EPCs, and
may explain the enhanced apopotosis displayed by EPC harvested from spontaneously
hypercholesterolemice Apoe
h/h
Ldlr
-/-
mice. Key words: oxidized low density lipoprotein, endo-
thelial progenitor cell, apoptosis, tyrosine nitration, PI3 kinase, Akt, p38
P372
Cellular and Molecular Mechanisms Mediating Blood Flow Recovery After
Acute versus Gradual Femoral Artery Occlusion Are Distinct in the Mouse
Yagai N Yang, Gale Tang, Jinglian Yan, Guodong Tie, Ari Hoffman, UCSF, San Francisco,
CA; Louis M Messina; Univ of Massachusetts, Worcester, MA
Introduction: Critical limb ischemia in humans results from gradual arterial occlusion caused
by atherosclerosis. Current mouse models of hindlimb ischemia use acute arterial occlusion
that does not accurately mimic the pathogenesis of human chronic critical limb ischemia. We
therefore developed the first mouse model of gradual arterial occlusion. Methods: Gradual
arterial occlusion was induced by placing ameroid constrictors on the proximal and distal left
femoral artery, and ligating the femoral artery branches (n34). Acute arterial occlusion was
accomplished by excising the left femoral artery (n34). Blood flow recovery, ischemia-
induced changes in gene expression, and immunohistochemical analysis were performed for
each animal model. Results: We identified two distinct patterns of gene expression, blood flow
recovery, SDF-1 expression, and macrophage and hemangiocyte recruitment after gradual
versus acute femoral arterial occlusion. Hypoxia-related genes increased significantly in the
calf (p<0.05), but not in the thigh (p0.05), after gradual versus acute femoral arterial
occlusion. Shear-stress dependent genes and inflammatory genes were upregulated immedi-
ately in the thigh only after acute femoral arterial occlusion (p<0.05). These differences in
gene expression were consistent with increased SDF-1a expression, recruitment of macro-
phages and hemangiocytes, and higher blood flow recovery after acute arterial occlusion
compared to gradual arterial occlusion (p<0.05). Conclusion: This is the first study to show
that the molecular and cellular mechanisms that regulate collateral artery enlargement and
blood flow recovery are critically dependent on the rate of arterial occlusion. After gradual
arterial occlusion, shear stress dependent and inflammatory genes were not upregulated. Also,
macrophage and hemangiocytes recruitment were lower than after acute occlusion. Models of
critical chronic limb ischemia, that more closely resemble the human condition, may provide
more accurate mechanistic insights with the objective of creating novel molecular therapies.
P373
Carotid 3-dimensional Ultrasound Atherosclerotic Plaque and Vessel Wall
Thickness Maps: Location-specific Changes After Intensive Statin
Treatment
Adam Krasinski, Micaela Egger, Bernard Chiu, J David Spence, Aaron Fenster, Grace
Parraga; Robarts Rsch Institute, London, Canada
Currently, carotid atherosclerosis progression and regression is typically measured using
plasma surrogates and regionally by measurement of intima media thickness from B-mode
ultrasound (US) images and using 3-dimensional (3D) measures of plaque and wall thickness
from 3D US images, intravascular ultrasound and MRI. Previously we studied 38 subjects, those
treated with atorvastatin for 3 months displayed a mean 14% decrease in 3DUS total plaque
volume and no change was displayed in the placebo treatment group. We have subsequently
developed a software analysis tool to create 2-dimensional (2D) maps of vessel wall and plaque
thickness from the measured vessel wall volume (VWV) contours. We hypothesize that these
maps will show location-specific changes in plaque and wall thickness in the atorvastatin group
2007 ATVB Poster Presentations e-101
by on March 25, 2011 atvb.ahajournals.org Downloaded from
over 3 months. As a proof of concept, we mapped the VWV contours of 5 subjects (3
atorvastatin, 2 placebo) and created thickness difference maps by subtracting the baseline and
3 month scan thickness maps. These maps show the spatial distribution of treatment-specific
changes in wall and plaque thickness. Changes were observed mainly in the common carotid
artery, with one of the subjects also showing changes in the internal carotid artery. In
conclusion, this analysis showed that thickness maps generated from VWV contours show
location-specific changes over a three month period for the atorvastatin group. Further analysis
of the entire study population using these maps will provide further insight into the temporal
and spatial dynamics of plaque progression and regression.
P374
Omega-3 Fish Oil Plus Ginkgo Biloba Impairs Alzheimer Nanoplaque
Formation and Size in Vitro
Lovisa Ringstad, Uppsala Univ Institute of Pharmacy, Uppsala, Sweden; Karl Winkler, Univ
Clinic Freiburg, Freiburg, Germany; Miguel Rodrguez, Charite , Campus Benjamin Franklin,
Berlin, Germany; Martin Malmsten, Uppsala Univ Institute of Pharmacy, Uppsala, Sweden;
Gu nter Siegel; Charite , Campus Benjamin Franklin, Berlin, Germany
The relationship between Alzheimers disease and coronary artery disease is striking:
Alzheimer plaques and arteriosclerotic plaques are basically very similar in their chemical
composition. The principal constituents of Alzheimer plaques are proteoglycans, lipoproteins
(preferentially VLDL and IDL), beta-amyloid and calcium, thus nearly extended by the
component A-beta as compared to arteriosclerotic plaques. Utilizing a patented arteriosclerosis
model (EP 0 946 876), we simulated in a biosensor assay Alzheimer nanoplaques [J. Colloid
Interface Sci. 276, 503506 (2004)] and measured in vitro their formation and size by
ellipsometric techniques, a laser-based physicochemical procedure. For the study with
omega-3 fish oil plus Ginkgo biloba (ProBrain

, SevenSeas, Hull, England), VLDL apoE4/E4 from


a cardiovascularly and stroke-endangered high-risk patient was used as lipoprotein. Further-
more, human A-beta42 (0.1 g/L) which inclines strongly to aggregation and fibrillogenesis, as
well as EPA (21.9 mg/L), DHA (15.4 mg/L) and Ginkgo biloba (0.28 mg/L) were applied in a
concentration as could be expected in the blood of probationers after the intake of one capsule.
The VLDL apoE4/E4 plasma fraction (30 mg/dL) showed beginning Alzheimer nanoplaque
formation already at a normal blood Ca
2
concentration. Fish oil plus ginkgo applied acutely in
the experiment, markedly slowed down this process of quaternary aggregational nanoplaque
complexation at all Ca
2
concentrations used. In a normal blood Ca
2
concentration of 2.52
mmol/L, the omega-3 ginkgo)-induced reduction of nanoplaque formation and molecular
size amounted to 10.1 1.3% (p 0.03) and 14.3 2.9% (p 0.02), respectively, as
compared to the controls. From these results, we concluded that the combination of omega-3
fish oil plus ginkgo hampered the docking of VLDL apoE4/E4 to the proteoglycan receptor and
of A-beta42 to VLDL apoE4/E4, and that altogether Alzheimer nanoplaque formation is
diminished as compared to control experiments without these substances. These in vitro
experiments provide a mechanistic explanation for a possible benefinial mode of action of fish
oil and ginkgo in Alzheimer, vascular or age-related cognitive decline.
P375
Salt Inactivates eNOS: A Mechanism of How Salt Contributes to
Hypertension
Juan Li, James White, Ling Guo, Min Chen, Zhiqing Song, William Everson, Jun Liu, Eric
Smart, Xiang-An Li; Univ of Kentucky, Lexington, KY
The contribution of a high salt diet to hypertension has been debated for decades. One pertinent
detail to this debate is that the molecular mechanisms of how salt induces high blood pressure
are not fully understood. Besides the kidney-volume system, vascular endothelial cells
constitute an important mechanism for regulating blood pressure. Endothelial nitric oxide
synthase (eNOS) catalyzes the generation of nitric oxide that causes blood vessel relaxation,
which results in decreased blood pressure. In this study, we report a surprising finding that
increases in salt levels acutely inactivates eNOS. By measuring eNOS activity using CHO-eNOS
cells and primary bovine endothelial cells, we demonstrated that a slight increase in salt levels,
as low as 5 mM NaCl, significantly suppressed eNOS activity. The eNOS activity was decreased
25%, 45% and 70% in the presence of 5mM, 10 mM and 20 mM of NaCl, correspondingly. The
eNOS activity was unaffected in the presence of equal milliosmoles of mannitol (10, 20, and
40 mM), which excludes osmotic effect. Interestingly, increases in salt levels also suppressed
activation of eNOS by A23187 or by acetylcholine, suggesting that salt induces changes in
eNOS conformation. In conclusion, we demonstrate that increases in salt levels acutely
inactivate eNOS which provides a novel mechanism of how salt contributes to hypertension.
P376
Glucose 6-phosphate Dehydrogenase Regulates Laminar Flow-induced
Endothelial Nitric Oxide Synthase Activation
Shi Pan, Christopher J Kovacs, Bradford C Berk; Univ of Rochester, Rochester, NY
Glucose 6-phosphate dehydrogenase (G6PD) is the rate-limiting enzyme in the pentose
phosphate pathway, where it metabolizes glucose 6-phosphate to 6-phosphogluconolactone
and generates NADPH. In endothelial cells, NADPH is an essential cofactor for dihydrofolate
reductase, which generates tetrahydrobiopterin. Because tetrahydrobiopterin is an essential
cofactor for endothelial nitric oxide synthase (eNOS), G6PD is very important for endothelial
function. Increases in blood flow activate eNOS. We hypothesized that G6PD plays an essential
role in flow-mediated eNOS activation. Using a cone and plate viscometer, we showed that flow
activated G6PD within 2 minutes. Small interference RNA knockdown of G6PD significantly
inhibited flow-mediated eNOS phosphorylation. In contrast, overexpression of G6PD increased
flow-mediated eNOS phosphorylation, while a G6PD mutant lacking enzymatic activity did not
alter eNOS phosphorylation. Our hypothesis was further supported by data that G6PD
expression increased significantly in cells exposed to laminar flow for 24 hours. In the Pretsch
mouse, which has much lower G6PD activity due to decreased G6PD protein expression, aortic
eNOS phosphorylation was dramatically decreased compared to wild type mice. These findings
demonstrate a novel role for G6PD in vascular homeostasis, by regulating eNOS function.
P377
WITHDRAWN
P378
Cardiovascular Targets of Dietary Isoflavones: Transcriptional Activation of
Endothelial Nitric Oxide Synthase and Antioxidant Genes in Human Fetal
Endothelial Cells
David J Rowlands, Francois Y Li, Patricia de Winter, Richard C Siow, Giovanni E Mann;
Kings College London, London, United Kingdom
Estrogen protects premenopausal women against coronary heart disease, however, hormone
replacement therapy following menopause has been linked with an increased incidence of
breast cancer and limited protection against cardiovascular disease. Dietary soy isoflavones,
such as genistein, daidzein and its metabolite equol, may serve as alternative estrogen receptor
modulators to reverse endothelial dysfunction by increased synthesis the vasodilator nitric
oxide. In our recent study in human endothelial cells, we reported that equol rapidly (2 min)
activates Akt and extracellular signal-regulated kinase (ERK1/2), leading to phosphorylation of
eNOS Ser1177 and association of the enzyme with the chaperone Hsp90. We have now
employed lucigenin chemiluminescence to document that genistein, daidzein and equol (100
nM) acutely stimulate superoxide anion (O
2
.-
) production in human umbilical vein endothelial
cells (HUVEC). Pretreatment with the eNOS inhibitor, L-NAME (100M), abrogated equol
stimulated O
2
.-
(n5, p0.01). Increased O
2
.-
production was associated with nuclear
translocation of the transcription factor Nrf2 (24 h) and a 2-fold upregulation of the antioxidant
stress protein heme oxygenase-1 (HO-1, n3, p0.05). Our findings suggest that isoflavones,
at low nanomolar concentrations, evoke rapid release of both NO and O
2
.
, leading to enhanced
Nrf2/antioxidant response element transcriptional activation of HO-1 expression. Dietary soy
isoflavones may thus afford protection against endothelial dysfunction in vascular diseases
associated with prolonged oxidative stress.
P379
Not All Endothelial Cells Are Equal Under Flow: Studies on Cell Adhesion
Molecule Expression
Mercedes Balcells, Heiko Methe, MIT, Cambridge, MA; Blanca Molins, Carmen Alegret,
Institut Quimic de Sarria, Barcelona, Spain; Elazer R Edelman; MIT, Cambridge, MA
Endothelial adhesion molecule expression can be modulated by mechanical stimuli which are
recognized through shear stress responsive elements that interact functionally with a number
of transcription factors. As endothelial cells (EC) in the human cardiovascular system may
exhibit multiple phenotypes in response to the complex flow patterns present in various
vascular geometries, we examined the differential effects of venous-like (VF) and coronary
artery-like flow (CAF) on regulation of adhesion molecule expression in human saphenous vein
(HSVEC) and coronary artery EC (HCAEC). Cells were seeded on fibronectin-coated silicone
rubber tubes and subjected to VF and CAF in a perfusion bioreactor. Immediately after 40h flow
e-102 Arterioscler Thromb Vasc Biol June 2007
by on March 25, 2011 atvb.ahajournals.org Downloaded from
exposure, cells were labeled with anti- ICAM-1, -E-selectin, and -VCAM-1 antibodies for flow
cytometry analysis. Monocyte adhesion studies were performed using calcein-AM labeled
mononuclear cells added to the perfusate for 45 min. Expression of E-selectin, VCAM-1, and
ICAM-1 was upregulated on cytokine-stimulated HSVEC after exposure to both VF (2.5, 1.1 and
1.9 fold) and non vascular bed-specific CAF (1.6, 1.5 and 2.5 fold). In marked contrast,
exposure of HCAEC to vascular bed-specific CAF and to non vascular bed-specific VF resulted
in downregulation of E-selectin and VCAM-1 expression when compared to expression levels
under static conditions. Whereas VF had no impact on ICAM-1 expression on cytokine-
stimulated HCAEC, CAF significantly increased ICAM-1 expression on HCAEC above the levels
measured on HSVEC (mean fluorescence intensity of 105214 and 814129, respectively,
p0.0005). To examine the functional relevance of our findings we quantified adhesion of
mononuclear blood cells to both EC types. Adhesion to cytokine-stimulated HSVEC significantly
exceeded adhesion to TNF--stimulated HCAEC when exposed to CAF (arbitrary fluorescence
units of 0.190.02 and 0.100.01, respectively, p0.001). These results may give further
rise to the notion of vascular bed specific endothelial molecular responsiveness: intrinsic
differences in the vascular beds that serve as the source for different EC may account for the
different effects of flow observed.
P380
Recovery of Endothelial Barrier Function by Kinin B1 Receptor-induced
Activation of Inducible Nitric Oxide Synthase
Viktor Brovkovych, Svitlana Brovkovych, Randal A Skidgel; Univ of Illinois College of
Medicine, Chicago, IL
Impaired endothelial barrier function can result from the release of inflammatory mediators in
the cardiovascular system. We previously showed that human lung microvascular endothelial
cells (HLMVEC) treated with cytokines (20 ng/ml interleukin-1 200 U/ml interferon- for
16 h) express kinin B1 receptors (B1Rs) and iNOS. Measurement of NO output in real time with
a porphyrinic electrode showed that 1 mM Arg alone generated prolonged (90 min)
iNOS-dependent high output NO (maximum 295 22 nM NO at 4060 min) whereas
addition of a B1R agonist, 100 nM des-Arg
10
-kallidin (DAKD), produced super-high output NO
(maximum 832 37 nM) also lasting 8090 min with a total output almost 3-fold higher
than Arg alone. To investigate the hypothesis that B1R activation of iNOS-dependent NO
production affects endothelial permeability, HLMVEC barrier function was continuously
assessed by electric cell-substrate impedance sensing. Cytokine treatment of HLMVEC as
above markedly decreased resistance to half the control value. Addition of 1 mM L-Arg to
stimulate basal iNOS activity resulted in partial recovery of cell resistance ( 25% increase from
the post-cytokine level) whereas addition of B1R agonist 100 nM DAKD to acutely activate iNOS
resulted in a significantly greater recovery (60 % increase). ACE inhibitor enalaprilat (100 nM)
also activated B1Rs and gave a response similar to that of DAKD. This effect fully developed
by 4060 min after agonist treatment, lasted for at least 2 h and was blocked by B1R
antagonist des-Arg
10
-Leu
9
-kallidin or by iNOS specific inhibitor N-(1-iminoethyl)-L-Lysine. The
role of NO was explored using the NO donor DETA-NONOate. Doses of DETA-NONOate (1 - 50
M) that generated NO in the same range as B1R agonist stimulation resulted in a 65%
increase in resistance whereas higher doses caused no change (100 M) or a further decrease
(0.5 mM - 5 mM) in resistance. Thus, B1R activation of iNOS restores endothelial barrier
function and could represent a novel target for repair of endothelial damage in inflammation.
This mechanism could also underlie some of the beneficial therapeutic effects of angiotensin
converting enzyme (ACE) inhibitors as these compounds are also direct agonists of B1Rs.
P382
A Critical Role for NF-B during FFA Impairment of Nitric Oxide Production
in Endothelial Cells
Matilda Pham, Ezekiel Maloney, Francis Kim; Univ of Washington, Seattle, WA
There is substantial evidence that endothelial vasodilation mediated by nitric oxide (NO)
production is impaired in animal models of diabetes and in humans with type 2 diabetes.
Increased free fatty acid (FFA) levels are often found in type 2 diabetes and are associated with
endothelial dysfunction. We have previously shown that excess FFA, glucose, or TNF- inhibits
endothelial NO production. In endothelial cells, FFA activates IKK, a regulatory kinase in the
NF-B inflammatory activation pathway and IKK is both necessary and sufficient to impair
insulin-mediated NO production. Objective: We hypothesized that NF-B activation is necessary
for FFA mediated impairment of endothelial insulin signaling. Results: Endothelial cells were
exposed to 100 M palmitate/BSA for 3 h and exhibited impaired insulin signaling and NO
production compared to the BSA treated control condition. FFA treatment resulted in activation
of IKK, NF-B and NF-B dependent- ICAM expression and IL-6 production. In order to assess
whether NF-B dependent transcription is necessary for FFA-mediated inhibition of endothelial
insulin signaling, we used a phosphorylation-resistant IB(ser32, 36/ala) construct (NF-B
super repressor) which blocks activation of NF-B without affecting FFA-mediated IKK
activation. The IB phosphorylation resistant construct was cloned into pBM-IRES-puro
retroviral vector and retrovirus was generated for transduction of HMEC. Overexpression of
IB was confirmed by Western blot analysis. Control endothelial cells were transduced with
a retroviral GFP construct. FFA treatment of HMEC transduced with the phosphorylation-
resistant IB construct did not impair insulin signaling or insulin-mediated NO production
when compared to the control GFP transduced cells. These experiments suggest that
FFA-mediated impairment of NO production is dependent on NF-B activation.
P383
Adiponectin and Its Receptors Increased Cholesterol Efflux in HEK293T
Cells
Ken Kitajima, Shin-Ichiro Miura, Fukuoka Univ Sch of Medicine, Fukuoka, Japan; Toshimasa
Yamauchi, Graduate Sch of Medicine, Univ of Tokyo, Tokyo, Japan; Yoshinari Uehara,
Fukuoka Univ Sch of Medicine, Fukuoka, Japan; Takashi Kadowaki, Graduate Sch of
Medicine, Univ of Tokyo, Tokyo, Japan; Keijiro Saku; Fukuoka Univ Sch of Medicine,
Fukuoka, Japan
A decrease in adiponectin secretion causes the early stage of atherosclerosis. High-density
lipoprotein (HDL) takes up cholesterol through ATP binding cassette A1 (ABCA1) as reverse
cholesterol transport (RCT). Recently, a new therapeutic strategy, reconstituted (r)HDL, has
been shown to enhance RCT. Therefore, we hypothesized that adiponectin increases the uptake
associated with ABCA1 and also enhances rHDL-induced uptake in human kidney cells
(HEK293T), which endogenously expressed ABCA1. We transfected adiponectin receptor 1 and
2 (AdipoR1 and AdipoR2) cDNA to HEK293T cells. The transfected cells were labeled with
3
H-cholesterol following cholesterol loading with or without adiponectin for 24 hours. The levels
of cholesterol efflux were analyzed using a liquid scintillation counter. AdipoR1- and
AdipoR2-transfected HEK293T cells (10.32.4 and 10.11.9 %, respectively) showed
significantly higher basal levels of efflux compared to mock-transfected cells (4.80.1%).
Treatment with adiponectin had additional effects on efflux (mock, 4.00.7; AdipoR1,
17.12.4; AdipoR2, 23.34.1 %) in transfected HEK293T. In addition, rHDL also increased
efflux (mock, 8.01.2; AdipoR1, 15.11.3; AdipoR2 25.95.1 %). Interestingly, rHDL-induced
cholesterol uptake increased in the presence of adiponectin (mock, 27.32.9; AdipoR1,
33.11.9; AdipoR2 30.15.8 %). In summary, adiponectin and its receptors increased
cholesterol efflux and also nhanced rHDL-induced efflux in transfected HEK293T cells. These
results suggest that adiponectin may enhance the RCT system and induce an anti-atherogenic
effect.
P384
Specificity in Interactions Between Matrix GLA Protein and Bone
Morphogenetic Proteins
Yucheng Yao, Esther Shao, Kristina I Bostrom; UCLA, Los Angeles, CA
Arterial calcification is ubiquitous in vascular disease, and contributes significantly to morbidity
and mortality. Prevention of calcification is mediated in part by the activity of matrix GLA protein
(MGP), an inhibitor of bone morphogenetic protein (BMP). MGP is characterized by the presence
of gamma-carboxylated glutamates, so-called GLA-residues, which are able to bind calcium
ions. However, it is not clear whether MGP inhibits multiple BMPs involved in vascular biology,
and what mechanism is used to bind BMP. We compared the effect of MGP on four different
BMPs that were expressed in bovine aortic endothelial cells (BAEC), BMP-2, -4, -6, and -7. All
four BMPs were similarly bound and inhibited by MGP as determined by a BMP-sensitive
luciferase reporter gene assay and co-immunoprecipitation, although MGP appeared to have
the highest affinity for BMP-2 as determined by an ELISA-based assay. We studied the binding
mechanism of MGP using mutagenesis of a FLAG-tagged MGP construct. The various mutations
were expressed at similar levels in BAEC, and were analyzed using luciferase reporter gene
assays, co-immunoprecipitation, and calcium binding. The results showed that binding of BMP
by MGP was dependent on a specific proline residue in the MGP protein localized in the center
of the GLA-rich region, which has been associated with BMP-binding. In addition, at least two
GLA-residues were required for BMP binding, one on each side of the proline residue.
Mutagenesis of the GLA-residues, but not the proline-residue, was associated with changes in
calcium binding. Together, our results suggest that MGP can interfere in multiple processes in
vascular biology where the tested BMPs are present. In addition, the results suggest that MGP
uses a BMP-binding mechanism that is in part similar to that of the BMP type I receptor and
Noggin, another BMP-inhibitor. These results may be important in providing guidance as to
appropriate directions for development of therapeutic interventions for arterial calcification.
P385
In Vivo Expression of Human Group II Secretory Phospholipase A2 Is
Associated with Increased Production of Biglycan and Macrophage Colony
Stimulating Factor
Mary Y Chang, Univ of Washington, Seattle, WA; Birgitta Rosengren, Mia Umaerus, Emelie
Dagnelid, Eva Hurt-Camejo, AstraZeneca, Molndal, Sweden; Alan Chait; Univ of Washington,
Seattle, WA
Secretory phospholipase A2 (sPLA2) digestion of lipoproteins leads to the formation of
lysophosphatidylcholine (lysoPC), a molecule that we previously have shown stimulates the
synthesis of biglycan and the proteoglycan form of MCSF (PG-MCSF) by arterial smooth muscle
cells. These matrix molecules have pro-atherogenic effects. To test whether increased lysoPC
generation in vivo is associated with increased synthesis of biglycan and PG-MCSF we used
transgenic mice overexpressing human group II sPLA2 (hsPLA2-IIA), which have increased
susceptibility to LPS-induced shock and to atherosclerosis when fed a high cholesterol diet.
Female hsPLA2-IIA mice were maintained for 4 weeks on (i) control diet, (ii) control diet with
LPS injection 48hrs prior to sacrifice, or (iii) western diet (n5 for each group). hsPLA2-IIA
mRNA levels were verified by qPCR to be significantly higher in liver (3.5-x), heart (6-x) and
aorta (1.4-x) in response to LPS. Similarly, hsPLA2-IIA protein levels (ng/mg tissue) were
significantly higher in liver (3.7-x), heart (4.2-x) and aorta (2.1-x) of LPS-treated mice. MCSF
mRNA levels correlated with expression of hsPLA2-IIA and were significantly higher in the liver
(2.6-x), heart (1.5-x) and aorta (2.2-x) of these mice. Biglycan mRNA levels also correlated with
expression of hsPLA2-IIA and were significantly higher in the liver (2-x) and aorta (2.8-x),
though not in the heart, of mice treated with LPS. Consumption of a western diet did not have
a marked effect in any organ on hsPLA2-IIA mRNA or protein levels, MCSF or biglycan mRNA
levels. Thus, increased in vivo expression of hsPLA2-IIA, which would generate lysoPC locally,
is associated with increased expression of both biglycan and MCSF. These findings support the
2007 ATVB Poster Presentations e-103
by on March 25, 2011 atvb.ahajournals.org Downloaded from
hypothesis that sPLA2-mediated production of lysoPC can influence synthesis of the
extracellular matrix molecules biglycan and PG-MCSF, with potential pro-atherosclerotic
consequences.
P386
Inhibition of Chemokine Receptor CCR2 in Bone Marrow Cells by siRNA
Lentiviral Treatment Leads to Reduced Aneurysm Growth
Vivian de Waard, Ilze Bot, Saskia de Jager, Erik AL Biessen, Theo JC van Berkel; Leiden
Amsterdam Cntr for Drug Rsch, Leiden, The Netherlands
Introduction: Abdominal aortic aneurysms (AAA) form a relatively common vascular disorder
among aging men and can be lethal if left untreated. Treatment consists mostly of surgery
when the expansion of the aorta has reached a certain diameter (5.5 cm). Before the
aneurysms become this large, treatment is scarcely available. It has been shown that
chemokine receptor CCR2 facilitates aneurysm formation in a murine AAA model, indicating
that macrophage recruitment plays a role in AAA. Hypothesis: Methods to block macrophage
recruitment by manipulation of CCR2 may lead to novel therapeutic approaches to prevent
aneurysm growth. Method: siRNA-mediated gene silencing of CCR2 was used to decrease
aneurysm formation. For delivery of the CCR2 siRNA to leukocytes, a bone marrow
transplantation experiment was performed in ApoE deficient (ApoE-/-) male mice. Bone marrow
cells were harvested from donor ApoE-/- mice and transduced with concentrated lentiviral
supernatant containing the CCR2 siRNA vector (siCCR2) or control vector. Lentiviral-transduced
cells (1.0x10
7
) were injected into irradiated recipient mice (single dose of 9 Gy X-ray total body
irradiation). After 6 weeks the mice were fed a Western-type diet. During the last 4 weeks of
diet, angiotensinII (AngII; 1.44 mg/kg/day) was given to induce AAA. Results: We observed 30%
(3/10) premature death after the onset of AngII perfusion in the control mice and 18% (2/11)
in the siCCR2 mice. In the surviving mice 29% (2/7) of the control mice had outward remodeling
of the aorta larger than 150% of the normal aorta diameter which qualifies as an aneurysm.
The siCCR2 group did not show any aneurysms (0/9) that met these criteria. However, smaller
micro-aneurysms were observed in both groups. These so-called micro-aneurysms are small
expansions of the vessel wall with elastic lamina breaks. When large and small aneurysms are
counted, the control mice contained 2.7 aneurysms per aorta, while the siCCR2 mice had a
significant decreased number of 1.4 aneurysms per aorta (p 0.04). Conclusion: CCR2 gene
silencing resulted in decreased aneurysm growth, showing that silencing of CCR2 by siRNA
lentiviral vectors forms a therapeutic approach to inhibit aneurysm formation.
P387
Role of Pericytes and PDGF-B in the Antileakage and Vascular Remodeling
Actions of Angiopoietin-1
Jonas Fuxe, Harras B Zaid, Tom Le, Erin Lashnits, Shaun OBrien, Univ of California, San
Francisco, San Francisco, CA; Gou Young Koh, Korea Advanced Institute of Science and
Technology, Daejeon, Republic of Korea; Donald M McDonald; Univ of California, San
Francisco, San Francisco, CA
Angiopoietin-1 (Ang1) is a clinically relevant mitogen for endothelial cells with important
anti-leakage and remodeling actions: it transforms distal capillaries of the microvasculature
into enlarged vessels that are leakage-resistant in the setting of acute inflammation. In
addition, Ang1 stabilizes blood vessels by promoting recruitment of pericytes during develop-
ment. Recently, the anti-leakage effect of Ang1 was suggested to be due to its ability to reduce
gap formation at intercellular junctions. However, little is known about the possible role of
pericytes in this process. Abluminal coverage of blood vessels by pericytes is requisite for
microvascular stability and is regulated by platelet-derived growth factor B (PDGF-B), a
chemotactic factor promoting recruitment of pericytes to the microvasculature. To investigate
the role of pericytes in the anti-leakage effect of Ang1, we tested the ability of Ang1 to reduce
inflammatory-induced microvascular leakage in the mouse trachea in the presence of a
PDGF-B inhibitor. We also studied the effect of Ang1 and the PDGF-B inhibitor on pericyte
coverage of tracheal blood vessels. Using high-resolution confocal microscopic analysis we
found, rather surprisingly, a 29% decrease in pericyte coverage in the microvasculature after
Ang1 treatment, despite a 40% reduction in leakage. PDGF-B inhibition did not affect vascular
leakage, even though pericyte coverage was reduced by 31%. Interestingly, PDGF-B inhibition
in combination with Ang1 treatment resulted in a substantial increase in leakage by 61%, and
a 55% reduction in pericyte coverage. Thus, Ang1 and the PDGF-B inhibitor had additive effects
on pericyte loss and the anti-leakage effect of Ang1 was completely reversed by inhibition of
PDGF-B. These results suggest that pericytes are involved in the anti-leakage actions of Ang1
and that a threshold level of pericyte coverage may be necessary for vascular stability. They
also indicate that crosstalk between Ang1 and PDGF-B pathways exists.
P388
Anticonnective Tissue Growth Factor Antibody Attenuates Vascular Fibrosis
and Prevents Passive Arterial Stiffness
Zhihua Wei, Hui Wang, Erin Zomber, Chris Jacob, Weihua Zhang, Parmjeet Sidhu, Kevin
Furstoss, Michael Barnes, Suzanne Spong, David Y Liu, George Coker, Al Lin, Ingrid
Langsetmo, Zhihe Li; Fibrogen Inc, South San Francisco, CA
Connective tissue growth factor (CTGF) mediates production of extracellular matrix (ECM) and
vascular remodeling that facilitates development of atherosclerosis, arteriosclerosis and
hypertension. We hypothesized that inhibition of CTGF might reduce vascular fibrosis and the
stiffness of vessel wall. Vascular fibrosis was induced by N

-nitro-L-arginine methyl ester


(L-NAME, 40 mg/kg/d in drinking water containing 1% NaCl) and angiotensin II (AngII, 120
ng/kg/min s.c.) for 3 weeks in SD rats. CTGF was inhibited by a fully human anti-CTGF antibody
(FG-3019, 10 mg/kg, 3x/week i. p) for 3 weeks starting simultaneously with administration of
L-NAME/AngII. Purified human IgG (hIgG, 10 mg/kg, 3x/week i. p.) and losartan (10 mg/kg/d
p.o) were used for negative and positive controls respectively. The thoracic aorta was collected
for evaluation of ECM proteins by Western blot, and carotid artery was dissected and
instrumented for measurement of passive arterial stiffness (PAS). The PAS, as assessed by
pressure/diameter curves was significantly increased with L-NAME/AngII treatment and there
was clear trend towards reducing the enhanced PAS by FG-3019 or losartan (Figure 1).
L-NAME/AngII induced an over production of fibronectin in the vessel wall and FG-3019
significantly prevented the enhancement of this protein (69.425.7 in hIgG vs 17.714.3 in
FG-3019 groups, p0.001). Data demonstrate that FG-3019 attenuates L-NAME/AngII-
mediated vascular fibrosis and prevents fibrosis-related passive arterial stiffness, suggesting
inhibition of CTGF is a potential therapeutic strategy for treatment of vascular remodeling
associated with atherosclerosis and hypertension.
P389
Biomechanical and Molecular Mechanisms of Early Abdominal Aortic
Aneurysm Formation
Kathryn A Maiellaro, Georgia Institute of Technology, Atlanta, GA; W Robert Taylor; Emory
Univ Sch of Medicine, Atlanta, GA
Abdominal aortic aneurysms (AAA) are characterized by localized vessel inflammation. This
localized vessel disease is likely caused by complex interplay between altered normal vessel
mechanics and gene/protein regulation. Our ongoing objective is to define the early molecular
and mechanical events that shift a healthy aorta into a dilating, aneurysmal aorta. The working
hypothesis for this study was that localized alteration of aortic wall mechanics directs the
spatial tendency for aneurysm formation and precedes molecular inflammatory events. Whole
aorta inflation tests were utilized to measure pressure-diameter relationships, with resulting
compliance as the mechanical metric. Our first model of AAA formation included 16 week
C57Bl/6 male mice treated with angiotensin II (angII), -aminoproprionitrile (APN), an inhibitor
of lysyl oxidases, and fed high fat chow. This experimental model has 100% occurrence of AAA
formation. The second model lacked APN, serving as a more physiologic model of AAA
formation. For the third model ApoE
-/-
mice treated with angII and high fat diet were also
investigated as a AAA model due to the hyperlipidemia-induced baseline inflammatory state.
Aortas were harvested at 3, 6, 9, & 12 days post treatment for inflation. A transmural pressure
ramp was applied from 5 to 135 mmHg and the concomitant diameter change was measured
at the level of the celiac artery and 7
th
intercostal arteries. Baseline maximum compliance
values of control aortas at the celiac artery and the 7
th
intercostal artery pair were 5.5x10
-3

0.0017 and 6.2 x10


-3
0.0021 mm/mmHg, respectively, corresponding with published
compliance measurements in male C57Bl/6 mice. In all AAA models, the pressure-diameter
relationships revealed no significant difference relative to control. However, histological results
revealed microstructural changes in elastin structure and collagen accumulation in all models
compared to control. The results, therefore, support the alternate hypothesis that early
molecular and microstructural events direct mechanical behavior in aneurysm formation. This
study represents a significant step toward thorough characterization of the early events that
occur during AAA formation.
P390
Circulating Inactive Human Matrix Gla Protein as a Biomarker for
Cardiovascular Disease
Leon J Schurgers, Ellen C Cranenburg, Univ of Maastricht, Maastricht, The Netherlands; Ralf
Koos, Vincent Brandenburg, Markus Ketteler, Univ Hosp Aachen, Aachen, Germany; Cees
Vermeer; Univ of Maastricht, Maastricht, The Netherlands
Matrix Gla protein (MGP) is a vitamin K-dependent protein and a strong inhibitor of vascular
calcification. In both animals and humans MGP deficiency results in extensive arterial
calcification. MGP activity depends on vitamin K-dependent carboxylation, hence vitamin
K-deficiency leads to undercarboxylated MGP (ucMGP) which is inactive. Recently it was shown
that ucMGP synthesis is strongly upregulated in calcified arteries, while in contrast local
GlaMGP expression was very low. Therefore, we developed an ELISA-based assay with which
ucMGP can be detected in the circulation. Serum ucMGP levels were measured in controls (n
54) and in 3 patient populations, those with aortic stenosis (n 25), chronic kidney disease
(CKD 5D) (n 40) and calciphylaxis (n 10). Neither age nor gender influenced the circulating
ucMGP concentrations. As compared to age- and sex-matched controls, all patient groups had
significantly decreased ucMGP levels. However, in calcification-prone patients with renal failure
virtually all patients had ucMGP levels below the control range. The renal patients underwent
MSCT (multi-slice computed tomography) scanning of the coronary arteries. After stratifying the
renal patients for tertiles of coronary calcification, ucMPG levels showed a significant decrease
with increasing amount of vasculare calcification: lowest tertile: 218 (79), intermediate tertile:
e-104 Arterioscler Thromb Vasc Biol June 2007
by on March 25, 2011 atvb.ahajournals.org Downloaded from
187 (46), and highest AS tertile 168 (49) nmol/L (p0.04). We conclude that the ucMGP assay
clearly identifies cardiovascular patients and/or those at high risk for developing vascular
calcification and that this assay may become an important tool in the diagnosis of
cardiovascular disease.
P391
Role of Lipid Rafts/Caveolae in Nongenomic C-Src Signaling by
Aldosterone in Vascular Smooth Muscle Cells
Glaucia E Callera, Augusto C Montezano, Alvaro Yogi, Univ of Ottawa, Ottawa, Canada; Rita
C Tostes, Univ of Sao Paulo, Sao Paulo, Brazil; Ernesto L Schiffrin, McGill Univ, Montreal,
Canada; Rhian M Touyz; Univ of Ottawa, Ottawa, Canada
Objective - We demonstrated c-Src-regulated p38 MAP kinase activation and NAD(P)H
oxidase-inducible generation of superoxide anion (O
2
-
) as novel nongenomic signaling
pathways for aldosterone in vascular smooth muscle cells (VSMC). These effects were inhibited
by eplerenone, mineralocorticoid receptor (MR) antagonist. c-Src is identified as lipid
raft-associated protein. Whether non-genomic c-Src-dependent signaling by aldosterone
involves the rafts and specialized caveolin-enriched membrane microdomains remains unclear.
Here we tested the hypothesis that aldosterone induces c-Src trafficking into caveolae/lipid
rafts. We also assessed whether these interaction influence NAD(P)H oxidase-derived O
2
-
.
Methods and results - VSMCs from WKY rats, caveolin 1 (Cav 1)
-/-
mice and wild-type Cav
1
/
mice were studied. siRNA was used for Cav 1 gene knockdown in mice cells. Membrane
cholesterol depletion/sequestration was achieved with 10 mM methyl--clyclodextrin or
50g/mL nystatin. Cholesterol-rich fractions from VSMC were obtained by sucrose-gradient
centrifugation and identified by flotillin-2 expression. Methyl--clyclodextrin and nystatin
abolished 0.1 M aldosterone-induced c-Src phosphorylation and O
2
-
generation in VSMCs.
Aldosterone significantly increased c-Src trafficking and phosphorylation into (by 12-fold,
p0.05) lipid rafts/caveolae. Aldosterone also stimulates p47phox translocation into these
cholesterol-rich fractions, indicating the activation of NAD(P)H oxidase (1 fold, p0.05).
Knockdown of Cav 1 inhibits aldosterone-induced c-Src and cortactin phosphorylation without
abolishing this effect. Similar results were observed in Cav 1 deficient cells. Aldosterone
induced c-Src translocation into cholesterol-rich domains in Cav 1
-/-
and Cav 1
/
VSMCs. MRs
were localized in flotillin-enriched fractions, indicating lipid raft-association. Conclusions- We
provided evidence for the functional and spatial importance of lipid rafts/caveolae in
MR-mediated aldosterone induced c-Src phosphorylation, which is required for redox signaling
in VSMCs. These findings highlight the importance of lipid rafts in non-genomic signaling by
aldosterone in VSMCs.
P392
Oxidative Stress Induces Vascular Smooth Muscle Cell Calcification via
AKT Signaling
Chang-Hyun Byon, Univ of Alabama at Birmingham, Birmingham, AL; Jay M McDonald, Univ
of Alabama at Birmingham and VA Med Cntr, Birmingham, AL; Yabing Chen; Univ of
Alabama at Birmingham, Birmingham, AL
Oxidative stress plays a critical role in the pathogenesis of atherosclerosis, including the
development of atherovascular calcification, a prominent feature of atherosclerosis. Enhanced
osteogenic differentiation of vascular smooth muscle cells (VSMC) is associated with oxidative
stress in vitro. Further, Akt phosphorylation has been associated with human VSMC calcification
in culture. Thus, we hypothesized that increased oxidative stress induces VSMC calcification
thought AKT signaling pathways. VSMC were explanted from the aorta of C57BL6 mice and
sorted by flow cytometry with smooth muscle specific -actin antibody. We found that a model
oxidant, H
2
O
2
, induced VSMC mineralization in a dose-dependent manner (fold increase at 0.05
mM1.50.7, 0.2 mM6.01.4, and 0.5 mM16.01.4 compared with control, n 4 for
each, p0.05). H
2
O
2
increased intracellular peroxide production as determined by dichlorofluo-
rescein fluorescence, indicating oxidative stress. The expression of bone-associated proteins
including alkaline phosphatase (ALP), type I collagen (Col I) and osteocalcin (OC) were induced
by 0.5 mM H
2
O
2
(fold increase in ALP8.71.2, Col I6.60.9, and OC5.31.4 compared
with controls, n 4 for each, p0.05). By contrast, the expression of smooth muscle specific
-actin was decreased (n4, p0.05). Taken together, the data suggest that oxidative stress
induces a phenotypic transition of VSMC into osteogenic cells. Furthermore, we identified that
H
2
O
2
induced Akt phosphorylation (n4). AKTI, a pharmacologic inhibitor of Akt, inhibited VSMC
calcification and the expression of bone-associated proteins in a dose-dependent manner.
These data demonstrate an important role of Akt signaling in mediating oxidative stress-
induced VSMC calcification. In summary, we found that oxidative stress induced transformation
of VSMC into osteogenic cells by increasing bone-associated proteins and decreasing SMC
-actin. Increased phosphorylation of AKT contributes to the osteogenic differentiation of
VSMC. Our results provide important insights into understanding the molecular mechanism of
oxidative stress induced vascular calcification.
P393
Rat Aortic Allografts Activate Progenitors for Smooth Muscle Cells in the
Adjacent Tissues
Monika Grudzinska, Piotr Religa, Karolinska Institutet, Stockholm, Sweden; Krzysztof
Bojakowski, Zbigniew Gaciong, Joanna Soin, Aleksandra Wasiak, Monika Staniak, Med Univ
of Warsaw, Warsaw, Poland; Cecilia Soderberg-Naucler; Karolinska Institutet, Stockholm,
Sweden
Introduction: Transplant arteriosclerosis is the major factor of late organ dysfunction after
transplantation. The pathology involves inflammation of the vessel with formation of the intimal
hyperplasia consisting of smooth muscle cells recruited into the vessel intima. The process
results in a progressive narrowing of the vessel lumen partly, due to a healing reaction in the
intima. Hypothesis: Smooth muscle cells contributing to the vascular narrowing are in large
extension derived from recipient of the graft. Evaluation of migration of tissue progenitor cells
from surrounding tissues was the aim of the study. Methods: Infrarenal aorta was transplanted
from female F344 to male Lewis rats and collected after different time points. Migration and
activation of host-derived SMCs and cells from surrounding tissue were analyzed by
immunohistochemistry and real-time polymerase chain reaction (PCR) for the SRY gene. The
samples were analyzed by immunohistochemistry with respect to cells present in the sections,
such as: SMCs, endothelium, leukocytes and activation of the cells (SM-actin, SM myosin, von
Willebrand factor, CD45, CD14, CD4, CD8, cyclinD1, PCNA). The samples were also analyzed
by electron microscopy. Results: Proliferating graft SMCs and infiltrating leukocytes were
observed in the intima and adventitia early after transplantation. The similar process was
observed also in the adjacent vessel, however in lower extension. With electron microscopy we
observed also phenotypic modulation of medial SMCs in the adjacent vessels. The cells from
the adventitia were major source of intimal cells migrating to the inima in the used model.
Conclusions: The tissue progenitor cells from the adjacent adventitia are important source of
the cells for formation of intimal lesion.
P394
Telomerase Reverse Transcriptase Is Expressed in Human Atherosclerosis
and Mediates Vascular Smooth Muscle Cell Proliferation and Cell Cycle
Progression
Elizabeth B Heywood, Florence Gizard, Takashi Nomiyama, Karrie L Jones, Dennis
Bruemmer; Univ of Kentucky, Lexington, KY
Emerging evidence indicates that telomerase controls key cellular functions including
replicative lifespan, differentiation, and cell proliferation. The rate-limiting telomerase reverse
transcriptase (TERT) confers the catalytic activity of telomerase. In the present study we
demonstrate expression of TERT in advanced human atherosclerotic lesions. Using confocal
microscopy, nuclear TERT expression colocalizes within neointimal vascular smooth muscle
cells (SMC). TERT mRNA and nuclear protein expression are negligible in quiescent SMC but
in response to stimulation with platelet-derived growth factor (PDGF) or serum TERT expression
is induced. TERT deficiency using siRNA reduced SMC proliferation in response to PDGF and
serum. Similarly, aortic SMC isolated from TERT-deficient mice revealed decreased mitogen-
induced SMC proliferation. The inhibition of SMC proliferation in cells transfected with TERT
siRNA occurred at the G1-S phase transition of the cell cycle and was associated with
decreased phosphorylation of the retinoblastoma protein. In conclusion, these experiments
demonstrate that TERT expression is required for SMC proliferation and cell cycle progression
and identify TERT as a previously unrecognized target for the treatment of cardiovascular
disease.
P395
Thymidine Phosphorylase Induces Intermediate Conductance,
Ca
2
-Activated Potassium Channel Expression in Rat Vascular Smooth
Muscle Cells
Wei Li, The Cleveland Clinic Foundation, Cleveland, OH; Hiroyuki Ando, Univ of Fukui, Fukui,
Japan; Hong Yue, The Cleveland Clinic Foundation, Cleveland, OH; Kuniyoshi Tanaka,
Shigetoshi Oiki; Univ of Fukui, Fukui, Japan
The intermediate conductance Ca
2
activated potassium channel (KCNN4) has been reported
to promote neointimal vascular smooth muscle cell (VSMC) proliferation, and its blockade could
prevent restenosis after angioplasty. We recently reported that thymidine phosphorylase (TP)
inhibited VSMC proliferation and migration. Here, we examined whether the effect of TP on
VSMC was via regulating KCNN4 expression. Phagemid vectors encoding human TP cDNA were
transfected into rat VSMC. TP overexpressing clones (C2 and C4) proliferated more slowly than
VSMC transfected with empty vector (PC) or parent VSMC. KCNN4 mRNA and protein
expression was significantly higher in C2 and C4 cells compared to PC cells and parent VSMC
(p0.001). KCNN1 expression was similar in all cells. KCNN2, 3 and Slo genes were not
expressed in VSMC. Patch clamping confirmed increased functional KCNN4 expression in the
plasma membranes of the C2 and C4 clones (Figure). Intracellular Ca [Ca
2
]i was higher in C2
and C4 cells at baseline and after ATP stimulation. The KCNN4 specific inhibitor clotrimazole
(CLT) and Tram 34 decreased [Ca
2
]i in C2 and C4 to the level in PC and VSMC cells, but did
not affect cell proliferation. Collagen gel contraction assays showed higher contractile ability in
C2 and C4 compared to PC and VSMC. This activity was lost in the presence of CLT. In
summary, TP gene transfer induces KCNN4 expression in VSMC. This contributes to increased
cellular contractile ability via elevating [Ca
2
]i, but does not alter the inhibitory effect on
proliferation. This study suggests that gene therapy with TP for vascular disease may be benefit
by preventing neointimal VSMC proliferation and regulating vascular tone.
2007 ATVB Poster Presentations e-105
by on March 25, 2011 atvb.ahajournals.org Downloaded from
P396
SERCA2a Gene Transfer Reduces Vascular Smooth Muscle Proliferation and
Restenosis in Humans: A New Target for Drug-Eluting Stents?
Larissa Lipskaia, INSERM UMR S 621, Paris, France; Ste phanie Lehoux, Bruneau Esposito,
INSERM CRCIL UMP S 689, Paris, France; Pascal Le Prince, Nicolas Bonnet, CHU
Pitie -Salpe triere, Paris, France; Isabelle Cantaloube, INSERM UMR S 621, Paris, France;
Claude Le Feuvre, CHU Pitie -Salpe triere, Paris, France; Roger Hajjar, CVRC MGH,
Charlestown, MA; Anne-Marie Lompre; INSERM UMR S 621, Paris, France
Ca
2
signalling is a key factor in the control of rat vascular smooth muscle cells (VSMC)
phenotype and proliferation. Here, we show that in human arteries 1) proliferation is associated
with changes in expression of Ca
2
handling proteins, 2) gene transfer of the sarco/
endoplasmic reticulum calcium ATPase, SERCA2a, prevents proliferation. Coronary arteries (CA)
from explanted heart and mammary arteries (MA) obtained by aorto-coronary bypass were
analyzed. In atherosclerotic plaques from CA SERCA2a, the ryanodine receptor (RyR) and a
marker of the terminal VSMC differentiation, smooth muscle myosin heavy chain 2 (SM2), were
expressed only in the media but not in the neointima whereas smooth muscle myosin heavy
chain 1 (SM1) and non-muscular myosin heavy chain-B (NM-B) were expressed in both media
and neointima. An ex-vivo model of MA injured and kept in organ culture under constant
pressure and flow was used to test the effect of SERCA2a on proliferation. After 15 days,
neointima formation was observed in injured MA. Furthermore, SERCA2a, RyR and SM2 were
absent from VSMC from the neointima. Adenovirus gene transfer of SERCA2a reduced vascular
remodeling and neointima formation (intima-media ratio was 0.100.04 in SERCA 2a-infected
and 0.530.17 in Gal-infected arteries). Freshly isolated human VSMC cultured in the
presence of serum loose SERCA2a, RyR and SM2 within 3 days. SERCA2a gene transfer
inhibited proliferation of VSMC in vitro. Inhibition of calcineurin/NFAT pathway by cyclosporine
A or by VIVIT strongly inhibited VSMC proliferation. Western blot analysis showed that gene
transfer of SERCA2a, VIVIT or cyclosporine treatment resulted in down regulation of cell cycle
controlling proteins cyclin D1 and Erg 1. In conclusions: SERCA2a controls proliferation of VSMC
and neointima formation in human via the transcription factor NFAT. These findings could have
potential implications for drug-eluting stents.
P397
Angiotensin II Decreases Cell Surface Expression of LRP in Abdominal
Smooth Muscle Cells via AT1 Receptors: Role of Receptor-associated
Protein
Vishwesh P Mokashi, Alan Daugherty; Univ of Kentucky, Lexington, KY
Objective: Infusion of angiotensin II (AngII) into hyperlipidemic mice promotes formation of
aneurysms to a specific region within the suprarenal aorta. This pathology is mimicked in
hyperlipidemic mice with smooth muscle cell (SMC)-specific deficiency of low density
lipoprotein receptor-related protein (LRP); a key regulator of extracellular proteolytic activity.
Hence, we hypothesized that AngII could decrease cell surface LRP in SMCs in aortic regions
that are susceptible to aneurysm development. Methods and Results: Previously, we had
shown that, infusion of AngII (1,000 ng/kg/min) into male C57BL/6 mice decreased LRP protein
abundance in the abdominal, but not the thoracic aortic region. We also demonstrated that
AngII decreased the abundance of LRP and its intracellular chaperone, RAP in ex vivo aortic
tissue in the abdomen, but not in the thorax. SMCs cultured from the abdominal region retained
the property of AngII-induced reductions of both LRP and RAP (p0.05), which was attenuated
by the AT1 receptor antagonist, losartan. AngII also reduced cell association and degradation
of the LRP ligand,
125
I-labeled alpha-2 macroglobulin, in cultured abdominal SMCs, but not in
thoracic SMCs. This reduction was attenuated by the AT1 receptor antagonist, losartan.
Real-time PCR demonstrated that AngII reduced abundance of RAP, but not LRP mRNA. In
addition, AngII also reduced RAP protein and mRNA abundance in abdominal SMCs deficient in
LRP. To investigate if AngII-induced reductions in RAP causes premature proteasomal
degradation of LRP, experiments were conducted with cyclohexamine (protein synthesis
inhibitor) and a combination of cyclohexamine and MG-132 (proteasomal inhibitor). AngII
reduced LRP protein in the presence of cyclohexamine, but not in the presence of MG-132,
whereas RAP protein expression was inhibited even in the presence of MG-132 (p0.05).
Conclusions: AngII decreased expression of LRP in abdominal aortic SMCs in both ex vivo
tissues and cultured cells in a AT1 receptor dependent manner. AngII reduced RAP
independently of LRP, and caused degradation of LRP but not RAP, in a proteasomal dependent
pathway. These data are consistent with AngII inhibiting RAP transcription to indirectly decrease
cell surface expression of LRP.
P398
Matrix Metalloproteinase Transactivation of Epidermal Growth Factor
Receptor Regulates Mitochondrial ATP Synthesis in Vascular Smooth
Muscle
Prabhakara Reddy Nagreddy, Univ of British Columbia, Vancouver, Canada; Fung L Chow, Li
Hao, Tamiko Nishimura, Univ of Alberta, Edmonton, Canada; Kathleen M MacLeod, John H
McNeill, Univ of British Columbia, Vancouver, Canada; Carlos Fernandez-Patron; Univ of
Alberta, Edmonton, Canada
Introduction: G-protein coupled receptors (GPCR) modulate vascular tone, at least, in part by
matrix metalloproteinase (MMP)-dependent epidermal growth factor receptor (EGFR) transac-
tivation. We previously have reported that vascular alpha-1 adrenoceptor mediated activation
of MMPs, such as MMP-7, and the EGFR engages mitochondrial redox processes to maintain
vascular tone. In the present study we investigated the hypothesis that maintenance of
adrenergic vascular tone by the MMP-EGFR pathway involves mitochondrial ATP synthesis.
Methods and Results: In rat vascular smooth muscle cells (VSMCs), stimulation of alpha-1
adrenoceptors with phenylephrine triggered ATP synthesis in a concentration and time
dependent manner. The increase in ATP synthesis was blocked by inhibitors of mitochondrial
ATP synthase (oligomycin), MMPs (GM 6001), EGFR (AG1478) and phosphoinositide-3-kinase
(PI3K)- dependent Akt phosphorylation (wortmannin and LY 294002). Further, inhibition of
MMPs or silencing EGFR expression blunted the phosphorylation of Akt and reduced GLUT4
translocation, effects that were also observed with PI3K inhibitors. In small rat mesenteric
arteries, exogenous ATP promoted activation of MMP-7, which was dependent on P2X but not
P2Y purinergic receptors. Downstream of alpha-1 adrenoceptors, the activation of MMP-7 was
blocked by inhibitors of ATP synthesis (oligomycin) or its bioavailability (apyrase). Moreover,
blockade of PI3K or ATP synthase dose-dependently inhibited adrenergic vascular tone.
Conclusions: Modulation of mitochondrial ATP synthesis by MMPs and promotion of MMP
activity by ATP are two new regulatory events in the signaling pathway of alpha-1
adrenoceptors. We suggest that metabolic and growth pathways merge to modulate MMP
activity and thereby impact vascular tone. Supported by an operating grant from the CIHR to
CFP. PRN was supported by a program grant from the HSFC and the MSFHR, Canada.
P399
Novel Effects of Lovastatin on Extracellular Matrix Gene Expression in
Vascular Smooth Muscle Cells
Diane Studzinski, Charles Shanley; Wayne State Univ, Detroit, MI
HMG CoA reductase inhibitors (statins) have been shown to reduce adverse cardiovascular
events. Moreover, the cardioprotective effects of these agents do not appear to be limited to
their lipid lowering capabilities. Other putative beneficial effects include inhibition of smooth
muscle cell proliferation and migration. Plaque stability is another critical component of
coronary and peripheral vascular disease. We examined the effects of a common HMG CoA
reductase inhibitor, lovastatin, on the expression of plaque stabilizing genes including collagen
Ia1 (COLI), collagen III (COL III), lysyl oxidase (LOX), thrombospondin-1 (TSP-1), and tropoelastin
(TROPO), in human umbilical artery smooth muscle cells (UASMC). In brief, UASMCs were
incubated with 1M lovastatin for up to 48 hours, RNA was extracted, reverse transcribed and
quantitative real time-PCR performed. Collagen I and III mRNA levels were not reduced (96 and
103 % of control); however, LOX mRNA levels were reduced to 67%, TROPO to 60% and TSP-1
to 62%. These effects were time and concentration dependent. Similar results for LOX, TROPO
and TSP-1 were seen following treatment with simvastatin and exposure to the MEK inhibitor,
PD98059. Simultaneous exposure to both lovastatin and PD98059 produced a synergistic effect
with a slightly greater decrease in all gene products. Relative levels of LOX protein appeared
slightly reduced in lovastatin-treated cells immunocytochemically stained with a LOX polyclonal
antibody. Initial western blot analyses of LOX protein levels (48hr) also showed a slight
reduction, suggesting a relatively long half life in this cell line under these conditions. Analysis
of protein levels at later time points are ongoing. These results suggest that LOX and collagen
are differentially regulated, whereas LOX and the elastin precursor tropoelastin appear to be
coordinately regulated. The mechanism for the lovastatin induced LOX and TROPO mRNA
reduction appears to be related to transcriptional control in the MAPK pathway. Taken together,
our results suggest a possible role for HMG CoA reductase inhibitors in extracellular matrix
remodeling and plaque stability.
e-106 Arterioscler Thromb Vasc Biol June 2007
by on March 25, 2011 atvb.ahajournals.org Downloaded from
P400
Hemodynamic Factors Accelerate Vascular Smooth Muscle Migration
Through IP3-Receptor: Implication for Atherogenesis of Hypertension
Tomohiro Tada, Jun Nawata, Huan Wang, Noriko Onoue, Doe Zhulanqiqige, Kenta Itoh,
Koichiro Sugimura, Yoshihiro Fukumoto, Hiroaki Shimokawa; Tohoku Univ Graduate Sch of
Medicine, Sendai, Japan
Background: High systolic blood pressure and wide pulsatile pressure are independent and
important risk factors for atherosclerosis. However, it is unknown whether these hemodynamic
factors directly influence migration of vascular smooth muscle cells (VSMCs), which is a critical
step for the initiation and development of atherosclerosis. In this study, we thus examined the
effect of pulsatile pressure on VSMC migration in vitro, using our original pressure loading
apparatus. Methods and Results: Cultured rat aortic VSMCs of 5 to 13 passages were
incubated in a Boyden chamber under variable pressures and pulse rates. High mean pressure
(180/90mmHg vs. 90/0mmHg), high pulse rate (120/min vs. 40/min) and wide pulse pressure
(190/110mmHg vs. 170/130 mmHg) all significantly accelerated VSMC migration (2.38, 1.27,
and 1.38-fold, respectively). Fura-2 fluorescence measurement of intracellular calcium showed
a correlation between intracellular calcium level and pressure. The pressure-promoted VSMC
migration was completely inhibited by pre-incubation with an intracellular calcium chelator,
BAPTA. The inositol 1,4,5-trisphosphate (IP3) receptor blockers, 2-aminoethoxydiphenyl borate
and xestospongin C, both significantly inhibited the VSMC migration, while the ryanodine
receptor blocker, ryanodine, had no effects. Calcium channel blocker (CCB), azelnidipine, and
angiotensin type-1 receptor blocker (ARB), olmesartan, also significantly and concentration-
dependently inhibited the VSMC migration. Conclusions: These results indicate that VSMC
migration is accelerated in response to the increase in mean pressure, pulse rate, and pulse
pressure, for which increased release of intracellular calcium from sarcoplasmic reticulum via
IP3-receptor may play an important role on the migration. They also suggest that the
anti-atherogenic actions of CCBs and ARBs are mediated not only by their blood pressure
lowering effects but also by their inhibitory effects on VSMC migration.
P401
TRPM7 and Its Kinase-sensitive Substrate Annexin-1 Are Differentially
Regulated by Stretch and Vasoactive Agents in Vascular Smooth Muscle
Cells
Rhian Touyz, Glaucia Callera, Tamara Paravicini, Ying He, Guoying Yao; Kidney Rsch Cntr,
OHRI, Ottawa, Canada
Introduction: Transient receptor potential melastatin cation channel 7 (TRPM7) is important in
vascular smooth muscle cell (VSMC) function. Mechanisms regulating TRPM7 are unclear.
Hypothesis: Mechanical stretch and vasoactive agonists, important in regulating vascular tone
and growth, modulate TRPM7 in VSMCs. Methods: Rat mesenteric VSMCs were studied.
Expression of TRPM7 mRNA and protein was assessed by RT-PCR and immunoblotting. Cellular
localization of TRPM7 was evaluated by immunofluorescence microscopy. Activation of
annexin-1, a TRPM7 kinase-sensitive substrate, was assessed by cytosol-to-membrane
translocation. TRPM7 was downregulated by siRNA. Cells were stimulated with vasoactive
agonists, Ang II (10
-10
-10
-7
mol/L), a vasoconstrictor, or bradykinin (10
-10
-10
-6
mol/L), a
vasodilator, or exposed to cyclic stretch using a FlexerCell system. Results: Immunofluores-
cence confocal microscopy demonstrated TRPM7 distribution along the cell membrane and
co-localization with flotillin-2, marker of lipid rafts, in VSMCs. In the basal state TRPM7 was
phosphorylated on serine/threonine residues. Ang II only modestly influenced TRPM7 expres-
sion and reduced phosphorylation. Bradykinin significantly increased TRPM7 expression and
phosphorylation (23 fold) and TRPM7-induced Mg
2
influx (3-fold, p0.05)). This was
associated with increased annexin-1 translocation (2-fold, p0.05). These effects were
abrogated in TRPM7 siRNA-transfected cells. Acute cyclic stretch significantly decreased
TRPM7 phosphorylation by 50% and attenuated annexin-1 activation. Our results demonstrate
that in VSMCs, TRPM7 is distributed in a network at the cell membrane and intracellularly
where it associates with cholesterol-rich domains. Whereas mechanical stretch and Ang II
tended to reduce TRPM7 activation, bradykinin upregulated TRPM7 expression and activity.
Conclusions: Our data suggest that TRPM7 is differentially regulated by vasoactive agents and
stretch. Association with cholesterol-rich domains might facilitate TRPM7- and Mg
2
-
dependent signaling in VSMCs. These novel findings further characterize vascular TRPM7 and
highlight the putative functional importance of this Mg
2
transporter in the regulation of VSMC
function.
P402
Intramural Delivery of Rapamycin with Molecularly Targeted Nanoparticles
Inhibits Stenosis Without Delaying Endothelial Healing After Angioplasty
Huiying Zhang, John S Allen, Ralph W Fuhrhop, Samuel A Wickline, Gregory M Lanza,
Tillmann Cyrus; Washington Univ, Saint Louis, MO
Drug eluting stents have demonstrated the anti-restenotic benefit of local antiproliferative drug
delivery following angioplasty. However, delayed re-endothelialization creates a risk for late
in-stent thrombosis. Furthermore, small vessels may not be amenable for stent placement. We
have shown that v3-integrin targeted perfluorocarbon nanoparticles deliver rapamycin into
the arterial wall and reduce stenosis after angioplasty. The objective of this study was to
determine whether intramurally targeted rapamycin nanoparticles impair endothelial repair.
Femoral arteries of NZW rabbits fed an atherogenic diet for 4 months were subjected to balloon
stretch injury. v3-intergrin targeted nanoparticles with 0.3-mol% rapamycin in the
surfactant were locally infused into one artery while the contralateral artery received saline or
v3-targeted nanoparticles without rapamycin as controls. Microscopic morphometric
analysis following Carstairs staining showed that endothelial recovery following rapamycin
treatment was not prolonged when compared with control at all time points (Figure). Intimal
plaque area observed en face was significantly smaller (P0.05) in arteries exposed to
rapamycin nanoparticles than that in controls (Figure). In conclusion, constrained intramural
delivery of rapamycin using v3- nanoparticles immediately after balloon angioplasty
diminishes acute plaque formation without impairing endothelial healing.
P403
Progenitor Cells and Secreted Paracrine Factors Induce Regional
Cardiomyocyte Hypertrophy Following Therapy for Acute Myocardial
Infarction
Brendan J Doyle, Paul Sorajja, Philip A Araoz, Paul Stalboerger, Dylan Miller, Cynthia Reed,
Jeffrey Schmeckpeper, Shaohua Wang, Chunsheng Liu, Andre Terzic, Stephen Riederer,
Noel M Caplice; Mayo Clinic, Rochester, MN
Administration of circulating progenitor cells (CPC) is a promising therapy for post-infarction
cardiac repair. However, the mechanisms that underlie apparent beneficial effects on
myocardial remodeling are unclear. We investigated the therapeutic effects of peripheral blood
progenitor cells, and the therapeutic effects of paracrine factors secreted by these cells, on
cardiomyocyte function in vitro and in vivo. In a porcine model of myocardial infarction
intracoronary injection of autologous CPC was associated with increased infarct territory mass
and improved regional ventricular systolic function at 2 months compared to control. Treatment
with conditioned media derived from autologous CPC was associated with similar improved
effects on infarct territory mass and function. Histologic analysis of the infarct border zone
revealed significantly increased cardiomyocyte size in CPC and conditioned media treated
groups, when compared to controls. A paracrine CPC effect was also verified in a pure
myocardial preparation in which cardiomyocytes devoid of fibroblast, neuronal and vascular
elements directly responded by increasing cell mass when exposed to the same conditioned
media. Analysis of conditioned media revealed elevated levels of TGF1 (human 267.311.8
pg/ml, porcine 57.16.1 pg/ml), a recognized mediator of hypertrophic signaling in the heart.
Neutralizing antibodies to TGF1 attenuated the pro-hypertrophic effect of conditioned media,
and use of recombinant TGF1 added to fresh media replicated the pro-hypertrophic effects
of conditioned media in vitro. These data demonstrate the potential of paracrine factors
including TGF1 secreted from circulating progenitor cells to induce cardiomyocyte hypertro-
phy contributing to increased infarct territory LV mass, with favorable medium term effects on
regional function following myocardial infarction.
P404
CD34

Cells Stimulate Vascularization, Inhibit Inflammation, and Increase


Cell Viability in Implantable Bioartificial Devices
Lindsay M Rodewald, Michelle R Sukup, Heidi Hansen, The Univ of Iowa, Iowa City, IA;
Victor G Rodgers, Univ of California, Riverside, Riverside, CA; Gina C Schatteman; The Univ
of Iowa, Iowa City, IA
For successful engraftment of allogenic cells using a bioartificial device, the device must
immuno-isolate cells, be immuno-invisible, exhibit good bulk transport characteristics, and be
rapidly vascularized. Current devices are not vascularized quickly enough to maintain adequate
cell viability and bulk transport, and device encapsulation is problematic. To address these
issues, we tested devices consisting of a pro-angiogenic scaffold surrounding a cell-
encapsulating alginate core in mice. Our goal was to prevent device encapsulation while
stimulating vascularization. We found that despite their pro-angiogenic nature, the scaffolds
acted as a physical barrier, inhibiting the approach of vessels to the alginate core. CD34

peripheral blood cells promote vascular growth in diabetic mice, so we also examined their
ability to induce vessel formation in the devices. Our data showed that they stimulated capillary
growth proximate to the device and reduced encapsulation. To determine if this translates to
improved cell viability in devices, pancreatic -cells were labeled with quantum dots. Retention
of quantum dots ( a measure of -cell viability) was also increased by inclusion of CD34

cells
in the device or injection of the cells near the device. Because CD34

cells from some patients


are dysfunctional, clinically, it may be beneficial to use allogenic CD34

cells. Thus, we
immunesuppressed mice prior to device implantation and CD34

cell injection. Surprisingly,


immunesuppression, inhibited vessel growth and increased encapsulation. Thus, CD34

cells
may help create an optimal milieu for device implantation. Work supported by the Juvenile
Diabetes Research Foundation
P405
ASS Nonresponder Rate Using Multiple Aggregation Tests Has High
Prevalence in CABG Patients
Inna Kammerer, Heart Cntr, City Hosp, Ludwigshafen, Germany; Peter Hellstern, Institute of
Hemostaseology and Transfusion Medicine, City Hosp, Ludwigshafen, Germany; Frank Isgro,
Heart Cntr, City Hosp, Ludwigshafen, Germany; Juergen Bach, Institute of Hemostaseology
and Transfusion Medicine, City Hosp, Ludwigshafen, Germany; Werner Saggau; Heart Cntr,
City Hosp, Ludwigshafen, Germany
Object : The predominant mechanism of early graft failure after coronary artery by-pass
grafting is associated with benefit of antiplatelet treatment of drugs like Acetyl-salicylic acid (
ASA ).The mechanisms for ASA resistance are multifactorial and can be screened by laboratory
tests. Methods : We determined platelet function using whole blood impedance aggre-gometry
( IA ) induced by ADP, arachidon acid ( AA ) and collagen ( Coll ), plateled aggregation ( PA ),
2007 ATVB Poster Presentations e-107
by on March 25, 2011 atvb.ahajournals.org Downloaded from
Platelet Function Analyzer-100 ( PFA-100). ASA 300mg was ad-ministered intravenously by
40patients on 7.9.post day after CABG. Non compliance administration was eliminated. Blood
was drawn immediately before, 60minutes and 24hours after ASA application. ASA Non-
Responsiveness (NR) was defined as maxi-mal AA-induced PA below 20mm, corresponding to
at least 66% inhibition. Results : ASA NR was observed in 9 patients (22% ) 1hr and 12 patients
( 30% ) 24 hrs after ASA application. Before ASA injection, patients had significant greater IA
values than 60 healthy controls ( p0,001 ) still PA determination was not different to control
values. Most ADP and Coll-induced IA and PA values were not reduced 1hour and 24hours after
ASA injection. Conclusion : One third of the patients after CABG are non responders to ASA
therapy. Screening for ASA resistance and optimised antiplatelet therapy may help to further
improve potency of bypass grafts.
P406
Testing the Multiscale Computational ToolKit for Modeling Thrombus
Development Using Near Real-time Confocal Imaging of Mesenteric
Vascular Injury
Malgorzata M Kamocka, Indiana Univ Sch of Med, Indianapolis, IN; Fang Qi, Zhiliang Xu,
Nan Chen, Mark Alber, Univ of Notre Dame, Notre Dame, IN; Elliot D Rosen; Indiana Univ
Sch of Med, Indianapolis, IN
To prevent the loss of blood following a break in blood vessels, components in blood and vessel
wall interact rapidly to form a thrombus to limit hemorrhage. This hemostatic response is rapid
and regulated, since excessive and inappropriate clotting reduces the patency of blood flow.
During the last century many of the components involved in the hemostasis have been
identified including proteins and cellular components in blood, elements in the vessel wall and
hydrodynamic factors Direct numerical simulation (DNS) is being used as a new tool to study
biological processes. The aim of this study is to develop a meaningful computational model of
clot development taking into account both physiological, biochemical and mechanical factors,
which usually occur at different length scales. This involves the development of a Multiscale
Computational ToolKit for Modeling Thrombus Development (MMTD). The proposed ToolKit
MMTD builds upon a combination of FronTier, a software framework using a front tracking
approach for the simulation of multiphase flows (developed by Dr. Glimm at SUNY at Stony
Brook), and a Cellular Potts Model-based (CPM) modeling environment, CompuCell3D
(developed by Dr. Alber). The predictions of the computational model are tested by high
resolution confocal monitoring of thrombus development following laser injury of the mesentery
vasculature. The model permits tracking platelet accumulation, fibrin deposition, leukocyte
incorporation and hydrodynamic parameters. The comparison between predictions of the
computational model and results of the experimental system permit refinement of the Toolkit
MMTD. Initial studies suggest the importance of hydrodynamic parameters in the heteroge-
neous domain structure of a developing thrombus. The development of a computational model
incorporating physiologically important parameters of thrombus development will enable one to
identify rate limiting, critically important, regulatory parameters for which small perturbations
result in large effects on thrombus development. Such improved understanding will make
significant contributions in the development of therapeutic strategies to treat thrombosis and
hemorrhage.
P407
Molecular Characteristics of Inactivated Human Tissue Plasminogen
Activator Association with Fibrin Implicate the Nature and Sites of the
Binding Interactions
Melvin E Klegerman, David D McPherson; Univ of Texas Health Science CntrHouston,
Houston, TX
Introduction. Recombinant tissue plasminogen activator (tPA) is the predominant thrombolytic
agent employed for clinical treatment of myocardial infarction and ischemic stroke. The
self-regulating nature of the thrombolytic process is mediated by two discrete fibrin-binding
sites in the tPA molecule, a higher affinity site in the finger domain and a lower affinity site in
the Kringle 2 domain. Using a two-stage fibrin pad ELISA protocol, we have been able to
delineate both affinities, obtaining K
D
values of 3.19 nM and 28.3 nM at 37
o
C. We have also
demonstrated that tPA (Activase) incorporated into echogenic liposomes (ELIP) fully retained its
thrombolytic and fibrin-binding properties. Purpose. To test the hypotheses that determination
of thermodynamic parameters associated with tPA-fibrin binding could provide information
about the molecular nature of the associations and that binding characteristics at 37
o
C would
be retained in serum. Methods. Testing of the latter hypothesis required that we use Activase
that had been inactivated using D-phe-pro-arg-chloromethylketone (PPACK). Fibrin pads were
prepared in 96-well microplates. After blocking exposed binding sites with an albumin solution,
various tPA/PPACK concentrations in buffer or rabbit serum were incubated in wells to
equilibrium at three temperatures. The contents of wells were removed and assayed for tPA
using a sandwich ELISA. From the free tPA fractions thus measured, Scatchard determination
of binding affinities was performed. Changes in standard enthalpy (dH
o
), free energy (dG
o
) and
entropy (dS
o
) were determined by vant Hoff analysis and equations of state. Results. The
associations were found to be highly exothermic (high affinity: dH
o
-21.7 kcal/mole, dS
o

-33.8 cal/mole
o
; low affinity: dH
o
-13.9 kcal/mole, dS
o
-9.9 cal/mole
o
), indicative of ionic
interactions involving 34 charged residues. A single affinity of tPA/PPACK-fibrin association at
37
o
C was observed (K
D
9.52 nM) in rabbit serum. Conclusions. tPA-loaded ELIP has clinical
utility for both therapeutic and diagnostic applications. We also identified a locus for the
high-affinity tPA-fibrin interaction.
P408
Polymorphism of Factor XIII and Gender-related Differences in Risk of
Cardiac Events Among Postinfarction Patients
Grzegorz Pietrasik, Wojciech Zareba, Daniel Ryan, Arthur J Moss; Univ of Rochester Med
Cntr, Rochester, NY
Background There is limited data on the effect of the FXIII V34L polymorphism in patients with
coronary artery disease. Methods We evaluated the effect of the Leu34 genotype of FXIII on
risk of recurrent cardiac events (cardiac death, nonfatal MI or unstable angina) in a cohort of
1012 post-infarction (MI) patients: 619 with the Leu34 genotype and 393 without the Leu34
genotype. There were 760 men and 252 women; gene frequencies were similar across the
genders (p0.73). Results In total population, the Leu34 genotype was not associated with
increased risk of event (adjusted HR 1.18, p0.20). The Leu34 genotype was associated
with higher rate of events among men, but not among women (Figure 1). After adjustment for
clinical covariates, the Leu34 genotype remained risk factor for recurrent events in men
(HR1.40, p0.03) but not in women (HR0.81, p0.38). Conclusions The V34L
polymorphism of FXIII gene is an independent risk factor for recurrent events among post-MI
men but not among women.
P409
The Intrinsic and Extrinsic Pathways in Hemostasis and Thrombosis
Christopher S Roberts, Amy L Zollman, Maria K Malgorzata, Elliot D Rosen; Indiana Univ,
Indianapolis, IN
Introduction Coagulation is essential to prevent life-threatening hemorrhage but can be
detrimental in instances of vascular thrombosis. Recent studies have indicated coagulation in
hemostasis and thrombosis may be initiated by separate pathways. Interestingly, mice deficient
in factors XII and XI of the intrinsic pathway exhibit major defects in thrombus formation
following FeCl
3
injury. Conversely, mice deficient in factor VII and tissue factor, the initiating
cofactors of the extrinsic pathway, die during embryogenesis or perinatally from massive
hemorrhage. The focus of this study is to compare the effects of the intrinsic and extrinsic
pathways of coagulation in several models of hemostasis and intravascular thrombosis.
Materials and Methods Three animal groups were analyzed in this study; wild type, factor
XII-inhibited, and factor VII deficient mice in a C57Bl/6 background. The injury models utilized
include a vascular puncture model of hemostasis and two intravascular injury models; ferric
chloride (FeCl
3
) and photochemical. The hemostasis model involves the puncture of the left
carotid artery by a 30 gauge needle. Total bleeding times were manually recorded and
hemoglobin absorption determined from collected blood. FeCl
3
injury was performed by adding
3ls of 10% FeCl
3
solution to a 5x1x0.2mm strip of filter paper, which was applied to the left
carotid artery for 3 minutes. Blood flow was measured for 30 minutes post-injury. Rose bengal
is a photoreactive dye that was administered intravenously and activated with a mercury lamp
to induce thrombosis in mesenteric vessels. Thrombus formation was analyzed by measuring
optical density of sequential images. Conclusion Factor XII-inhibited mice exhibited no defects
in hemostasis but failed to develop occlusive thrombi following FeCl
3
or photochemical injury.
In contrast, FVII deficient mice deficient exhibited impaired hemostasis but minimal defects in
thrombosis. These results suggest that FXII inhibition may provide a target for treating
thrombosis without hemorrhagic complications.
Figure 1B Probability of event in women by the Leu34 genotype.
Figure 1A Probability of event in men by the Leu34 genotype.
e-108 Arterioscler Thromb Vasc Biol June 2007
by on March 25, 2011 atvb.ahajournals.org Downloaded from
P410
Hematopoietic CellDerived Tissue Factor Accelerates Thrombosis in
C-Reactive Protein Transgenic Mice
Jianbo Wu, Elizabeth A Grunz, Jordan M Brown, Tammy Strawn, Univ of Missouri-Columbia,
Columbia, MO; Nigel Mackman, The Scripps Rsch Institute, La Jolla, CA; William P Fay;
Univ of Missouri-Columbia, Columbia, MO
Elevated plasma levels of C-reactive protein (CRP) are associated with increased risk of
myocardial infarction, and transgenic mice expressing human CRP demonstrate accelerated
thrombosis after vascular injury. However, the mechanisms underlying the prothrombotic effect
of CRP are poorly defined. We tested the hypothesis that CRP promotes thrombosis via effects
on TF expression by circulating cells of bone marrow origin. We irradiated male CRP-transgenic
(CRP-Tg) mice and transplanted them with bone marrow cells (BMC) from wild-type (WT) mice
or mice completely deficient in mouse TF, but expressing a human TF transgene at a very low
level (approximately 1% normal human TF levels, termed low-TF mice). Six weeks after
transplant we subjected mice to photochemical carotid artery injury and measured the time
required to form an occlusive thrombus. Mean time to occlusion was 13.22.3 min in CRP-Tg
mice transplanted with WT BMC (n10) vs. 19.12.1 min in CRP-Tg transplanted with low-TF
BMC (n7; p0.08; additional transplant experiments are in progress). Plasma TF activity was
significantly higher in CRP-Tg mice transplanted with WT BMC (n3) vs. CRP-Tg mice
transplanted with low-TF BMC (n4) (2.740.19 pM vs 1.360.18 pM, respectively,
p0.01). In contrast, we have shown previously that WT mice transplated with WT BMC exhibit
no differences in thrombosis times compared to WT mice transplanted with low-TF BMC. These
results suggest that CRP increases the risk of thrombosis by up-regulating circulating TF
activity. They also support the hypothesis that circulating TF is prothrombotic, and may help to
explain the association between elevated plasma CRP and increased risk of ischemic
cardiovascular events.
P412
The EP3 Receptor for Prostaglandin E2 in Peripheral Arterial Disease
Maria Bjarnadottir, Thorkell Andresson, Kolbeinn Gudmundsson, deCODE genetics,
Reykjavik, Iceland; Mark Gurney; deCODE genetics, Woodridge, IL
Background. Our genetic studies in Iceland link the PTGER3 gene encoding the EP3 receptor
for PGE2 to increased risk for PAD. EP3 acts oppositely to the IP receptor for PGI2 to regulate
cAMP in platelets. Production of PGE2 in atherosclerotic plaque may enhance platelet
responsiveness to co-aggregants present in the damaged vessel wall, thereby tilting the
balance of pro- and anti-thombotic signals such that genetic variants in EP3 increase the risk
for disease particularly in the arteries of the lower limbs as in PAD. Methods. Platelets were
collected from 10 young controls, 20 patients with PAD, and 20 age-matched elderly controls.
pVASP was measured by EIA. Results. cAMP-dependent phosphorylation of VASP is the major
check-point regulator of platelet cytoskeletal reorganization. Iloprost increases pVASP,
preventing platelet activation, while sulprostone (an EP3 agonist) with a co-aggregant (e.g.,
collagen) decreases pVASP. The action of sulprostone is blocked by DG-041, an EP3 antagonist.
Baseline platelet samples from young controls have approximately 2x greater levels of pVASP
than do elderly controls or PAD patients (p0.0002). Thus, there is a strong, age-dependent
decrease in platelet pVASP. PAD patients have 15% lower pVASP than elderly controls.
Stimulation with iloprost increases pVASP 100 fold, with both elderly and young controls
reaching the same phosphorylation levels. However, iloprost increases pVASP in PAD patients
significantly less (p 0.01). Conclusions. Production of PGE2 in atherosclerotic plaque may
push platelets to a more activated state characterized by decreased pVASP, in part due to
signaling through the PGE2/EP3 pathway. Our therapeutic hypothesis is that an EP3 antagonist
may return platelets to a less activated state by restoring the balance of pro- and
anti-thrombotic signaling through the PGE2/EP3 and PGI2/IP pathways, respectively. Therefore,
DG-041, a potent and selective EP3 antagonist, was developed as a novel antiplatelet agent
and is currently in Phase II clinical studies.
P413
EP3 Receptors: A New Target for Inhibition of Platelet Function in
Inflammatory Thrombotic Disease
Stan Heptinstall, Univ of Nottingham, Nottingham, United Kingdom; Bob Anders, Mark
Gurney, deCODE genetics, Inc, Woodridge, IL; David Hermann, Dan Hartman; deCODE
genetics, Inc, Brighton, MI
Background. Platelets have receptors for natural agents that mediate platelet activation
including TXA2 and ADP. Drugs that target these receptors (e.g. aspirin or clopidogrel) reduce
platelet activation and are useful antithrombotic agents. Platelets also have EP3 receptors for
PGE2, a naturally occurring prostanoid. Based on genetic findings we hypothesized that EP3
receptors may participate in the thrombotic process and that EP3 antagonists may be of value
as antithrombotic agents. Here we have examined the effects of the selective EP3 agonist
sulprostone on platelet function and the inhibitory effects of the EP3 antagonist DG-041.
Methods. Studies were performed in vitro in PRP or blood from human subjects. Platelet
functional studies were performed by aggregometry (light absorbance, platelet counting and
impedance [Multiplate]) and/or flow cytometry. Results. Sulprostone alone did not induce
platelet aggregation in PRP but enhanced aggregation induced by other agonists including
collagen, TRAP, PAF, U46619, serotonin and ADP. Its effect was concentration-dependent up
to 1M. With some agonists used at low concentrations (e.g. collagen and U46619)
aggregation increased from virtually zero up to an irreversible response. In blood sulprostone
alone caused a small aggregation response, and markedly enhanced the aggregation induced
by other agonists. Sulprostone (1M) enhanced P-selectin expression induced by various
agonists and increased platelet-monocyte and platelet-neutrophil conjugate formation. Sulpr-
ostone exerted its effects via enhanced Ca2 mobilization (measured using Fluo-3) and via
inhibition of adenylate cyclase (measured via VASP phosphorylation). DG-041 (used at
concentrations up to 3M) prevented the effects of sulprostone on platelet aggregation,
P-selectin expression, Ca2 mobilization and VASP phosphorylation. Sulprostone potentiated
platelet function even in the presence of aspirin (an inhibitor of TXA2 synthesis) and AR-C69931
(a P2Y12 antagonist) and DG-041 prevented this. Conclusions. EP3 receptors on platelets
potentiate platelet activation and platelet interaction with blood leukocytes. An EP3 antagonist
may be of therapeutic value in inflammatory thrombotic disease.
P414
Platelet Thromboxane Assay Is a Good Predictor of Aspirin Response
Andrew O Maree, Massachusetts General Hosp, Boston, MA; Patrick Dicker, The Royal
College of Surgeons in Ireland, Dublin, Ireland; Ronan J Curtin, Cleveland Clinic, Cleveland,
OH; Hani Jneid, Massachusetts General Hosp, Boston, MA; Peter Crean, Saint James Hosp,
Dublin, Ireland; Dermot Cox, The Royal College of Surgeons in Ireland, Dublin, Ireland; Igor
F Palacios, Kenneth A Rosenfield, Massachusetts General Hosp, Boston, MA; Desmond J
Fitzgerald; Univ College Dublin, Dublin, Ireland
Introduction: Persistent normal platelet function despite aspirin therapy, referred to as aspirin
resistance, has been associated with a threefold increased risk of major cardiovascular events.
Hypothesis: We hypothesised that newer point-of care aspirin resistance assays, such as the
platelet function analyzer (PFA) -100, may be a less sensitive and specific measure of
cycloxygenase pathway inhibition when compared with laboratory assays of aspirin response.
Methods: Patients (n199) with stable cardiovascular disease taking aspirin (75300mg) daily
were screened by (PFA) -100 (collagen/epinephrine cartridge). Twenty-five aspirin-resistant
and 25 matched aspirin-sensitive patients were recalled for further analysis. Assays of serum
thromboxane (TX) B
2
, platelet TXB
2
generation in response to exogenous arachidonic acid (AA)
(platelet TX), urinary TX, platelet aggregation to AA (1.6mM), epinephrine (5uM), collagen
(0.5ug/ml) and (TRAP) (5uM) were performed. Results: Fifteen percent of patients were
determined aspirin resistant, failing to prolong the PFA-100 closure times (193 seconds). The
PFA-100 correlated with weak but not strong platelet agonists or serum TX generation (Table
1). The platelet TX assay was the most sensitive measure of aggregation response to both weak
and strong platelet agonists. Conclusion: In a stable cardiovascular population, the PFA-100
point-of-care assay correlated with weak but not strong platelet agonists nor serum
thromboxane B
2
generation. Platelet thromboxane generation correlated closely with most
assays of platelet function and may represent a sensitive assay of platelet cyclooxygenase
inhibition and thus aspirin resistance.
TABLE 1. ASPIRIN ASSAY CORRELATION; RELATIVE RISK (95% CI); P-VALUE* FROM
FISHERS EXACT TEST.
PFA-100
(< 193 sec)
Serum TX
(>2.2ng/ml)
Platelet TX
(>90ng/ml)
PFA-100 (< 193 sec) 1.15 (0.41 to 3.24)
p1.000
2.57 (1.18 to 5.61)
p0.0853
Serum TX (>2.2ng/ml) 1.19 (0.34 to 4.17)
p1.000
- 12.00 (3.07 to 46.88)
p0.0001
Platelet TX (>90ng/ml) 3.69 (1.02 to 13.34)
p0.0853
8.86 (2.96 to 26.52)
P0.0001
-
Urinary TX ( >130
pg/mg )
3.00 (0.68 to 13.27)
P0.2404
1.40 (0.52 to 3.74)
P0.7360
2.39 (0.75 to 7.57)
p0.1560
AA Aggregation (1.6mM)
(>/20%)
3.94 (1.24 to 12.54)
p0.0282
6.94 (2.96 to 16.29)
P0.0001
32.00 (4.65 to 220.37)
p0.0001
Epinephrine
Aggregation
(5uM) (>/40%)
7.62 (1.76 to 32.89)
p0.0026
3.63 (1.58 to 8.35)
p0.0030
7.33 (2.40 to 22.39)
p0.0001
Collagen Aggregation
(0.5ug/ml) (>/20%)
4.5 (1.53 to 13.23)
p0.0212
1.88 (0.81 to 4.37)
P0.2240
3.47 (1.67 to 7.22)
p0.0070
TRAP Aggregation
(5uM) (>/70%)
1.61 (0.37 to 6.94)
P.7027
3.22 (0.82 to 12.60)
p0.0618
6.50 (0.94 to 45.11)
p0.0164
P415
Higher Platelet Activity Is Present in Patients with Restenosis After
Percutaneous Coronary Intervention but Not in Patients with an Occlusion
of Coronary Artery Bypass Graft
Pavel P Osmancik, Frantisek Bednar, Leona Pavkova, Petr Stros, Karel Jirasek, Petr
Widimsky; CardioCntr, 3rd Med Sch, Charles Univ in Prague, Prague, Czech Republic
Background: Platelet activity plays an important role in acute coronary syndromes as well as
in the progression of atherosclerosis. The aim of the study was to compare platelet activity in
patients with two different types of ischemic complications after coronary revascularization:
with an occlusion of bypass graft after coronary artery bypass grafting surgery (CABG) and with
a restenosis after percutaneous coronary intervention (PCI). Methods: Forty-five patients with
coronary artery disease were studied in a cross-sectional designed study. Fifteen of them were
patients with the worst bypass graft patency from Prague-4 study (control protocol-driven
coronary angiography was performed at one-year after surgery; originally 47 bypass grafts
implanted, 94% of venous grafts occluded), 15 patients were patients with the best graft
patency from Prague-4 study (51 grafts implanted, 100% patent). The resting 15 were patients
with restenosis 612 months after PCI. Blood samples were drawn at least 4 weeks after
coronary angiography, or 5 weeks after PCI if it was performed. No patients was at dual
antiplatelet treatment at the time of blood sampling. Platelet activity was determined by
membrane expression of platelet antigen CD62P (P-selectin, % of positive cells) by flow
cytometry, aggregability by ADP-aggregometry. Data are expressed as meanSEM. Results:
All three groups were similar with respect to age, body mass index and presence of diabetes
mellitus. No patient suffered from acute coronary syndrome. Membrane expression of CD62P
antigen was significantly higher in the patients with restenosis compared to patients with
occluded or patent bypass grafts (1.960.07 vs. 0.770.03 vs. 0.570.03, p0.001,
Kruskal-Wallis test). CD62P expression was not different between patients with occluded vs.
patent grafts. ADP-aggregometry was not different between groups (55.51.1 vs. 56.10.8
2007 ATVB Poster Presentations e-109
by on March 25, 2011 atvb.ahajournals.org Downloaded from
vs. 55.91.1). Conclusion: Platelet activity play more important role in the development of
restenosis after PCI compared with the occlusion of bypass graft after CABG, at least in the
period up to 1 year after revascularization. More-aggressive anti-thrombotic treatment should
be considered for patients with history of restenosis after PCI.
P416
Mechanism of Paradoxical Platelet Activation Induced by Blockers of
Platelet Integrin
IIb

3
(GPIIb/IIIa)
Nicole Bassler, Baker Heart Rsch Institute, Melbourne, Australia; Christoph Loeffler, Univ
Hosp, Freiburg, Germany; Pierre Mangin, Yuping Yuan, Monash Univ, Melbourne, Australia;
Meike Schwarz, Univ Hosp, Freiburg, Germany; Christoph E Hagemeyer, Steffen U
Eisenhardt, Ingo Ahrens, Baker Heart Rsch Institute, Melbourne, Australia; Christoph Bode,
Univ Hosp, Freiburg, Germany; Shaun P Jackson, Monash Univ, Melbourne, Australia;
Karlheinz Peter; Baker Heart Rsch Institute, Melbourne, Australia
Introduction:
IIb

3
blockers provide benefits when applied intravenously (although with
limitations) but failed as oral drugs. We developed a model describing the current concept of
ligand-mimetic integrin blockade as potential reason for paradoxical platelet activation.
Methods: Platelet activation was determined by flow cytometry, immunofluorescence micros-
copy and Ca
2
measurements. Results: As an experimental model, we show activation of
platelets in solution induced by ligand-mimetic
IIb

3
blockers consisting of 3 components: 1.
Pre-stimulation (ADP 1M), 2. Induction of ligand-bound conformation of
IIb

3
(binding of

IIb

3
blockers, RGD-peptides and anti-LIBS antibodies) and 3.
IIb

3
clustering (via antibodies).
Platelet adhesion on collagen represents an in vivo correlate of platelet pre-stimulation and
receptor clustering, in which the presence of ligand-mimetic
IIb

3
blockers results in platelet
activation as detected by P-selectin expression (mean fluorescence SEM: no addition
6.130.49 vs. eptifibatide 18.251.02, p0.001) CD63 and CD40L expression as well as by
measuring Ca
2
(Ca
2
[M] SEM: no addition 18.464.65 vs. eptifibatide 54.3010.62,
p0.05). This paradoxical platelet activation can be inhibited by ADP (P2Y12) receptor blockers
(e.g. clopidogrel). Conclusion: We describe a mechanism of
IIb

3
blocker-induced paradoxical
platelet activation, which may explain major limitations of
IIb

3
blockers. These findings
suggest co-medication with ADP-receptor blockers and moving beyond the initial approach of
ligand-mimetic blockade towards the development of allosteric or activation-specific integrin
blockade.
P417
Assays Evaluating Platelet Inhibition Provided by Clopidogrel Yield
Significantly Different Results and Correlate Poorly Among Themselves
Chantal Pharand, Marie Lordkipanidze , Thuy Anh Nguyen, Donald A Palisaitis, Hopital
Sacre -Coeur, Montreal, Canada; Jacques Turgeon, Universite de Montreal, Montreal,
Canada; Erick Schampaert, Jean G Diodati; Hopital Sacre -Coeur, Montreal, Canada
Background: The level of inhibition of platelet aggregation provided by clopidogrel is subject
to important inter-individual variations; however, it has been reported in various populations,
using different platelet function tests, thus making the results difficult to compare. Hypothesis:
We assessed the hypothesis that inhibition of platelet aggregation by clopidogrel is subject to
variability, with respect to the platelet function test used in its evaluation. Methods: One
hundred and twenty patients suffering from stable coronary artery disease were recruited prior
to diagnostic angiography. They received clopidogrel for 1 to 7 days before the procedure.
Blood samples were obtained before clopidogrel initiation and at the time of diagnostic coronary
angiography using the following platelet function tests: optical aggregometry (LTA; adenosine
diphosphate [ADP] 5 and 20 M as the agonist), electrical impedance in whole blood (WBI; ADP
5 and 20 M), drop in platelet count (ADP 5 and 20 M), PFA-100

(ADP cartridge) and


VerifyNow

P2Y
12
. Results: The correlation between different platelet function tests was
generally low. However, the same test performed at different concentrations of ADP yielded
highly correlated results. Conclusion: Platelet function tests are not equally effective in
measuring clopidogrels antiplatelet effect. When compared to the current gold standard
(LTA-ADP), aggregation in whole blood shows moderate correlation, while point-of-care assays
correlate poorly. Thus, platelet function assays should not be used interchangeably.
TABLE: PEARSONS CORRELATION COEFFICIENTS
TESTS
LTA,
ADP
20 M
WBI,
ADP
5 M
WBI,
ADP
20 M
Platelet count
drop, ADP
5 M
Platelet count
drop, ADP
20 M
PFA-100

ADP
Verify Now

P2Y
12
LTA,
ADP 5 M
0.94* 0.37* 0.47* 0.51* 0.53* -0.31 0.43
LTA, ADP 20 M 0.36* 0.48* 0.40 0.53* -0.30 0.37
WBI, ADP 5 M 0.92* -0.05 0.06 -0.29 0.16
WBI, ADP 20 M 0.11 0.06 -0.27 0.31
TESTS
LTA,
ADP
20 M
WBI,
ADP
5 M
WBI,
ADP
20 M
Platelet count
drop, ADP
5 M
Platelet count
drop, ADP
20 M
PFA-100

ADP
Verify Now

P2Y
12
Platelet count
drop,
ADP 5 M
0.69* -0.28 0.62
Platelet count
drop,
ADP 20 M
-0.12 0.55
PFA-100

-0.31
*: p0.05
P418
Prolonged Prophylactic Administration of High-dose Clopidogrel Before
Elective Percutaneous Coronary Intervention Is More Effective than the
Standard 300-mg Bolus in Inhibiting Platelet Aggregation
Chantal Pharand, Thuy Anh Nguyen, Marie Lordkipanidze , Donald A Palisaitis, Hopital
Sacre -Coeur, Montreal, Canada; Jacques Turgeon, Universite de Montreal, Montreal,
Canada; Erick Schampaert, Jean G Diodati; Hopital Sacre -Coeur, Montreal, Canada
Background : Effective platelet inhibition at the time of PCI reduces the risk of periprocedural
thrombosis. In patients with stable angina who undergo elective PCI, the issue of optimal
loading dose and time of clopidogrel administration remains controversial. Hypothesis : We
assessed the hypothesis that increasing the cumulative clopidogrel dose administered before
PCI would result in better inhibition of platelet aggregation. Method : One hundred and twenty
patients were prospectively randomized in a double-blind, placebo-controlled fashion into one
of 4 groups of clopidogrel dosing regimens 1 week prior to PCI (300 mg on the day prior to PCI;
600 mg on the day prior to PCI; 300 mg followed by 75 mg daily started one week before PCI;
and 300 mg followed by 150 mg daily started one week before PCI). Platelet function was
assessed at baseline, at the time of diagnostic coronary angiography, and 2 hours after stenting
by optical aggregometry (LTA) induced by 20 M of ADP. Results: All regimens significantly
reduced platelet aggregation at the time of angiography, as well as 2 hours following stenting
when compared to baseline (p 0.0001; Figure). The 300 mg bolus followed by 150 mg daily
showed the greatest inhibition of platelet aggregation, while a single 300 mg bolus resulted in
the least inhibition, an absolute difference of 30% at the time of angiography (p 0.007),
which increased to 36% 2 hours post-stenting (p 0.007) Conclusion: In choosing a
clopidogrel regimen, it is important to effectively block the surge in platelet activity induced by
PCI. The 300-mg bolus and 150-mg daily regimen seemed most effective in achieving and
maintaining such a level of platelet inhibition.
P419
Altered Reactive Oxygen Species Generation and Scavenging in Platelets
from Patients with Heart Failure
Ashish Shah, Eugenia Gkaliagkousi, Kings College London, London, United Kingdom; Julia
DeCourcey, Ruth Buckley, Kings College Hosp, London, United Kingdom; James Ritter,
Albert Ferro; Kings College London, London, United Kingdom
Heart failure (HF) is characterised by increased oxidative stress, which results in a reduction
in bioactive nitric oxide (NO) as well as increased platelet activation leading to increased
thrombotic events. Platelets have the capacity both to produce and to scavenge reactive oxygen
species (ROS). However, the production and scavenging of ROS by platelets in HF has not been
well characterised. We therefore sought to determine whether platelets from patients with HF
have higher ROS production and/or defective ROS scavenging capacity. Twenty five HF patients
(mean age 58.3 3.3 years; 20 male, 5 female; 7/9/4/5 patients in NYHA classes I/II/III/IV) and
19 healthy controls of similar age and sex distribution (mean age 57.1 2.9 years; 12 male,
7 female) were studied. ROS production was measured in gel-filtered platelets by pholasin
chemiluminescence, in the presence of horseradish peroxidase, both at baseline and for 30
minutes after stimulation with collagen (8 g/ml). Responses were expressed as arbitrary light
units/10
5
platelets. In other experiments, the decrease in light signal over 10 minutes was
measured following the addition of gel-filtered platelets to Tyrode solution, as a measure of
ROS-scavenging capacity, and this was expressed as percentage decrease in luminescence. All
data were expressed as mean SEM, and were analyzed by paired or unpaired Students t
test as appropriate, with p 0.05 (two tailed) taken as significant. Platelets from HF subjects
exhibited greater ROS production than did those from controls, both basally (13320 1665 vs.
8062 1293 light units/10
5
platelets respectively, p0.023) and after stimulation with
collagen (36020 9863 vs. 11120 2176 light units/10
5
platelets respectively, p0.036).
On the other hand, the reduction in pholasin luminescence by platelets was less for HF subjects
than for controls (65.9 4.8 vs. 88.2 2.3 % reduction in light signal respectively,
e-110 Arterioscler Thromb Vasc Biol June 2007
by on March 25, 2011 atvb.ahajournals.org Downloaded from
p0.0005). Our results suggest that, in HF, platelets produce more ROS both at baseline and
after collagen stimulation, and also exhibit an impaired capacity to scavenge ROS. These
factors may contribute to increased platelet activation, which in turn may contribute to the
increase in thrombotic events observed in this condition.
P420
An Amphiphilic Lipid Compound Is an Effective Biosealant Agent and
Bacterial Deterrent
George L Adams, Roberto J Manson, Luther Milton, Dana Giangiacomo, Jonathan S Ellison,
Jeffrey H Lawson; Duke Univ, Durham, NC
Introduction: More than 500,000 percutaneous interventions are performed in the United
States annually. Cardiologists and surgeons aim to maintain balance between hemostasis and
thrombosis to prevent adverse events. However, peripheral arterial access complications occur
in 1.5% to 9% of patients. Hypothesis: We assessed the hypothesis that a novel amphiphilic
lipid compound would: 1) achieve hemostasis more effectively than control when injected into
a swine liver biopsy tract and 2) inhibit common percutaneous procedure pathogens. Methods:
Glycerol mono-oleate (GMO) is a cubic phase-forming amphiphilic lipid, which when exposed
to excess aqueous fluid at physiologic temperature, undergoes a spontaneous phase transition
from a fluid-like lamellar phase to a cubic phase. Seven anticoagulated swine (ACT250)
underwent ten open liver biopsies with a 14-gauge needle; five injected with GMO (intervention)
and five injected with nothing (control). Thirty seconds, 2 minutes, 5 minutes and 10 minutes
after the procedure, bleeding was objectively graded; 0 no bleeding and 1 bleeding. Also,
GMO was injected into four plates containing culture media for Enteroccocis faecalis,
Staphylococcus aureus, Escherichia coli, and Klebsiella pneumoniae. When injected, GMO
converted to a cubic phase with definitive margins in the culture media. Each bacterium was
then coated over their respective media and GMO. Results: There was a significant (p0.017),
treatment effect on each success/failure bleeding outcome at 30 seconds (p0.0001), 2
minutes (p0.0001), and 5 minutes (p0.0038) based on a multiple logistic regression
analysis controlling for initial bleeding, pig, and liver biopsy site. At 10 minutes, the bleeding
results were not significant (p0.0917), likely explained by a pigs innate ability to clot at this
time period. For the bacteria experiment, there was no growth of bacteria on the GMO for any
of the plates. Specifically, the Staphylococcus aureus plate displayed a 200 micron halo
containing no bacterial growth surrounding the GMO. Conclusions: These results illustrate a
significant biosealant effect at multiple time points using GMO. Furthermore, GMO acts as a
bacterial deterrent especially with Staphylococcus aureus.
P421
Lack of von Willebrand Factor Release with Different Blood Flow Patterns
Generated During Rhythmic Lower Limb Exercise
Joaquin U Gonzales, Barry W Scheuermann; The Univ of Toledo, Toledo, OH
The potential for vascular endothelial cell injury caused by hemodynamic stress during physical
activity in humans is unknown, but may provide insight into the flow stimulus that improves
endothelial cell function following exercise training. The purpose of the present study was to
assess the hypothesis that different muscle contraction-produced shear forces could stimulate
the release of von Willebrand factor (vWF), a marker of endothelial cell injury. Methods: Eight
healthy young men [25.6 3.1 (SD) y] performed 20 min of single-leg knee extension exercise
at two contraction rates: fast (FR, 11% duty cycle) and slow (SR, 50% duty cycle). To control
for metabolic demand of blood flow, work rate was held constant (15.25 W) between FR and
SR. Common femoral artery blood flow was measured at rest and during knee extension
exercise using Doppler ultrasound. Rocket immuno-electrophoresis was used to measure
plasma levels of vWF from venous blood collected by venipuncture before, immediately after,
and 60 min following exercise. High-intensity cycling exercise was performed on a separate
day as a control condition since previous studies have shown post-exercise increases in vWF
with this mode of exercise. Results: The FR and SR exercise protocols produced significantly
different blood flow patterns with the FR resulting in a larger retrograde flow component (100
mlmin
-1
) than the SR which consisted solely of antegrade flow. As a result, the magnitude of
blood velocity oscillations experienced by the vascular endothelium was different between
contraction rates (FR: 154.5 34.1 vs. SR: 112.7 21.6 cms
-1
, P 0.05) with the average
calculated shear rate being greater for FR than SR (383.1 147.7 vs. 337.6 127.3 s
-1
, P
0.01). Since workrate was held constant, mean blood flow was similar between FR (1890.7
739.7 ml min
-1
) and SR (1734.4 789.1 ml min
-1
). Plasma levels of vWF were similar
between FR and SR at rest and failed to change following exercise. In contrast, vWF increased
by 16% (P 0.05) in plasma collected at 60 min following cycling. Conclusion: The different
levels of hemodynamic stress that accompany lower limb exercise, at least during moderate
intensity, do not induce endothelial cell injury as assessed by plasma levels of vWF.
P422
Physiological Testosterone Stimulates Tissue Factor Pathway Inhibitor
Expression in Human Umbilical Vein Endothelial Cells Via the Androgen
Receptor
Hong Jin, Yu-guang Li, Geng Peng, Dong-ming Wang; Shantou Univ Med college, Shantou,
China
Aims: The aim of this study was to evaluate the effect of testosterone with varied
concentrations on antigen and mRNA levels of tissue factor pathway inhibitor (TFPI) released
by human umbilical vein endothelial cell (HUVEC) and to investigate the mechanism of this
regulation. Methods: HUVEC within 23 passages were cultured in 96-well plates and 25 mm
flasks. The cells were incubated in the presence or absence of testosterone (3, 30, 3000,
30000 nmol/L) for 48 h. After the incubation TFPI levels of media were measured by IMUBIND
Total Elisa kit. And RT-PCR was carried out to compare each groups TFPI mRNA level. Then
experiments were repeated with HUVEC incubated in androgen receptor antagonist (flutamide
10 mol/L) for 3 h previously . Results: testosterone at physiologic concentrations (3,30
nmol/L) stimulated the secretion of TFPI significantly (P0.05). However, TFPI antigen and
mRNA levels were markedly reduced at a larger dose (30000nmol/L). Flutamide attenuated
testosterones effects (p0.05). Conclusion: Our results demonstrated that testosterone, at
physiological concentrations, has beneficial influence on hemostatic system by enhancing the
anticoagulant activity through stimulating the TFPI levels secreted by the endothelium, and that
the vascular androgen receptor is involved in the processes. Figure 1 Testosterones effect
on TFPI gene expression in absence or presence of flutamide( S, n4). (A) M, marker;
1, control; Lane 2, 3nM T; Lane 3, 30nM T; Lane 4, 3000nM T; Lane 5, 30000nM T; Lane 6,
F and 3nM T; Lane 7, F and 30nM T; Lane 8, F and 3000nM T; Lane 9, F and 30000nM T. (B)
(T: testosterone F: flutamide)
P423
ACE2 Confers Endothelial Protection and Attenuates Atherosclerosis
Fina Lovren, Yi Pan, Adrian Quan, Guilin Wang, Hwee Teoh, Subodh Verma; St. Michaels
Hosp, Toronto, Canada
One of the main effectors of endothelial dysfunction is angiotensin-II (AngII), and pharmaco-
logical approaches to limit AngII bioactivity remain the cornerstone of current cardiovascular
therapeutics. Recently, ACE2 has been identified as a critical negative modulator of AngII
bioactivity, which serves to counterbalance the effects of ACE in determining net tissue AngII
levels. Although ACE2 is highly expressed within the endothelium, the functional significance
of this is unknown. Using gain and loss of function strategies respectively, we systematically
evaluated the role of ACE2 in endothelial regulation and vascular inflammation. Furthermore,
since atherosclerosis represents a clinically relevant end-point of progressive endothelial
dysfunction we examined the potential of ACE2 to limit experimental atherosclerosis. Isolated
aortae from ACE2 deficient mice exhibited impaired endothelium-dependent relaxation.
Furthermore, over-expression of ACE2 in human endothelial cells promotes endothelial cell
migration and tube formation, and limited monocyte and cellular adhesion molecule expression;
effects that are reversed in ACE2 gene silenced cells. Endothelial cells isolated from ACE2
deficient mice exhibited impaired tube formation and monocyte adhesion. Importantly, ACE2
promoted capillary formation and neovessel maturation in-vivo and profoundly reduced
atherosclerosis in ApoEKO mice. Statins, an integral component of atherosclerosis treatment
increased endothelial ACE2 expression. These data indicate a critical role of ACE2 as a mediator
of endothelial homeostasis, a fundamental biological process, and suggest that ACE2-based
treatment approaches may limit aberrant vascular responses and atherothrombosis.
P425
Early Major Adverse Cardiac Events and Long-term Survival in Patients
Treated with Bare Metal Stents and Drug-eluting Stents
Muhammad S Munir, Amany H Ahmed, Craig M DeLaughter, KJ Shankar, Vei-Vei Lee,
MacArthur A Elayda, Texas Heart Institute, Houston, TX; S W Casscells, Univ of Texas
Health Science Cntr, Houston, TX; James M Wilson; Texas Heart Institute, Houston, TX
Background Randomized controlled trials have reported a similar procedural risk and a
reduced rate of restenosis with DES when compared with BMS. We sought to examine the
relative performance of BMS and DES in an unselected patient population referred for coronary
revascularization. Methods We examined in-hospital and long-term follow-up data from
unselected patients who underwent isolated primary revascularization by DES or BMS at our
institution. Two case-matched groups were created using the BMS and DES patients
propensity score. Early (30 day) MACE, including death, myocardial infarction, stroke and
mid-term survival, using the Kaplan-Meier method, are reported. Outcomes were compared,
using Chi-Square and Log-rank statistics. Results For 2101 patients receiving DES, a
propensity matched comparator was obtained from the BMS population. Three-year follow-up
data was obtained. Early MACE were more frequent after DES implantation (DES 3.71% vs BMS
2.05%; p0.01). This was attributable to more frequent peri-procedural myocardial infarction
in the DES group compared with the BMS group (DES 1.52% vs BMS 0.43%; p0.01). Despite
the difference in early risk, Kaplan-Meier estimated mortality after 3 years was not different
(DES 8% vs BMS 7.5%; pNS). Conclusion In this retrospective, propensity-matched patient
population, peri-procedural myocardial infarction was more frequent after DES implantation
when compared with BMS implantation. Long-term outcome was comparable in patients
receiving either BMS or DES. Given the relative lack of difference in early risk reported from
2007 ATVB Poster Presentations e-111
by on March 25, 2011 atvb.ahajournals.org Downloaded from
randomized controlled trials, this finding may reflect treatment of more complex lesions with
DES as opposed to BMS.
P426
Glutathione Peroxidase Activity in Cerebral Vasospasm After Subarachnoid
Hemorrhage
Gail J Pyne-Geithman, Danielle N Caudell, Matthew Loftspring, Joseph F Clark; Univ of
Cincinnati, Cincinnati, OH
An oxidizing environment has long been implicated in the etiology of cerebral vasospasm (CV)
after subarachnoid hemorrhage (SAH). This environment has been variously postulated to be
generated by immune, blood or brain cells. Using human CSF we have previously shown that
heme oxygenase 1 and lipid peroxide levels are increased with CV. Here, we directly correlate
CSF glutathione peroxidase (GPx) activity with occurrence of CV after SAH in humans. Human
CSF was obtained as part of standard of care in an IRB exempt manner from SAH patients with
or without CV, and from obstructive hydrocephalus patients (control CSF). A commercially
available GPx activity kit (ZeptoMetrix Corp.) was used to assess activity, monoclonal human
anti-GPx was obtained from AbCam for the western blot analysis and other chemicals were
obtained from Sigma. CSF from vasospastic patients (CSF
V
, n5), non-vasospastic patients
(CSF
C
, n7) and non-hemorrhagic patients (Control, n10) were subjected to the following
analyses: GPx activity, GPx protein levels, copper levels and iron levels. Hemoglobin, bilirubin
and lipid peroxidation levels had previously been determined for the same patient samples. GPx
activity levels (U/L) were 32 2.9, 98 9.1 and 341 29.7 for Control, CSF
C
and CSF
V
respectively. We then performed western blot analysis to determine whether the difference in
activity was due to increased levels of GPx protein. We found no significant difference in band
intensity between the three groups. Hemoglobin concentrations did not differ significantly
between hemorrhagic CSF groups. Both copper and iron can act as pseudoperoxidases
(non-enzymatic peroxidation). Copper(I) concentrations (expressed as g copper per g
hemoglobin) did not differ significantly between CSF
V
and CSF
C
(38.50 3.6 vs. 40.78
5.28). Similarly, Fe
3
concentrations (M) were not significantly different in both SAH CSF
groups (CSF
C
, 29.7 9; CSF
V
, 18 29.6). These results suggest an increase in GPx activity
that is indicative of CV after SAH. Further studies will reveal if this observation can be
predictive, or if GPx could be a viable therapeutic angle for this serious complication.
P427
Flow-mediated Changes in Pulse Wave Velocity in Patients with
Hypertension
Ghazanfar Qureshi, Gregory Jean-Noel, Louis Salciccioli, Jason Lazar; State Univ of New
York Downstate Med Cntr, Brooklyn, NY
Background: Flow mediated changes in brachial artery diameter are widely used to assess
endothelial dysfunction. Recently, a new measure of endothelial function was proposed using
hyperemic changes in pulse wave velocity (PWV). Objective: The objective of this study was
to compare changes in PWV upon hyperemia in healthy and hypertensive subjects (HTN).
Method: We measured flow-mediated changes in PWV in 16 healthy and 10 HTN subjects.
Baseline blood pressures (BP) and arterial stiffness (augmentation index-AI and PWV) by
applanation tonometry were measured. A right upper arm Hokensons cuff was inflated 50
mmHg above systolic BP for 5 minutes. Post 1 min deflation and at 2 min intervals, reactive
hyperemic response was measured by PWV. Results: Healthy subjects were younger (4017.3
vs 544 (p.015). BPs and HR were similar between the 2 groups. Baseline arterial stiffness
parameters including AI (22.1 17.4 % vs 28.3 10.4 %, p.27) and PWV (8.6 1.4 m/s vs
8.7 1.4 m/s, p.75) were similar between the 2 groups. In healthy subjects, flow-mediated
hyperemia decreased PWV at 1 minute relative to baseline by 21.8 % (8.61.4 m/s to
6.761.7, p.0001). There was no significant change in the PWV in patients with HTN
(8.71.4 to 8.91.1, p.64). Hyperemic PWV response differed between the healthy and HTN
groups (-21.8 % vs 2.6%, p.0001). HTN group showed a delayed hyperemic response
where PWV reduced by 10.8% relative to baseline from 8.71.4 to 7.51.5 m/s (p.15) at
3 min and more rapidly returned back to baseline at 7min post hyperemia (8.81.5 m/s). In
healthy subjects, PWV levels returned to baseline by 11 min (8.21.1). On multivariate
analysis, HTN was independently related to hyperemic PWV response (r
2
.49, p.002) when
age and HTN was selected in enter model. Conclusion: In HTN, hyperemia results in a decline
PWV at 1 min in healthy but not in HTN subjects. The temporal pattern of PWV changes also
differed. As compared to healthy subjects, the hypertensive group showed a delayed and
diminished decline in PWV upon hyperemia and more rapid recovery to baseline. Differential
responses might reflect endothelial dysfunction in HTN, which merits further study.
P428
Combining Paclitaxel and Sirolimus Favorably Improves Endothelial
Cytotoxicity and Platelet Proaggregatory Effect of Either Agent Alone
Chanwit Roongsritong, Sandra Rodriguez, Jan Simoni, Ashwani Kumar; Texas Tech Univ
HSC, Lubbock, TX
Drug eluting stents (DES) have over the past few years emerged as a mainstay of percutaneous
coronary intervention (PCI) due to its superior patency rate over bare metal stents. With
widespread utilization of DES, late thrombotic risk associated with these stents has increasingly
become a public concern. Paclitaxel and sirolimus are the two agents used in the currently
available DES in the US. Our previous research has shown significant differences between the
effects of paclitaxel and sirolimus on human coronary artery endothelial cells (HCAEC) and
human blood components. We found that while paclitaxel exerted toxic effect on HCAECs, its
systemic presence effectively blocked platelet aggregation. Conversely sirolimus was found to
have cytoprotective effect on endothelium but caused slight platelet activation. We hypothe-
sized that combining the two agents may lead to more desirable overall effects than either
agent alone for DES. Methods In this study the platelet anti- and pro-aggregatory effects were
tested using different dose combinations of both drugs. The cytotoxic effects were evaluated
using the RBC fragility model. Results We found that the platelet pro-aggregatory effect of
sirolimus can be attenuated with paclitaxel. Furthermore, the cytotoxic effect of paclitaxel was
diminished in the presence of sirolimus. The most desirable effect in terms of platelet
aggregation and endothelial cytotoxicity was observed at a 500 to 1 paclitaxel to sirolimus
molar ratio. Conclusion This study suggests that combination therapy could be effective in
avoiding certain pathological responses to paclitaxel or sirolimus alone. However, the effect of
paclitaxel and sirolimus combination on smooth muscle cell proliferation in comparison to
either agent alone is not yet known. Further study to explore this new concept is warranted.
P429
Heme Oxygenase-1: A Novel Key Player in the Development of Tolerance in
Response to Organic Nitrates
Philip Wenzel, II Medizinische Klinik, Johannes-Gutenberg-Univ Mainz, Mainz, Germany;
Matthias Oelze, Meike Coldewey, Labor f Molekulare Kardiologie, Mainz, Germany; Dirk
Stalleiken, Actavis Deutschland GmbH, Langenfeld, Germany; Thomas Munzel, II
Medizinische Klinik, Johannes-Gutenberg-Univ Mainz, Mainz, Germany; Andreas Daiber;
Labor f Molekulare Kardiologie, Mainz, Germany
Objective-Nitrate tolerance is likely due to an increased production of reactive oxygen species
(ROS) leading to an inhibition of the mitochondrial aldehyde dehydrogenase (ALDH-2),
representing the nitroglycerin (GTN) and pentaerythrityl tetranitrate (PETN) bioactivating
enzyme, and to impaired nitric oxide bioactivity and signaling. We tested whether differences
in heme oxygenase-1 (HO-1) induction might explain why PETN but not GTN therapy is devoid
of nitrate and cross-tolerance. Methods and Results-Wistar rats were treated with PETN or GTN
(10.5 or 6.6A

a A

g/kg/min for 4d). In contrast to GTN, PETN did not induce nitrate
tolerance or cross-tolerance as assessed by isometric tension recordings in isolated aortic
rings. Vascular protein and mRNA expression of HO-1 and ferritin were increased in response
to PETN but not GTN. In contrast to GTN therapy, NO signaling, ROS formation (as determined
by chemiluminescence) and the activity of ALDH-2 (as assessed by an HPLC based method)
were not significantly inhibited by PETN. Inhibition of HO-1 expression by apigenin induced
tolerance to PETN whereas HO-1 gene induction by hemin prevented tolerance in GTN treated
rats. Conclusions- HO-1 expression and activity appear to play a key role in the development
of nitrate tolerance and might represent an intrinsic antioxidative mechanism of therapeutic
interest.
P430
Adequately Treated Type 2 Diabetes Is Associated with Lower Wall Shear
Rate of the Common Carotid Artery
Eva Chytilova, Zdislava Kasalova, Jan Malik, Radka Dolezalova, Tomas Stulc, Richard Ceska;
General Faculty Hosp, Prague, Czech Republic
Introduction: Arterial sites with low wall shear stress (WSS) are more prone to the
development of atherosclerotic plaques, as was observed in carotid arteries in subjects with
atherosclerosis risk factors. Diabetes mellitus, a strong risk factor, could be modified by statins
and angiotensin-converting enzyme inhibitors (ACEI). The aim of our study was to discover if
type 2 diabetes mellitus (DM) subjects compensated by metformin, with established statin and
ACEI therapy, still have lower WSS in common carotid arteries than healthy controls. Methods:
We enrolled 26 compensated DM subjects aged 62 10 years, treated by metformin, statins
and ACEI for more than 6 months, and 16 age-matched healthy controls. Ultrasound
examination was targeted to distal 1 cm of common carotid arteries, where maximal and mean
velocities were measured. Internal diameter (ID) and intima-media thickness (IMT) were
analyzed off-line by a specialized software. Wall-shear rate (maximal and mean) was used as
a measure of WSS, calculated according to the following formula: WSR 4x velocity/ ID
Differences between groups were analyzed by unpaired t-test. Results: Diabetic subjects had
significantly lower WSR, because of both thinner lumen and slower blood flow velocities. Lower
WSR was accompanied by higher IMT. Conclusion Adequately treated subjects with compen-
sated DM still have atherogenic hemodynamic profile.
DM Controls p-value
Total cholesterol (mmol/l) 4.60.9 5.00.8 0.05
Triglycerides (mmol/l) 2.01.8 1.20.5 0.01
ID (mm) 6.80.9 5.70.5 0.00006
WSRmax(s
-1
) 40.011.2 51.69.9 0.002
WSRmean(s
-1
) 19.76.3 28.96.5 0.00002
P431
Predictive Equations for Estimation of Body Composition in Healthy Asian
Indians, with DEXA and MRI as Standard Methods
Nidhi Gupta, Kashish Goel, Maulana Azad Med College, New Delhi, India; Pawan Kumar, R
M Pandey, N K Vikram, All India Institute of Med Sciences, New Delhi, India; J S Wasir,
Anoop Misra; Fortis Hosp, New Delhi, India
INTRODUCTION: Estimation of percentage body fat (%BF), total abdominal fat (TAF), subcuta-
neous adipose tissue (SCAT), and intra-abdominal adipose tissue (IAAT) is important, especially
in an ethnic group like Asian Indians who are more prone to insulin resistance, the metabolic
syndrome and type 2 diabetes mellitus. However, it involves expensive methodology, which is
often not available in developing countries. HYPOTHESIS: Equations developed from simple
anthropometric measures would have a fair degree of accuracy in predicting %BF, TAF, SCAT
and IAAT. METHODS: Anthropometry (height, weight, multiple skinfold thickness, circumfer-
ences), %BF by DEXA scan, TAF, IAAT and SCAT by single slice MRI at L
34
level were
measured in 171 healthy subjects (95 males and 76 females). The mean age was 32.21
9.5 y, and mean BMI 22.95 kg/m
2
. Cross-sectional data analysis and multiple regression
equations were used on the training data set (70% sample) to develop predictive equations, by
taking combinations of demographic and anthropometric variables as potential predictors.
e-112 Arterioscler Thromb Vasc Biol June 2007
by on March 25, 2011 atvb.ahajournals.org Downloaded from
Prediction equations thus developed were applied on validation data set (30%). RESULTS: The
simplest equation for predicting %BF included age, gender, BMI, triceps skinfold and waist
circumference (R
2
84.4%). Replacing BMI with weight and height reduced the overall
variance (R
2
86.4%). BMI showed a strong correlation with all abdominal fat sub-
components, but it best correlated with SCAT(r 0.79). The most precise predictive equation
for estimation of IAAT included age, gender, BMI, hip circumference and waist circumference
(R
2
52.1%). Waist circumference was the strongest predictor of IAAT (r 0.69) and hip
circumference of SCAT(r 0.80). Irrespective of age and gender, BMI and hip circumference
explained 66.8% variability in SCAT. CONCLUSION: The following predictive equations would be
clinically relevant for adult Asian Indians: 1) %BF 42.4 0.003*age 7.04*gender -
0.22*weight - 0.42*height 0.42*triceps skinfold 0.29*waist circumference. 2) TAF -
45485 1627.4*BMI 367.8*waist circumference. 3) IAAT -238.7 16.9*age
934.2*gender 578.1*BMI 434.2*waist circumference - 441.1*hip circumference. 4)
SCAT - 51669.5 707.7*BMI 519.7*hip circumference.
P432
Diet-Induced Obesity in Male Apolipoprotein E-Deficient Mice Increases
Adipose Serum Amyloid A Expression and Atherosclerosis
Victoria L King, Maria deBeer, Frederick deBeer, Nicholas Hatch, Joanne Wrobleswski,
Nathan Whittaker, Lisa R Tannock; Univ of Kentucky, Lexington, KY
The current epidemic of obesity affecting Westernized nations is accompanied by an increase
in atherosclerotic diseases. The links between obesity and atherosclerosis include hyperlipid-
emia, diabetes and chronic inflammation, yet the mechanisms linking obesity to accelerated
atherosclerosis are not fully understood, in part due to a paucity of models of obesity
accelerated atherosclerosis. Similar to humans, apolipoprotein E deficient (apoE-/-) mice
spontaneously develop atherosclerosis over their lifetime. Therefore, we sought to determine
if diet induced obesity accelerated atherosclerosis in apoE-/- mice. Eight week old male
apoE-/- mice were fed diets containing 10 or 60% kcal from fat for 17 weeks. Mice fed the
high fat diet had increased body weight gain (3.3 fold, p0.001) and plasma total cholesterol
levels (17%, P0.05) compared to mice fed the low fat diet. Atherosclerosis was increased in
high fat fed mice compared to low fat fed mice (0.473 0.084 vs. 0.307 0.062 mm2,
P0.05). Furthermore, high fat fed mice had increased adipose tissue serum amyloid A (SAA)
expression, elevated systemic SAA concentrations (2.6 fold, P0.05). Moreover, SAA was
associated with VLDL, LDL and HDL in mice fed the high fat diet compared to those fed the low
fat diet, in which SAA was primarily associated with HDL. Thus diets enriched in saturated fat
induce obesity accelerated atherosclerosis in male apoE-/- mice, which may in part be
mediated via increased adipose SAA synthesis.
P433
Role of Postconditioned Medium from Glucose Primed Endothelial Cells on
SMCs
Thomas Ledet, Lars M Rasmussen, Univ of Aarhus, Aarhus C, Denmark; Sren S Srensen,
RigsHospet, Copenhagen, Denmark; Lise Wogensen; Univ of Aarhus, Aarhus C, Denmark
Objective: Glucose is considered to be implicated in the development of cardio-vascular
disease in diabetes. Changed interactions between endothelial and human arterial smooth
muscle cells (HuVSMCs) may instigate arterial disease. Consequently, adhesion, migration,
proliferation, expression of integrins and collagen types from HuVSMCs are studied after
treatment with post-conditioned medium (post-CM
Glucose
) from glucose primed HUVECs (human
umbilical vascular endothelial cells) in order to avoid direct effect of high glucose level and NO.
Methods: Post-CM
Glucose
from HUVECs is obtained in the subsequent 18 hours period with 5,5
mM glucose, after 18 hours primary growth at 16 mM. Integrins or collagens are identified by
immunoprecipitation, quantified with an ELISA, adhesion, migration or proliferation measured
on HuVSMCs. Results: Adhesion decreases to collagen I, III and IV, but increases to type VI after
post-CM
Glucose
treatment. Equimolar sorbitol has no effect. Antibody to
2
- or
v
/
3
- integrins
reduces adhesion to type I collagen. Post-CM
Glucose
decreases the expression of
2
or
v
/
3
-
integrin but elevates the release of collagen I, III, IV and VI, while direct addition exclude PDGF-
AA or BB chains, TGF-, bFGF or endothelin as candidates. Migration or proliferation of
HuVSMCs is unaffected after post-CM
Glucose
addition
.
Conclusion: Glucose stimulates HUVECs
to release substance(s), leading HuVSMCs to reduced integrin expression and decreased
adhesion to collagens. The increased collagen secretion may be a compensatory reaction to
reduced integrin expression. The secreted factor(s) do not belong to well-known cytokines. The
mechanisms imposed by glucose, may lead to diabetic macroangiopathy and subsequently
increased incidence of atherosclerosis.
P434
Are Fats Created Equal in Obese and Lean Mice?
Mengting Li, Henry Clay High Sch, Lexington, KY; Xiang-An Li, Gregory Graf, Eric J Smart;
Univ of Kentucky, Lexington, KY
INTRO: Obesity is defined as the excessive accumulation of lipids in adipocytes. These
lipid-filled cells were considered inert blocks until a discovery in 1995 revealed that they
contain metabolism-regulating proteins which have profound effects on the development of
diabetes. Fatty acids compose 85% of adipocytes (and triglycerides make up 98%), yet this
major component of adipocytes and its role in obesity is largely unexplored. AIM/HYPOTHESIS:
In this study, we assessed the hypothesis that a significant discrepancy exists between the fatty
acid composition of obese and lean mice. METHOD: We obtained epididymal fat from 10 adult
db/db mice and 10 control wildtype mice. We extracted fatty acids with chloroform and added
heptadecanoic acid as an internal standard. We then quantified fatty acid composition by gas
chromatography equipped with an omega wax 250 capillary column. RESULTS: We found that
levels of palmitic and stearic acid were significantly higher in the epididymal fat of obese mice,
while the amounts of linoleic acid were lower compared to that of lean mice (refer to table
below). CONCLUSION: In this study, we found that the adipose tissue of obese mice contains
significant more saturated fatty acids and less unsaturated fatty acids than that of lean mice.
PROSPECTIVE: The diverse types of fatty acids play important roles. Our study indicates a
possible connection between fatty acid functions and obesity.
Palmitic acid Stearic acid Linoleic acid
Obese 29.880.61% 6.210.65% 27.232.12%
Lean 22.450.81% 3.200.25% 38.230.96%
* % is out of total fatty acid composition
P435
Interferon Induces Insulin Resistance in Adipocytes via Induction of
Suppressors of Cytokine Signaling
Fiona C McGillicuddy, Elise H Chiquoine, Christine C Hinkle, Muredach P Reilly; Univ of
Pennsylvania, Philadelphia, PA
Introduction: Chronic inflammation is thought to be a key mediator of obesity-induced insulin
resistance. Macrophage infiltration of adipose, with increased secretion of TNFalpha and IL-6,
has been well documented. Recent reports suggest a shift toward TH1 lymphocyte phenotype
in obesity, characterized by increased secretion of IFNg. Little is known about the effects of
IFNg on insulin sensitivity. Hypothesis: We hypothesized that IFNg would induce insulin
resistance in adipocytes in part through induction of suppressor of cytokine signaling proteins
(SOCS). Methods: We compared the effects of IFNg to TNFa, a well established mediator of
insulin resistance, on insulin signaling in 3T3-L1 adipocytes. Results: Pre-incubation of
adipocytes for 4 h with both IFNg (-44.93 8.1 %, n4) and TNFa (-45.39 4.8 %, n4)
attenuated insulin-induced phosphorylation of Akt, a downstream marker of insulin sensitivity.
Furthermore, IFNg (-61.09 5.4 %, n4) and TNFa (-45.5 13.4 %, n4) also decreased
tyrosine phosphorylation of IRS-1. The effect of IFNg and TNFa on the expression of SOCS 1
and 3 was investigated as previous reports implicate SOCS in cytokine induced insulin
resistance. Both SOCS1 and 3 mRNAs were induced (9.9 2.0 and 4.4 1.4 fold
respectively, n4) after 4h treatment with IFNg, but not with TNFa (1.6 0.2 and 0.59 0.1
fold change, n4). In contrast, TNFa induced marked down-regulation of IR, IRS-1 and Glut4
mRNA levels (21 2.5 %, 9.8 1.11 % and 15 5 % of control, n4), whereas IFNg had
no effect on IR or Glut4 and had modest effects on IRS-1 mRNA (63 1.45 % of control, n4).
Finally we confirmed that transient overexpression of SOCS3 in 3T3L1s attenuated insulin-
induced phosphorylation of Akt to a similar extent as IFNg. Conclusion: We demonstrate for the
first time that IFNg, most likely via SOCS proteins, induces insulin resistance to a similar extent
as TNFa. However, IFNg and TNFa induce insulin resistance in adipocytes through differential
signaling mechanisms. These findings suggest a potential role for IFNg, a TH1 derived cytokine,
in adipose and systemic insulin resistance.
P436
Decreased Plasma Adiponectin Concentration in Major Depression
Leo Roberto, Di Lorenzo Giorgio, Tesauro Manfredi, Fortuna Enzo, Bianchi Francesco,
Razzini Cinzia, Siracusano Alberto, Lauro Renato, Romeo Francesco; Univ Rome Tor
Vergata, Rome, Italy
Objective: Adiponectin is the most abundant adipose-derived plasma protein. Recently
adiponectin levels have been linked to most variables of metabolic syndrome and conventional
risk factors for cardiovascular disease. However, its relation with major depression is yet
unclear. Therefore we examined plasma adiponectin levels in drug-nave first-episode MD
patients without conventionalrisk factors for cardiovascular disease to answer the following
questions: 1) are adiponectin levels different between MD patients and healthy subjects? 2) Is
there any relation between plasma adiponectin concentrations and MD? Method: We evaluated
plasma adiponectin levels in 32 first-episode drug-nave major depression (DSM-IV-TR)
patients without conventional cardiovascular risk factors and 32 matched healthy subjects.
Results: As expected, MD patients had significantly higher HAM-D score than controls
(t37.355, P0.001). ANOVA showed a strong diagnostic group effect: MD patients had
significantly lower adiponectin levels than controls (9.14.3 vs 13.85.4 g/ml, P0.001).
Moreover, there was a significant gender effect on the adiponectin levels: women had higher
levels than men both in control group (14.96.1 vs 11.75.1 g/ml, P0.01) and in MD
patients (9.74.1 vs 7.33.2 g/ml, P0.01). A significant negative correlation was found
between plasma adiponectin levels and HAM-D in MD group (r-0.757, P0.001). Conclu-
sions: The present study is the first showing decreased adiponectin plasma levels in these
patients, and relating hypoadiponectinemia to MD severity.
P437
Testing National Cholesterol Education Program-Adult Treatment Panel III
(NCEP-ATP III) Guidelines for Central Obesity in African Americans
Sabyasachi Sen, Carson C Chow, Madia Ricks, Nancy G Sebring, Barbara A Frempong,
Anne E Sumner; NIDDK, National Institutes of Health, Bethesda, MD
Due to the interrelationship between central obesity, insulin resistance, vascular disease and
diabetes, the definition of central obesity is important. NCEP-ATPIII has defined central obesity
in men and women as waist circumference (WC) 102 and 88 cms respectively. Validity of
these thresholds in African Americans is unknown. Our goal was to determine the WC which
best predicts insulin resistance, dyslipidemia and glucose intolerance in African Americans.
Subjects were 131 African Americans( 68M, 63W ) age 358y, BMI 30.97.5 range
18.554.7 kg/m
2
, WC 9818, range 69173cm. All subjects had oral glucose tolerance tests
and fasting lipid profiles. Insulin resistance was determined from the insulin sensitivity index
(S
I
) using minimal model analyses. Insulin resistance was defined as S
I
2.0.To determine the
WC cut-off which best predicts insulin resistance the following were calculated for 1cm
increments of WC from 80110cm: receiver operating characteristic (ROC) curves, sensitivity,
specificity, odds ratio (OR) and diagnostic accuracy. Diagnostic accuracy is percent of correct
2007 ATVB Poster Presentations e-113
by on March 25, 2011 atvb.ahajournals.org Downloaded from
decisions. For men, a single WC (102cm) had both the highest area under ROC curve
(0.760.06, 95%CI 0.640.88) and highest diagnostic accuracy,78% with sensitivity 73%,
specificity 80% and OR 10.46.6, 95%CI 3.136.1. For women, a single WC (99cm) had
both highest area under ROC curve (0.810.05, 95%CI 0.710.91) and highest diagnostic
accuracy,80% with sensitivity 86%, specificity 77% and OR 19.213.5, 95%CI 4.778.9. Lipid
and glucose parameters above and below WC cut offs of greatest risk are shown below. In
conclusion for African Americans the WC which best predicts insulin resistance, dyslipidemia
and glucose intolerance were WC102cm for men and WC99cm for women. Therefore in
African American women the WC which best identifies risks associated with central obesity
may be higher than is currently recommended by the NCEP-ATPIII guidelines.
LIPID AND GLUCOSE PARAMETERS ACCORDING TO THE WC OF GREATEST RISK (TOTAL
68M, 63W)
WC<102
(45 Men)
WC>102
(23 Men)
WC<99
(35 Women)
WC>99
(28 Women)
TG (mg/dL) 7738 9533* 4819 6734**
CHOL-(mg/dL) 16232 19444*** 16134 18036*
HDL(mg/dL) 4711 458 5511 519
HDL size(nm) 8.920.48 8.610.24** 9.360.35 9.150.42*
LDL (mg/dL) 9929 13041** 9729 11632*
LDL particle no. 1194420 1482503** 1139309 1342393*
Glucose Intolerant 12% 44%** 6% 43%***
*P0.05, **P0.01, ***P0.001
P438
Population-based Response Estimates for Extended-release
Niacin/Simvastatin Versus Other High-potency Dyslipidemia Therapy in
Patients with the Metabolic Syndrome
Eric J Stanek, Kos Pharmaceuticals, Cranbury, NJ; Ralph A Quimbo, Mark J Cziraky,
HealthCore, Inc, Wilmington, DE; Ron A Weathermon, Scott L Charland; Kos
Pharmaceuticals, Cranbury, NJ
Background: The atherogenic dyslipidemia of the metabolic syndrome (MetSyn) is character-
ized by low HDL-C, elevated triglycerides (TG), and elevated non-HDL-C. This dyslipidemia may
be treated with statins, niacin, or the combination of statin plus niacin. We compared the
effects of extended-release niacin/simvastatin (ERN/S) with other high-potency lipid altering
agents in a MetSyn population model. Methods: Patients were selected from a 2.1 million
record managed care plan database if they had a baseline lipid panel between 1/1/0012/
31/01, no concomitant lipid therapy, and continuous plan eligibility for 24 months. Patients with
MetSyn were identified by ICD-9 code (277.7) or by the presence of 3 or more clinical criteria
for the MetSyn. Percent achieving optimal values for LDL-C, HDL-C, TG, nonHDL-C, TG/HDL-C
and combined LDL-C, HDL-C, and TG were modeled from individual patient baseline values
using current product labeling (assuming additive effect for ERN/S). ERN/S 2g/40mg (ERN/S
2/40) was compared to, atorvastatin 80mg (A80), simvastatin 80mg (S80), simvastatin
80mg/ezetimibe 10mg (S/E 80/10), and rosuvastatin 40mg (R40). Results: We analyzed
23,773 MetSyn patients. Mean (SD) age was 6413 years; 12,236 (51%) female; 16,788
(71%) HTN; 5,650 diabetes mellitus (24%); and 11,191 (47%) CHD or risk equivalent. Modeled
treatment effects are provided in the table. Conclusions: In this MetSyn population model,
ERN/S achieved greater overall projected optimal lipid value achievement compared to other
high-potency lipid-altering therapy.
(N23,773)
% LDL-C,
100/130
mg/dL
% HDL-C
40 men;
50 women
% TG
150
% nonHDL-C
160/130 per
risk level
% TG/HDL-C
3.5
% LDL, HDL,
TG optimal
Baseline
a
35.2 28.5 34.1 28.5 36.2 4.0
ERN/S 2/40 87.4
b
100
b
62.9
b
91.2
b
90.6
b
55.2
b
A80 88.4 55.4 55.4 76.6 60.1 30.9
S80 80.8 71.7 48.5 72.0 59.1 31.1
S/E 80/10 88.4 61.1 52.3 75.9 59.1 32.1
R40 89.7 80.6 50.6 80.8 63.4 39.6
a
p0.05 all vs baseline;
b
p0.05 vs A,S, S/E, R;
P439
WITHDRAWN
P440
Testosterone-dependent Endothelial Dysfunction Following Insulin
Resistance Is Mediated by Regulation of 20-Hydroxyeicosatetranoic Acid
Synthesis
Harish Vasudevan, Jihong Jiang, John H McNeill; The Univ of British Columbia, Vancouver,
Canada
Endothelial dysfunction plays a key role in the development of hypertension following insulin
resistance (IR), a major cause for concern in todays society. In high fructose-fed rats, a model
of IR and hypertension, testosterone is essential for the development of endothelial dysfunction.
Testosterone-dependent regulation of Cytochrome P450 (Cyp) 4A controls the synthesis of the
arachidonic acid-derived vasoconstrictor 20-HETE. Cyp2C-catalyzed epoxyeicosatrienoic acids
(EETs) and nitric oxide (NO) are vasodilators that counteract the actions of 20-HETE. As EETs
and NO levels are decreased during IR, we suspected a role for testosterone-dependent
vasoactive pathways in impairing endothelial relaxation. We hypothesized that following insulin
resistance, testosterone-dependent alterations in 20-HETE synthesis contribute to the devel-
opment of endothelial dysfunction and hypertension. To test this hypothesis, we conducted 2
separate studies on male Wistar rats. In study 1, sham-operated or gonadectomized rats were
fed with 66% fructose for 9 weeks. Subsequently, the superior mesenteric arteries were
isolated and assessed for changes in endothelium-dependent relaxation to acetylcholine in the
presence or absence of 1-aminobenzotriazole (ABT), a Cyp4A blocker. In study 2, following 9
weeks of fructose feeding, rats were treated for 3 weeks with 25 mg/kg ABT (i.p.) or the
anti-androgen flutamide (8 mg/kg; s.c.). Blood pressure was measured prior to fructose feeding
and at the end of 9 weeks. We also measured plasma testosterone and the insulin sensitivity
index. Fructose induced insulin resistance but did not affect testosterone levels. In vitro, ABT
improved the relaxation to acetylcholine in the blood vessels of intact rats with IR. Both
gonadectomy and flutamide decreased blood pressure in IR rats. Treatment with ABT did not
affect insulin sensitivity but decreased the blood pressure. Further studies are in progress to
identify and quantify the specific Cyp4A and Cyp2C isoforms associated with the IR-induced
switch in vascular arachidonate metabolism. In conclusion, our studies point to the involvement
of androgen receptor-mediated increase in 20-HETE synthesis in the development of
endothelial dysfunction and subsequent hypertension.
P441
Group X Secretory Phospholipase A
2
Plays a Role in the Regulation of
Adrenal Glucocorticoid Synthesis
Preetha Shridas, Nancy R Webb; Univ of Kentucky, Lexington, KY
Numerous in vitro studies have implicated Group X secretory phospholipase A
2
(GX sPLA
2
) in
important biological processes. The enzyme potently hydrolyzes cell membranes to release
bioactive lipid mediators, including free fatty acids and lysophospholipids. Hydrolysis of low
density lipoprotein (LDL) and high density lipoprotein (HDL) by GX sPLA
2
has been speculated
to lead to the production of pro-atherogenic particles. In addition, the enzyme is also a high
affinity ligand for the M-type sPLA
2
receptor. To elucidate the physiological function of this
enzyme in vivo, we recently developed C57BL/6 mice with targeted deletion of the GX sPLA
2
gene. Mice deficient in GX sPLA
2
have approximately 50% higher plasma corticosterone
concentration compared to wild type mice. Analysis by real-time RT-PCR show that expression
of Steroidogenic Acute Regulatory Protein (StAR), a rate limiting protein in corticosteroid
production, is approximately 25-fold higher in adrenals of GX KO mice compared to wild-type
(WT) mice. Our data that GX sPLA
2
and M-type receptor mRNAs are both expressed in mouse
adrenals suggest that GX sPLA
2
may directly regulate adrenal corticosteroid production. To
investigate this possibility, we overexpressed the enzyme in mouse adrenocortical tumor (Y-1)
cells, which endogenously express Group X sPLA
2
and the M-type receptor. Consistent with our
findings in GX sPLA
2
-deficient mice, progesterone production by Y-1 cells stably transfected
with GX sPLA
2
cDNA is 3040% lower compared to control Y-1 cells. StAR expression is also
suppressed in Y-1 cells as a result of GX sPLA
2
over-expression. Treatment of Y-1 cells with
Indoxam, an inhibitor of sPLA
2
activity as well as a potent blocker of sPLA
2
binding to the
M-type receptor, results in the restoration of progesterone production to normal levels. In
conclusion, these in vivo and in vitro results indicate that GX sPLA
2
has a direct effect on
adrenal cells to regulate glucocorticoid production, possibly by regulating the expression of
StAR. The mechanism of how these factors are regulated by GX sPLA
2
is currently under
investigation.
P442
Apolipoprotein A-IDependent Cholesterol Efflux Is the Predominant
Pathway of Cholesterol Export from Macrophages in Vitro and in Vivo
Nigora Mukhamedova, Wilissa DSouza, Genevieve Escher, Zigmundt Krozowski, Baker Heart
Rsch Institute, Melbourne, Australia; Dmitri Sviridov; Baker Heart Rsch Inst, Melbourne,
Australia
Objective: The objectives of this study were to establish contribution of various pathways of
cholesterol efflux to the efflux to apolipoprotein A-I (apoA-I) and HDL and to establish
contribution of these pathways to cholesterol export from macrophages in vitro and in vivo.
e-114 Arterioscler Thromb Vasc Biol June 2007
by on March 25, 2011 atvb.ahajournals.org Downloaded from
Results: Eight genes responsible for rate-limiting steps of various pathways of cholesterol
efflux were overexpressed alone or in combination with each other in RAW 264.7 macrophages
followed by assessment of cholesterol efflux. When apoA-I was used as an acceptor,
overexpression of the combination of ABCA1, CYP27A1, PLTP and SR-B1 (Combination I) was
the most active enhancing the efflux by up to 8 times. This combination caused 2-fold increase
of the efflux to plasma, but no increase of the efflux to isolated HDL. When HDL was used as
an acceptor, overexpression of combination of caveolin-1 and SR-B1 (Combination II) was the
most active doubling the efflux to HDL, but not affecting the efflux to apoA-I. ABCA1, ABCG1,
caveolin-1 and two combinations were then tested in in vivo model of cholesterol efflux. Mouse
macrophages overexpressing these genes were cholesterol-loaded and labeled and trans-
planted intraperitoneally into mice. Appearance of labeled cholesterol in blood, liver, fecal bile
acids and sterols was measured after 24 h. Combination I and to a lesser extend ABCA1 alone
caused elevation of cholesterol export from macrophages to plasma, liver and feces. ABCG1,
caveolin-1 and combination II did not affect export of cholesterol from the macrophages in vivo.
Conclusions: Pathways of cholesterol efflux to apoA-I work synergistically and represent the
predominant pathway of cholesterol export from macrophages in vitro and in vivo.
P443
LCAT Deficiency Accelerates Cholesterol Accumulation of Liver in ABCA1
Null Mice
M Anwar Hossain, Nobukatsu Akita, Fumihiko Kobayashi, Shinji Yokoyama, Maki Tsujita;
Nagoya City Univ, Nagoya, Japan
Aim: To reveal the cholesterol homeostasis on hypoalphacholesteroamia in mice, both 1) the
diffusion mediated efflux pathway which accelerate by LCAT reaction and 2) apoAI/ABCA1
mediated cellular cholesterol releasing pathway were abolished in mutant mice. Method: Six
genotypes, wild, LCAT(), LCAT(-), ABCA1(-), LCAT()/ABCA1(-) and LCAT(-)/ABCA1(-), of 22
week-old mice were examined. Euthanatized mouse were perfused by PBS with subsequent
fixation by 4% formaldehyde. Cholesterol content of each tissue was measured by enzymatic
color detection method (Kyowa Med.). Results: Tissue TC of liver (43.38.7 g/mg) from male
mice were increased in LCAT(-) (134.7710.4 g/mg), ABCA1(-) (79.87.44 g/mg) and in
LCAT(-)/ABCA1(-) (197.316.34 g/mg). Liver TC from female mice (44.576.6 g/mg) were
also increased in LCAT(-) (60.125.5 g/mg), ABCA1(-) (62.5824.8 g/mg) and in
LCAT(-)/ABCA1(-) (80.315.4g/mg). On the other hand, CE content in steroidogenic tissue
was decreased in LCAT(-)/ABCA1(-). TC level of spleen and brain were also significantly
decreased in all of ABCA1(-) back ground mice. No significant differences of TC contents were
observed in kidney, thymus, heart, and lungs. Conclusion: These results demonstrate that
ABCA1 null mice show high cholesterol content in their liver and it was accelerated in
ABC/LCAT double deficient mice. This result indicates that one of cellular non-specific pathway
was also highly involved to maintained cholesterol homeostasis in liver. Involvement of ABCG1
in this phenomenon will also be examined.
P444
Triglyceride Alters Cholesterol Metabolism and ER-Stress Pathways in
Cholesterol EsterLaden Macrophage Foam Cells
Jody C Ullery, Jerod S Denton, Brian E Cox, W G Jerome; Vanderbilt Univ Sch of Medicine,
Nashville, TN
Macrophage foam cells are prominent in atherosclerotic lesions. In late stage disease, much
of the cholesterol accumulation in these foam cells is found in large, swollen, lysosomes.
Tissue culture models using human macrophages incubated with various modified LDLs
indicate that cholesterol accumulation within lysosomes can disrupt lysosome function leading
to foam cells with significant lysosomal free and esterified cholesterol, similar to cells found in
atherosclerotic lesions. The cholesterol is trapped and not accessible for efflux, even in the
presence of strong efflux promoters. In the artery wall, however, the foam cells are bathed not
only in modified LDLs but other lipid particles as well, including triglyceride-rich particles (TRP),
such as VLDL. Little is known about how metabolism of these TRP might affect cholesterol
metabolism and, specifically, the formation of cholesterol-rich macrophage foam cells. Our
studies explore the effect of TRP on intracellular cholesterol metabolism. Results show that
triglyceride (TG), delivered to the cell as a component of VLDL or TG-rich lipid dispersions,
reduces cholesteryl ester (CE) accumulation by 50% in THP-1 macrophage foam cells. Reduced
CE accumulation occurs in response to increased TG levels within the cell particularly within
lysosomes. TG, delivered to the cell as a component of TRP, decreases the volume of
lysosomes providing further evidence of increased lysosomal cholesterol clearance. Cholesterol
accumulation in lysosomes inhibits acidification of lysosomes but lysosomal TG reduced this
inhibition and maintained lysosome activity. The maintenance of an acidic lysosomal
environment would enhance the degradation and clearance of internalized CE, facilitating the
movement of cholesterol out of the lysosome, to sites where it can be utilized for cholesterol
efflux. However, the presence of excess TG within the macrophage also activates ER-stress
proteins, such as CHOP and Grp78, and induces the phosphorylation of eIF2A, indicating the
activation of the unfolded protein response. Our results show that excess TG in CE-laden foam
cells has multiple effects, the balance of which would influence the atherogenic potential of the
foam cell.
P445
The Absence of abcg1 in Alveolar Macrophages Triggers Pulmonary
Inflammation
Allison J Wojcik, Suseela Srinivasan, Borna Mehrad, Catherine C Hedrick; Univ of Virginia,
Charlottesville, VA
The ATP-binding cassette transporter G1 (abcg1) effluxes cholesterol from macrophages and
plays an important role in pulmonary lipid homeostasis. Deletion of abcg1 in mice results in
pulmonary lipidosis, consisting of accumulation of lipid-filled type 2 pneumocytes containing
altered surfactant composition, and changes in lipid metabolism genes. We hypothesize that
alveolar and monocyte-macrophages contribute to pulmonary lipidosis in these mice by
triggering inflammation in the lung. To study the early development of pulmonary lipidosis in
abcg1-/- mice, alveolar and monocyte-macrophages and dendritic cells were isolated from the
lungs of 10-week old mice by bronchoalveolar lavage (BAL). BAL was used for RNA expression
analysis, cytokine bio-plex arrays or flow cytometry. Alveolar macrophages isolated from
abcg1-/- mice were lipid-filled, and larger in size. Furthermore, abcg1-/- alveolar macrophages
showed 5-fold or greater increased expression of proinflammatory cytokines MIP-2, IL-6, and
IL-1beta compared to wildtype mice. Flow cytometry analysis of abcg1-/- BAL lymphocytes
showed a 10-fold increase (2,725 abcg1-/- monocyte-macrophages, 265 wt) in the number of
newly recruited monocyte-macrophages (CD11c-low CD11b-high MHCII-low) compared to wild
type prior to phenotypic onset of lipidosis. When abcg1-/- mice received an intraperitoneal
injection of 2mg/kg of LPS, macrophages isolated in BAL after 4 hours showed increased
expression of proinflammatory cytokines TNFalpha, IL-6, KC and increased expression of
COX-2. Cytokine secretion into BAL fluid was measured using a Bio-plex suspension array and
abcg1-/- mice produced at least twice as much TNFalpha, IL-6, MIP-1alpha, IL-12(p40) and KC
compared to wild type. These data suggest that alveolar macrophages contribute to the
pulmonary disease of abcg1-/- mice by secreting more proinflammatory cytokines and
recruiting monocyte-macrophages to the inflamed lung in the very early stages of lipidosis.
Thus, we provide a novel link between macrophage abcg1 function and regulation of
inflammation. Understanding the role of abcg1 in alveolar macrophages and its link to
inflammation will aid in developing therapies for various pulmonary diseases and other
inflammatory diseases.
P446
Proteomic Profiling of Prechylomicron Transport Vesicles Involved in
Assembly and Secretion of ApoB48-containing Chylomicrons by the
Intestine
Diana M Wong, The Hosp for Sick Children, Toronto, Canada; Joseph Macri, Hamilton
Regional Hosp, Hamilton, Canada; Elaine Xu, Mark Naples, Khosrow Adeli; The Hosp for
Sick Children, Toronto, Canada
Intestinal lipoprotein overproduction, caused by an overproduction of apolipoprotein B
(apoB)-48-containing lipoproteins in the small intestine, is increasingly recognized as an
important feature of dyslipidemia of insulin resistant states. Chylomicrons are transported from
the endoplasmic reticulum (ER) to the Golgi using a specialized compartment called the
prechylomicron transport vesicle (PCTV). In normal hamsters, the small intestine constitutively
secretes small lipoprotein particles containing apoB-48 to prime for fat ingestion. However, in
fructose-fed hamsters, lipoproteins are overproduced. Fructose-feeding induces the assembly
of intestinal lipoproteins which may change the formation of PCTVs. In this study, we report
proteomic profiles of PCTVs isolated from the enteric ER of normal, chow-fed hamsters, as well
as the fructose-fed hamster; an established model of diet-induced insulin resistance. PCTVs
were examined under both fasting and postprandial (fat load) conditions. Using tandem mass
spectrometry and 2D gels, we have characterized the PCTV and developed proteomic profiles
of PCTV-associated proteins from insulin-resistant and control enterocytes, with the intention
of identifying proteins involved in insulin signaling attenuation and lipoprotein overproduction.
A number of PCTV-associated proteins were found to be differentially expressed including
factors involved in lipoprotein assembly, namely microsomal triglyceride transfer protein (MTP)
and apoB-48, as well as proteins involved in vesicular transport, such as Sar1 GTPase and
VAMP7. Enhanced expression of these markers was observed in the fructose-fed/insulin
resistant state as well as following an oral fat load. Detailed proteomic profiles of intestinal
PCTVs under various dietary conditions will be presented. We postulate that upregulated
expression of Sar1 GTPase and MTP in the fructose-fed hamster intestinal enterocytes may
contribute to increased PCTV formation and chylomicron assembly. These findings have
increased our understanding of the intracellular assembly and transport of nascent chylomi-
crons and potential cellular factors responsible for lipoprotein overproduction in insulin-
resistant states.
P447
PCSK9 and LDLR Trafficking in Hepatocytes
Da-wei Zhang, Rita Garuti, Tom Lagace, Jay Horton, Jonathan C Cohen, Helen H Hobbs; UT
Southwestern Med Cntr, Dallas, TX
Genetic variation in the proprotein convertase PCSK9 contributes to inter-individual variation of
plasma levels of LDL-cholesterol (LDL-C). Gain-of-function mutations in PCSK9 are associated
with elevated levels of LDL-C, whereas loss-of-function mutations gene cause hypocholester-
olemia. PCSK9 affects LDL-C level by altering the expression levels of LDL receptors (LDLR)
post-translationally. The mechanism by which expression of PCSK9 modifies LDLR number is
not known. Lagace et al. (J Clin Invest 116:2995) have demonstrated that addition of PCSK9
to cells results in a decrease in LDLRs. To determine if PCSK9 acts at the cell surface, we
examined the effects of PCSK9 on LDLR levels in primary hepatocytes from wild-type mice and
from mice lacking ARH, an endocytic adaptor protein required for internalization of the LDLR.
In primary hepatocytes from wild-type mice, treatment with recombinant PCSK9 markedly
reduced cellular LDLR levels within 4 h. In contrast, no reduction in LDLR levels was observed
in hepatocytes from mice that expressed no ARH, an adaptor protein required for LDLR
internalization. To determine the fate of the LDLR after internalization, we performed
immunofluorescence confocal microscopy at timed intervals after addition of PCSK9 to examine
the distribution of LDLR in WIF-B cells. Prior to the addition of PCSK9, the LDLR was located
predominantly on the cell surface. Within 1 h of addition of PCSK9 to the media, the
immunodetectable LDLR co-localized with an early endosome protein (early endosome antigen
1), followed by co-localization with a late endosomal (endolin 78) and lysosomal protein
(Cathepsin D). Western blotting confirmed that cellular levels of LDLR were markedly reduced
at 4 h in the PCSK9 treated cells. These studies are consistent with LDLR internalization being
required for PCSK9 action and that PCSK9 promotes rerouting of the LDLR from a recycling
pathway to a degradative pathway.
2007 ATVB Poster Presentations e-115
by on March 25, 2011 atvb.ahajournals.org Downloaded from
P448
Whole Genome Expression Profiling Reveals a Significant Role for Immune
Function in Human Abdominal Aortic Aneurysms
Guy M Lenk, Gerard Tromp, Shantel Weinsheimer, Wayne State Univ, Detroit, MI; Zoran
Gatalica, Creighton Univ, Omaha, NE; Ramon Berguer, Univ of Michigan, Ann Arbor, MI;
Helena Kuivaniemi; Wayne State Univ, Detroit, MI
Abdominal aortic aneurysm (AAA) is a common disorder of the aged population, with
approximately 10% of those 65 years or older harboring an AAA. Most AAAs are asymptomatic
until rupture, that often leads to a sudden death. Global gene expression profiling of aneurysmal
(n7) and control abdominal aorta (n7) was carried out using two distinct microarray
platforms; Illumina Sentrix-6 and Affymetrix U133plus2.0. The analysis of the data included
differential expression and a determination of enrichment in the Kyoto Encyclopedia of Genes
and Genomes (KEGG) biological pathways as well as Gene Ontology (GO) categories to provide
information about possible functional changes in AAA tissue samples. After a correction for
multiple testing using False Discovery Rate (FDR), there were 3,274 distinct genes that had an
FDR 0.05. The 10 most enriched KEGG pathways included six immunity-related pathways
including the natural killer cell mediated cytotoxicity (hsa04650; p-value: 3.60e-07) and
leukocyte transendothelial migration (hsa04670; p-value: 8.06e-07) pathways, and four that
were general biological pathways. The top 10 GO categories included six immune-related and
four general cell signaling categories. Taken together, the gene expression patterns strongly
indicate the involvement of the immune system in AAA. A more detailed follow up of the
changes in gene expression in the two most enriched KEGG pathways implicates these
immune-related biological processes in the pathogenesis of AAA.
P449
A Polymorphism in the Protease-like Domain of Apolipoprotein(A) Is
Associated with Severe Coronary Artery Disease
May M Luke, Celera, Alameda, CA; John P Kane, Univ of California, San Francisco, San
Francisco, CA; Dongming M Liu, Charles M Rowland, Dov Shiffman, Celera, Alameda, CA;
June Cassano, Cleveland Clinic Foundation, Cleveland, OH; Joseph J Catanese, Celera,
Alameda, CA; Clive R Pullinger, Univ of California, San Francisco, San Francisco, CA; Diane
U Leong, Andre R Arellano, Carmen H Tong, Celera, Alameda, CA; Irina Movsesyan,
Josephina Naya-Vigne, Univ of California, San Francisco, San Francisco, CA; Curtis
Noordhof, Nicole T Feric, Queens Univ, Kingston, Canada; Mary J Malloy, Univ of California,
San Francisco, San Francisco, CA; Eric J Topol, Cleveland Clinic Foundation, Cleveland, OH;
Marlys L Koschinsky, Queens Univ, Kingston, Canada; James J Devlin, Celera, Alameda,
CA; Stephen G Ellis; Cleveland Clinic Foundation, Cleveland, OH
Genetic variants reproducibly associated with severe coronary artery disease (CAD) could
improve risk stratification and shed light on disease mechanism. In case-control studies of
white subjects whose severity of CAD had been assessed by angiography, we previously tested
12,077 single nucleotide polymorphisms (SNPs) and found 5 SNPs that were nominally
associated with severe CAD in 2 studies: Study-1 (781 cases, 603 controls) and Study-2 (471
cases, 298 controls). We have now tested the hypotheses that the risk alleles of these 5 SNPs
may be associated with severe CAD in a third study (554 cases, 373 controls). We found that
the risk allele of one of these 5 SNPs, LPA Ile4399Met (rs3798220), was associated with severe
CAD. LPA encodes apolipoprotein(a), a component of lipoprotein(a). Ile4399Met is located in the
protease-like domain of apolipoprotein(a). Compared with noncarriers, carriers of the 4399Met
risk allele (2.7% of controls) had an adjusted odds ratio for severe CAD of 3.14 (P 0.005).
This association remained significant after correcting for multiple testing. Carriers had higher
plasma lipoprotein(a) levels (P 0.003) and smaller apolipoprotein(a) isoforms (P 0.001).
After adjusting for apolipoprotein(a) size, the association of LPA Ile4399Met with severe CAD
and lipoprotein(a) levels remained significant. In conclusion, the LPA 4399Met risk allele is
associated with elevated lipoprotein(a) levels and increased risk of severe CAD.
P450
Temporal Gene Expression of Prosthetic Graft Neointima Isolated by Laser
Capture Microdissection
Junaid Y Malek, Thomas S Monahan, Nicholas D Andersen, Sheng-Qian Wu, Sowmya
Senani, Mauricio A Contreras, Frank W LoGerfo; Beth Israel Deaconess Med Cntr, Boston,
MA
Background: Vessel injury incurred during prosthetic arterial grafting triggers the formation of
anastomotic intimal hyperplasia (AIH), which limits the patency of all prosthetic vascular grafts.
Previous investigations using microarray analysis have been limited to the study of whole vein
homogenate including adventitia, media and neointima. The present investigation uses laser
capture microdissection (LCM) to collect only neointimal tissue from the distal anastomosis of
prosthetic arterial grafts for temporal microarray analysis. Methods: Bilateral expanded
polytetrafluoroethylene (ePTFE) carotid interposition grafts (n15) were implanted into dogs.
Distal anastomotic segments were harvested at 1, 7, 14, 30, or 60 days (n3 per time point).
Unaltered proximal common carotid artery served as control. Total RNA was isolated from
LCM-harvested anastomotic neointima and control carotid artery media. RNA was probed with
second generation canine gene chips to analyze differential gene expression between
neointimal and control medial tissue at each time point. MAS 5.0 statistical analysis was used
to identify gene expression changes of 2-fold or greater in all 3 samples per time point. Results:
Two-hundred forty-two genes were found to be up-regulated and 446 genes were found to be
down-regulated at various time points, including many novel genes not previously implicated
in AIH. Eighteen genes were found to be up-regulated at more than one time interval. These
include collagen type 1 alpha-1, cathepsin S, vascular cell adhesion molecule-1, and
thrombospondin-2. Seventeen genes were found to be down-regulated at more than one time
interval. These include insulin-like growth factor 2 receptor and mitochondrial ribosomal
protein S30. Conclusions: LCM can be used to harvest specific cell populations, in this case the
neointima, for microarray analysis to define gene expression changes induced by pros-thetic
arterial grafting. Continued analysis of identified gene expression patterns will provide insight
into the cellular pathways responsible for the formation of AIH and aid in the identification of
targets for therapy.
P453
Variation at the PCSK9 and LDLR Locus, LDL Levels, and Heart Disease in
PROSPER
Eliana Polisecki, Tufts Univ, Boston, MA; Hind Muallem, Nobuyo Maeda, Univ of North
Carolina, Chapel Hill, NC; Stella Trompet, Wouter Jukema, Leiden Univ Med Cntr, Leiden,
Netherlands Antilles; Alex McMahon, Michele Robetson, Ian Ford, Univ of Glasgow, Glasgow,
United Kingdom; Gerard Blauw, Leiden Univ Med Cntr, Leiden, Netherlands Antilles; Michael
Murphy, Univ College, Cork, Ireland; James Shepherd, Univ of Glasgow, Glasgow, United
Kingdom; Ernst Schaefer; Tufts Univ, Boston, MA
Genetic variation at the low density lipoprotein (LDL) receptor (LDLR) and the proprotein
convertase subtilisin/kexin type 9 serine protease (PCSK9) genes loci have been reported to
affect LDL cholesterol (C) levels and coronary heart disease (CHD) risk. Both PCSK9 and LDLR
gene products are regulated by intracellular cholesterol levels and affect LDL clearance. In
order to analyse the association between variation at these gene loci, LDL C levels, and CHD,
we examined DNA from the PROSPER study, 5804 men and women (mean age 75 years) of
whom 43% had vascular disease at baseline, and were randomized to placebo or pravastatin
40 mg/day and followed for a mean of 3.2 years. We have evaluated 2 functional single
nucleotide polymorphisms (SNPs) at the PCSK9 locus (rs708272 (R46L) and rs505151 (E607G))
and 2 at the 3 UTR LDLR locus (rs1433099 (U3 CT) and rs2738466 (U3 AG). The rare
allele at the PCSK9 R46L SNP had a prevalence of 3.7% in this population with mean 8% lower
LDL C values, no significant relationship to LDL lowering response or presence of vascular
disease at baseline, and no relationship to the development of CHD on trial. The E607G rare
allele at PCSK9 was present in 6% of the population, with no association with LDL levels or
CHD. At the LDLR locus we noted allele frequencies of 0.26 and 0.24 for the U3 CT and
U3 AG respectively. No significant lipid effects in carriers of the U3 CT, but significant
protection from CHD on trial in all subjects, in men, and especially in men on pravastatin (HR
0.67, p0.016) was noted. For the U3 AG variant there was a mean 8% reduction in LDL
C values in all subjects at baseline; however there was an increased risk of developing a CHD
endpoint on treatment (HR 1.5, p0.003). Conclusion: The PCSK9 R46L and the LDLR
U3 AG SNPs may be related with higher LDLR synthesis and greater LDL clearance, since
they are related to lower LDLC levels. In contrast to the ARIC study we saw no effect of the
PCSK9 R46L variant on CHD, possibly because our population was elderly. Our data indicate
that genetic variation at the LDLR and the PCSK9 gene loci can affect LDL C levels and CHD
risk.
P454
Identification of Thrombosis Modifier Genes in Mice
Qila Sa, Erika P Hart, Cleveland Clinic Lerner Rsch Institute, Cleveland, OH; Annie Hill,
Joseph Nadeau, Case Univ Sch of Medicine, Cleveland, OH; Jane Hoover-Plow; Cleveland
Clinic Lerner Rsch Institute, Cleveland, OH
Thrombosis is the leading cause of mortality and morbidity of cardiovascular diseases in
Western countries. Susceptibility to thrombosis varies in human populations as well as different
inbred mouse strains. Genetic factors identified for thrombotic risk account for only a small
percent of the variation, suggesting many genes have yet to be identified. The aim of this
research is to uncover additional genes associated with thrombotic risk, utilizing mouse
genetics in two inbred strains A/J and C57BL/6J (B6) with marked differences in arterial
occlusion time in a thrombosis model, and rebleeding time in a tail bleeding/rebleeding assay.
Previously, using the tail bleeding/rebleeding assay as a surrogate reporter for hemostasis and
thrombosis, a panel of 21 chromosome substitution strains (CSS) carrying individual A/J
chromosomes in the B6 background, were screened. Quantitative Trait Loci (QTL) associated
with the rebleeding phenotype are located on chromosome 5, 11 and 17, and a QTL analysis
was performed in F2 mice (B6 X CSS5, CSS-11, or CSS-17). A significant locus (P0.00086)
was identified at marker D5Mit338 on chromosome 5 (LOD3.0), accounting for 16% of the
variance in rebleeding time using 79 F2 progeny. A suggestive locus was identified on
chromosome 11 (LOD1.7, explaining 10% of the variance) with 76 F2 mice and chromosome
17 (LOD1.7, explaining 10% of the variance) with 149 F2 mice. On the mouse QTL on Chr
17, 22 genes were identified in a region homologous to a previously identified QTL for
thrombotic risk on the human Chr 18. Six of the mouse genes were differentially expressed in
liver of A/J and B6 mice, and 10 of these genes have SNPs determined from the mouse SNP
database. One candidate gene, Emilin2, a secreted glycoprotein, is preferentially expressed in
leukocytes, and binds to proteins in the vessel wall. Emilin2 is also differentially expressed in
lung and bone marrow cells of the B6 and A/J mice. The candidate genes in the mouse QTLs
have not previously been identified as modifiers of thombosis, and determination of the
causative genes has the potential to identify novel determinants of thromboitc risk.
P455
Effect of Inhibiting the 5-Lipoxygenase Pathway with Montelukast and
Low-dose Theophylline on Cardiovascular Disease Risk Factors in
Asthmatics
Hooman Allayee, Jaana Hartiala, Won Lee, USC Keck Sch of Medicine, Los Angeles, CA;
Margarete Mehrabian, David Geffen Sch of Medicine at UCLA, Los Angeles, CA; Charles
Irvin, Univ of Vermont College of Medicine, Burlington, VT; David Conti, USC Keck Sch of
Medicine, Los Angeles, CA; John Lima; Nemours Childrens Clinic, Jacksonville, FL
Recent studies have implicated the 5-lipoxygenase (5-LO) pathway in cardiovascular disease
(CVD), which may have important clinical applications since several drugs that target this
pathway are currently used to treat asthma. We sought to determine whether montelukast, a
5-LO pathway inhibitor, and low-dose theophylline, affected serum inflammatory and lipid CVD
e-116 Arterioscler Thromb Vasc Biol June 2007
by on March 25, 2011 atvb.ahajournals.org Downloaded from
risk factors in a recently completed clinical trial of asthmatics. In patients with moderate to
severe asthma, serum C-reactive protein levels (CRP) were significantly lower in the
montelukast group (n60) compared to placebo (n73) after one month (1.7mg/L vs.
3.2mg/L, respectively; P 0.006) and six months of treatment (2.3mg/L vs. 3.5mg/L,
respectively; P 0.04). All serum lipid levels were also significantly lower in both montelukast
and theophylline groups compared to placebo (P 0.050.0001) but these effects were
primarily observed in individuals who were also using inhaled corticosteroids as monotherapy
for asthma. We conclude that asthmatics taking montelukast and, to some extent, low-dose
theophylline have lower inflammatory and lipid CVD risk factors. These observations suggest
that inhibition of the 5-LO pathway may have potential use as a novel treatment strategy for
CVD in asthmatics, and perhaps the general population, although the effects on high-density
cholesterol levels should also be taken into consideration.
P456
Differential Regulation of C-Reactive Protein and Lipoprotein-Associated
Phospholipase A2 in Human Inflammatory Syndromes
Roshanak Bagheri, Megan Wolfe, Nehal Mehta, Univ of Pennsylvania Sch of Medicine,
Philadelphia, PA; Reshmaal Gomes Cumaranatunge, Univ of Connecticut Health Cntr,
Farmington, CT; Atif Qasim, Jennifer Tabita-Martinez, Manthodi Faisal, Robert Wilensky,
Daniel J Rader, Muredach P Reilly; Univ of Pennsylvania Sch of Medicine, Philadelphia, PA
Background: Plasma levels of C-Reactive Protein (CRP) and Lipoprotein-associated phospho-
lipase A2 (Lp-PLA2) are biomarkers of cardiovascular disease (CVD). However, studies
comparing acute and chronic CVD provide conflicting results. We hypothesized that these
biomarkers are differentially regulated during human inflammation. To test this hypothesis we
examined CRP and Lp-PLA2 induction during two distinct inflammatory syndromes, experi-
mental endotoxemia and acute coronary syndromes (ACS). Method: A human endotoxemia
model (n20, 50% male, 80% Caucasian, mean age 27.34.8, inpatient GCRC protocol) was
used. Serial whole blood Lp-PLA2 mRNA levels (quantitative PCR) and plasma levels of CRP and
Lp-PLA2 mass and activity were measured for 24 hours prior to and following intravenous
administration of 3 ng/kg endotoxin (LPS). Time matched analysis of variance (ANOVA) was
applied to this data. We also compared plasma levels of CRP and Lp-PLA2 in patients with ACS
(N223) to age, gender and race matched controls (N249) without coronary artery disease,
nested in a large angiographic cohort (N3,800). Findings: Following LPS, Lp-PLA2 mRNA
decreased transiently (ANOVA F6.77, p0.01) with greatest decrease of 82% at 2 hours
(p0.001). Plasma levels of Lp-PLA2 mass declined (by 18% at 6 hours, p0.007) although
activity did not change significantly. In contrast CRP level increased by almost 100-fold
(baseline 0.47 mg/L, peak 42.4 mg/L at 24 hours; F90, P0.001) in endotoxemia. In either
ACS cases or controls there was no correlation between plasma CRP level and Lp-PLA2 mass
or activity. In models adjusted for age, gender, race, Framingham Risk Scores, and
medications, plasma CRP levels (mean SD) were markedly higher in ACS cases compared
to controls (4.71 2.5 mg/dL vs 2.8 2.3 mg/dL; p0.001) whereas no significant
difference was found in plasma levels of Lp-PLA2 mass (p1.0) or activity (p0.07) between
ACS cases and controls. Conclusions: Unlike CRP, plasma Lp-PLA2 mass and activity as well
as mRNA do not increase and may even decline during acute human inflammation. This
differential in vivo regulation may account for conflicting epidemiological findings and influence
utility as CVD risk predictors in different clinical setting.
P457
The Role of Src Kinase in Regulating 15-Lipoxygenase Expression in
Primary Human Monocytes
Ashish Bhattacharjee, Srabani Pal, Lerner Rsch Institute, Cleveland Clinic, Cleveland, OH;
Gerald M Feldman, Food and Drug Administration, Bethesda, MD; Martha K Cathcart; Lerner
Rsch Institute, Cleveland Clinic, Cleveland, OH
IL-13 is a cytokine secreted by Th2 lymphocytes that is capable of inducing expression of
15-lipoxygenase (15-LO) in primary human monocytes. Our studies have defined the functional
IL-13 receptor complex, association of Jaks with the receptor components and the tyrosine
phosphorylation of specific Stat molecules, Stat1, Stat3, Stat5 and Stat6 in response to IL-13.
We have also observed important roles for both IL-13-stimulated Ser727 as well as Tyr705
phosphorylation of Stat3 and that prior to its nuclear translocation Stat3 resides in a cytosolic
signaling complex with p38MAPK and PKC, two additional critical regulators of 15-LO gene
expression. Recently we have shown that IL-13 induces Src kinase phosphorylation and Src
tyrosine kinase activity is required for both IL-13 induction of 15-LO mRNA as well as protein
expression. We demonstrated that Src is present in the signalosome along with p38MAPK and
PKC and Src kinase activity has no role in regulating the docking of any of these three
components to the molecular complex. Our data further showed that Src kinase activity
regulates IL-13-induced p38MAPK tyrosine phosphorylation which is required for regulating
Stat1 and Stat3 serine 727 phosphorylation, an important step in IL-13-induced 15-LO
expression in human monocytes. Taken together, the results of this study provide new insights
as to how 15-LO expression is regulated by various signaling molecules in human monocytes
and help us to better understand the role of 15-LO and cytokines in the pathogenesis of chronic
inflammatory disease such as asthma and atherosclerosis.
P458
Increased Interferon-Gamma Production in Response to Beta-2GPI
Immunization in B Cell-Deficient Apoe
-/-
Mice
Nicole A Braun, Adam C Morgan, Amy S Major; Vanderbilt Univ Med Cntr, Nashville, TN
B cells have been shown to be protective in atherosclerosis. Our laboratory and others have
demonstrated that absence of B cells increases atherosclerosis in LDLr
-/-
and apoE
-/-
mice.
Immunization of atherosclerosis-susceptible animals with oxidized LDL elicits antibodies
against modified lipoprotein and protects against atherosclerosis. Conversely, immunization of
mice with another atherosclerosis-associated antigen, -2 glycoprotein I (-2GPI) exacerbates
atherosclerosis in a T helper cell-dependent manner. Because B cells have been shown to
regulate T helper cell responses to specific antigen, we hypothesized that immune responses
to -2GPI by T helper cells could be regulated by B cells. To test this hypothesis, we compared
the T helper cell immune response to -2GPI in apoE
-/-
mice to apoE
-/-
mice deficient for B cells
(MT.apoE
-/-
). In these experiments, we immunized 10 apoE
-/-
and 10 MT.apoE
-/-
mice with
10 g of purified human -2GPI with adjuvant (TiterMax Gold) subcutaneously. Control mice
received adjuvant only. Two weeks following the initial immunization, mice were boosted using
the same protocol. Six weeks following the antigen boost, flow cytometry demonstrated no
difference in absolute numbers of CD4

T helper cells among the groups. However, compared


to -2GPI immunized MT.apoE
-/-
mice, immunized apoE
-/-
mice had an increased percentage
of CD4

(20% 3 vs 6%1) and CD8

(20% 3 vs. 9%2) T cells expressing the activation


marker CD69. In vitro restimulation of splenocytes with 10 g/ml of -2GPI, demonstrated that
MT.apoE
-/-
splenocytes had increased production of IFN- compared to apoE
-/-
splenocytes
(3,499 1,755 vs. 10,530 1,869 pg/ml). Proliferation of splenocytes in response to -2BPI
was not different among the groups. These data suggest that B cells may function to modulate
CD4

T cell responses to atherogenic antigens such as -2GPI and that in the absence of B
cells, CD4

T cells increase production of pro-inflammatory cytokines.


P460
Human C-Reactive Protein Gene Promoter Expression Is Inhibited by
Helix-loop-helix Factor Id3 in Mouse Aortic Smooth Muscle Cell Culture
Hamid Deliri, Rohit Malhotra, Univ of Virginia Health System, Charlottesville, VA; Alex J
Szalai, Univ of Alabama, Birmingham, AL; Nahum Meller, Coleen A McNamara; Univ of
Virginia Health System, Charlottesville, VA
Introduction: Human C - reactive protein (hCRP), is implicated as a causal factor in the
pathogenesis of atherosclerosis. hCRP contributes to atherosclerosis through multiple mech-
anisms including induction of vascular smooth muscle cell (VSMC) proliferation. The helix-
loop-helix transcription factor Id3 is expressed during vascular lesion formation, acts as a
dominant inhibitor of specific gene expression, and induces VSMC proliferation. Four single
nucleotide polymorphisms (SNPs) of the hCRP promoter gene, 409G/390C (GC), 409G/390T
(GT), 409A/390T (AT), and 409G/390A (GA), affect baseline hCRP levels. hCRP SNPs are in the
region of transcription factor binding E-box elements that may be regulated by Id3. In this study
we tested the hypothesis that Id3 influences hCRP promoter gene expression. Methods: Aortic
VSMC cultures of Id3 wild type (Id3
/
), and Id3 knockout (Id3
-/-
) mice were co-transfected with
an empty vector (EV) or a vector expressing Id3 (p-Id3) and one of the four different hCRP
promoter-luciferase constructs harboring the E-box polymorphisms. Luciferase activity was
measured after 484 hours of incubation. All experiments were performed three times in
triplicate. Results: Summarized in the table. Conclusion: This study implies that Id3 is a
regulator of hCRP gene expression. Studies to determine specific mechanisms and the impact
of this pathway on VSMC growth may provide important insight into the molecular mechanisms
of vascular lesion formation.
RELAIVE LUCIFERASE ACTIVITY SD OF FOUR DIFFERENT SNP HAPLOTYPES OF HCRP
GC GT GA AT
Id3
-/-
VSMC Culture EV 5.00.6 8.51.6 4.31.1 5.82.1
p-Id3 1.70.1 2.10.5 1.10.4 1.30.7
Id3
/
VSMC Culture EV 2.70.4 4.81.2 3.20.3 3.61.4
p-Id3 1.30.4 1.70.4 1.60.5 1.50.3
P461
Aldose Reductase Expression and Activity Are Induced in Human
MonocyteDerived Macrophages by Oxidized LDL
Christian A Gleissner, Univ of Virginia, Charlottesville, VA; HyungJun Cho, Univ of Korea,
Seoul, Republic of Korea; Dane M Dunson, Klaus Ley; Univ of Virginia, Charlottesville, VA
Aldose reductase (AR) is the rate-limiting enzyme of the polyol pathway, which utilizes excess
glucose and reduces it to sorbitol and fructose. AR activity has been associated with
retinopathy, nephropathy, and neuropathy in diabetic patients. Overexpression of human AR
has been shown to increase atherosclerotic lesions in LDL receptor-knockout mice. Using 22
Affymetrix gene chips (HG-U133A) we measured gene expression in human peripheral blood
mononuclear cells (PBMCs), monocytes and monocyte-derived macrophages as well as foam
cells induced by native LDL, minimally modified LDL (mmLDL) or oxidized LDL (oxLDL).
Statistical analysis was performed using local pooled error test (LPE) and heteregenous error
model (HEM). A significant increase of AR gene expression could be demonstrated in
macrophageas after 48 hours of treatment with LDL and oxLDL, however not with mmLDL.
Simultaneously, several genes associated with increased oxidative stress were upregulated
(e.g. genes of the glutathione or thioredoxin system or metallothioneins). Upregulation of AR by
oxLDL was confirmed by real-time PCR. Furthermore, an increase of AR enzyme activity could
be shown as measured by increased NADPH consumption. oxLDL-dependent induction of AR
activity was reduced when macrophages were treated with the AR inhibitor epalrestat. We
hypothesize that oxLDL-induced expression of AR in foam cells represents a novel pro-
inflammatory mechanism and a potentially relevant therapeutic target in atherosclerosis.
P462
Plasmin Activation of MMP-9 Regulates Peritoneal Macrophage
Recruitment by Preventing Cellular Accumulation in the Mesothelium
Yanqing Gong, Erika Hart, Jane Hoover-Plow; Cleveland Clinic, Cleveland, OH
Inflammation plays a critical role in the initiation of cardiovascular, respiratory and gastroenteric
diseases. Based on studies using deficient mice, plasminogen (Plg) has been shown to play a
major role in inflammatory cell recruitment; however, the mechanism underlying its partici-
2007 ATVB Poster Presentations e-117
by on March 25, 2011 atvb.ahajournals.org Downloaded from
pation is unknown. In a thioglycollate-induced peritonitis model conducted in Plg-deficient
mice, a significant (P 0.01) number of macrophages accumulated (4-fold) and collagen IV
deposition was increased by 3-fold in the peritoneum precluding macrophage trans-mesothelial
migration. MMP-9 was activated in Plg
/
mice but not in Plg
-/-
mice at 72 h, the peak time
of macrophage migration to the peritoneal cavity. Mice deficient in MMP-9 and mice treated
with MMP-9 neutralizing antibody had significantly (P 0.01) impaired (50%) macrophage
migration in this model. Plg deficient mice reconstituted locally with an active form of MMP-9,
but not its inactive proenzyme, significantly (P 0.01) rescued macrophage migration. In
addition, in vitro macrophage migration assays showed that MMP-9 deficiency or MMP-9
neutralization blocked macrophage migration by 90% across matrigel and collagen IV in
response to Plg. Thus, a major role of Plg in inflammatory cell recruitment depends on its
activation of MMP-9, which in turn controls matrix turnover.
P463
Proinflammatory Activities of C-reactive Protein and
Lysophosphatidylcholine on Human Macrophages Are Selectively
Suppressed by Mutual Complex Formation
Ki Hoon Han, Asan Med Cntr, Seoul, Republic of Korea; Mi-Kyung Chang, Univ of California,
San Diego, La Jolla, CA; Jewon Ryu; Asan Med Cntr, Seoul, Republic of Korea
Background; Both C-reactive protein (CRP) and oxidized LDL (oxLDL) trigger pro-inflammatory
activities by macrophages during the process of atherosclerosis. We previously reported that
CRP calcium-dependently binds to phosphorylcholine (PC) head group of oxLDL in vitro.
However, the functional relevance of the complex formation on the progression of atheroscle-
rosis is not clear. Methods and Results; Chemiluminescent immunoassay and HPLC confirmed
that lysophosphatidylcholine (LPC), a main PC-bearing component of oxLDL, calcium-
dependently formed stable complex with CRP. Binding assay showed that the specific binding
of CRP-LPC complex to FcRIA or FcRIIA-transfected 293FTcells was less efficient than CRP
pentamer, in which calculated Kd value for CRP-LPC complex was 35 fold lower than that of
CRP binding. Moreover, confocal microscopic images clearly showed the co-localization of CRP
and LPC in human macrophages, and the direct delivery of CRP-LPC complex into the cytosol
without activating FcRs triggered less potent activation of AP-1 and NFB than those activated
by CRP and LPC alone, suggesting the mutual inhibition of CRP and LPC through maintaining
complex formation in the cytosol, as well as less FcR activation. CRP did not alter the overall
uptake of DiI-labeled oxLDL to human macrophages. However, CRP was also found to suppress
pro-inflammatory activities of oxLDL mixture and vice versa, in which co-presence of CRP and
oxLDL induced less potent activation of AP-1 and production of pro-inflammatory mediators
and cytokines than those elicited by CRP and oxLDL alone. On the other hand, oxLDL-
stimulated expressions of genes that are responsible for cholesterol efflux, such as ABCA1,
CD36 and PPAR proteins, by human macrophages were preserved in the presence of CRP.
Conclusions; We conclude that the formation of complexes between CRP and PC-bearing
oxidized phospholipids may selectively inhibit pro-inflammatory effects of CRP and oxLDL on
macrophages in the arterial wall and may eventually contribute to the retardation of the
progression of atherosclerosis.
P464
Immunodominant and Atheroprotective MDA-derived Epitopes
Karsten Hartvigsen, Univ of California, San Diego, La Jolla, CA; Christoph J Binder, Med
Univ of Vienna, Vienna, Austria; Apais Rafia, Richard Harkewicz, Lotte F Hansen, Joseph L
Witztum; Univ of California, San Diego, La Jolla, CA
Immunization of atherosclerosis-prone animals with malondialdehyde (MDA)-modified homol-
ogous LDL is atheroprotective. Previously, we showed that MDA-LDL immunization leads to a
MDA-LDL-specific Th2 biased cellular and humoral response. The mechanisms as well as the
chemical identity of the MDA-derived epitopes are largely unknown. MDA-modification of
biomolecules results in diverse molecular structures, both simple and complex, with and
without inter- or intra-molecular cross-linking. Our goal is to define immunodominant epitopes
in MDA-LDL that lead to atheroprotective immunity. Here, we immunized mice with candidate
antigens and controls and determined optimal immune responses and impact on atheroscle-
rosis. Modified mouse serum albumin (MSA) was used as a homologous protein carrier of the
MDA-derived adducts in order to test whether LDL-lipids or apoB are essential for
atheroprotection. Male LDLR
-/-
mice were divided into 7 groups (n15); of which 6 were
immunized with Freunds adjuvant (FA) and the 7th with PBS. Mice immunized with MDA-LDL,
two preparations of MDA-modified MSA (with simple or complex MDA-derived adducts), and
propanal-modified (PA)-MSA (carrier-adduct control) showed elevated IgM and IgG1IgG2c
titers to their respective immunogens, suggesting Th2 biased responses. Antigen-specific
cellular responses confirmed this Th2 bias. The mice were fed cholesterol-enriched regular
chow for 28 weeks. Cholesterol levels were not different (800 mg/dL). En face analysis of the
entire aorta revealed that immunization with preparations of MDA-modified MSA significantly
reduced lesion area compared to the control immunizations with PA-MSA, MSA, and FA
(30%; all p0.05) or PBS (38%; p0.01). Although cross sectional analysis at the aortic
origin showed the same atheroprotective trend, statistical significance was not obtained. In
summary, these data suggest that 1) a strong non-relevant Th2 response alone (i.e. to PA-MSA)
was not atheroprotective, 2) neither the modified LDL per se nor the apoB carrier are necessary
for atheroprotection and 3) the molecular structure of the adduct provides the atheroprotective
immunity. Work is underway to identify the fine structural features of the atheroprotective
epitopes.
P465
Medial Macrophages That Accumulate in Abdominal Aortic Regions Prone
to Angiotensin II-induced Aneurysms Are Not Primarily Derived from
Blood-borne Cells
Hong Lu, Debra L Rateri, Lisa A Cassis, Alan Daugherty; Univ of Kentucky, Lexington, KY
Objective: Angiotensin II (AngII)-infusion causes the formation of abdominal aortic aneurysms
(AAA) in hyperlipidemic mice. Initially, AngII-infusion leads to macrophage accumulation in the
medial layer of the abdominal aorta prior to AAA formation. Defining the origin of macrophages
that accumulate in the aortic media has mechanistic implications in the evolution of AAAs.
Thus, the purpose of this study was to use mice expressing allelic variants of CD45 (CD45.2
vs. CD45.1) to determine if medial macrophages originate from blood borne cells during AngII
infusion. Methods and Results: Bone marrow transplantation studies were performed by
repopulating irradiated recipient LDL receptor -/- male mice expressing CD45.2 (68 week old,
male) with donor bone marrow-derived cells from mice of a congenic strain expressing CD45.1.
After a 4 week engraftment interval, 80% of blood monocytes expressed the donor (CD45.1)
genotype. At 4 weeks, tissues were acquired and immunostained for allelic variants of CD45.
Donor-derived CD45.1 variant predominated in thymus and spleen, consistent with successful
repopulation. Aortas were dissected free and sectioned throughout the AAA prone region.
Aortas from non-infused mice had few macrophages in the adventitia and none were detected
in the media. Another group of engrafted mice were placed on a fat-enriched diet (21% milk
fat, 0.15% cholesterol) and infused with AngII (1,000 ng/kg/min) for intervals prior to AAA
formation (between 3 to 5 days). Abundant macrophages clustered near the medial-adventitial
interface of abdominal aortas and medial accumulation of macrophages were observed in the
AAA prone region. Macrophages that accumulated in both the adventitia and media of the
abdominal aorta following AngII infusion stained predominantly for the recipient CD45.2 variant.
Conclusions: Macrophages that accumulate in the media of the aorta in the early stages
following AngII-infusion primarily arise from resident macrophages, consistent with an
adventitial origin rather than from the blood.
P466
Resistin Gene Variation Is Associated with Plasma Resistin Levels and
Inflammatory Markers but Not Atherosclerosis or Metabolic Syndrome in
Nondiabetic Caucasians
Atif N Qasim, Thomas Metkus, Mahlet Tadesse, Stephanie Restine, Megan Wolfe, Daniel
Rader, Muredach P Reilly; Univ of Pennsylvania, Philadelphia, PA
Introduction Resistin is an adipokine which has been linked to inflammation, insulin resistance
and atherosclerosis in mice, however a similar role in humans has been debated. We have
shown previously that plasma resistin levels are associated modestly with markers of
inflammation and atherosclerosis but not with metabolic syndrome or insulin resistance.
Human studies of resistin gene variation and these parameters are lacking. Therefore, we
hypothesized that a gain of function resistin promoter SNP and associated haplotypes would be
associated with resistin levels, inflammatory markers and atherosclerosis but not with lipid
parameters or the metabolic syndrome. Methods We examined the relationship of 3 resistin
SNPs -420CG, -1092AG and 156TC and their associated haplotypes with plasma levels
of resistin, TNF alpha and CRP, as well as lipids, NCEP-defined metabolic syndrome and
atherosclerosis as measured by coronary calcium scores in Caucasians in the Study of Inherited
Risk of Coronary Atherosclerosis (n840). Results Plasma resistin levels were higher,
dose-dependently with the G allele of the -420 polymorphism (CC 5.8 2.7ng/mL , GC 6.5
4.0 ng/mL and GG 7.5 5.1 ng/mL), after adjusting for age, gender and BMI. (p0.003).
There was no association of -420CG with coronary calcium scores, BMI, lipid variables or the
metabolic syndrome. Estimated haplotypes, based on the 3 SNPs, that contained the G allele
from the -420 variant were positively associated with resistin levels (p0.047), CRP (p0.03)
and TNF alpha (p0.02) but not the presence of the metabolic syndrome or coronary
calcification. When considered alone, the -1092AG and 156TC SNPs had no association
with resistin levels, coronary calcium, inflammatory markers, metabolic syndrome or BMI.
Conclusion Genetic variation in resistin was not associated with coronary atherosclerosis or
the metabolic syndrome. The -420CG resistin polymorphism accounted for a small but
significant amount of variation in plasma resistin levels and -420G haplotypes were associated
with CRP and TNF alpha levels. These findings are consistent with the concept that resistin is
an inflammatory adipokine but may not be a strong predictor of atherosclerosis, adiposity or
metabolic syndrome in humans.
P467
Increased Presence of Macrophages Secreting Resistin in Epicardial Fat of
Patients with Acute Coronary Syndrome
Giacomo Ruotolo, Silvia Langheim, San Raffaele Scientific Institute, Milano, Italy; Lorella
Dreas, Ospedali Riuniti, Trieste, Italy; Lorenzo Veschini, Francesco Maisano, Chiara Foglieni,
San Raffaele Scientific Institute, Milano, Italy; Bartolo Zingone, Ospedali Riuniti, Trieste,
Italy; Ottavio Alfieri, Elisabetta Ferrero, Attilio Maseri; San Raffaele Scientific Institute,
Milano, Italy
Aim of the present study was to evaluate the expression of several adipocytokines in epicardial
fat of patients undergoing CABG surgery for acute coronary syndrome (ACS, n23) as
compared to that of age- and BMI-matched patients with chronic stable angina (CSA, n26).
Patients undergoing cardiac surgery for valvular defects, but with angiographically normal
coronary arteries served as reference group (n20). The local expression and protein secretion
(24h culture medium) of adipocytokines from epicardial fat biopsies was evaluated by
Real-Time PCR (adiponectin, leptin, resistin, visfatin, IL-6, IL-8, IL-10, CRP, MCP-1, PAI-1, MIF),
and multiplexed fluorescent immunoassay (adiponectin, resistin, leptin, IL-6, PAI-1, MCP-1),
respectively. Immunohistochemical stainings of epicardial fat slides were also performed to
show presence of inflammatory cells (T-lymphocytes, macrophages, mast cells). The 24h
medium from epicardial adipose tissue culture was also tested in experiments of endothelial
e-118 Arterioscler Thromb Vasc Biol June 2007
by on March 25, 2011 atvb.ahajournals.org Downloaded from
cell (HUVEC) permeability and proliferation in vitro. A significantly higher expression of resistin,
CD68, and MCP-1 genes was observed in epicardial fat biopsies of ACS as compared to CSA
patients. ACS patients also showed significantly increased levels of resistin in 24h culture
medium of epicardial adipose tissue. In addition, these changes were paralleled by a
significantly increased presence of CD68 cells in epicardial fat depots of ACS patients.
Finally, a significantly increased permeability of endothelial cells was also observed after
incubation with culture medium from epicardial fat of ACS patients.In conclusion, an increased
presence of macrophages is associated with higher expression and secretion of resistin in
epicardial adipose tissue of patients with ACS.
P468
CD4

T-Helper Cell Distributions and Associations with Atherosclerosis:


Results from the Multi-Ethnic Study of Atherosclerosis (MESA)
Russell P Tracy, Nancy Swords Jenny, Margaret F Doyle, Sally A Huber, Univ of Vermont
College of Medicine, Colchester, VT; Bruce M Psaty, Richard A Kronmal; Univ of
Washington, Seattle, WA
Introduction: CD4

T Helper (Th) cells are key effectors of adaptive immunity and important in
atherosclerosis development in mice. Less is known about their role in human atherosclerosis.
Methods: We examined this association in 524 white, black, Hispanic and Chinese MESA
participants. Mean age was 59 years (range 4484); 56% were women. Fresh blood samples
were shipped overnight to the Central Laboratory. CD4

cells in peripheral blood mononuclear


cell preparations were stimulated with ionomycin/phorbol myristate acetate. Th1 cells were
defined by flow cytometry as CD4

/interferon-

and Th2 cells were CD4

/interleukin-4

. Th1
and Th2 cells were expressed as percent of CD4

cells. Total CD4

cells were expressed as


percent of lymphocytes. Results: The mean (standard deviation) for %CD4

, %Th1 and %Th2


cells were 43.5% (13.4%), 14.5% (7.6%) and 0.7% (0.8%), respectively. Neither %CD4

nor
%Th1 cells were significantly correlated with inflammation markers interleukin-6 and
C-reactive protein. In age, sex and ethnicity adjusted regression models, %CD4

cells were
higher with older age (1.4%/10yrs), female sex (4%), and white ethnicity (2.69.6% higher
than others). However, %Th1 cells were lower with age (1%/10yrs) and female sex (1.2%).
%CD4

cells were associated with cytomegalovirus (CMV) serology markers (p0.05). %Th1
cells were associated with markers for CMV and hepatitis A (both p0.05). In stepwise
regressions of coronary calcification (modeled as ln-transformed Agatston score in those with
a positive score, n237) with age, sex, ethnicity, cardiovascular disease risk factors and T cell
indices, %Th1 remained significantly positively associated with degree of coronary calcification
(p0.001). Conclusions: In a multi-ethnic population of men and women, a Th cell distribution
biased towards Th1 cells was associated with increased infectious burden and coronary
calcification, suggesting a role for Th1 cells in human atherosclerosis.
P469
Natural Killer T-Cells Influence Both Atherosclerosis and Plasma Lipid
Levels in Low-Density Lipoprotein ReceptorDeficient Mice
Paul A VanderLaan, Catherine A Reardon, Godfrey S Getz; Univ of Chicago, Chicago, IL
Natural killer T (NKT) cells are a subset of T-lymphocytes that respond to lipid antigens
presented in the context of CD1 molecules and have been shown to be proatherogenic in a
number of mouse models through either exogenous stimulation or genetic deficiency. To
examine the effects of altering absolute NKT cell numbers in an otherwise immune competent
murine model of atherosclerosis, the low density lipoprotein receptor (LDLR) deficient mouse
was crossed with either the V14J18 T-cell receptor transgenic mouse possessing an
increased proportion of NKT cells or the CD1d knockout mouse which lacks NKT cells. When
fed a Western-type diet for 12 weeks, the V14tgLDLR
-/-
mice had significantly higher plasma
triglyceride levels compared to CD1d
-/-
LDLR
-/-
mice (1,36480 vs 1,02795 mg/dL for males,
p0.01; 57372 vs 33034 mg/dL for females, p0.03; n1012 per group). Furthermore,
the NKT cell deficient mice had higher HDL cholesterol levels assessed by FPLC fractionation
(968 vs 13912 mg/dL for males, p0.01; 9410 vs 11515 mg/dL for females, pns).
The V14tgLDLR
-/-
mice had larger atherosclerotic lesions than the CD1d
-/-
LDLR
-/-
mice in both
the innominate artery (46,68016,923 vs 19,0745,373 m
2
for males, pns;
43,24815,906 vs 5,4143,183 m
2
for females, p0.02) and ascending aortic arch
(63,8418,817 vs 36,0082,866 m
2
for males, p0.03; 74,05316,585 vs
32,9243,425 m
2
for females, p0.01). No changes in aortic root atherosclerosis were
noted, though earlier time points are currently being examined. In sum, NKT cells have the
potential to subtly impact atherogenesis by affecting systemic lipid levels in addition to any
local actions in the atherosclerotic plaque.
P470
Leukocyte Core 2 16-N-glucosaminyltransferase-I (C2GlcNAcT-I) Deletion
Decreases Neointimal Formation After Arterial Injury in Apolipoprotein
E-deficient Mice
Huan Wang, Rong Tang, Univ of Minnesota, Minneapolis, MN; Jamey D Marth, Univ of
California, La Jolla, CA; Klaus Ley, Univ of Virginia, Charlottesville, VA; Yuqing Huo; Univ of
Minnesota, Minneapolis, MN
Vascular inflammation after arterial injury causes neointimal hyperplasia, which is one of major
reasons for restenosis after percutaneous transluminal coronary angioplasty (PTCA). Selectins
and their receptors are critically involved in the interactions of leukocytes and platelets with
injured vessel wall. Leukocyte C2GlcNAcT-I is an enzyme modifying several molecules
including P-selectin glycoprotein ligand-1 (PSGL-1), CD43, CD44 and CD45. It modulates
PSGL-1 binding activity by adding specific O-linked oligosaccharides terminating with sialyl
Lewis-X moieties, which is crucial for PSGL-1 optimal binding to P-selectin. In this study, we
investigated whether and how leukocyte C2GlcNAcT-I influences neointimal formation using a
mouse carotid artery wire injury model. We crossed C2GlcNAcT-I deficient mice with
apolipoprotein-E deficient (apoE
-/-
) mice to generate double knockout mice. These mice and
their littermate controls were fed a western diet for 2 weeks, followed by wire injury on their
left carotid arteries. Three, 5 and 7 days after arterial injury, immunostaining was performed
to examine accumulation of leukocytes and platelets to injured arteries and Evans blue staining
was conducted to assess endothelial repair. Four weeks after arterial injury, injured arteries
was collected and Movats pentachrome was used to stain cross-sections of arteries for overall
features of neointimal hyperplasia. We found that C2GlcNAcT-I deletion in apoE
-/-
mice
suppressed neutrophil and platelet adhesion to injured arteries and markedly benefited artery
endothelization, resulting in a significant decrease in the size of neointima after arterial injury.
This study demonstrates that leukocyte C2GlcNAcT-I plays an important role in the formation
of arterial neointima and suggests that inhibition of leukocyte C2GlcNAcT-I may be a novel
approach in the treatment of restenosis after PTCA.
P471
-Smooth Actin Positive Particles Are Observed with Macrophage
Adhesion to Endothelial Cells in Vivo
Kosuke Azuma, Hirotaka Watada, Tomoya Mita, Ryuzo Kawamori; Juntendo Univ, Tokyo,
Japan
Background. Atherosclerosis is a chronic inflammatory condition characterized by the
accumulation of lipids and macrophages. Adhesion of circulating monocytes to the endothelial
surface seems to be involved in the lesion initiation. However, the pathological changes
observed with monocyte/macrophage adhesion in vivo have not been fully elucidated yet. Aim.
The aim of this study is to elucidate the pathological changes with monocyte adhesion to
endothelial cells. Methods. Wild type, C57 BL6 mice and Apolipoprotein (apo) E knock-out mice
at the age of 7 weeks were subjected to quantitation of monocyte adhesion to the thoracic aorta
by new En face method for optimal observation of endothelial surface (NEMOes) (Azuma et al
2006 ATVB) using Mac2 as a marker of activated macrophage and conventional histological
evaluation. Results. In wild type mice, NEMOes occasionally identified mac-2 positive cells on
endothelial cell surface at the atherosclerosis prone sites, such as branching of intercostal
artery or intimal thickening area. The macrophages were with many protuberances and were
mostly on endothelial surface. Surprisingly, alpha-smooth muscle actin positive particles whose
diameters are approximately 1 to 3 micrometers were also found at the atherosclerosis prone
sites. Most of these particles were located just above the fenestrated intra elastica lamina. A
few particles were found on the endothelial cell surface. Surrounding the particles on the
endothelial surface, we frequently found activated macrophages. In addition, the smaller
alpha-smooth muscle actin positive particles were observed around the macrophages. These
smaller particles seem to be the debris of large particles. These features are reminiscence of
interaction between these particles and macrophages. In apo E knock-out mice, we found
profoundly more adherent macrophages. In addition, we found more particles around the
macrophages. Conclusion. Our data suggest the pathological role of alpha-smooth muscle
actin positive particles. The interaction between these particles and macrophages seems to be
involved in the lesional initiation of the progression of atherosclerosis.
P472
Overexpression of Glutathione Peroxidase 4 Reduces Atherosclerosis in
Apolipoprotein E-deficient Mice
Lichun Zhou, Zhongmao Guo, Hong Yang; Meharry Med College, Nashville, TN
Accumulation of oxidized lipids in the arterial wall is believed to give rise to atherosclerosis.
Glutathione peroxidase 4 (GPx4) is a peroxide scavenger that removes oxidative modifications
from lipids, e.g., free fatty acids, cholesterols and phospholipids. The primary goal of this study
is to assess the effect of overexpressing or downexpressing GPx4 on atherosclerosis in
apolipoprotein E-deficient (ApoE
-/-
) mice. Our data demonstrated that the atherosclerotic lesions
in the aortic tree and the aortic sinus of the ApoE
-/-
mice overexpressing GPx4 (hGPx4Tg/
ApoE
-/-
) were significantly smaller than those of the ApoE
-/-
control mice. Almost all of the
ApoE
-/-
control mice at 4 - 5 months of age developed both early stages of atherosclerotic
lesions (e.g., foam cells and free lipids) and advanced lesions (e.g., fibrous caps and acellular
areas) in the aortic sinus. In contrast, only about two third of the hGPx4Tg/ApoE
-/-
mice
developed acellular areas in the atherosclerotic lesions. We also observed that overexpression
of GPx4 reduced the aorta F2-isoprostane levels in ApoE
-/-
mice, attenuated 7-ketocholesterol
(7-Kc)-induced apoptosis and lysophosphatidylcholine-induced necrotic death in mouse aorta
endothelial cells and macrophages, reduced oxidized low-density lipoprotein (oxLDL)- and
7-Kc-induced adhesion of monocytes to endothelial cell, and suppressed the oxLDL- and
7-Kc-induced expression of vascular cell adhesion molecule-1 (VCAM-1) and intercellular
adhesion molecule-1 (ICAM-1). These data suggest that overexpression of GPx4 inhibits
atherogenesis, and that reducing lipid oxidation and suppressing the sensitivity of vascular cells
2007 ATVB Poster Presentations e-119
by on March 25, 2011 atvb.ahajournals.org Downloaded from
to oxidized lipids are the mechanisms by which GPx4 inhibits atherogenesis. Unexpectedly,
heterozygous mutation to GPx4 did not increase atherosclerotic lesions and oxidized lipids in
ApoE
-/-
mice.
P473
Mac-1 Mediates CD40L-induced Atherogenesis
Andreas Zirlik, Christoph Maier, Univ of Freiburg, Freiburg, Germany; Norbert Gerdes,
Karolinska Institute, Stockholm, Sweden; Lindsey MacFarlane, Brigham and Womens Hosp,
Boston, MA; Juliana Soosairajah, Baker Heart Institute, Melbourne, Australia; Udo
Bavendiek, Univ of Hannover, Hannover, Germany; Ingo Ahrens, Baker Heart Institute,
Melbourne, Australia; Sandra Ernst, Univ of Freiburg, Freiburg, Germany; Nicole Bassler,
Baker Heart Institute, Melbourne, Australia; Anna Missiou, Zsofia Patko, Univ of Freiburg,
Freiburg, Germany; Masanori Aikawa, Brigham and Womens Hosp, Boston, MA; Uwe
Scho nbeck, Boehringer Ingelheim Pharmaceuticals, Ridgefield, CT; Christoph Bode, Univ of
Freiburg, Freiburg, Germany; Peter Libby, Brigham and Womens Hosp, Boston, MA;
Karlheinz Peter; Baker Heart Institute, Melbourne, Australia
Strong evidence supports a role for CD40L as marker and mediator of inflammatory diseases
such as atherosclerosis. Despite extensive characterization of CD40, the classical receptor for
CD40L, in immune defense, its role in inflammatory diseases remains uncertain. This study
aimed to characterize the contribution of CD40 signaling to atherogenesis. Surprisingly, mice
deficient in both CD40 and the low-density lipoprotein-receptor (LDLR) do not develop smaller
lesions in the aortic arch, root, and thoraco-abdominal aorta compared to LDLR single-deficient
mice that consumed an atherogenic diet for 8 and 16 weeks. Lesions in these two groups of
mice also had similar composition. Based on previous reports demonstrating functional binding
of GPIIbIIIa to CD40L in thrombosis, we investigated other integrins as potential alternative
receptors for CD40L. We demonstrated that CD40L interacts with the integrin Mac-1 on human
monocyte/macrophages (using flow cytometry, radioactive binding assays, and immunopre-
cipitation), resulting in Mac-1-dependent adhesion and migration of inflammatory cells as well
as myeloperoxidase release in vitro. Furthermore, mice deficient in CD40L show significantly
reduced thioglycolate-elicited accumulation of inflammatory cells in the peritoneal cavity
compared with mice deficient in CD40 and wild-type controls. Inhibition of Mac-1 in
LDLR-deficient mice attenuates lesion development and reduces lesional macrophage accu-
mulation. These observations identify the interaction of CD40L and Mac-1 as an alternative
pathway for CD40L-mediated inflammation. This novel mechanism expands understanding of
inflammatory signaling during atherogenesis and has implications regarding novel anti-
inflammatory therapies.
P474
Structure-Function of Apolipoprotein A-I
Milano
and Related Variants
Eric T Alexander, Michael C Phillips, Childrens Hosp of Philadelphia, Philadelphia, PA;
Daniel J Rader; Univ of Pennsylvania Med Cntr, Philadelphia, PA
Epidemiological studies have shown that plasma apolipoprotein A-I (apoA-I) and high density
lipoprotein (HDL) cholesterol levels have an inverse relationship with the risk of developing
atherosclerosis. The naturally occurring mutation R173C apoA-I, or apoA-I
Milano
, belies that
correlation in that individuals heterozygous for this mutation have reduced plasma levels of
apoA-I and HDL, but are not at a greater risk for cardiovascular disease. The hypothesis being
investigated here is that the altered cardioprotective ability of apoA-I
Milano
is specifically related
to the introduction of a cysteine residue into the apoA-I primary sequence at position 173 and
not the loss of the arginine residue. To address this question, we engineered two mutations of
apoA-I, R173K apoA-I and R173S apoA-I. R173K apoA-I is a conservative mutation that
maintains the positive charge at position 173 whereas R173S apoA-I closely mimics the
apoA-I
Milano
mutation in that the structure of serine is very similar to cysteine, except for the
presence of a hydroxyl group rather than a sulfhydryl group. The proteins have been physically
characterized. ApoA-I
Milano
is less stable than WT apoA-I and has less alpha-helical content.
R173S apoA-I resembles apoA-I
Milano
while R173K apoA-I is almost identical to WT apoA-I.
Adeno-associated viruses containing the mutant apoA-I genes were generated and used to
infect apoA-I -/- mice. The plasma lipoprotein profile of mice expressing R173K apoA-I was
indistinguishable from that of mice expressing WT apoA-I. However, mice expressing either
R173S apoA-I or apoA-I
Milano
had reduced apoA-I levels and HDL cholesterol levels compared
to WT apoA-I. Mice expressing WT apoA-I had plasma HDL cholesterol concentrations of 80
13mg/dl while mice expressing apoA-I
Milano
had only 31 2mg/dl. Interestingly, mice
expressing R173S apoA-I displayed an intermediate phenotype with plasma HDL cholesterol
levels of 48 5mg/dl. In summary, the altered structure and function of apoA-I
Milano
is due both
to the loss of a positive charge at position 173 and to the introduction of the cysteine residue
which allows the formation of disulfide bonds.
P475
A C-Terminal Truncated Human Apolipoprotein A-V Displays Unique
Structural and Lipid-Binding Properties
Jennifer A Beckstead, Childrens Hosp, Oakland Rsch Institute, Oakland, CA; Kasuen Wong,
Univ of California, Berkeley, Berkeley, CA; Vinita Gupta, Childrens Hosp, Oakland Rsch
Institute, Oakland, CA; Chung-Ping L Wan, California State Univ, Long Beach, Long Beach,
CA; Victoria R Cook, Richard B Weinberg, Wake Forest Univ Sch of Medicine,
Winston-Salem, NC; Paul M Weers, California State Univ, Long Beach, Long Beach, CA;
Robert O Ryan; Childrens Hosp, Oakland Rsch Institute, Oakland, CA
Human apolipoprotein A-V (apoA-V) is a potent modulator of plasma triacylglycerol (TG) levels.
To probe different regions of this 343 amino acid protein, 4 single Trp apoA-V variants were
prepared. The variant with a Trp at position 325, distal to the tetra-proline sequence at residues
293296, displayed an 8 nm blue shift in wavelength of maximum fluorescence emission upon
lipid association. To evaluate the structural and functional role of this 51-residue C-terminal
segment, a truncated apoA-V, comprising amino acids 1292, was generated. Far UV circular
dichroism spectra of full-length apoA-V and apoA-V(1292) were similar with 50% alpha
helix content. In guanidine HCl denaturation experiments, both full-length and truncated apoA-V
yielded biphasic profiles consistent with the presence of two structural domains. The
denaturation profile of the lower stability component, but not the higher stability component,
was affected by the truncation. In fluorescent dye binding experiments, apoA-V(1292)
contained fewer solvent exposed hydrophobic sites than full-length apoA-V. Truncated apoA-V
displayed an attenuated ability to solubilize DMPC phospholipid vesicles compared to full-length
apoA-V yet it bound to a triolein/water interface with faster kinetics. Taken together, the data
support the concept that the 51 amino acid segment C-terminal to the tetra-proline sequence,
is not required for apoA-V to adopt a folded protein structure yet functions to modulate apoA-V
lipid binding activity and thereby, may be relevant to the mechanism whereby it influences
plasma TG levels.
P476
Disruption of Glycosylation at Asparagine-116 of Endothelial Lipase
Enhances Activity and Lipoprotein Lipid Hydrolysis in Vitro and in Vivo
Robert J Brown, Gwen C Miller, Nathalie Griffon, Christopher J Long, Daniel J Rader; Univ
of Pennsylvania, Philadelphia, PA
We previously identified that four of five putative N-linked glycosylation sites of human
endothelial lipase (EL) are utilized, and showed that substitution of the carbohydrate-linked
Asn-116 with Ala (N116A) increased the hydrolytic activity of EL. We expanded this observation
by first assessing the catalytic activities of additional mutants of the Asn-Asn-Thr glycosylation
sequence: Asn-116 to Thr (N116T), Asn-117 to Ala (N117A), and Thr-118 to Ala (T118A). The
specific activities of N116T- and T118A-EL were significantly enhanced toward dipalmi-
toylphosphatidyl choline (250 30% and 372 33%, respectively, p0.001) versus both
wild-type (WT)- and N117A-EL (100 25% and 121 11%, respectively). The specific
activities of N116T- and T118A-EL toward triolein were also significantly greater (294 14%
and 269 6%, respectively, p0.001) versus both WT- and N117A-EL (100 20% and
100 26%, respectively). These data demonstrate that it is the loss of glycosylation at
Asn-116, and not a structural effect of specifically the N116A mutant, that results in enhanced
catalytic activity of EL. We next assessed the hydrolysis of native lipoprotein lipids by
N116A-EL. Compared to WT-EL, the N116A mutant exhibited a significant 5-fold increase in
low density lipoprotein (LDL) hydrolysis and a 1.8-fold increase in high density lipoprotein (HDL)
2 hydrolysis; HDL
3
hydrolysis was unchanged. Consistent with these observations, adenoviral-
mediated expression of N116A-EL (3 x 10
10
virus particles per mouse) in LDL-receptor-null
mice significantly reduced levels of both HDL cholesterol (8.3 1.9 mg/dl, p0.03) and
non-HDL cholesterol (37.8 9.7 mg/dl, p0.03) beyond the reductions observed by the
expression of WT-EL alone (30.8 9.7 and 81.8 13.8 mg/dl, respectively). Finally, we
introduced Asn-116 of EL into the analogous positions within lipoprotein lipase (LPL) and
hepatic lipase (HL), resulting in N-linked glycosylation at this site. Glycosylation at this site
significantly suppressed LPL hydrolysis of synthetic substrates, LDL, HDL
2
, and HDL
3
, but had
little effect on HL activity. Overall, these data show that N-linked glycosylation at Asn-116 of
EL, or the analogous site of LPL, reduces hydrolysis of lipids within synthetic and lipoprotein
substrates.
P477
Normal Endothelial Function in Carriers of the Apolipoprotein A-IMilano
Mutant Despite Low HDL-cholesterol Levels
Monica Gomaraschi, Paola Conca, Damiano Baldassarre, Sonia Eligini, Cesare R Sirtori,
Guido Franceschini, Laura Calabresi; Univ of Milano, Milano, Italy
Carriers of the apolipoprotein A-IMilano (apoA-IM) mutant show severe reductions in the
plasma concentration of antiatherogenic HDL but do not present with preclinical atherosclerosis
and premature CHD. Aim of the present study was to investigate endothelial function in A-IM
carriers, since low HDL-C levels have been associated with features of endothelial dysfunction.
Plasma concentrations of soluble cell adhesion molecules (sCAMs) and forearm arterial
compliance (FAC) during reactive hyperemia were evaluated in 21 A-IM carriers, 21 healthy
subjects with low HDL-C, and 42 controls. Low HDL-C subjects had significantly higher plasma
sCAM levels than controls (sVCAM-1: 656.349.3 vs 502.625.5 ng/ml; sICAM-1:
335.621.5 vs 267.08.9 ng/ml; sE-selectin: 62.94.1 vs 47.93.0 ng/ml); on the contrary,
no differences were detected between A-IM carriers (sVCAM-1: 550.632.1 ng/ml; sICAM-1:
309.826.9 ng/ml; sE-selectin: 52.34.3 ng/ml) and controls. Low HDL-C subjects had lower
FAC than controls, while no differences were detected between A-IM carriers and controls.
These results suggest that HDL from A-IM carriers may be more efficient than control HDL in
modulating endothelial function. To test this hypothesis, plasma HDL were isolated from 6 A-IM
carriers and 6 controls, and their ability to inhibit VCAM-1 expression and to induce eNOS was
tested in cultured endothelial cells. A-IM HDL were two times more effective than control HDL
in reducing TNFalpha-induced VCAM-1 expression; the inhibition occurred at a transcriptional
level, as demonstrated by RT-PCR. In addition, cells exposed to A-IM HDL showed higher
expression of eNOS than cells treated with control HDL. In conclusion, despite the very low
HDL-C levels, A-IM carriers do not display features of endothelial dysfunction, such as the
increase of circulating sCAM levels and the impairment of arterial compliance, probably
because of a superior ability of A-IM HDL to protect the endothelium.
P478
The Interhelical Sequence Between Helices 7 and 8 of Human
Apolipoprotein A-I Influences High-Density Lipoprotein Subclass
Association and Generation
Ronald Carnemolla, Andrew Djunaidi, Catherine A Reardon, The Univ of Chicago, Chicago,
IL; Jiajun Wang, Wayne State Univ, Detroit, MI; Godfrey S Getz; The Univ of Chicago,
Chicago, IL
Humans exhibit a heterogeneous HDL profile (i.e. HDL
2
and HDL
3
), in contrast to other species
(e.g. mouse) that display a monophasic profile. HDL
2
is thought to be more atheroprotective
e-120 Arterioscler Thromb Vasc Biol June 2007
by on March 25, 2011 atvb.ahajournals.org Downloaded from
than HDL
3
. Human apoA-I is a major determinant of the biphasic distribution of HDL. ApoA-I is
made up of 10 repeating amphipathic -helices (11 or 22 a.a.) interrupted in most cases by
proline residues, except between helices 7 and 8. We generated apoA-I mutants in which the
interhelical sequence (IHS) between helices 7/8 (7 residues) was substituted with each of the
remaining IHS found in huA-I. The mutant and wild-type (wt) proteins did not significantly differ
in physical and functional properties examined. In in vitro association assays involving
recombinant huA-I and HDL subclasses, we observed that wt huA-I has a near equal affinity
for HDL
2
(1.42M) and HDL
3
(1.63M). In contrast, huA-I mutants with IHS3/4 or IHS9/10
replacing IHS7/8 preferentially associated with HDL
2
and displayed higher K
d
association values
for HDL
2
(0.49 M and 0.27M respectively) versus HDL
3
(11.2M and 4.6M respectively).
The huA-I mutant in which IHS4/5 replaced IHS7/8 preferentially associated with HDL
3
and
displayed a higher K
d
association constant for HDL
3
(0.63M) versus HDL
2
(5.4M). To
examine if the mutant huA-I proteins generate nascent HDL particles of different densities, we
stably transfected rat hepatoma cells expressing wt and mutant huA-I proteins. Using cell lines
expressing comparable levels of huA-I protein and species-specific huA-I antibodies, we found
that wt huA-I forms two major nascent HDL particles with peaks at 1.0901 g/mL and 1.1557
g/mL. In contrast, huA-I (IHS7/8IHS3/4) generates a single nascent HDL between 1.1037
1.1191 g/mL. The peak of the nascent HDL formed by huA-I (IHS7/8IHS4/5) mutant is 1.1362
g/mL, but also appears to have a shoulder at a lower density. The distribution of the
endogenous rat apoA-I on the nascent HDL followed that of the huA-I protein in each case. In
conclusion, the IHS placed between helices 7 and 8 in huA-I can influence affinity for HDL
subclasses and can modify the type of HDL profile secreted from stably transfected rat
hepatoma cells. This focuses new attention on the interhelical sequences in controlling the
properties of apoA-I.
P479
Effect of Oxidation on Rate of Apolipoprotein A-IHDL Exchange
Giorgio Cavigiolio, Michael N Oda; Childrens Hosp Oakland Rsch Institute, Oakland, CA
In the atherosclerotic plaque, reverse cholesterol transport begins with ABCA1 transporter-
mediated transfer of cholesterol from cholesterol-laden macrophages to lipid-poor apolipopro-
tein A-I (apoA-I). Lipid-associated apoA-I is a very poor substrate for cholesterol uptake from
ABCA1; interestingly, the vast majority of circulating apoA-I is lipid associated on spherical HDL.
Curtiss et al. have proposed that cholesterol efflux to HDL is facilitated primarily by the release
of lipid-poor apoA-I from spherical HDL and as a result may be a key event in the mobilization
of cholesterol from the atherosclerotic plaque. Oxidized HDL (specifically apoA-I) is observable
in patients with established atherosclerosis and in vitro severely impairs cholesterol efflux by
ABCA1. We hypothesized that oxidation of apoA-I impairs its ability to exchange between
lipid-free and lipid-bound states, which leads to depletion of lipid-poor apoA-I as substrate for
ABCA1. The rate of apoA-I - HDL exchange was measured, using a fluorescent apoA-I
construct, which exhibited fluorescence resonance energy transfer (FRET) only in the lipid-poor
state. We have used this construct to assay the rate of apoA-I - HDL exchange for 9.6 nm and
7.8 nm rHDL particles. Rates of exchange were monitored by addition of excess lipid free
apoA-I, which displaces fluorescent apoA-I off rHDL composed of lipid and fluorescent apoA-I
construct. The rate of AEDANS fluorescence intensity increase is a measure of the apoA-I - HDL
exchange, as lipid bound-fluorescent apoA-I exchanged into the lipid-poor state. The rates of
7.8 nm and 9.6 nm rHDL exchange require 50 seconds and 18 minutes to achieve 50%
transfer, respectively, indicating that apoA-I - HDL exchange rates on 9.6 nm rHDL are at least
18-fold slower than on 7.8 nm HDL. These results confirm that our assay is sensitive enough
to measure biologically relevant differences in HDL exchange rate properties. When myelo-
peroxidase oxidized apoA-I was used to displace the fluorescent apoA-I variant from HDL, we
found that oxidized apoA-I - HDL exchange rates were reduced. While this result requires
further validation, it is an indication that impairment of apoA-I exchangeability by oxidation may
be a key molecular mechanism for oxidation in atherogenesis.
P480
Electronegative LDL Circulating in Smokers Impairs Endothelial Progenitor
Cell Differentiation by Inhibiting Akt Phosphorylation via the LOX-1
Receptor
Daming Tang, Jonathan Lu, Jeffrey P Walterscheid, Hsin-Hung Chen, Baylor College of
Medicine, Houston, TX; David Engler, Univ of Texas-Houston Med Sch, Houston, TX; Tatsuya
Sawamura, National Cardiovascular Rsch Institute, Osaka, Japan; Henry J Pownall, Marco
Marcelli, Chao-Yuh Yang, Chu-Huang Chen; Baylor College of Medicine, Houston, TX
Endothelial progenitor cells (EPCs) play an important role in endothelial regeneration and
vasculogenesis. Chronic smoking is associated with reduced EPCs, but the mechanism was
unclear. Here, we examined how cigarette smoking inhibits EPC differentiation through the
bioactivity of circulating electronegative low-density lipoprotein (LDL). Using anion-exchange
chromatography, we purified the LDL of chronic smokers into 5 subfractions, L1-L5, where L5
is the most electronegative. L5 was not present in matched, nonsmoking subjects. Under
normal conditions, human circulating monocytes expressed CD31 and KDR by day 3 and
differentiated into mature EPCs by day 21 in culture. L5 inhibited CD31 and KDR expression and
EPC differentiation, whereas L1-L4 had no effect. L5 also inhibited telomerase activity to
accelerate EPC senescence, as demonstrated by enhanced cytosolic acidic -galactosidase
activity. These effects inversely correlated with reduced Akt phosphorylation. Transfection of
EPCs at day 3 with dominant-negative Akt constructs (Akt-DN) mirrored the effects of exposure
to L5 by inhibiting CD31 and KDR expression, stalling EPC differentiation, and promoting early
senescence. In contrast, transfection with constitutively-active Akt (Akt-CA) rendered the EPCs
resistant to L5 exposure, allowing for normal maturation. Moreover, L5 upregulated the
lectin-like oxidized LDL receptor-1 (LOX-1) expression in EPCs at day 3, and pretreatment of
EPCs with JTX20, a LOX-1 neutralizing antibody, prevented L5-mediated inhibition of EPC
differentiation. Furthermore, internalization of DiI labeled L5 (DiI-L5) at the plasma membrane
was blocked by JTX20. These findings suggest that cigarette smoking is associated with the
formation of L5, which inhibits EPC differentiation by impairing Akt phosphorylation via the
LOX-1 receptor.
P481
Intensive Statin Therapy from Vulnerable Plaque to Prevention of
Progression of CAD in Patients with High Levels of Lp(a)
Giorgio Corinaldesi, Medicina Generale ASL 7, Ancona, Italy; Christian Corinaldesi; Universita`
Politecnica Delle Marche, Ancona, Italy
Many cardiovascular risk factors for CAD show familiar clustering, in particular we have studied
a family from Jordany where 9 people showed a severe increase of Lp(a) 40mg/dl with an
high recurrence of multivessel CAD, stroke, and lower limbs vascular disease; all of them
showed a genetic variant of gene (6q2.62.7), consisting on a polymorphism C93T, presence
of allele 8, and presence of small isoforms, which are clearly linked to a increased synthesis
of Lp(a). It has been demonstrated that Lp(a) levels greater than 30 mg/dl are responsible for
about 15% of CAD, for this reason it seems to constitute an independent risk factor of
athero-thrombotic disease. Notoriously there are not drugs that can lower the levels of Lp(a),
except rapamycin, high dosed probucol, and l-asparaginase, for this reason we have used high
doses of rosuvastatin (40mg/die). Statins, other than inhibiting HMGCoA reductase, present a
pleiotropic effect. In fact, they inhibit the synthesis of isoprenoid units (farnesol, and geraniol)
with an anti-proliferative effect, they also inhibit MMP-9, they inhibit TF, they increase e-NOS
synthesis, and they have an anti-oxidant effect. Statins are also characterised by an
anti-inflammatory property, as they reduce level of VCAM-1, ICAM-1, E-selectin, IL-6, PCR.
Among all our patients (aged between 9 and 54 years) two showed an anamnesis of recurrent
angina and coronary artery by-pass grafting, two of them stable angina, three of them were
oligosymptomatics; all of our patients had long coronary stenosis (18 mm), multiple stenosis
in vessel with a diameter lower than 2.5mm, two patients had previous acute myocardial
infarction and bifurcation stenosis. In the present study all patients were treated with
rosuvastatin 40 mg/die for 18 months, and we have observed an average reduction of
plasmatic concentration of Lp(a) of 24%, with a significant decrease of PCR (35%), MMP-9,
MMp-12 and elastase; all these effects appeared to be independent to the LDL-cholesterol
lowering effects (38 %). These findings appear to be very suggestive for the treatment of these
very problematic patients and for the control of coronary stenosis progression.
P482
Evidence of Increased Secretion of Apolipoprotein B48-Containing
Lipoproteins in Subjects with Type 2 Diabetes
Jean-Charles Hogue, CRML, CHUL du CHUQ, Que bec, Canada; Benot Lamarche, INAF, Laval
Univ, Que bec, Canada; Andre J Tremblay, Jean Bergeron, Claude Gagne , Patrick Couture;
CRML, CHUL du CHUQ, Que bec, Canada
Patients with type 2 diabetes have high levels of triglyceride-rich lipoproteins (TRL), including
apolipoprotein (apo) B-48-containing TRL of intestinal origin but the mechanism leading to
overaccumulation of these lipoproteins remains to be fully elucidated. Therefore, the objective
of this study was to examine the in vivo kinetics of TRL apoB-48 in type 2 diabetic subjects
(N13) and in controls (N11) using a primed-constant infusion of [5,5,5-D
3
]-L-leucine for 12
hours in the fed state. Diabetic subjects had significantly higher fasting glycemia (8.72.0 vs.
4.80.4 mmol/L, P0.0001), higher fasting insulinemia (8745 vs. 5714 pmol/L, P0.03),
higher plasma TG (4.601.73 vs. 1.230.67 mmol/L, P0.0001) and lower HDL-C levels
(0.860.16 vs. 1.190.24 mmol/L, P0.0009) than controls. As compared with controls,
diabetic subjects had a higher TRL apoB-48 pool size (16265 vs. 3223 mg, P0.0001),
reduced TRL apoB-48 fractional catabolic rate (5.81.6 vs. 7.8 2.0 pools/d, P0.01) and
elevated TRL apoB-48 production rate (PR) (11.05.2 vs. 3.01.9 mgkg
-1
d
-1
). Furthermore,
multiple linear regression analyses revealed that the diabetic/control status was an indepen-
dent predictor of TRL apoB-48 PR and represented nearly 35% of its variance. These results
suggest that overaccumulation of apoB-48-containing TRL seen in patients with type 2 diabetes
is not only due to decreased catabolism of TRL apoB-48 but also to increased production rate
of these lipoproteins.
P483
Estrogen Receptor 1 (ESR1) Polymorphism Is Associated with Plasma
Triglyceride Metabolism in Caucasians of the Rochester Family Heart Study
Kathy L E Klos, Univ of Texas Health Science Cntr at Houston, Houston, TX; Robert E
Ferrell, Univ of Pittsburgh, Pittsburgh, PA; Alanna C Morrison; Univ of Texas Health Science
Cntr at Houston, Houston, TX
Sex steroid hormones, mediated by estrogen receptors, are thought to influence plasma lipid
profile through the control of lipase and apolipoprotein (apo) gene expression. We hypothesized
that genotypes of the ESR1 SNPs rs2234693 (PvuII) and rs1801132 would predict measures of
triglyceride (TG) metabolism in the community-based Rochester Family Heart Study of
pedigrees ascertained without regard for health through households with two or more children
enrolled in primary and secondary schools of Rochester, MN. Plasma TG was measured by
standard enzymatic methods. Plasma ApoCII, ApoCIII, and ApoE were measured by radioim-
munoassay. Individuals who had not fasted for at least 8 hours prior to the clinic evaluation
were excluded. Plasma variables were log transformed, and adjusted for age and BMI within
gender (children separately from adults) prior to analysis. A generalized estimating equation
(GEE) approach was used to test for association among related children. A general linear model
was used to evaluate association in unrelated individuals of the parent and grandparent
generation strata. In female children, rs1801132 rare allele homozygotes had higher levels of
all four traits compared to other genotypic classes (p0.01). The female rs1801132 rare allele
homozygotes of the parent generation had higher levels of ApoC-II, ApoC-III and TG, but
p-values were only suggestive (p0.13, 0.05 and 0.07 respectively). In males, rs2234693 was
associated with ApoC-II, ApoC-III and TG levels in the parent generation (p0.01) with higher
levels seen in rare allele homozygotes. In males of the grandparent generation, rare rs2234693
allele homozygotes had higher TG levels (p0.01) and trended towards higher ApoC-II and
ApoC-II levels (p0.13 and 0.14, respectively). Among these individuals, DNA variation in the
2007 ATVB Poster Presentations e-121
by on March 25, 2011 atvb.ahajournals.org Downloaded from
ESR1 gene region appears to influence measures of plasma TG metabolism in a gender- and
age-specific manner.
P484
Lecithin:Cholesterol Acyl Transferase Can Rescue the Abnormal Phenotype
Produced by the Natural Apolipoprotein A-I Mutations (Leu141Arg)
Pisa
and
(Leu159Arg)
FIN
Georgios Koukos, Boston Univ Med Sch, Boston, MA; Angeliki Chroni, National Cntr for
Scientific Rsch Demokritos, Athens, Greece; Adelina Duka, Boston Univ Med Sch, Boston,
MA; Dimitris Kardassis, Univ of Crete, Heraklion, Greece; Vassilis I Zannis; Boston Univ Med
Sch, Boston, MA
Objective: To explain the etiology and find mode of therapy of low HDL observed in humans
harboring two natural apoA-I mutations, (Leu141Arg)
Pisa
and (Leu159Arg)
FIN
. Methods and
Results: We have generated recombinant adenoviruses expressing apoA-I(Leu141Arg)
Pisa
and
apoA-I(Leu159Arg)
FIN
and studied the properties of the mutant proteins in vitro and in vivo. Both
mutants were secreted efficiently from cells but had diminished capacity to activate LCAT in
vitro. Adenovirus-mediated gene transfer of either of the two mutants in apoA-I deficient mice
(apoA-I
-/-
) resulted in greatly decreased total plasma cholesterol, apoA-I, high density
lipoprotein (HDL) cholesterol levels, cholesteryl ester to total cholesterol ratio (CE/TC),
preponderance of pre1-HDL and small size 4-HDL particles and generated only few
spherical HDL particles, as compared to mice expressing WT apoA-I. Simultaneous treatment
of the mice with adenoviruses expressing either of the two mutants and human LCAT,
normalized the plasma apoA-I, HDL cholesterol levels, and the CE/TC ratio, restored normal
pre- and -HDL subpopulations and generated spherical HDL. Conclusion: The study
establishes that apoA-I(Leu141Arg)
Pisa
and apoA-I(Leu159Arg)
FIN
inhibit an early step in the
biogenesis of HDL due to inefficient esterification of the cholesterol of the pre1-HDL particles
by the endogenous LCAT. Both defects can be corrected by treatment with LCAT.
P485
Identification and Molecular Characterization of New PCSK9 Missense
Mutations Associated with Familial Hypercholesterolemia
Vivienne Homer, Canterbury Health Laboratories, Christchurch, New Zealand; David Marais,
Univ of Cape Town, Cape Town, South Africa; Andrew Laurie, Canterbury Health
Laboratories, Christchurch, New Zealand; Francesca Charlton, The Heart Rsch Institute,
Camperdown, Australia; David Sullivan, Royal Prince Alfred Hosp, Camperdown, Australia;
Philip Barter, Kerry-Anne Rye, The Heart Rsch Institute, Camperdown, Australia; Peter
George, Canterbury Health Laboratories, Christchurch, New Zealand; Gilles Lambert; The
Heart Rsch Institute, Camperdown, Australia
Proprotein Convertase Subtilisin Kexin Type 9 (PCSK9) is a bona fide inhibitor of the
LDL-receptor. In humans, PCSK9 gain of function mutations are associated with FH, whereas
mutations inactivating PCSK9 are associated with reduced plasma LDL and cardiovascular
events. Characterization of the naturally occurring mutations reported to date has provided
some insights into PCSK9 mechanisms of action but it has not been possible to distinguish the
phenotypic effect of some gain of function from some loss of function mutations based on their
autocatalytic cleavage and secretion pattern. In the present study, we analysed the PCSK9
exons and intronic junctions of FH patients found to be non LDL-receptor or apolipoprotein
B100 mutation carriers. The previously reported S127R French mutation was found in a
South-African family, whereas new heterozygous missense mutations D129G and A168E were
found in two families from New Zealand. Except for the A168E, these mutations modify a highly
conserved residue. Segregation with the FH phenotype was also incomplete in the A168E
family. PCSK9 overexpression studies in HuH7 hepatoma cells shows that both S127R and
D129G missense PCSK9 mutants have 75% reduced autocatalytic activity compared to wild
type, whereas the A168E mutant is processed normally. The S127R and D129G mutants were
not secreted in the culture media, unlike both the A168E mutant and wild type PCSK9. Cellular
LDL binding was decreased by 2530% in cells overexpressing S127R and D129G mutants
compared with those overexpressing wild-type PCSK9. Overexpression of the A168E mutant
resulted in a non-significant 10% decrease in cellular LDL binding vs. wild-type. The cell
surface LDL receptor levels paralleled those of cellular LDL binding. Our study indicates that
(1) the region within the prodomain of PCSK9 encompassing the S127 and D129 residues is
critical for PCSK9 autocatalytic activity and secretion, and that (2) two non-secreted naturally
occurring mutants of PCSK9 inhibit LDL receptor expression and activity, suggesting that
PCSK9 mediated inhibition of the LDL receptor in the liver occurs intracellularly.
P486
Antisense Inhibition of Apolipoprotein B-100 Significantly Reduces LDL
Cholesterol and Glucose Levels in Ob/Ob Mice
Kristina Lemonidis Tarbet, Charles P Whipple, Mark J Graham, Rosanne M Crooke; Isis
Pharmaceuticals, Carlsbad, CA
In previous studies, we demonstrated that intraperitoneal (i.p.) administration of a mouse-
specific apolipoprotein B-100 (apoB-100) antisense oligonucleotide (ASO), ISIS 147764,
produced dose- and time-dependent reductions in hepatic apoB mRNA and protein and serum
apoB-100, total cholesterol and LDL-C levels in hyperlipidemic mice. These pharmacological
effects have been confirmed in multiple animal models and most importantly, in humans during
Phase 1 and 2 clinical trials. As a large number of individuals with diabetes also suffer from
dyslipidemia and coronary artery disease, we administered ISIS 147764 to ob/ob mice on a
high fat/cholesterol diet in order to determine the effects of inhibiting apoB-100 in
insulin-resistant mice. Treatment of these animals with 50 mg/kg/wk of ISIS 147764 for 6
weeks resulted in a 65% reduction in hepatic apoB mRNA with a commensurate reductions in
total cholesterol (34%), VLDL-C (61%), LDL-C (55%) and serum triglycerides (21%). Serum
glucose levels were also reduced by 33% when compared to those in saline-treated diabetic
mice. As observed in other murine hyperlipidemia models, reductions in serum apoB-100 did
not cause hepatic steatosis as determined by Oil Red O staining of livers and quantitation of
liver triglyceride levels. Furthermore, ASO administration in these mice was well tolerated.
These and other data from our laboratory and the clinic continue to validate the safety and
efficacy of antisense inhibitors to apoB-100 in various dyslipidemic states.
P487
The N-Terminal 50 Amino Acid Residues of the
1
Domain of
Apolipoprotein B Induce Instability in Lipoprotein Particle Assembly
Medha Manchekar, Univ of Alabama at Birmingham , Birmingham, AL; Richa Kapil, Zhihuan
Sun, Jere P Segrest, Nassrin Dashti; Univ of Alabama at Birmingham, Birmingham, AL
Apolipoprotein B (apoB)-100, essentially the only protein component of the atherogenic LDL,
has a pentapartite structure, NH2-
1
-
1
-
2
-
2
-
3
-COOH. The domains contain multiple
amphipathic strands and the domains contain multiple amphipathic helices. The
1
domain, like lamprey lipovitellin (LV), is a globular composite of -helices and -sheets. We
previously proposed that the apoB particle assembly is initiated when the
1
domain,
comprising of the first 1000 amino acid residues of apoB (designated apoB:1000), folds into a
LV-like lipid pocket to form the apoB lipid pocket. We demonstrated that in stable
transformants of McA-RH7777 cells, apoB:1000 is secreted as a stable monodisperse
phospholipid-rich particle. We also showed that apoB:1200, containing 200 residues of the
1
domain of apoB, is secreted predominantly as a lipid-poor particle with only a fraction of the
protein as a relatively lipid-rich particle. To map the effect of each domain of amphipathic
strands within the N-terminal region of the
1
domain on the relative levels of secretion of large
versus small particles, we made sequential truncations of region between apoB:1200 and
apoB:1000 to produce apoB:1050, apoB:1100, and apoB:1150. Characterization of the secreted
particles by metabolic labeling of stable transformants of McA-RH7777 cells with [
3
H]glycerol
and their isolation by non-denaturing gradient gel electrophoresis, demonstrated that the
presence of only 50 amino acid residues of the N-terminal region of the
1
domain causes
instability in the particle. Thus, in contrast to apoB:1000, apoB:1050 was secreted in two
forms, a large lipidated particle and a small lipid-poor particle. Inclusion of residues 1050 to
1100 caused further destabilization in the particle. ApoB:1100 appeared to form at least four
particles, suggesting that the domain between residues1050 and 1100 might be more
destabilizing than the previous 50 residues. ApoB:1150 formed particles that were similar to
those formed by apoB:1050, suggesting that the domain between residues 1100 and 1150
might partially restore particle stability. In conclusion, our results suggest that not all the
sequences in the N-terminal 200 residues of the
1
domain are equal in their effects on particle
stability.
P488
ABCA1 and SR-BI Are Significant Contributors to in Vivo Reverse
Cholesterol Transport from Macrophages
Ming-Dong Wang, Vivian Franklin, Michel Gallant, Yves L Marcel; Univ of Ottawa Heart
Institute, Ottawa, Canada
BACKGROUND: Cholesterol transporters, ABCA1, ABCG1 and SR-BI, are key mediators of
cholesterol efflux to apoA-I and HDL, which represents the first step of reverse cholesterol
transport (RCT) in vivo. However the individual contributions of each transporter to in vivo RCT,
defined here as transport from macrophages to liver and further into bile have not been
thoroughly measured. Here we first measured the in vivo RCT from cholesterol labeled
macrophages with either ABCA1 or SR-BI deficiency. METHODS AND RESULTS: Bone marrow
derived macrophages from abca1 (-/-) or sr-b1 (-/-) or control mice were labeled with
3H-cholesterol-AcLDL or 3H-cholesterol-LDL and injected into control mice. After injection,
return of 3H-cholesterol from labeled abca1 (-/-) or sr-b1(-/-) macrophages to serum, liver, bile
and feces, was measured. ABCA1 deficiency in macrophages reduced cholesterol return from
macrophages either labeled with LDL or acLDL by up to 50% percent compared to control cells,
dependent on the readout (plasma, liver or bile radioactivity). SR-BI deficiency only reduced
cholesterol return from LDL labeled macrophages, but not from acLDL labeled macrophages,
consistent with the role of SR-BI in efflux to HDL and not to apoA-I. CONCLUSION: These results
indicate that both ABCA1 and SR-BI play significant roles in efflux and in vivo RCT, consistent
with their protective functions against development of atherosclerotic lesions.
e-122 Arterioscler Thromb Vasc Biol June 2007
by on March 25, 2011 atvb.ahajournals.org Downloaded from
P489
Diurnal Transcriptional Regulation of Microsomal Triglyceride Transfer
Protein and Plasma Lipid Levels
Xiaoyue Pan, M Mahmood Hussain; SUNY Downstate Med Cntr, Brooklyn, NY
Several cardiovascular diseases such as atherosclerosis and coronary heart disease exhibit
circadian variations. High plasma lipid levels are risk factors for these diseases and plasma
lipids also show diurnal variations. Our aim was to identify molecular mechanisms controlling
diurnal variations in plasma lipid levels in rats and mice maintained in a 12-h photoperiod with
free access to chow diet. Plasma triglyceride and cholesterol levels were high in the dark than
in the light phase in these animals. These variations were mainly due to changes in
apoB-lipoproteins, as HDL levels did not show circadian rhythm. Intestinal lipoprotein
production studies revealed that the absorption of [
3
H]triolein or [
3
H]cholesterol was high at
24:00 h than at 12:00 h. Similar observations were made using in situ rat intestinal loops
indicating that intestinal lipoprotein production, independent of gastric emptying, shows diurnal
variation. Since MTP is critical for apoB-lipoprotein biosynthesis, we investigated intestinal MTP
activity and protein levels at different times in rats and mice. Both MTP activity and protein
showed diurnal variations and these changes were correlated with changes in the biosynthesis
of MTP. MTP synthesis by enterocytes isolated at 24:00 h was higher than those isolated at
12:00 h. To assess whether diurnal variations in MTP synthesis occurred at the transcriptional
level, we measured steady state mRNA levels. MTP mRNA levels showed diurnal variations and
were higher in the dark phase than in the light phase. In contrast, intestinal and liver GAPDH
protein and mRNA levels did not change significantly during 24 h. To understand mechanisms
underlying the changes in mRNA, w measured gene transcription rates in the nuclei isolated
from intestinal mucosa at 12:00 and 24:00 h. The mttp gene transcription was 4-fold higher
in the midnight than at midday. To examine tissue specificity, we also measured MTP in the
liver. Similar to the small intestine, rat and mouse hepatic MTP activity, protein, and mRNA
levels exhibited diurnal variations. These studies show that the intestinal and liver MTP
expression undergoes diurnal regulation at the transcriptional level and suggest that changes
in MTP contribute to diurnal variations in plasma lipid levels.
P491
Quantifying the Effects of Cholesteryl Ester Transfer Protein on
Lipoproteins in Vitro with a Mathematical Model
Laura K Potter, GlaxoSmithKline, Rsch Triangle Park, NC; Dennis L Sprecher, Max C Walker,
Frank L Tobin; GlaxoSmithKline, King of Prussia, PA
Cholesteryl ester transfer protein (CETP) plays a pivotal role in reverse cholesterol transport,
although the exact consequences of its activity on lipoprotein metabolism are not as yet well
understood. Using mathematical modeling, the dynamics of CETP activity and the effects of
CETP inhibition can be explored in silico to gain additional insight on the plausibility of the
transfer mechanisms and to motivate new experiments that will further elucidate these
mechanisms. We have developed a mathematical model for in vitro CETP activity that captures
the kinetics of CETP binding and the lipid exchange with lipoproteins. The model includes the
binding and transfer dynamics of CETP, different classes of lipoprotein particles, and the
changes in triglyceride (TG) and cholesterol ester (CE) contents of the lipoproteins. The model
is built upon the conceptual shuttle model where the activity of CETP facilitates the transfer
of TG and CE between lipoproteins by swapping a lipid molecule with a single lipoprotein at a
time. Equilibrium is attained when the core ratios of CE and TG in the interacting lipoproteins
are equal. Model parameters were estimated using standard optimization techniques that
require the model to simultaneously fit eight published experimental data sets. The resulting
simulations reasonably match the in vitro data, which include time course measurements and
dose response data with respect to CETP and lipid levels. The calibrated model was used to
simulate CETP activity and CETP inhibition in vitro. Simulations of isolated HDL, LDL and VLDL
incubated with purified CETP predict that CE and TG transfers increase with particle size, with
the greatest sensitivity to changes in VLDL particle size. The model also was used to compare
theoretical mechanisms of action for CETP inhibition, including binding of inhibitor to CETP only,
inhibitor binding to one or more lipoprotein classes, and inhibitor binding to CETP and
subsequent binding to lipoproteins. The model predicts that the latter mechanism, similar to a
noncompetitive inhibitor, is the most efficient of these mechanisms at blocking CETP transfer
activity. Such predictions exemplify how this model can be used in silico to help explore and
understand the dynamics of CETP activity and inhibition.
P492
Overexpression of ER60 Induces Apolipoprotein B Degradation and the
Secretion of an 80 kDa Fragment in HepG2 and McRH 7777 Cells: Role of
ER60 in Nonproteasomal apoB Turnover
Wei Qiu, Rita Kohen Avramoglu, Ning Huang, Khosrow Adeli; The Hosp for Sick Children,
Toronto, Canada
We have previously shown that overexpression of ER60 in HepG2 cells increases intracellular
degradation of apolipoprotein B (apoB) (Qiu et al., Biochemistry 2004, 43:48194831). Here we
report the identification of an ER60-induced proteolytic fragment of apoB that is secreted
following ER60 overexpression. ApoB stability was found to be significantly reduced with ER60
overexpression in McA-RH7777, HepG2 or COS-7 cells transiently transfected with human
apoB48. Interestingly, an 80 kDa fragment of apoB was detected and found to be
predominantly secreted into the media of both McA-RH7777 and HepG2 cells following ER60
overexpression. The generation and secretion of the 80 kDa apoB fragment was detected in the
presence or absence of MTP based on MTP transfection studies in COS-7 cells. Importantly,
generation of the 80 kDa apoB fragment was only observed with ER60 overexpression and not
other ER chaperones such as GRP78, which has been shown to promote proteasomal apoB
degradation. The secreted 80 kDa apoB fragment was insensitive to the inhibition of ALLN,
latacystein, MG132 or pCMB, which previously showed partial inhibitory activity toward a 50
kDa apoB fragment generated in permeabilized HepG2 cells overexpressing ER60. Using [35S]
methionine labeling, immunoprecipitation and immunoblotting, we confirmed that the secreted
80 kDa apoB fragment is derived from the N-terminus of the apoB molecule and may be
co-secreted with ER60 into the culture medium. Taking together, our data supports a novel
ER60-mediated degradative pathway that leads to the generation of a secretory N-terminally-
derived 80 kDa apoB fragment. We postulate that ER60 induces, directly or indirectly, an
ER-associated proteolytic mechanism leading to the generation and release of an 80 kDa apoB
fragment.
P493
Acquisition of Triacylglycerol Transfer Activity by Microsomal Triglyceride
Transfer Protein During Evolution
Paul Rava, M Mahmood Hussain; SUNY Downstate Med Cntr, Brooklyn, NY
Microsomal triglyceride (TG) transfer protein (MTP) is essential for the assembly of neutral lipid
rich apolipoprotein B (apoB)-lipoproteins. Previously we reported that the Drosophila MTP does
not transfer TG but transfers phospholipids. In contrast, human MTP transfers both lipids. To
explore the acquisition of TG transfer activity by MTP during evolution, we obtained protein
sequences from birds, amphibians and insects that were homologous to human MTP.
Sequence comparison revealed a MTP specific sequence. Using this sequence we identified
homologous proteins in nematodes. Based on phylogenetic analysis, we divided these proteins
into four groups (mammals, fish, insects, and nematodes). Structural analysis demonstrated
that all MTP possess similar secondary and tertiary structures. In addition to their structural
similarities, MTP homologs were found to share several biochemical properties. Expression of
candidate proteins from each group as FLAG chimeras showed that all proteins were associated
with protein disulfide isomerase in the endoplasmic reticulum as well as in the Golgi, and
supported assembly and secretion of apoB-lipoproteins. In vitro lipid transfer assays revealed
that invertebrate MTP (nematode and insect) were unable to transfer TG in vitro. However these
studies demonstrated that TG transfer activity first appeared in fish, matured during the
evolution of amphibians and birds, and was conserved in mammals. We conclude that TG
transfer activity was acquired during a transition from invertebrates to vertebrates coincident
with the use of apoB-lipoproteins capable of carrying large amounts of neutral lipids. We
speculate that this acquisition might have provided a significant advantage to the evolution of
larger and more complex organisms.
P494
Adenoviral Overexpression of Hormone-sensitive Lipase and Adipose
Triglyceride Lipase Promotes Fatty Acid Oxidation and Reduces Hepatic
Steatosis Without Affecting VLDL Secretion in ob/ob Mice
Brendan N Reid, Gene P Ables, Oleg A Otlivanchik, Ira J Goldberg, Robert F Schwabe,
Columbia Univ, New York, NY; Streamson C Chua, Jr, Albert Einstein College of Medicine,
New York, NY; Li-Shin Huang; Columbia Univ, New York, NY
Hepatic steatosis is often associated with the insulin-resistant state. We demonstrated that
hormone-sensitive lipase (HSL) and adipose triglyceride lipase (ATGL), two enzymes critical for
lipolysis in adipose tissues, could mobilize hepatic triglycerides (TG) in vivo and in vitro without
adverse effects. Adenoviral overexpression of HSL and ATGL in ob/ob mice decreased lipid
droplet sizes and reduced liver TG mass by 5060% but had no effects on fasting plasma TG
levels or TG and apoB secretion rates. There was no apparent effect on the expression of genes
involved in fatty acid (FA) uptake (CD36) and synthesis (FAS). Plasma 3-hydroxybutyrate levels
and the expression of FA oxidation genes (PPARa, AOX, CPT1) were increased in these animals,
suggesting an increase in beta-oxidation. As in ob/ob mice, adenoviral overexpression of HSL
and ATGL in oleate-fed rat hepatoma McA-RH7777 cells reduced cellular TG by 60%, which
could be attributed to increased beta-oxidation and reduced TG synthesis. HSL overexpression
also reduced cellular diacylglycerol as well as synthesis and secretion of cholesteryl esters in
McA-RH7777 cells, whereas ATGL overexpression increased cellular diacylglycerol. These
observations are consistent with the respective substrate specificities of these two enzymes.
HSL overexpression also increased TG secretion and decreased intracellular apoB degradation
resulting in an increase in apoB secretion, an effect unseen in vivo. In summary, hepatic
overexpression of HSL or ATGL promotes FA oxidation and reduces hepatic steatosis without
affecting VLDL secretion; the discrepant in vivo and in vitro effects of HSL overexpression on
VLDL secretion possibly result from differences in cellular machinery available in vivo or in vitro.
Overall, this report identifies HSL and ATGL as potential therapeutic targets for ameliorating
hepatic steatosis associated with insulin resistance and obesity.
P495
Postprandial Lipid Effects of a 1,3 Diacylglycerol Oil versus Triacylglycerol
Oil in an Insulin-Resistant Population
Gissette Reyes, Koichi Yasunaga, Eileen Rothestein, Wahida Karmally, Stephen F Holleran,
Rajasekhar Ramakrishnan, Henry Ginsberg; Columbia Univ Med Cntr, New York, NY
Background: Postprandial (PP) hypertriglyceridemia is common in individuals with insulin
resistance with or without concomitant type 2 diabetes mellitus, and has been associated with
the presence of both coronary and carotid artery atherosclerosis. Studies in rodents and
humans suggest that fatty acids from dietary DAG are not efficiently incorporated into
chylomicrons after absorption from the intestinal lumen. Some studies with diets enriched in
1,3-diacylglyerol (DAG) oil have shown reduced PP triglyceride (TG) levels compared with diets
containing triacylglycerol (TAG) oil. It is important to identify new diet approaches that would
benefit populations with dyslipedemia. Methods: We enrolled 25 insulin resistant (HOMA
2.5), non-diabetic subjects in a double-blind, randomized crossover trial to test acute and
chronic effects of a DAG diet compared with a TAG diet. Subjects received DAG oil or TAG oil,
in food products, for five weeks and were then crossed-over to the other oil after a one-week
washout period. At the end of each five-week diet period, subjects had two separate PP fat-load
tests, one with each oil. Results: PPTG areas under the curve (AUC) over 8 hours (mean SD
2007 ATVB Poster Presentations e-123
by on March 25, 2011 atvb.ahajournals.org Downloaded from
h.mg/dl) were determined for each of the four postprandial studies. Fasting lipids were similar
after 5 weeks of a DAG or TAG diet. We found no statistically significant acute or chronic effects
of DAG in our two primary comparisons. Thus, neither DAG PPTG on chronic TAG (AUC: 503
439) nor TAG PPTG on chronic DAG (AUC: 517 638) was significantly different from TAG
PPTG on chronic TAG (AUC: 565 362). In a subgroup-analysis, subjects with fasting TG levels
lower than 200mg/dl had significantly smaller AUC during DAG PPTG compared with TAG PPTG
on a TAG background diet (p.02). However, no significant difference in AUCs was found in
the subjects with fasting TG greater than 200mg/dl. Conclusion: We conclude that a DAG
enriched diet had no acute or chronic effects on PPTG in the overall group of insulin resistant
subjects, but we did observe an acute PPTG-lowering effect in subjects with fasting TG less
than 200mg/dl. Further studies are necessary to understand the absence of DAG effects on lipid
metabolism in individuals with high fasting TG.
P496
ApoA-I FIN Partially Prevents Cholesterol Deposition and Inflammation in
Skin and Lymph Nodes of Diet-fed LDLr-/-, ApoA-I-/- Mice
Manish S Bharadwaj, Manal Zabalawi, John S Owen, Michael J Thomas, Wake Forest Univ,
Winston-Salem, NC; Vassilis I Zannis, Adelina Duka, Boston Univ Sch of Medicine, Boston,
MA; Mary G Sorci-Thomas; Wake Forest Univ, Winston-Salem, NC
The mutation L159R apoA-I (apo A-IFIN) is associated with a dominant negative phenotype
which manifests as severe hypoalphalipoproteinemia and an increased risk for atherosclerosis
in humans. To investigate whether this mutation substitutes for the absence of wild-type apoA-I
in hypercholesterolemic mice we conducted the following studies. Mice lacking both mouse
apoA-I and LDL receptor, designated, apoA-I-/-, LDLr-/- DKO were crossed for 7 generations
with mice transgenic for human L159R apoA-I, designated Fin-DKO and compared to
transgenic human wild-type (Wt) apo A-I mice or Wt-DKO. Fin-DKO mice were found to have
a1.5-fold higher total plasma cholesterol but a 10-fold lower plasma apoA-I concentration than
Wt-DKO mice. FPLC analysis showed that HDL cholesterol was dramatically reduced in Fin-DKO
as compared to Wt-DKO mice, while 2-D gels showed that the reduction in HDL was mainly
due to a lack of alpha migrating HDL particles. When Wt-DKO, Fin-DKO and DKO mice were fed
0.1% cholesterol 10% palm oil for 12 wk, DKO mice displayed a previously reported 12-fold
enrichment of cholesterol-enriched macrophages in the skin, while Wt-DKO mice did not. In
diet-fed DKO mice the only other tissue besides skin that was cholesterol enriched were
peripheral lymph nodes as compared to Wt-DKO mice, suggesting a link between cholesterol
accumulation in the lymph nodes and skin. Interestingly, Fin-DKO mice showed only partial
cholesterol enrichment in these tissues despite the very low concentration of the mutant apoA-I
in plasma. These data suggest that even at low plasma concentration apoA-I Fin may partially
protects against events leading to macrophage activation and inflammation.
P497
Effect of Reconstituted HDL on Cholesterol Efflux from Tissues in Vivo
Scott Turner, Jason Voogt, Marc Hellerstein; KineMed, Emeryville, CA
The first step in reverse cholesterol transport (RCT) is efflux of free cholesterol from peripheral
tissues into the bloodstream. Plasma HDL concentrations do not accurately reflect efflux, so a
direct, in vivo, measurement is required. We have developed an in vivo method for quantifying
the efflux of cholesterol from peripheral tissues into the rapidly exchanging cholesterol pool in
blood and liver. Efflux of cholesterol is measured by the constant infusion isotope-dilution
technique during an IV infusion of 13C-cholesterol. In rodent studies, IV infusions of human
reconstituted HDL (rHDL) resulted in an immediate decrease in free cholesterol enrichment and
an increase in cholesterol efflux. Subsequent studies demonstrated that prolonged continuous
infusion of rHDL increased cholesterol efflux in a dose-dependant manner. A 7 hour infusion
of rHDL (19mg/kg/hr, 135mg/kg total) increased cholesterol efflux from 15mg/kg/hr to
20mg/kg/hour. Similarly, a 24 hour infusion of rHDL (12mg/kg/hour, 290mg/kg total) increased
efflux from 15mg/kg/hr to 23mg/kg/hour. Consistent with increased efflux rates, plasma
cholesterol concentrations increased dramatically during rHDL infusions. Results of these
studies support the model of rapid-turnover pool in communication with a large store of extra
hepatic cholesterol. Moreover they demonstrate that cholesterol acceptor capacity (HDL), not
the activity of transporters (ABCA1, etc.), is rate limiting for whole body cholesterol efflux in
rats. Application of this technique in both rodents and humans could provide new insight into
the effects of rHDL treatment on cholesterol efflux and RCT.
P498
Beneficial Effects of Pioglitazone on Lipoprotein Profiles in Nondiabetic
Patients: Initial Clinical Observations
Patricia Vassallo, Rush Univ, Chicago, IL; Ron Blankstein, Morton Arnsdorf, Univ of Chicago,
Chicago, IL; Steven Feinstein; Rush Univ, Chicago, IL
Background: Pioglitazone (PIO) favorably affects lipid patterns in patients (pts) with diabetes
and the metabolic syndrome, but the effects on nondiabetic pts is unknown with limited clinical
trials. We evaluated the short term effects of PIO on the lipoprotein profile of nondiabetics
already on aggressive medical therapy. Methods: Retrospective chart review was used to
identify nondiabetic pts with dyslipidemia who failed to achieve goals despite treatment. Pts
were started on PIO 15mg while other lipid therapies remained constant. Paired t-tests were
used for statistical analysis. Results: 16 pts fulfilled the study criteria. Pts were male with a
mean age of 61 yrs and a mean weight of 221 lbs. At baseline, 100% were treated with both
a statin and ezetimide and 75% were treated with niacin. After mean follow up of 11.2 months,
44% (7/16) of pts shifted their pattern size from type B (small, dense) to type A (large, buoyant).
Mean HDL increased by 13% while LDL particle number decreased by 15%, and LDL particle
size increased by 2% (see figure). There was no significant change in TC, LDL, and TG. No side
effects were reported. Conclusions: The addition of PIO to nondiabetic pts already on niacin,
statins, and ezetimide resulted in a significant increase in HDL and a modest decrease in LDL
particle number and an increase in LDL particle size. Larger randomized clinical trials are
needed to confirm these findings, and if confirmed, to assess the therapeutic benefits.
P499
Measurement of Cholesterol Efflux and Global Reverse Cholesterol
Transport Rates in Vivo with Stable Isotopes
Jason Voogt, Salena Killion, Mohamad Awada, Lisa Murphy, Marc Hellerstein, Scott Turner;
KineMed, Inc., Emeryville, CA
RCT, the transport of cholesterol (C) from peripheral tissues and out of the body, is the only
route for reducing C burden in tissues, including vascular wall macrophages. RCT is the leading
explanation for the cardioprotective effects of HDLc. Accordingly, interventions that stimulate
flux through the RCT pathway represent a therapeutic target. There had not previously been a
method for measuring the first arm of the pathway (efflux of C from tissues) or for measuring
RCT rates in humans. We have developed an in vivo method for quantifying efflux rate and flux
through the global RCT pathway. The method involves non-radioactive (stable) isotopes, is
non-invasive and can be used in humans. Efflux is measured by the isotope dilution principle,
through a constant IV infusion of
13
C
2
-cholesterol. A mono-exponential rise-to-plateau in free
C was observed in humans and rats. Efflux rates were 7.2 1.3 mg/kg/hr in healthy humans,
with a rapidly exchanging free C pool size of 7.6 /- 2.0 g. Intra-individual variability during
-repeat studies was 10%. Efflux rates in rats were 15 2 mg/kg/hr. Administration of an
LXR agonist for 14 days (TO901317, 1020 mg/day) increased efflux rates, and ezetimibe
non-significantly increased efflux. By multiplying efflux rates by the fractional recovery of label
in the stool over 4 -7days, global RCT (flux from tissue cholesterol to fecal sterols) was
determined. Administration of an LXR agonist to rats increased global RCT flux two-fold.
Ezetimibe treatment also increased global RCT in rats. Measurements of de novo cholesterol
synthesis rates in tissues with
2
H
2
O were consistent with increases in RCT. In summary, efflux
rates of C from tissues in humans are 12 g/day, representing a much greater quantitative
value than whole body C balances (1 g/d) or apoB-mediated transport of C (1 g/d).
Anti-atherogenenic interventions alter RCT fluxes in animals. Application of this technique in
rodents and humans could provide new insights into the physiological and pharmacological
control of C efflux and RCT.
P500
EP 80318: A Novel CD36 Ligand with Hypocholesterolemic and
Anti-Atherosclerotic Properties in Apoe-Null Mice
Vale rie L Bessi, Kim Bujold, Sylvie Marleau, Huy Ong; Universite de Montre al, Montre al,
Canada
Introduction. Macrophage scavenger receptor CD36 is known to mediate internalization of
oxidized lipoproteins at the level of the intima in arteries with the formation of foam cells as
the first step of atherosclerotic lesion development. We have previously reported that EP 80317,
a growth hormone-releasing peptide (GHRP) analog with selective CD36 binding properties
exerts significant CD36-dependent anti-atherosclerotic effect in mice. Whether this anti-
atherosclerotic effect is shared by other members of GHRP family is not known. A new GHRP
analog EP 80318 (Atab-D-MeTrp-D-Lys-Trp-D-Phe-Lys-NH
2
) with specific binding affinity
towards CD36 at 2.5 M was used to document anti-atherosclerotic properties in apoE-null
mice used as atherosclerotic murine model. Methods. ApoE-null mice fed a HFHC from 4
weeks old were administered a daily s.c. dose of either one of two selective CD36 ligands, EP
80317 or EP 80318 (300 g/kg) or 0.9% NaCl, from 6 to 18 weeks of age (n 69 per group).
Blood was withdrawn from the subclavian vein and aortas were isolated from the aortic arch
to the iliac bifurcation and cut longitudinally under stereomicroscope. Neutral lipids were
colored by oil red O staining and the percentage of atherosclerotic lesions was determined by
morphometric analysis. Plasma lipids were quantified by enzymatic methods. Results. A
chronic treatment with EP 80318 or EP 80317 reduced the percentage of total aortic lesions
by 30% (p 0.01) and 41% (p 0.01) compared to 0.9% NaCl, respectively. This effect was
associated with a hypocholesterolemia, as shown by a reduction of 31% (p 0.05) of total
plasma cholesterol in mice treated with EP 80318 and of 26 % (p 0.05) for mice treated with
EP 80317 (22.6 2.2 mmol/L in controls, 15.5 1.3 mmol/L and 16.8 2.1 mmol/L in mice
treated with EP 80318 and EP 80317, respectively). In contrast, neither triglycerides, nor HDL
cholesterol plasma concentrations were modulated by either one of the treatments. Conclu-
sion. Our results support the potential application of GHRP derivatives targeting CD36 for the
prevention of atherosclerotic lesions development.
P501
Antibody Response to Several Different Autoantibodies Is Strongly
Associated with Acute Coronary Syndromes
Ivana Burazor, Clinical Cntr, Nis, Serbia and Montenegro; Aristo Vojdani, Immunosciences
Lab. Inc, Los Angeles, CA; Mirko Burazor; Clinical Cntr, Nis, Serbia and Montenegro
Over the last several years, the idea that immune processes play a key role in atherosclerosis
and its complications, acute coronary syndromes (ACS), has received attention. We assessed
e-124 Arterioscler Thromb Vasc Biol June 2007
by on March 25, 2011 atvb.ahajournals.org Downloaded from
the hypothesis that different auto-antibodies might be involved in the plaque formation and
instability and lead to appearance of ACS. Methods and Results: The study included 211
participants; of whom 111 were patients with ACS (60.8 4.57 years of age, 60% males) and
100 were aged matched controls with no known coronary artery diseases - Blood donors.
Patients with previous infection, surgery, trauma or concomitant reumathological diseases
were excluded from the study. Blood was sampled, frozen on - 400C and sent on dry ice to
Immunosciences Lab. Inc (USA) for analyzes. All traditional risk factors were noted. Antibodies
(IgG) against endothelial cells, oxidized LDL, beta 2 glycoprotein 1, plasmin, platelet membrane
glycoprotein and bacterial HSP 70 were determined as well as levels of interleukin 6, high
sensitivity CRP, homocysteine, lipoprotein (a) and protein C. Our data showed significant
prevalence of examined antibodies in patients with ACS. The levels of circulating antibodies and
novel risk factors were significantly higher in patients. Linear regression confirmed that
immune - inflammatory activation and thrombosis might be due to autoimmune reactions. In
conclusion, seropositivity for several auto-antibodies appears to be very sensitive and specific
marker of ACS. It causal involvement and its diagnostic role in ACS and atherothrombosis itself
deserve further study.
P502
Acetaldehyde Increases Endothelial Cell Chemokine mRNA Expression
John P Cullen, David Morrow, Eileen M Redmond; Univ of Rochester Med Cntr, Rochester,
NY
A biphasic effect of alcohol on the incidence of cardiovascular disease has been documented,
where moderate consumption of alcohol exerts a protective effect while chronic alcohol abuse
and/or binge drinking are associated with a higher incidence of cardiovascular disease. The
subtle balance between alcohol and its primary metabolite, acetaldehyde, has a crucial impact
on the physiological consequences of alcohol consumption. Inflammatory mediators have been
shown to play an important role in the progression and destabilization of the atherosclerotic
plaque. We hypothesize that binge alcohol consumption exacerbates cardiovascular disease, in
part by elevated concentrations of its primary metabolite, acetaldehyde, causing an increase in
chemokine expression. Methods: Human umbilical venous endothelial cells (HUVEC), passages
35, were treated with acetaldehyde (5100 M) for 6h. HUVEC MCP-1, TNF and IL-6 mRNA
mRNA levels were determined by quantitative real time PCR (QRTPCR). Results: At acetalde-
hyde concentrations of 5, 10 and 25 M, there was a 2.5 fold significant increase (p0.05,
n4) in TNF mRNA expression. In the presence of 50 M acetaldehyde, TNF mRNA levels
were further increased by 7.851.93 fold (p0.05, n4). Acetaldehyde at concentrations
25 M had no effect on HUVEC MCP-1 or IL-6 mRNA. However, acetaldehyde at
concentrations of 50 and 100 M, increased the expression of HUVEC MCP-1 and IL-6 mRNA:
1.880.56 and 4.50.79-fold increases (n3), respectively, observed at 50 M. Conclusion:
Acetaldehyde, at higher concentrations, equivalent to those seen in the blood of binge drinkers
and alcoholics, i.e. 50 M, significantly increased the expression of HUVEC MCP-1, TNF
and IL-6 mRNA. These effects of acetaldehyde may contribute, in part, to the increase in
coronary heart disease that is associated with binge patterns of alcohol consumption.
P503
Deficiency of AT1a Receptors Profoundly Reduces Sidestream Cigarette
SmokeAugmented Atherosclerosis in LDL Receptor -/-
-
Mice
Alan Daugherty, Deborah A Howatt, Sung G Han, C G Gairola; Univ of Kentucky, Lexington,
KY
Objective: The renin-angiotensin system has been implicated in the development of the
atherosclerotic vascular diseases through mechanisms that are independent of blood pressure.
Administration of angiotensin type 1a (AT1a) receptor antagonists or a deficiency of AT 1a
receptors, markedly reduces hypercholesterolemia-induced atherosclerosis. We have previ-
ously shown that inhalation exposure to cigarette smoke increases atherosclerotic lesion
formation in hypercholesterolemic mice. To determine if RAS plays a role in sidestream
cigarette smoke-augmented atherosclerosis, we examined the formation of aortic lesions in
AT1a receptor -/- mice. Methods and Results: AT1a receptor / and -/- mice in an LDL-/-
background, maintained on a diet enriched in saturated fat diet, were exposed to sidestream
cigarette smoke for 4 hrs/day for 5 days/week for 16 weeks. Control groups were exposed to
ambient air. Significant increases in urinary cotinine and lung tissue CYP 1A1 levels in exposed
animals confirmed their effective exposure to cigarette smoke. Concentrations of oxidative
stress biomarker 8-OHdG were significantly increased in the urine of smoke-exposed mice.
Plasma cholesterol concentrations and lipoprotein cholesterol distributions were not signifi-
cantly influenced by either the AT1a receptor genotype or the exposure to sidestream cigarette
smoke. The exposure to sidestream cigarette significantly increased the size of atherosclerotic
lesions in AT1a receptor / mice. Deficiency of AT1a receptors produced a dramatic
reduction in the atherosclerotic lesion sizes in both groups exposed to either ambient air or
sidestream cigarette smoke. Conclusions: These results are consistent with AT1a receptors
having a profound effect on atherogenesis promoted by both hypercholesterolemia and
sidestream cigarette smoke.
P504
Id3 Deficiency Increases Atherosclerosis in ApoE Knockout Mice
Amanda C Doran, James W Chumley, R P Slayton, Stephanie N Oldham, Alexandra
Tichotsky, Nahum Meller, Coleen A McNamara; Univ of Virginia, Charlottesville, VA
Background: Inhibitor of differentiation-3 (Id3) has recently emerged as an important regulator
of vascular response to injury, although nothing is known about its role in atherosclerosis. To
examine this, we crossed Id3
-/-
mice to atherosclerosis prone ApoE
-/-
mice to produce
Id3
-/-
/ApoE
-/-
double knockout mice (DKO). Methods: Beginning at eight weeks of age, male
ApoE
-/-
or DKO mice were fed either standard chow or Western diet for 16 or 32 weeks. Mice
were euthanized by ketamine/xylazine overdose and pressure-perfused with saline prior to
removal of the whole aorta. Aortic arches were paraffin embedded, sectioned in 5 m intervals
from the cusp to the bifurcation of the brachiocephalic artery and stained using Russells
modified Movat method. Descending aortas were stained for lipid content using Sudan IV,
pinned open longitudinally and analyzed by enface. Lesion sizes from both the arch and the
descending aorta were quantified using ImagePro software. The two-tailed Mann-Whitney U
test was used for analysis and p values 0.05 were judged to be significant. Results:
Atherosclerotic lesions in DKO mice were increased three-fold in both the aortic arch
(p0.0007) and the descending aorta (p0.01) compared with ApoE KO mice after 16 weeks
on diet. After 32 weeks on diet, lesion size in the descending aorta was two-fold larger
(p0.005) than in ApoE mice. No significant differences were found in weight, total cholesterol,
LDL, HDL, triglycerides, insulin or glucose between ApoE
-/-
and DKO mice when fed the same
diet. Conclusions: These studies suggest that Id3 has a protective effect on the vasculature.
Further studies will be needed to determine the mechanism for the observed difference in
atherosclerotic lesion formation.
P505
Absence of CD36 and SR-AI/II Protects Against Atherosclerosis in ApoE
Knockout Mice: Protection Is Equivalent to Absence of CD36 Alone
Maria Febbraio, Sai Kuchibhotla, DiFernando Vanegas, Cleveland Clinic, Cleveland, OH; Ella
Guy, Weill Med College of Cornell Univ, New York, NY; George Nimako, Richard E Morton,
Roy L Silverstein; Cleveland Clinic, Cleveland, OH
Objective: The role of scavenger receptors in atherogenesis has become controversial as a
result of conflicting reports and a recent proposal that suggested that scavenger receptor
absence would enhance the pro-inflammatory, pro-atherogenic milieu in the artery wall. The
purpose of this study was to determine the effect of combined absence of scavenger receptors
CD36 and SRA I/II on atherosclerosis lesion development in the apoEo model. Methods: We
created background matched strains of apoEo, SRAo/apoEo, CD36o/apoEo and CD36o/SRAo/
apoEo that were greater than 99% C57Bl/6. These mice were fed a Western diet at 4 weeks
of age for 12 weeks. Results: Our results confirm a pro-atherogenic role for CD36 in this model.
There was a 61% and 74% decrease in aortic tree lesion area in CD36oapoEo males and
females, respectively, compared with apoEo controls. Combined absence of CD36 and SRA
provided no further protection in either gender. Absence of SRA was protective (32% decrease
in lesion) in female mice. Importantly, combined absence of CD36 and SRA did not result in
increased atherosclerosis as would be predicted by a recent hypothesis.
P506
Sphingosine 1-Phosphate Reduces Oxidized LDLMediated Upregulation of
Macrophage CD36
Pawel E Ferdek, Catherine C Hedrick; Univ of Virginia, Charlottesville, VA
CD36 is highly expressed on macrophages where it recognizes oxidized lipids that are present
in apoptotic cells and in oxidized LDL (oxLDL). CD36 mediates lipid accumulation and
macrophage foam cell formation and is associated with atherosclerosis. Macrophages from
C57BL/6J (B6) mice were isolated by peritoneal lavage, and incubated with oxLDL (50ug/ml),
S1P (500nM) or oxLDL plus S1P for 4, 12 and 24h. CD36 mRNA and protein levels were
measured. We observed that the timecourse of macrophage CD36 mRNA expression is
significantly delayed compared to ABCG1 in response to OxLDL. There were no changes in
CD36 mRNA or protein after 4h or 12h treatments with OxLDL and/or S1P. However, incubation
of B6 macrophages for 24h with OxLDL showed a significant induction of CD36 mRNA and
protein. Incubation of B6 macrophages with S1P for 24h showed approximately a 50%
reduction in basal CD36 mRNA and protein. Additionally, S1P inhibited oxLDL-mediated
upregulation of CD36. To investigate the mechanisms for the S1P regulation of CD36
expression, mRNA and protein stability assays were performed using S1P, actinomycin D and
cyclohexamide. No significant differences were found, suggesting that S1P does not affect
CD36 mRNA or protein stability. 12/15LO products have been shown to regulate PPAR, which
in turn, regulates CD36. We excluded the involvement of 12/15LO in the regulation of CD36
expression by comparing CD36 expression in B6 mice and 12/15LO-/- mice, in which we found
no differences in macrophage CD36 expression. Further, we did not observe changes in
12/15LO activity in B6 macrophages treated with S1P. However, we observed a significant
induction of Akt phosphorylation in B6 macrophages treated with S1P for 24h. Akt
phosphorylation promotes macrophage survival and regulates CD36 expression, both of which
are critical for regulating macrophage foam cell formation and apoptotic cell uptake in the
vessel wall. The increase in Akt phosphorylation is consistent with regulation of CD36
expression; thus, we anticipate that Akt is directly involved in the S1P-mediated regulation of
CD36. S1P may be a novel regulator of macrophage function in atherosclerosis by promoting
macrophage cell survival and controlling macrophage apoptotic cell uptake and foam cell
formation by CD36.
P507
Contribution of Myocardial Small Arteries in the Development of Human
Coronary Atherosclerosis
Tigran H Ghevondyan; National Institute of Health of Armenia, Yerevan, Armenia
Background. The solution of IHD problem is hidden in its prevention. Do we know all risk
factors? Aim. The purpose of the study was to look for IHD risk factors in myocardial
microcirculatory bed. Material and methods. The study was carried out on heart of 80 people
(42 healthy in age of 2487 years died of the violent reasons and 38 died from the first acute
myocardial infarction in age of 5089 years). Methods of autopsy, morphometry, x-ray
microangiography, histology, stereology, statistics have been used. With developed method the
volume density of intramyocardial arterial bed of the left ventricle wall (Vvart.) on histological
slides was measured. The stage and intensity of atherosclerotic lesion in main coronary
arteries, the degree of stenosis were evaluated. Results. In hearts prepared for angiography
the index Vvart. exceeded in 9 times the value of a similar index for the hearts not subjected
to injection. In average the value of Vvart. of hearts with a myocardial infarction was 28 %
2007 ATVB Poster Presentations e-125
by on March 25, 2011 atvb.ahajournals.org Downloaded from
below of healthy hearts index. There was a significant variation of the value of Vvart. both
among healthy people and patients. The distribution of indices Vvart. was different. In group of
healthy young and middle-aged persons the indices Vvart. were distributed with comparatively
regular intervals between extremes. The Vvart. of elderly and senile healthy persons was pilled
up mainly at the top borders of the scale. In group of myocardial infarction the indices Vvart.
were accumulated near to bottom border of scale. Significant negative correlation between
value of Vvart. and coronary atherosclerosis intensity was found out both in healthy people and
at died of myocardial infarction. Conclusion. Due to quantification of morphological changes
and anatomical features of heart an earlier unknown risk factor of IHD was disclosed. It has two
negative influences. At low value of volume density of heart intramural arterial bed both
intramyocardial blood supply suffers and development of coronary atherosclerosis is acceler-
ated. Combined using of radiological (MRI, CT, ultrasound) and developed morphological
methods could reveal correlation between results of two methods. Afterwards with only
non-invasive methods indicated risk factor of IHD in once lifetime can be exposed.
P508
WITHDRAWN
P509
A Systemic ACAT Inhibitor, 18-Methylesterferruginol, Ameliorates
Atherosclerotic Lesion in Cholesterol-fed LDLr
-/-
Mice Without Affecting
Plasma Cholesterol Levels
Sojin An, Jong-Min Han, Min-Jung Kim, Yong-Dae Park, Yue-Jan Jin, Hyung-Jae Jeong,
Woo S Lee, Tea-Sook Jeong; KRIBB, Daejeon, Republic of Korea
Acyl-coenzyme A:cholesterol acyltransferase (ACAT) plays an important role in production of
apolipoprotein B-containing lipoproteins and formation of foam cells. In the current study, we
investigated anti-atherosclerotic effects of 18-methylesterferruginol (18-MEF), a novel ACAT
inhibitor in LDLr
-/-
mice fed western diet. 18-MEF isolated from Torreya nucifera leaves
inhibited ACAT activities in the intact cells stably expressed hACAT1 or 2 with an IC
50
values
of 11.9 and 2.3 M, respectively. Treatment with 18-MEF (0.05 %, w/w) for 8 weeks markedly
reduced macrophage-derived foam cell formation and intestinal and hepatic ACAT activities.
Interestingly 18-MEF did not cause change in the plasma content of cholesterol, but decreased
plasma TG level by around 35%. This phenomenon is presumably due to defective lipoprotein
assembly in enterocyte or hepatocyte by ACAT inhibition, because the plasma level of apoB48
and apoE, apoprotein markers of chylomicron or VLDL, were decreased by 0.3 fold and 0.5 fold,
respectively. Immunohistochemical analysis demonstrated that 18-MEF significantly reduced
the macrophage accumulation in aortic lesion. Thus it is reasonable that the level of
macrophage scavenger receptors in aorta, i.e. CD36 (0.4), LOX-1 (0.6) and MSRA (0.8), was
decreased. 18-MEF improved the inflammatory state by decreasing the level of TNF- (0.1) in
plasma and the expression of IL-1 (0.6), TNF- (0.7), ICAM-1 (0.5), VCAM-1 (0.6), COX-2 (0.6)
and iNOS (0.6) in aorta. Notably, 18-MEF highly induced cholesterol 7-hydroxylase (CYP7A1)
and oxysterol 7-hydroxylase (CYP7B1) in aorta by 1.4 and 5 fold, respectively. Accordingly,
it is speculated that ACAT inhibition promotes cholesterol catabolism via cytochrome p450
pathway in aorta. In addition, intestinal ABC1 and I-BABP, which function in cholesterol
reabsorption, was nearly vanished in 18-MEF-treated group suggesting promoted cholesterol
excretion. In conclusion, 18-MEF reduced atherosclerotic lesion and improved inflammatory
condition via the systemic ACAT inhibition in aorta, liver, and intestine without affecting plasma
cholesterol level.
P510
Dietary 7-ketocholesterol Does Not Alter the Extent of Atherosclerosis in
Male LDL ReceptorDeficient Mice Despite a Decrease in Hepatic
Paraoxonase-1 Expression
Atil Y Kargi, Chang Yeop Han, Timothy McMillen, Mohamed A Omer, Shari A Wang, Univ of
Washington, Seattle, WA; Andrea DeBarber, Oregon Health and Science Univ, Portland, OR;
Robert D Steiner, Doernbecher Childrens Hosp, Oregon Health and Science Univ, Seattle,
WA; Tsuyoshi Chiba, Alan Chait; Univ of Washington, Seattle, WA
Dietary oxysterols have been implicated in the pathogenesis of atherosclerosis. Diets enriched
in oxysterols decrease activity of paraoxonase-1 (PON-1), a circulating atheroprotective protein,
and lead to enhanced atherosclerosis despite decreased plasma cholesterol levels. Whether
individual oxysterols have similar effects is unknown. The most abundant dietary oxysterol is
7-ketocholesterol (7KC). Culturing AML12 mouse hepatocytes for 24 hours with 10M of 7KC,
but not unoxidized cholesterol or other oxysterols, decreased PON-1 and apoA-1 production
and increased the inflammatory protein, serum amyloid A (SAA). These changes were mediated
by NFB activation and might decrease the atheroprotective effects of HDL. We hypothesized
that adding 7KC to an atherogenic diet would lead to similar changes in the hepatic expression
of these proteins in vivo, and increase atherosclerosis. To test this hypothesis, we replaced
10% of the cholesterol with 7KC in an atherogenic diet (21% saturated fat and 0.15%
cholesterol) fed to male LDLR-/- mice for 12 weeks and measured hepatic apoA-1, PON-1 and
SAA expression, plasma lipids and atherosclerosis. At the end of the study, 7KC was present
in substantial amounts in the plasma of 7KC-fed mice, but not in the control group. However,
there was no significant difference among the two groups in weight, plasma triglycerides and
SAA, or lipoprotein distribution by FPLC. 7KC consumption was associated with lower plasma
cholesterol levels, (608 11 vs 570 10 mg/dl; p 0.02) and 33% lower hepatic PON-1
expression (p 0.04), with no difference in hepatic expression of apoA-I or SAA.
Atherosclerotic lesion area at the aortic sinus was not different among the groups. We conclude
that despite altering hepatic production of apoA-1, PON-1 and SAA in vitro, 7KC, when added
to an atherogenic diet, had similar effects only on hepatic PON-1 expression, but did not
increase atherosclerosis in male LDLR -/- mice. The lack of a difference in atherosclerosis may
in part be explained by the adverse effect on PON-1 being offset by a reduction in plasma
cholesterol. Higher doses of 7-ketocholesterol may be required to achieve the type of
inflammatory changes observed in vitro.
P511
Native C-Reactive Protein Does Not Increase Atherosclerotic Lesion
Formation in Apolipoprotein E-deficient Mice
Miguel A Ortiz, Marcelo J Sosa, Candace L Walker, Gonzalo L Campana, George
Boguslawski, Colin Terry, Hector L DiCarlo, Carlos A Labarrere; Methodist Rsch Inst,
Indianapolis, IN
Background: Elevated C-reactive protein (CRP) levels are associated with endothelial activation
and development of atherosclerosis. However, the conclusive proof of this association in vivo
remains lacking. Some researchers reported a pro-atherosclerotic effect of CRP in ApoE-
deficient mice (apoE
-/-
) while others did not. We assessed the hypothesis that continuous
administration of azide- and endotoxin-free human native-CRP (n-CRP) to apoE
-/-
mice would
increase atherosclerotic lesion formation. Methods and Results: Twelve-week old male apoE
-/-
mice (n22) were used. Half of the animals received a continuous infusion of human n-CRP
(20.4 g /mice /day) for four weeks using osmotic pumps; the other half received CRP vehicle
buffer alone. Mice were fed Purina 5001 rodent diet ad libitum. Then atherosclerosis was
evaluated in aortic roots after Oil Red-O staining, and in complete aortas after Sudan IV
staining. The extent of atherosclerotic lesion in the aortic root was not significantly different
between the groups (CRP: 29.60 % vs. Control: 29.4 %, p 0.967). However, when measured
throughout the whole aorta, the size of the lesion in mice treated with CRP was significantly
smaller compared to control animals (CRP: 7 % vs. Control: 9.2%, p 0.0029). Serum levels
of soluble ICAM-1, VCAM-1, E-selectin and P-selectin were not significantly different between
groups (p 0.117). Conclusions: We demonstrated that continuous infusion of human n-CRP
to apoE
-/-
mice did not increase atherosclerotic lesion formation, but, surprisingly, was
associated with reduction of lesion formation throughout the whole aorta but not in the aortic
root.
P512
Local Production of Lp-PLA
2
and Lysophosphatidylcholine in the Coronary
Circulation: Association with Early Coronary Atherosclerosis and
Endothelial Dysfunction in Humans
Shahar Lavi, Joseph P McConnell, Charanjit S Rihal, Abhiram Prasad, Verghese Mathew,
Lilach O Lerman, Amir Lerman; Mayo Clinic, Rochester, MN
Background: Lipoprotein-associated phospholipase A
2
(Lp-PLA
2
) is a novel biomarker and
participant in vascular inflammation. Inflammation is also associated with coronary athero-
sclerosis. We tested the hypothesis that local coronary production of Lp-PLA
2
is enhanced in
patients with early coronary atherosclerosis and associated with local endothelial function.
Methods: Coronary angiography, blood flow, flow reserve, endothelial function, and intravas-
cular ultrasound with volumetric analysis were obtained in 30 patients without significant
coronary artery disease. Plasma samples were collected simultaneously from the left main
coronary artery and the coronary sinus for measurement of Lp-PLA
2
, its active byproduct and
mediator lysophosphatidylcholine (lysoPC), and C-reactive protein (CRP). Results: Fifteen
patients had mild coronary atherosclerosis and 15 had no evidence of atherosclerosis by
ultrasound (controls). Hemodynamic parameters and cholesterol profile were similar in both
groups. Arterial Lp-PLA
2
levels were similar in early atherosclerosis and controls (24619 vs.
23019 ng/ml). Contrarily, Lp-PLA
2
net production across the coronary circulation was higher
in patients with early atherosclerosis compared to controls (1097403 vs. -487404 ng/min,
p0.001) and correlated with the percent atheroma volume (r
s
0.37, p0.04). LysoPC net
e-126 Arterioscler Thromb Vasc Biol June 2007
by on March 25, 2011 atvb.ahajournals.org Downloaded from
production was also higher in patients compared to controls (74273 vs. -786273, p0.03)
and correlated with coronary endothelial dysfunction (r
s
0.5, p0.005). CRP net production
was not significantly different between the groups. Conclusion: Early coronary atherosclerosis
in humans is characterized by local production of Lp-PLA
2
. Local coronary generation of lysoPC,
a product of Lp-PLA
2
, is then associated with coronary endothelial dysfunction. These results
support the role for Lp-PLA
2
in the mechanism of regional vascular inflammation and early
atherosclerosis in humans.
P513
The Effect of Sphingomyelin Biosynthesis Inhibition on Lipid Absorption in
Mice
Zhiqiang Li, Yan Li, Xian-Cheng Jiang; SUNY Downstate Med Cntr, Brooklyn, NY
Serine palmitoyltransferase (SPT) is the rate-limiting enzyme in the sphingomyelin (SM)
biosynthesis pathway. Mammalian SPT is composed of 2 different subunits, Sptlc1 and Sptlc2.
Our group and others have found that mice orally treated with myriocin, a specific SPT inhibitor,
reduces SM, cholesterol, and triglyceride levels in the circulation, while intraperitoneal
treatment reduces only SM levels. Since oral administration of myriocin has a direct effect on
the gastrointestinal tract, as suggested by a previous report, we hypothesized that the decrease
of cholesterol and TG levels in the circulation might be due to a reduction in lipid absorption.
We carried out cholesterol absorption studies on wild-type and apoE knockout (KO) mice, with
or without oral myriocin administration, using the conventional fecal dual-isotope ratio method.
The approach involved the gavage of a single bolus of 0.1 uCi [
14
C] cholesterol and 0.2 uCi [
3
H]
sitostanol, together with 1 mg cold cholesterol in 30 ul olive oil. We observed that, after
myriocin treatment, wild-type or apoE KO mice absorbed significantly less radiolabeled
cholesterol than their controls (40% and 61%, respectively). The amount of radiolabeled
cholesterol assimilated into the circulation was significantly reduced in these mice, compared
to their controls (69% and 82%, respectively). Moreover, myriocin treated mice absorbed
significantly less radiolabeled tirglyceride than their controls (56% and 49%, respectively).
More importantly, we found that heterozygous Sptlc2 KO mice, with less than 50% SPT activity
in the small intestine, absorbed significantly less cholesterol (44%, p0.01) and contained
significantly less radiolabeled cholesterol and [
3
H]glycerolipids in the circulation (46% and 51%,
respectively) than control mice. These results indicate that SPT is involved in intestinal
absorption of cholesterol and triglycerides. Manipulation of SPT activity by either specific
inhibitors or gene therapy might provide a novel alternative treatment for dyslipidemia.
P514
Spontanous Atherosclerosis in Old LPL-Deficient Mice with Severe
Hypertriglyceridemia on a Normal Chow Diet
Xiaohong Zhang, Rong Qi, Xunde Xian, Wei Huang, Peking Univ, Beijing, China; Jianglin Fan,
Yamanashi Univ, Tokyo, Japan; Colin Ross, Michael R Hayden, Univ of British Columbia,
Vancouver, Canada; George Liu; Peking Univ, Beijing, China
Whether severe hypertriglyceridemia (HTG) in lipoprotein lipase (LPL)-deficiency is pro- or
anti-atherogenic remains a unresolved question. To investigate this issue we pursued a long
term observation of development of atherosclerotic lesions in a unique LPL gene deficient
mouse model and compared with those in wild type counterparts fed normal rodent chow diet.
At 4 month of age homozygous LPL (LPL-/-) deficient mice exhibited severe HTG but no sign
of any aortic lesion. However, these mice developed foam cell-rich lesions at the aortic root at
age over 15 months, while wild-type and heterozygous (LPL) mice were lesion-free at the
same age. In searching for causes of such unexpected development of spontaneous
atherosclerosis we found plasma malondialdehyde (MDA) levels in old LPL-/- mice were
significantly higher than those of LPL and wild-type mice. Electron spin resonance (ESR)
analysis showed a marked increase in oxidative susceptibility of chylomicrons (CMs) from the
old LPL-/- mice, as compared with those from young mice. Incubation of CMs from old LPL-/-
mice with cultured human umbilical vein endothelial cells (HUVECs) showed significantly
increased upregulation of VCAM-1 and MCP-1, and enhanced actual adherence of human
THP-1 mononuclear cells over those from young ones. In conclusion, our results demonstrate
an unexpected occurrence of spontaneous atherosclerosis in old LPL deficient mice and
suggest a mechanism involving oxidation of chylomicrons in formation of aortic lesions via
activation of endothelial cells.
P515
Dexamethasone Induces MCP-1 mRNA Destabilizing Activity in Vivo in Rat
Aortas
Yi Liu, Latika Dhawan, Bin Liu, Mark B Taubman; Univ of Rochester, Rochester, NY
Introduction: Monocyte chemoattractant protein-1 (MCP-1) plays an important role in the
pathogenesis of atherosclerosis. We have previously shown that glucocorticoids specifically
destabilize MCP-1 mRNA in cultured vascular smooth muscle cells (SMC) by a glucocorticoid
receptor (GR)-dependent mechanism. The purpose of this study was to define whether this
phenomenon also occurs in vivo. Methods: Sprague-Dawley rats were treated with Dexameth-
asone (Dex) 1mg/kg I.P. 24hrs and 1hr before sacrifice. Aortic media were isolated and S-100
cytoplasmic extracts were obtained as done for cell culture. Extracts were incubated with in
vitro-transcribed radiolabeled MCP-1 mRNA and analyzed on 4% denaturing polyacrylamide
gels. Protein content was determined by Bio-Rad assay. Results: Extracts from Dex-treated rats
induced a more rapid degradation of radiolabeled MCP-1 mRNA (t1/210min) as compared to
extracts containing equal amount of proteins from untreated rats (t1/2 60min). Experiments
were done to determine whether the Dex-sensitive activities in rat aorta were similar to that
described in cell culture. We previously demonstrated that the initial 224 nucleotides of the
MCP-1 mRNA contain the Dex-sensitive region. Similarly, extracts from Dex-treated rats
degraded the 1224 region, but not other Dex-insensitive region. Like in cell culture, the Dex
sensitive degradative activity was heat unstable and sensitive to proteinase K. We have
previously shown that addition of exogenous GR blocked the degradative activity of Dex-treated
cultured VSMC. Similarly, addition of GR blocked the degradation of MCP-1 mRNA in aortic
extracts in a concentration-dependent fashion. Conclusion: These results show that Dex
treatment induces an MCP-1 mRNA degradative activity in rat aorta in vivo. Further elucidation
of this mechanism may provide novel approaches to regulate vascular MCP-1 expression.
P516
Changes of Renin Angiotensin System in Visceral Adipose Tissues in Rats
with Metabolic Syndrome
Ma Lq, Liu Dy, Li Zb, Luo Zd, Zhang Ll, Wang Lj, Cao Tb, Zhu Zm; Certer for Hypertension
and Metabolic Disease,Chongqing Institute of Hypertension,Third Military Med Univ,
Chongqing, China
Recent studies showed that renin agiotensin system (RAS) in adipose tissue play an unique role
in the regulation of insulin sensitivity and adipocyte differentiation. It is unclear the changes of
RAS in abdominal fat in metabolic syndrome (MS). This study aims to investigate the RAS of
visceral adipose tissues in rat with MS and effect of angiotensin II on adipocyte differentiation.
Methods: thirty male Wistar rats were divided into rats on normal chow or high-fat diet. The
rats on high-fat diet for 24 weeks developed the MS compared with rats on normal diet. The
mRNA and protein expression of RAS in mesenteric fat tissue were measured by RT-PCR and
Western blot. Lipid droplet in 3T3-L1 preadipocytes and mature adipocytes were observed
using oil-red staining. Cytosolic free calcium level was measured by the fluorescence
technique. Results The mRNA expressions of angiotensinogen, angiotensin-converting enzyme
I, angiotensin II type 1 receptor in mesenteric fat were significantly increased in rats with MS
compared with those in rats on normal diet (P0.05). After administration of angiotensin II, less
lipid droplets in mature adipocytes were observed in vitro. In contrast, dominate lipid droplets
in mature adipocyte was found after administration of captopril and candesartan. It was
reported that adipocyte differentiation was associated with the increase of cytosolic free
calcium concentration. We found that angiotensin II increased the cytosolic free calcium
concentration in preadipocytes (ratio 340/380: 0.390.05 vs 0.51 0.07, P0.01), which
was blocked by captopril (0.280.02) and candesartan(0.120.02). In contrast, angiotensin II
induced the increase of cytosolic free calcium levels was reduced in mature adipo-
cyte(0.310.03 vs 0.190.02). Candesartan, but not captopril, can recover the angiotensin
II-mediated the increase of cyotosolic free calcium levels in mature adipocyte (0.210.04 for
captopril, 0.510.03 for candesartan). Conclusions it concluded that RAS in the visceral
adipose tissues was activated in rats with MS, and antagonizing of RAS can recover the
lipogenesis of adipocyte, which may be associated with diminishing of ectopic lipid distribution
and improving the insulin sensitivity.
P517
Increased Oxidative Stress Precedes Leaflet Restriction in Early Aortic
Valve Disease
Jordan D Miller, Kristine M Serrano, Robert M Weiss, Yi Chu, Christopher J Berry, Robert M
Brooks, Donald D Lund, Wayne E Richenbacher, Jeffrey E Everett, Univ of Iowa, Iowa City,
IA; Stephen G Young, Univ of California-Los Angeles, Los Angeles, CA; Donald D Heistad;
Univ of Iowa, Iowa City, IA
Oxidative stress is evident in atherosclerotic plaques, but its role in calcific aortic valve stenosis
(AVS) is unknown. We tested the hypothesis that oxidative stress is increased in human valves
with calcific aortic valve stenosis (AVS), and that increases in oxidative stress precede valve
dysfunction in a mouse model of AVS. Dihydroethidium and dichlorofluorscein fluorescence
indicated that both superoxide and hydrogen peroxide levels are markedly increased near the
calcified regions of explanted aortic valves from humans with AVS (n 7). Quantitative
real-time RT-PCR showed no changes in Nox1, Nox2, or Nox4 expression in calcified regions
versus noncalcified regions. In contrast, expression of extracellular superoxide dismutase (SOD)
(p 0.05), copper zinc SOD (p 0.05), and catalase (p 0.05) were reduced in regions with
high oxidative stress near calcium deposits. We recently reported that Ldlr
-/-
/Apob
100/100
mice
fed a normal chow diet develop AVS at 1822 months of age (Circulation, 2006, 114: 20659),
and that superoxide levels were elevated in the valves of mice with severe AVS. Here,
hypercholesterolemic male Ldlr
-/-
/Apob
100/100
/Mttp
fl/fl
/Mx1Cre
/
mice fed a high fat (HF) diet for
6 months had increased superoxide levels in the aortic valve versus normocholesterolemic
control mice given pI:pC at 1 month (Tempol-inhibitable fraction of DHE: control 6 2 RLU
(Mean SE), HF 14 4 RLU, p 0.05), despite no significant reduction in aortic valve
opening (leaflet separation measured by echocardiography: control 1.0 0.1 mm, HF
0.9 0.1 mm). However, MRI assessment of aortic valve function showed evidence of aortic
valve regurgitation in 75% of the fat-fed mice (versus none of the normocholesterolemic
controls), indicating aortic valve sclerosis/dysfunction. Histological examination of the valves
revealed small but significant increases in leaflet calcification (Alizarin Red: control 0.2
0.2%, HF 1.8 0.7%, p 0.05) and marked lipid deposition (Oil Red O: control 6
4%, HF 18 5%). Collectively, these data suggest that oxidative stress occurs early in
aortic valve disease. We speculate that reducing oxidative stress is a potential therapeutic
strategy to slow progression of calcific aortic valve stenosis.
P518
Neuronatin: A New Inflammation Gene Expressed on the Aortic
Endothelium of Diabetic Mice
Nino Mzhavia, Shuiqing Yu, Hayes M Dansky, Ira Goldberg; Columbia Univ Med Cntr, New
York, NY
The mechanisms by which diabetes accelerates atherosclerosis are not well understood. To
determine effects of hyperglycemia/insulin resistance on arterial wall biology, aortic gene
expression profiles were generated using aortas from db/db, high fat diet fed mice and their
respective nondiabetic controls. For each model expression analysis was done on samples from
3 distinct RNA pools generated by pooling aortas from 36 mice. Among the differentially
expressed genes, the neuronatin gene, a gene of unknown function originally identified in the
2007 ATVB Poster Presentations e-127
by on March 25, 2011 atvb.ahajournals.org Downloaded from
developing rat brain, was found to be upregulated by approximately 10 fold in the aorta of
db/db and by 2 fold in the aorta of high fat diet fed mice. The neuronatin protein was selectively
increased in the aortic endothelium of db/db mice, but expression in other tissues (pituitary,
brain, heart, kidney) was not altered in diabetic mice. Infection of human aortic endothelial cells
with a nnat encoding adenovirus resulted in increased expression of a panel of NFB regulated
inflammatory cytokines, chemokines, and adhesion molecule genes. Neuronatin expression
also augmented TNF- induced IL-6 production in endothelial cells. Neuronatin expression
activated Erk kinase in endothelial cells, which may be a mechanism by which neuronatin
activates the NFB program of inflammation. In summary, we have discovered that neuronatin
expression leads to stimulation of specific inflammation genes in endothelial cells that have
direct relevance to the pathogenesis of atherosclerosis. The upregulation of neuronatin in the
vasculature of diabetic mice may be a mechanism by which diabetes accelerates atheroscle-
rosis in vivo.
P519
Activated Factor XII Type A Is an Independent Predictor of All-cause
Mortality in Patients Admitted with Chest Pain
Volker Poenitz, Trygve Brugger-Andersen, Stavanger Univ Hosp, Stavanger, Norway; David
Pritchard, Axis-Shield, Dundee, United Kingdom; Heidi Grundt, Dennis W Nilsen; Stavanger
Univ Hosp, Stavanger, Norway
Background: Activated Factor XII (XIIa) is a predictor of recurrent coronary ischemic events in
patients following a myocardial infarction (MI). Recently, novel in-vivo types of XIIa have been
described. We assessed the relation between admission levels of activated factor XII type A
(XIIaA) and long-term all-cause mortality in a large, consecutive cohort of patients admitted
with chest pain. Methods: Blood samples for XIIaA determination were obtained immediately
following admission in 871 patients admitted with chest pain suspected of having a MI. Plasma
XIIaA concentrations were determined by ELISA at admission. All cause mortality within each
quartile of XIIaA was compared for a 24-month follow-up period. Results: After a follow-up
period of 24 months, 130 patients (14.9%) had died. The unadjusted risk ratio for death of
patients with XIIaA in the highest quartile (Q4) was 2.92 (95% CI 1.724.95; p0.01)
compared with patients with XIIaA in Q1. In a multivariate Cox regression model, XIIaA (HR 3.31,
95% CI 1.766.23; p0.01) added prognostic information for all-cause mortality additional to
that of all other parameters associated with mortality such as age, history of heart failure or
coronary heart disease and BNP. Conclusion: XIIaA is a powerful and independent indicator of
long-term all-cause mortality in patients admitted with chest pain and provides prognostic
information above and beyond to that provided by conventional risk factors.
P520
Lower Levels of Matrix Gla Protein in Patients with Moderate to Severe
Hypertension in Comparison to Normotensive Subjects
Roger Rennenberg, Univ Hosp Maastricht, Maastricht, The Netherlands; Leon Schurgers,
Cees Vermeer, Maastricht Univ, Maastricht, The Netherlands; Peter de Leeuw, Bram Kroon;
Univ Hosp Maastricht, Maastricht, The Netherlands
Background: Medial vascular calcifications are common among patients with hypertension. The
vitamin K-dependent protein matrix Gla-protein plays an important role in preventing arterial
calcification. We hypothesized that in subjects at risk for calcification serum levels of the
protective matrix Gla protein are lower. In this study we investigated circulating matrix Gla
protein in moderate to severe hypertensive patients compared to normotensive control
subjects. Methods and results: From 90 moderate to severe hypertensive patients and 90 age
and sex matched healthy control subjects venous blood was taken to measure matrix Gla
protein. Hypertensive patients had significantly lower circulating serum matrix Gla protein as
compared to control subjects (5.5 nM versus 7.2 nM, P 0.0001). Conclusions: In this study
we demonstrate that moderate to severe hypertensives have lower circulating matrix Gla
protein levels compared to age and sex matched healthy controls.
P521
Acute Phase Serum Amyloid A Induces Expression of the Rat Type IIA
Phospholipase A2 Gene
Christopher P Sullivan, Boston Univ Sch of Medicine, Boston, MA; Michel Raymondjean,
Universite Pierre et Marie Curie, Paris, France; Barbara M Schreiber; Boston Univ Sch of
Medicine, Boston, MA
Atherosclerosis is a multifactorial disease characterized by inflammatory lesion formation in the
vasculature. An elevation in circulating acute phase proteins is an indicator of disease. Serum
amyloid A (SAA) is one such indicator but its function remains unclear. SAA, which is abundant
in atherosclerotic lesions, might be deposited from the circulation or synthesized locally by
macrophages and smooth muscle cells within the developing lesion. Interestingly, treatment of
neonatal rat aortic smooth muscle cells with SAA causes movement of endogenous cholesterol
to the endoplasmic reticulum. It was of interest to determine the mechanism by which SAA
facilitates this trafficking. Gene microarray and Western blot analyses showed that secretory
phospholipase A2, group IIA (sPLA2) mRNA and protein expression are elevated in SAA-treated
smooth muscle cells. To determine the potential contribution of transcriptional activation,
transfection experiments were performed using a plasmid containing a 488bp fragment of the
rat sPLA2 promoter and SAA-induced increases in promoter activity were shown. Previous
reports in the literature show that CAAT-enhancer binding protein beta (C/EBP) and NFB
up-regulate sPLA2 transcription and their role in SAA-induced sPLA2 gene expression was
explored. C/EBP mRNA and protein increase in smooth muscle cells exposed to SAA. Promoter
constructs with deleted or mutated C/EBP-binding sites are not responsive to SAA. NFB
accumulates in the nucleus in cells treated with SAA and an NFB element in the promoter is
responsive to SAA. Electrophoretic mobility shift assays confirm the findings that C/EBP and
NFB contribute to the SAA-induced increase in expression of the sPLA2 gene. These results
demonstrate that SAA increases transcription of the sPLA2 gene and suggest that the activity
of sPLA2 may play a role in the ability of SAA to traffic cholesterol.
P522
Arterial Macrophage Colony Stimulating Factor Influences Atherosclerotic
Lesion Size by Regulating Monocyte Migration and Apoptosis
Zory Shaposhnik, Xuping Wang, Mohamad Navab, Aldons Lusis; UCLA, Los Angeles, CA
Introduction: Macrophage colony stimulating factor (M-CSF) regulates monocyte/macropage
survival, differentiation, proliferation and migration. M-CSF is highly induced by oxidized lipids
in endothelial cells, smooth muscle cells and monocyte/macrophages. It was found that M-CSF
deficient mice on an apoE-/- or low-density lipoprotein receptor null (LDLR-/-) null backgrounds
show in excess of a 50-fold decrease in the size of atherosclerotic lesions. However, the
mechanism by which the M-CSF deficiency contributes to lesion development is unclear.
Objective: We examined the basis of the resistance to atherosclerosis of mice deficient in
M-CSF and the contributions of macrophage-derived M-CSF on lesion formation. Methods:
Since mice homozygous for a null allele of M-CSF do not breed on an inbred C57BL/6J
background and exhibit various pathologies, we studied atherosclerosis in M-CSF heterozygous
mice bred onto a hyperlipidemic LDL receptor null background. Atherosclerotic lesions were
examined for measures of apoptosis, proliferation and chemotaxis. The source of M-CSF
influencing lesion development was examined using bone marrow transplantation. Results:
Heterozygous mice on a C57BL/6J LDLR -/- background exhibited a 50% decrease in lesion
size (257,785 um
2
28,565 Wt; n7 vs. 153,812 um
2
9,511 Het; n12). The mice had
a 61% increase in the fraction of apoptotic (TUNEL positive) macrophages per unit of
macrophage positive lesion area (0.310 0.06 Wt; n6 vs. 0.189 0.023 Het; n7). No
differences in the frequencies of mitotically active cells (2.38 0.76% Wt; n4 vs. 4.22
0.789% Het; n6) were detected. In vitro studies indicated that neutralizing M-CSF decreased
monocyte chemotaxis 37% (n9 per group). Bone marrow transplantation studies showed that
the source of the bone marrow, whether /, , or -/- for M-CSF, had no effect on the
development of atherosclerosis, suggesting that endothelial cell-derived M-CSF is responsible
for the effect on atherosclerosis. Conclusion: Our studies indicate that likely mechanisms for
the effect of M-CSF on atherosclerosis include monocyte recruitment and macrophage survival
and that sources of M-CSF other than monocyte-macrophages contribute to lesion formation.
P523
Plasma Expression of Functional scFv Antibody Against OxLDL in C57BL/6
Mice Following Adenoviral Mediated Gene Delivery
Peter X Shaw, Atsushi Miyanohara, UC San Diego, La Jolla, CA; Chin N Lai, Veterans Affairs
San Diego Healthcare System, San Diego, CA; Sotirios Tsimikas, Kirk Hammond, Joseph
Witztum; UC San Diego, La Jolla, CA
Much evidence indicates that the oxidation of LDL plays an important role in the atherogenic
process. The unregulated uptake of OxLDL by macrophage scavenger receptors is believed to
accelerate atherosclerosis, and in vitro studies have shown that certain antibodies to OxLDL
can inhibit the binding and uptake of OxLDL by macrophages. However, the in vivo
consequences of directly expressing in plasma high titers of intact antibody or antibody
fragments capable of blocking the uptake of OxLDL have not been tested. Raising plasma titers
of an antibody to OxLDL using gene delivery of its cDNA would allow us to directly determine
the long term in vivo impact of such a strategy on atherogenesis. In this report, we converted
a human monoclonal antibody IK-17, which binds to MDA-LDL and Cu-OxLDL and blocks the
macrophage uptake of OxLDL, into the single chain format (scFv) by molecular engineering. We
demonstrated that it was secreted into the culture media and was biologically active when a
eukaryotic expression vector harboring scFvIK-17 was transfected into HEK-293 cells. We
further generated an adenovirus vector encoding the cloned scFvIK-17 gene and demonstrated
that scFvIK-17 can be efficiently expressed and secreted into the plasma of C57BL/6 mice after
intravenous gene transfer of 2.5 X 10
11
virus particles. The plasma scFvIK-17 bound specifically
to MDA-LDL and Cu-OxLDL but not to unmodified LDL, and immunostained atherosclerotic
lesions, similar to its parental Fab form. In summary, achieving plasma expression of an
antibody using gene transfer technology is a novel way to study the effects of antibodies to
OxLDL on atherosclerosis progression and could provide insights into mechanisms by which
immunological mechanisms modulate atherogenesis. This strategy could eventually have
therapeutic consequences.
e-128 Arterioscler Thromb Vasc Biol June 2007
by on March 25, 2011 atvb.ahajournals.org Downloaded from
P524
Structural Transition from Pentamer to Monomer: A Mechanism That Finely
Regulates the Reactivity and Behavior C-Reactive Protein Exerts in the
Inflammatory Process of Atherosclerosis
Shang-Rong Ji, Yi Wu, Lanzhou Univ, Lanzhou, China; Lawrence A Potempa, Immtech
Pharmaceuticals Inc, Vernon Hills, IL; Li Zhu, Yu-Heng Liang, Qiang Qiu, Jing Zhao; Lanzhou
Univ, Lanzhou, China
C-reactive protein (CRP) is an ancient protein with high phylogenetic conservation; no
deficiency or amino acid sequence polymorphism of CRP has yet been identified in humans.
Emerging evidence indicates that CRP has at least two conformationally distinct isoforms, i.e.
pentameric CRP (pCRP) and monomeric CRP (mCRP). Both CRP isoforms play roles in
inflammation and presumably atherosclerosis(13). However, the origin of mCRP in situ and
how a typical acute phase protein, pCRP, could function like a fine modulator of sophisticated
cellular or physiological systems remains elusive. The basic function of pCRP is largely
dependent on surface or multivalent ligand binding, thus membranes enriched in phospho-
choline ligands could be important sites for pCRP function. Our results(4) showed that upon
membrane binding, pCRP first converted to a meta-stable intermediate with partly retained
native conformation, i.e. mCRP
m
, which could subsequently detach from membrane with a slow
dynamics leading to formation of mCRP. Such a stepwise structural transition is accompanied
with obviously enhanced capacity to exert classical or modified CRP bioactivities. We
hypothesize that pCRP isoform represents a rest state of CRP, while mCRP
m
and mCRP
represent functional state of CRP. The rapid pCRP3mCRP
m
transition may be a reliable
mechanism that amplifies the classical CRP functions and could contribute to acute phase
response. The subsequent slow mCRP
m
3mCRP transition represents the secondary phase of
CRP function, and exert modified activities as shown by us and others (3,5,6). The abundance
of available pCRP ligands, the transition dynamics, and the proteolytic sensitivity of mCRP, all
restrict pCRP3mCRP
m
3mCRP
s
to be primarily a local process occurring in inflammatory loci
and can serve as a buffering mechanism allow this protein to be fine regulator without
unwanted violent stimulation. 1. Pepys, MB, and Hirschfield, GM (2003) JCI 111:1805 2. Verma,
S, Devaraj, S, and Jialal, I (2006) Circulation 113:2135 3. Schwedler, SB, Filep, JG, Galle, J et
al (2006) Am J Kidney Dis 47:212 4. Ji, S-R, Wu, Y, Zhu, L et al (2007) Faseb J 21:doi:10.1096
5. Ji, S-R, Wu, Y, Potempa, LA et al (2006) ATVB 26:935 6. Ji, S-R, Wu, Y, Potempa, LA et al
(2006) IJBCB 38:648
P525
MMP-9 Deficiency Does Not Influence Atherosclerosis but Promotes
Abdominal Aortic Aneurysms in Both Hypercholesterolemic and
Angiotensin II-infused Mice
Xiaojie Xie, Deborah A Howatt, Lisa A Cassis, Alan Daugherty; Univ of Kentucky, Lexington,
KY
Objective: The role of matrix metalloproteinase-9 (MMP-9) in the development of atheroscle-
rosis remains to be clarified. However, MMP-9 has been implicated in the development of
experimental abdominal aortic aneurysms (AAAs). The purpose of this study was to define the
role of MMP-9 deficiency on hypercholesterolemia- and AngII-induced atherosclerosis and AAA
formation in ApoE-/- mice. Methods and Results: ApoE -/- mice were developed that were
either wild type or deficient in MMP-9, with all comparisons being performed between
littermates. Mice were infused with either saline or AngII (1,000 ng/kg/min) via osmotic pumps
for 28 days and fed a normal laboratory diet. MMP-9 deficiency had no effect on plasma
cholesterol concentrations, lipoprotein-cholesterol distributions, or systolic blood pressure
during saline or AngII infusion. MMP-9 deficiency had no effect on the size of atherosclerotic
lesions during either saline or AngII infusion. Unexpectedly, AAAs were observed in the
supra-renal aorta (P 0.027) of saline-infused MMP-9 -/- mice. Furthermore, infusion of AngII
led to significantly increased AAA formation (P 0.006) and increased death due to rupture
of the abdominal aorta (P 0.004). To determine whether MMP-9 deficiency led to structural
changes, the AAA prone region was studied for cellular content and matrix integrity. We also
determined whether MMP-9 deficiency led to functional defects by performing contractility
studies in aortic rings. No structural or functional changes were discernable in the AAA prone
region that could account for the exacerbated disease. Conclusion: These studies provided the
unexpected result that deficiency of MMP-9 lead to AAA formation in mice with hypercholes-
terolemia alone, and increased the incidence and severity of AngII-induced AAAs. The lack of
MMP-9 during the development of the mice may lead to circumstances that promote AAA
formation.
P526
Cilostazol and Atorvastatin Have Synergistic Effects on Endothelial Nitric
Oxide Synthase Phosphorylation and Protection Against
Ischemia-Reperfusion Injury
Yumei Ye, Saraswathy Manickavasagam, Yu Lin, Ming-He Huang, Jose R Perez-Polo, Yochai
Birnbaum; Univ of Texas Med Branch, Galveston, TX
Background: Atorvastatin (ATV) limits myocardial infarct size (IS) by upregulating Akt and
protein kinase A (PKA) which activate endothelial nitric oxide synthase (eNOS) via phosphor-
ylation at Ser
1177
. However, when given orally, high doses of ATV (10 mg/kg/d) are needed to
achieve maximal protective effect. Cilostazol (CIL) is a phosphodiesterase III inhibitor, used for
treating patients with peripheral arterial disease. Several studies have suggested that CIL
stimulates NO production by activating PKA. Hypothesis: CIL and ATV may have synergistic
effects on eNOS phosphorylation and IS limitation. Methods: Sprague-Dawley rats received
3-day oral: 1) water; 2) ATV 2mg/kg/d; 3) CIL 20mg/kg/d: 4) ATVCIL. Rats underwent 30min
coronary artery occlusion and 4h reperfusion (n10 in each group), or hearts were explanted
for immunoblotting without being subjected to ischemia. Area at risk (AR) was assessed by blue
dye and IS by triphenyl-tetrazolium-chloride (TTC). Results: Body weight and the size of AR
were comparable among groups. There were no significant differences among groups in mean
blood pressure and heart rate. CIL, but not ATV, reduced IS. The ATVCIL group had IS that
was significantly smaller than in the other three groups (p0.001 for each comparison)
(Figure). ATV, CIL and their combination did not affect total eNOS expression. ATV at 2 mg/kg/d
did not affect Ser
1177
P-eNOS levels, whereas CIL increased it (25815%). Myocardial P-eNOS
levels were higher in the ATVCIL group (4067%). Conclusions: ATV and CIL have
synergistic effect on eNOS phosphorylation and in IS limitation. By increased activation of
eNOS, CIL may augment the pleiotropic effects of statins.
P527
WITHDRAWN
P528
Sirolimus Attenuates Angiotensin II Plus Diet-accelerated Atherogenesis
and Abdominal Aortic Aneurysm Formation in ApoE-deficient Mice
Lei Zhao, Cordis Corp, a Johnson & Johnson company, Warren, NJ; Michael Lazar, Jessica
Gerbavac, Consumer & Personal Product Worldwide, Div of Johnson & Johnson Consumer
Companies Inc, Skillman, NJ; Jonathon Zhao, Cordis Corp, a Johnson & Johnson company,
Warren, NJ; Kathy Wenzel, Miri Seiberg, Consumer & Personal Product Worldwide, Div of
Johnson & Johnson Consumer Companies Inc, Skillman, NJ; Robert Falotico; Cordis Corp, a
Johnson & Johnson company, Warren, NJ
Background: Sirolimus is a macrolide inhibitor of mTOR kinase that markedly attenuates
transplant vasculopathy and neointimal hyperplasia in animal models and humans. The effects
of sirolimus on atherosclerotic lesions and abdominal aortic aneurysm (AAA) formation require
further characterization. Objective: We investigated the effects of sirolimus on atherosclerotic
lesion development and AAA formation in apolipoprotein E deficient (apoE
-/-
) mice on a high lipid
diet receiving an angiotensin II (angII)-infusion to accelerate vascular pathology. Methods: Male
apoE
-/-
mice were placed on a proatherogenic diet for 4 wks and given a continuous infusion
of angII (1 g/kg/min) via osmotic minipump. Sirolimus (0.5, 1.0, 4.0 mg/kg, ip) or vehicle were
administrated once daily for 4 wks. In a second study, apoE
-/-
mice received an angII infusion
and proatherogenic diet for 8 wks. After 4 wks of induction, sirolimus (1.0 mg/kg, ip) or vehicle
were given once daily for the remaining 4 wks to assess the potential for lesion regression.
Atherosclerotic lesion area and histology, AAA characterization and plasma cytokine levels were
assessed. Results: Daily ip injection of sirolimus significantly reduced atherosclerotic plaque
area (en face analysis) in a dose-dependent manner at 4 wks (sirolimus: 3.1 0.4 % at 1.0
mg/kg, vs vehicle: 12.9 1.8 %, P 0.0005) and attenuated progression of established
lesions at 8 wks (sirolimus: 18.4 2.3% vs vehicle: 37.6 2.7 %, P 0.0001). Sirolimus
2007 ATVB Poster Presentations e-129
by on March 25, 2011 atvb.ahajournals.org Downloaded from
markedly reduced the incidence (sirolimus: 0 %, vs vehicle: 32 %, P 0.005) and severity of
AAA as determined by aortic diameter (sirolimus: 0.74 0.02 mm vs vehicle: 1.38 0.25
mm, P 0.005). Sirolimus prevented elastin disruption and reduced CD68

inflammatory cell
infiltration in aneurysmal tissue. These effects were associated with an altered Th1/Th2
cytokine profile in vivo. Conclusion: Our data suggest that sirolimus reduces the development
of atherosclerotic lesions and AAA in angII-infused apoE
-/-
mice and may prove to be beneficial
in modulating the severity of inflammatory vascular diseases.
P529
Quantitative Annual Effect of Atorvastatin on Size and Content of
Noncalcified Plaques of Coronary Arteries Following Atorvastatin Treatment
by Multislice CT
Nobusada Funabashi, Masae Uehara, Yoko Mikami, Yumi Shiina, Issei Komuro; Chiba Univ,
Chiba, Japan
Background: Intensive lipid-lowering treatment with Atorvastatin reduced progression of
coronary atherosclerosis, confirmed by IVUS. To quantitate the annual effect of Atorvastatin on
size and content of non calcified coronary plaques (NCP) using MSCT and comparing LDL
cholesterol levels. Methods: 21 subjects (16 males, 3579 years, median 69) with NCP by
MSCT (Light Speed Ultra 16) were enrolled. All were asymptomatic to differentiate thrombi from
NCP in coronary arteries. Following LDL measurements, all were given 10mg of Atorvastatin (2
were 5mg as LDL levels were already lower than 70mg/dl) for 1 year, and MSCT and LDL
measurements were repeated. One remarkable NCP was selected in each subject and
evaluated as representative of effect of Atorvastatin. The area and CT values of NCP, excluding
calcified portions, were manually measured from axial or multiplanar reconstruction images
under the same conditions. Results: 21 NCP (18 LAD, 2 LCx, and 1 RCA) were evaluated. The
mean LDL level was 122 mg/dl at the first scan and significantly decreased to 96 mg/ml at the
second scan (P0.05). The areas of NCP were 231 mm2 (mean 11.8) at first-scan, and 232
mm2 (12.6) at second-scan. The mean areas of NCP were not significantly different between
the both-scans (11.8 at first and 12.6mm2 at second scan). The averages of CT values were
55HU at first scan and 62HU at second scan and the mean of SDs of CT values were 40HU at
first and 45HU at second scan and both were significantly higher in the second scan (P0.05).
There was a significant positive correlation between ratios (%) of annual change in area to
baseline area at first scan of NCP (y) and LDL cholesterol levels (x) after one year of Atorvastatin
treatment (y0.0106x-0.7265, R20.1514, P0.05). Conclusion: Using MSCT, we could
quantitate the effect of Atorvastatin to the size and content of NCP and compare those with LDL
cholesterol levels. Atorvastatin may decrease area of NCP if LDL levels are sufficiently
decreased. Also, it may increase CT values, which could suggest a change in NCP components.
LDL levels may be an important factor in decreasing the area of NCP. Further studies are
needed using 64-slice MSCT in a larger population with sufficient decreases in LDL levels.
P530
Cyclic Bending Is a Major Contributor to Critical Stress Conditions Leading
to Coronary Plaque Rupture: 3D MRI-Based FSI Models and Mechanical
Image Analysis
Chun Yang, Beijing Normal Univ, Beijing, China; Jie Zheng, Pamela K Woodard, Washington
Univ, St. Louis, MO; Shunichi Kobayashi, Shinshu Univ, Nagano, Japan; Gregorio A Sicard,
Washington Univ, St. Louis, MO; Dalin Tang; Worcester Polytechnic Institute, Worcester, MA
Introduction Mechanical forces play an important role in the complicated process of
atherosclerotic plaque rupture. We have developed MRI-based 3D multi-component models
with fluid-structure interactions (FSI) in order to perform stress/strain analysis for atheroscle-
rotic plaques and to identify possible mechanical and morphological indices for accurate plaque
vulnerability assessment. Hypothesis Cyclic bending of coronary arteries caused by heart
motion may be a major contributor to critical stress conditions of atherosclerotic plaque leading
to increased plaque rupture risk. Method A 3D multi-component FSI model was introduced to
evaluate the effects of cyclic bending on stress/strain distributions in coronary plaques using
geometry re-constructed from a 3D ex vivo high resolution MRI data set (36 slices) acquired
from a human coronary plaque. Blood flow was assumed to be laminar, Newtonian, and
incompressible. Both vessel and plaque component materials were assumed to be hyper-
elastic and isotropic. Cyclic arterial bending secondary to cardiac motion was introduced into
the computational model by specifying a region of asymmetric repeat displacement. The
displacement was adjusted to achieve desirable curvature changes. In vitro flow experiments
using hydrogel stenotic tubes with cyclic bending were conducted to validate our models.
Results Computational simulations were conducted using the hydrogel stenosis model and the
coronary plaque sample under a 70130 mmHg physiological pulsating pressure. Stress
behaviors tracked at selected critical sites (thin cap and maximal stress sites) showed that
cyclic bending caused 100400% higher stress variations. Multi-component plaque structure
and cyclic bending led to nonuniform compression/expansion and higher stress variations in the
plaque. Conclusions Our initial study indicates that cyclic bending affects stress variations in
coronary plaques to the extent that it plays at least as important a role as blood pressure does
and must be included in coronary models for accurate mechanical analysis and stress-based
plaque vulnerability assessment. Additional studies using this new model are warranted.
P531
Peroxisome Proliferator-Activated Receptors and Angiogenesis: Direct
Comparative Analysis of Selective Agonists
Federico Biscetti, Eleonora Gaetani, Andrea Flex, Flavia Angelini, Paolo Pola, A Gemelli Univ
Hosp, Catholic Univ Sch of Medicine, Rome, Italy; John J Castellot Jr, Tufts Univ Sch of
Medicine, Boston, MA; Colin N Palmer, Ninewells Hosp and Med Sch, Dundee, United
Kingdom; Roberto Pola; A Gemelli Univ Hosp, Catholic Univ Sch of Medicine, Rome, Italy
Objective: In recent years, there has been increasing appreciation of the fact that peroxisome
proliferator-activated receptors (PPARs) might be important modulators of angiogenesis.
However, whether PPAR and PPAR, the two main members of the PPAR family, act as
inhibitors or inducers of the biological mechanisms that underlie angiogenesis still remains a
controversial issue. Importantly, all the studies investigating the effects of these receptors on
angiogenesis have been carried out with molecules such as thiazolidinediones, endogenous
prostaglandins, and fibrates, that, in addition to act as PPAR ligands, are also able to activate
a number of PPAR-independent pathways. In this study, we used highly-selective PPAR and
PPAR agonists and performed a direct comparative analysis of their in vitro and in vivo
angiogenic properties. Methods and Results: For the in vitro study, a co-culture model of
human endothelial cells and interstitial cells was used. For the in vivo studies, the murine
corneal model of angiogenesis was utilized. We found that WY14643 and GW1929, two
selective agonists of PPAR and PPAR, respectively, had the ability to induce tube formation
in vitro and neoangiogenesis in vivo. PPAR- and PPAR-induced angiogenesis was associated
with increased expression of vascular endothelial growth factor (VEGF) and did not differ in
extent and morphology from that induced by VEGF. Conclusions: This is the first study
performing a direct comparison of the angiogenic properties of highly-selective PPAR agonists.
Our findings suggest that a careful revision of data obtained by using non-selective and
non-specific ligands of PPAR and PPAR and indicating an anti-angiogenic effect of these
receptors is needed.
P532
WITHDRAWN
P533
Postischemic Neovascularization Is Modulated by the A
1
Adenosine
Receptor
Ayoub Mirza, Themis Karaoli, Univ of Virginia, Charlottesville, VA; Bertil Fredholm, Karolinska
Inst, Stockholm, Sweden; Amy L Tucker; Univ of Virginia, Charlottesville, VA
Arteriogenesis and angiogenesis play a critical role cardiovascular development and in adaptive
responses to tissue ischemia associated with arterial stenosis. Peripheral arterial disease (PAD)
affects over 7 million Americans and claims the limbs of over 100,000 of these annually.
Adenosine is a purine nucleoside metabolite of ATP degradation released by all ischemic
tissues, where it acts to limit tissue injury via multiple mechanisms, including angiogenesis.
Many of the effects of adenosine are mediated through its interaction with four subtypes of G
protein-coupled cell surface receptors: A
1
, A
2A
, A
2B
, and A
3
. Proangiogenic effects mediated by
A
2
adenosine receptors (AR) have been described, but the role of the A
1
AR is unexplored. The
aim of our study was to identify the role of the A
1
AR in neovascularization of the ischemic
hindlimb. We compared neovascular responses between wild type (WT) mice and mice with
gene-targeted A
1
AR-deficiency (A
1
KO) following femoral transection. While this technique
does not result in distal tissue necrosis in WT mice, 50% of the A
1
KO animals developed digital
gangrene on the ischemic side. Laser doppler perfusion imaging (LDPI) of the distal extremity
on post-operative day 7 demonstrated a 40% reduction in perfusion index (ischemic/non-
ischemic flow ratio) in the A
1
KO (294%, n7) versus WT mice (528%, n8, p0.03).
These observations were supported using endothelial staining in histologic sections of
gastrocnemius muscle, in which we observed 55% fewer capillaries in A
1
KO sections than in
WT (p0.03). Delivery of A
1
AR antagonist (WRC 0571) in WT mice following ischemia, resulted
in a 30% attenuation in LDPI index (control 4211%, n4 vs. WRC 312%, n5) and a 33%
decrease in capillary number, suggesting that impaired neovascularization in A
1
KO mice is the
result of receptor deficiency and not artifact from manipulation of the targeted locus. Further
support comes from contrast-enhanced ultrasound imaging in the proximal hindlimb day 3
following ischemia showing a 42% lower perfusion index in A
1
KO (414%, n4) versus WT
(714%, n4, p0.05) muscle. We conclude that the A
1
AR plays a significant role in
stimulation of ischemia-mediated neovascularization and is a potential therapeutic target for
PAD.
e-130 Arterioscler Thromb Vasc Biol June 2007
by on March 25, 2011 atvb.ahajournals.org Downloaded from
P534
Bone MarrowDerived Stem Cells Engineered to Overexpress Prostacyclin
Synthase Survive Under Hypoxia Condition and Enhanced Capillary
Assembly Around Homing Site in Hind Limb Ischemia Model
Yasushi Numaguchi, Masakazu Ishii, Ryuji Kubota, Chinatsu Hattori, Toyoaki Murohara;
Nagoya Univ, Nagoya, Japan
Mesenchymal stem cell (MSC) delivery contributes to collateral formation through cell
incorporation into vessels and secretion of angiogenic cytokines like HGF, VEGF, and MCP-1 in
a paracrine manner. Prostacyclin is a proangiogenic prostanoid which has a multifactorial
function such as antiapoptosis and antiaggregation in endothelial cells. To tested hypothesis
that cell therapy with MSC overexpressed PGIS would enhance proangiogenic effect to lead to
accelerated recovery from hindlimb ischemia to the delivery of MSC alone. Methods and
Results: Murine MSC was harvested by flushing femurs and transfected with adenoviral vector
encoding GFP alone or GFP and PGIS (AdGFP and AdBiGP; 150 MOI each). Cell cycle analysis
assessed by FACS revealed that, under hypoxia condition (5%O2), apoptosis rate was reduced
by 68% in GFPPGIS-transfected MSCs and promoted cell proliferation by 52% in accordance
with increase of bcl-2 and PPAR protein expression confirmed by western blotting.
C57BL6/J mice received gene or MSC injection to the adductor muscle 1 day after femoral
artery liagation. Mice were divided into 4 groups (n8 each) following injected content; vehicle,
AdBiGP(AdGP, 4x109 PFU), MSC with AdGFP (MG), and MSC with AdBiGP(MGP)
(MSC;1x106 cells/200A

L each). Laser Doppler perfusion imaging demonstrated increased


blood flow recovery in mice of MG and MGP groups (Ischemic/Non ischemic limb at 1, 2, 3
weeks; p0.01 vs. control) and MGP mice reached to peak blood flow by 7 days after
surgery earlier than MG (p0.05), while AdGP group had no significant blood flow increase
with edema in the legs. In mice of MGP, collateral formation at 2 weeks was accelerated than
MG mice (p0.05). (Table) Histological analysis of ischemic tissues revealed that capillary
assembly was enhanced around homing sites with positive staining of HGF and VEGF.
Conclusion: Local delivery of MSC overexpressed PGIS enhanced capillary assembly around
homing sites via paracrine mechanism.
BENEFICIAL EFFECTS OF MSCPGIS DELIVERY
Control Ad-GFPPGIS MSC-GFP MSC-GFPPGIS
Blood Flow Recovery
(Ischemic/Non ischemic
limb at 3 weeks)
0.520.03 0.560.04 0.820.03# 0.840.02#
Capillary Density
(/ HPF, at 2 weeks)
998 12413* 3604# 41232#
Data: meanSE, *p0.05 vs control, #p0.01 vs control, p0.05 vs
MSCGFP
P535
Segetalin Inhibits Angiogenesis via a VEGF-dependent Process
Roger Ourouda, Sophie Nadascimento, Marlene Gallet, Faculte de Medecine DAmiens,
Amiens, France; Jean-Daniel Brion, Universite Paris XI, Paris, France; Pascal Sonnet,
Jacques Rochette, Carole Amant; Faculte de Medecine DAmiens, Amiens, France
Introduction Angiogenesis is a physiological vascular process that can be implicated in tumour
growth and metastasis. Growth factors such as Vascular Growth Factor (VEGF) are implicated
in angiogenesis process. Segetalins are cyclic peptides isolated from Vaccaria segetalis
(caryophyllaceae) seeds. Segetalins are phytoestrogens used in Chinese pharmacopoeia for
amenorrhea treatment. Recently, epidemiologic and experimental studies showed that
phytoestrogen consumption reduced cardiovascular disease risk. Hypothesis : Segetalin
molecules could inhibit angiogenic process and tumour growth by inhibiting VEGF secretion
from breast cancer cells. Methods and results : Natural Segetalin (SA) and S1 (segetalin
analog) were synthesized in our laboratory. S1 was obtained from the natural compound by an
alanine tryptophan substitution. 1) Cell proliferation tests were performed using Human
Umbilical Endothelial Cells (HUVEC), and breast cancer cell lines MCF7 and MDA-MB-231. No
difference between SA or S1 treated cells and vehicle treated cells was observed. 2) At 10
-5
M
and 10
-6
M, S1 only inhibits tube formation in a matrigel assay (an in vitro angiogenesis model).
3) Using cell culture supernatants, we observed that SA and S1 (10
-5
M and 10
-6
M) decreased
by 60% VEGF production in MCF7 and MDA-MB-231 vs vehicle treated cells (p0.008) (VEGF
ELISA). Conclusion : SA and S1 do not modify HUVEC, MCF7 and MDA-MB-231 proliferation
but inhibit tumour angiogenesis by decreasing VEGF production from MCF7 and MDA-MB-231.
S1 but not SA inhibits endothelial cell differentiation.
P536
Identification of Genes Homing to Developing Collaterals in Ischemic
Tissue
Marcel O Schmidt, Georgetown Univ, Washington, DC; Stephan Zbinden, Mary Susan
Burnett, Stephen E Epstein, Washington Hosp Cntr, Washington, DC; Anton Wellstein;
Georgetown Univ, Washington, DC
Collateral development (collaterogenesis) is a critical compensatory response to coronary and
peripheral arterial obstructive disease, but natural mechanisms do not restore normal flow
capacity. Clinical trials of angiogenic agents have failed to demonstrate important improvement
in collaterogenesis. One possible strategy to enhance collaterogenesis is to specifically target
delivery of collaterogenic agents to developing collaterals, resulting in high local concentrations
without systemic toxicity. To achieve this, specific molecules homing to developing collaterals
must be identified. We employed a newly developed phage display technique to identify such
molecules. Methods and Results: ApoE mice underwent femoral artery ligation to create
hindlimb ischemia. A T7 phage display cDNA library from human bone marrow cells was
injected intravenously at several time points following ligation. Phages bound to developing
collaterals were amplified and successively re-injected into another mouse. After several
enriching rounds we identified the cDNA inserts of the phage, which preferentially bound to the
endothelium of developing collaterals. Phage expressing these gene fragments on their surface
bound to collaterals with up to 130 times higher affinity than to tissue of control mice. We
enriched for 13 different cDNA inserts coding for unknown and known genes. In an in vitro
assay, selected phage activated an endothelial cell monolayer resulting in increased
susceptibility to monocyte invasion. Expression studies of cDNA libraries of various tissues
showed that the cDNA inserts are expressed preferably in undifferentiated bone marrow tissue.
Conclusions: We have demonstrated proof-of-concept that our phage display strategy identifies
molecules that home preferentially to developing collaterals. With this approach we identified
13 different gene fragments coding for ligands that not only bind specifically to developing
collaterals, but also induce endothelial activation. We anticipate using such molecules to deliver
therapeutics to developing collaterals, and as activating ligands that may induce collateral
development.
P537
Type 2 Diabetes Impairs Collateral Artery Enlargement More than Type 1
Diabetes in Response to Hind Limb Ischemia in Mice
Jinglian Yan, Guodong Tie, Yagai Yang, Brian D Park, Robert L Raffai, Univ of California,
San Francisco, San Francsico, CA; Louis M Messina; Univ of Massachusetts Med Sch,
Worcester, MA
Objective: Diabetes is an important major risk factor for peripheral arterial disease. Whether
Type 1 or Type 2 diabetes has similar effects on the vascular response to limb ischemia is
unknown. We investigated the effects of type 1 versus type 2 diabetes on blood flow recovery,
arteriogenesis, and angiogenesis after induction of hindlimb ischemia. Methods: Ischemia was
induced in streptozotocin (STZ)-treated (Type 1 diabetes), db/db (Type 2 diabetes), and wild
type C57BL/6 mice (control). Serial Laser Doppler perfusion studies followed by angiography
were performed in all three groups to determine blood flow recovery, and the number of
collateral arteries. Histochemical analysis of muscle was also performed to determine capillary
density, collateral diameter and fat infiltration. Results: Blood flow recovery after hindlimb
ischemia was less complete in both Type 1 and 2 diabetes than in wild type mice (p0.05).
Both types of diabetes showed significantly fewer collateral arteries and smaller diameters than
did controls (p0.05). However, Type 2 diabetic mice showed a greater impairment in blood
flow recovery than Type I diabetic mice (p0.05). This difference was not due to fewer
numbers of collateral arteries or lower capillary density (pNS), despite lower pre-ischemic
capillary density in type 2 diabetic mice (P0.05). Rather, Type 2 diabetic mice had collateral
arteries of significantly smaller diameters than Type 1 diabetic mice (p0.05). Despite very low
perfusion in the ischemic limb, no gangrene was observed in type 2 diabetic mice. In contrast,
muscle weight was significantly increased in type 2 diabetic mice due to extensive fat
infiltration unlike in type 1 diabetic or control mice. Conclusions: Type 2 diabetic mice
displayed a distinctly different response to hindlimb ischemia than Type 1 diabetic mice. Type
2 diabetic mice had a greater impairment in blood flow recovery than Type 1 diabetics because
of less collateral artery enlargement. The extensive fatty infiltration of the ischemic muscle of
type 2 diabetic mice has not been documented in any other disease model. The molecular
mechanisms responsible for these differences in blood flow recovery and collateral artery
enlargement between Type 1 & 2 diabetes remain the focus of ongoing studies.
P538
In Vivo Imaging of Murine Vasodynamics by Fourier Domain Optical
Coherence Tomography
Gregor Muller, Sven Meissner, Alexander Krueger, Birgit Eichhorn, Ursula Ravens, Edmund
Koch, Henning Morawietz; Univ of Technology Dresden, Dresden, Germany
In vivo imaging of vessels can provide new insights in the regulation of vasodynamics. In
contrast to established isometric force measurements, fourier domain optical coherence
tomography (OCT) imaging allows the investigation of vasoconstriction and vasodilatation in the
anatomical context of the vessel without preparation traumata. The murine saphenous artery
is a suitable model for the in vivo examination particularly due to the small diameter, the
significant response of the blood vessel to vasomotor stimuli and the advantageous anatomical
position for transluminal imaging. Male C57BL/6 mice (8 weeks of age) were fed a control and
a high-fat diet for 10 weeks. Changes in blood vessel diameter were induced by dermal
application of the vasodilator sodium nitroprusside (SNP) and the vasoconstrictor potassium.
OCT images were acquired with a two-axis galvanometer scanner head which operated in 2D
or 3D mode. Three dimensional image stacks were used to determine the morphology of the
saphenous artery, vein and nerve. Time series (3 frames per second, 300512 pixel per frame)
of cross-sectional images were analysed with image processing software measuring the time
course of the vessel lumen dynamics. The results of this feasibility study are summarized in
the table (n4, meanSEM). In conclusion, fourier domain optical coherence tomography
allows the imaging of the vasodynamics of murine vessels in vivo. Further studies using
high-fat diet in atherosclerosis-sensitive mice models will extend our knowledge about
diet-specific changes in vasodynamics.
2007 ATVB Poster Presentations e-131
by on March 25, 2011 atvb.ahajournals.org Downloaded from
control diet high-fat diet P value
inner diameter, m 174.813.3 137.017.1 0.132
-after vasoconstriction 65.813.5 698.9 0.848
-after vasodilatation 250.520.8 242.83.8 0.726
time to half-maximal constriction, sec 5.60.9 6.21.4 0.760
time to half-maximal dilatation, sec 6.61.1 9.51.3 0.144
flow resistance, (Pa s)/m
3
2.41 10
8
8.60 10
7
7.38 10
8
2.46 10
8
0.105
-after vasoconstriction 2.43 10
10
1.40 10
10
1.28 10
10
5.58 10
9
0.466
-after vasodilatation 5.66 10
7
1.66 10
7
5.32 10
7
3.38 10
6
0.849
wall tension, mN/mm 0.720.02 0.680.06 0.552
-after vasoconstriction 4.420.41 3.440.35 0.115
-after vasodilatation 0.530.02 0.560.05 0.625
P539
Cryoimaging of Atherosclerotic Lesions for 3D Microscopic Identification
Mike Nguyen, Olivier Salvado, Robert Hoffman, David L Wilson; Case Western Reserve Univ,
Cleveland, OH
Our objective is to obtain 3D microscopic characterization of large atherosclerotic vessel
specimens in less time and expense than standard histological methods, the gold-standard for
identifying tissue types. These data sets can be used to validate other imaging modalities such
as MRI or for high throughput animal model studies. We used cryo-imaging developed at Case
Western Reserve University where block face of frozen samples are imaged with bright-field
and autofluorescence imaging. We hypothesized that the main tissues in atherosclerotic
lesions, e.g., lipid, fibrosis, calcification, can be accurately identified using cryoimaging. A
whole vessel segment up to 5 cm can be imaged and a 3D microscopic volume generated. To
test this hypothesis we conducted a validation study. Common iliac arteries from 10 cadavers
were removed at autopsy and frozen within 4 hours into a block of cryo-histological medium
gel (OCT). Blocks were positioned on a large cryomicrotome stage and 2 mm rings were cut
after imaging the block face with an episcopic microscope at 7.5x magnification (39 m x 39
m resolution) using bright-field and autofluorescence imaging. For each ring, standard
histological preparations were done: either H&E, elastic van Giesson, and Mallory trichrome, or
oil-red-O. A total of 192 matched cryoimages and histological sections were collected. From
the comparison between cryoimaging and histology, we established rules to identify tissue
types. Color/autofluorescence levels were adventitia: red-textured/, media: pink/,
solid calcification: white-textured/, lipid: yellow/0, cholesterol cleft: yellow-brown/
, and connective tissue: pale-pink/.
P540
Excess Plasma Cholesterol in Patients with Single Cardiovascular Risk
Factor, Increases Select Phenotype Endothelial Microparticles Expression
and Directly Correlates With Elevated Circulating Levels In Blood
Marian T Calfa, Lucia M Mauro, Alexander C Ferreira, Hannah J Dodson, Wenche Jy, Yeon
S Ahn, Eduardo De Marchena, Joaquin J Jimenez; Univ of Miami, Miami, FL
Background: In response to injury, endothelial cells (EC) were shown to release membrane-
derived microparticles CD 31/42b- and CD 62E (EMP). Elevated circulating EMP levels in
blood were documented with multiple cardiac risk factors. Nonetheless, the link between EMP,
markers for endothelial injury and plasma cholesterol was not independently studied.
Therefore, we analyzed in patients with no interfering coronary disease risk factors the impact
of high (HC) as opposed to normal cholesterol on EMP release, circulating levels respectively
the correlation with plasma cholesterol level and low-density lipoprotein (LDL). Methods: High
and normal cholesterol participants, age (45 years) and gender matched, exhibited no
associated major coronary risk factor. EMP counts in fresh blood samples was assayed by flow
cytometry using anti-CD31, 42b, 62E antibodies. Coronary artery endothelial cells (CAEC) were
cultured and treated with plasma from each group. All methods employed were previously
reported. Statistical analysis was performed considering a p0.05 of significance. Results
(see table): Cholesterol and LDL were significantly higher in the HC group (each p0.001).
Circulating levels of CD31/42b- EMP but not CD 62E were increased with HC as opposed
to control (p0.001). CD 31/42b- release from CAEC in culture was induced with HC plasma
compared to normal cholesterol and negative control (each p0.01). There was no effect on
CD 62E induction (p0.62). Correlation analysis between cholesterol, LDL levels and
circulating EMP count revealed a consistently positive association only with CD 31/42b- and
not with CD 62E phenotype. Conclusion: Cholesterol rich plasma affects EC and induces
select phenotype EMP release. HC, respectively LDL is independently and directly associated
with increased CD 31/42b-EMP phenotype level, in contrast to CD 62E. Therefore, elevated
count and EMP phenotype reflect the endothelial injury pattern associated with HC.
STUDY RESULTS TABLE
Groups
n17
Cholesterol
level
(mg/dL)
LDL
level
(mg/dL)
CD31/
42b-
level
(x10
6
/mL)
CD 62E
level
(x10
6
/mL)
CAEC
released
CD31/
42b-
count
(x10
6
/mL)
CAEC
released
CD
62E
count
(x10
6
/mL)
CD31/
42-
correlation
analysis
CD62E
correlation
analysis
High
Choles-
terol
242.3544.09 182.0713.76 1.190.61 0.290.10 0.750.24 0.260.07 Positive
With
LDL
R0.71
p0.0001
Choles-
terol
R0.60
p0.001
No
relation
With
LDL
R0.25
p0.19
Choles-
terol
R0.12
p0.49
Normal
Choles-
terol
140.0017.59 85.5711.34 0.340.08 0.260.11 0.230.1
Negative
control
0.190.07
0.240.6
Negative
control
0.210.4
P541
Nonhematopoietic NADPH Oxidase Regulates VCAM-1-dependent Leukocyte
Migration in Vivo: A Role for Endothelial Cell NADPH Oxidase
Joan M Cook-Mills, Hiam Abdala-Valencia, Northwestern Univ Feinberg Sch of Medicine,
Chicago, IL; Marsha Wills-Karp; Cincinnati Childrens Hosp Rsch Foundation, Cincinnati, OH
Leukocytes migrate from the blood into tissue by binding to and migrating across endothelial
cells. One of the endothelial cell adhesion molecules that mediate leukocyte binding is vascular
cell adhesion molecule-1 (VCAM-1). VCAM-1 is expressed on endothelial cells at sites
predisposed to atherosclerosis and the VCAM-1 knockout mouse is an embryonic lethal. In
addition, the infiltration of eosinophils into the lung in experimental asthma is dependent on
VCAM-1. We have reported that ligand binding to VCAM-1 activates endothelial cell NADPH
oxidase for the generation of reactive oxygen species (ROS). These VCAM-1-stimulated ROS are
required for VCAM-1-dependent leukocyte migration in vitro. Therefore, we examined whether
endothelial-derived NADPH oxidase modulates VCAM-1-dependent leukocyte recruitment in
vivo. To examine non-hematopoeitic NADPH oxidase function in vivo, mice deficient in NADPH
oxidase (CYBB mice) were irradiated and received wild type hematopoietic cells to generate
chimeric CYBB mice. To examine VCAM-1-dependent leukocyte migration, the mice were
challenged with the antigen chicken ovalbumin (OVA) as this induces VCAM-1-dependent
eosinophilia. In response to OVA, the chimeric CYBB mice had increased numbers of
eosinophils bound to the endothelial luminal surface as well as reduced eosinophilia in the lung
tissue and bronchoalveolar lavage. This occurred without changes in VCAM-1 expression or
cytokine/chemokine levels (IL-5, IL-10, IL-13, IFN, or eotaxin) in the lavage fluids or lung
tissue of OVA-challenged mice. There was also no change in leukocyte infiltration into the lung
that occurs independent of VCAM-1. Interestingly, the OVA-challenged chimeric CYBB mice had
reduced airway hyperresponsiveness (AHR). The AHR in OVA-challenged chimeric CYBB mice
was restored by bypassing the endothelium with intratracheal administration of eosinophils.
These data suggest that VCAM-1 induction of NADPH oxidase in the endothelium is necessary
for the VCAM-1-dependent leukocyte recruitment during inflammation. Moreover, these studies
provide a basis for targeting VCAM-1-dependent signaling pathways in anti-inflammatory
therapies.
P542
Mst1 Plays an Important Role in TNF-induced Apoptosis of Vascular
Endothelial Cell
Toshihiro Ichiki, Hideki Ohtsubo, Kenji Sunagawa; Kyushu Univ Grad Sch of Med Sciences,
Fukuoka, Japan
Background: Apoptosis of endothelial cells (ECs) is observed in unstable atherosclerotic plaques
and believe to induce erosion of plaque that leads to formation of thrombus and development
of acute coronary syndrome. Mst1, a member of mammalian sterile20-like family protein
kinase, is cleaved and activated during apoptosis. Although staurosporine and genotoxic agents
are reported to activate Mst1, no physiological stimuli reported to date activate Mst1 except for
Fas/FasL signal. Tumor necrosis factor- (TNF-) is one of the mediators inducing apoptosis,
however, the role and activation of Mst1 in TNF-- induced EC apoptosis have never been
e-132 Arterioscler Thromb Vasc Biol June 2007
by on March 25, 2011 atvb.ahajournals.org Downloaded from
examined. Aim: We examined whether TNF- activates Mst1 in EC. Results: Western blot
analysis for Mst1 showed that TNF- induced cleavage of Mst1 in a time- and dose-dependent
manner, which indicated the activation of Mst1 kinase activity. TNF--induced Mst1 activation
was significantly attenuated by pretreatment with Z-DEVD-FMK, a caspase3 inhibitor,
confirming the previous report showing that Mst1 is activated by caspase 3. However,
downregulation of Mst1 by siRNA did not affect TNF--induced activation of caspase 3, which
is not consistent with the previous data that Mst1 and caspase 3 constitute a positive feed back
loop. Inhibitors for mitogen activated protein kinases (ERK, p38MAPK and JNK) did not affect
TNF--induced Mst1 activation. Diphenyleneiodonium, a NADPH oxidase inhibitor, and
N-acetylcysteine, an antioxidant, also suppressed TNF--induced activation of Mst1 and
caspase3, suggesting a role of reactive oxygen species in the activation of Mst1. Nuclear
staining with Hoechst33258 and fluorescence activated cell sorting (FACS) analysis showed
that TNF- and overexpression of Mst1 induced apoptosis of ECs. TNF--induced EC apoptosis
was attenuated by introduction of siRNA for Mst1 but not by scramble RNA. Conclusion: These
results suggest that TNF- induces Mst1 activation through activation of caspase3 and
oxidative stress, and that Mst1 plays an important role in the induction of TNF--induced
apoptosis of ECs. Therefore, inhibition of Mst1 in unstable plaque may reduce apoptosis of ECs
and stabilize vulnerable plaque.
P544
Flow Differentially Regulates the mTOR Pathway in Cocultured Endothelial
and Smooth Muscle Cells
Carla Olive, Marina Santacana, MIT, Cambridge, MA; Angelo A Cardoso, Univ of Indiana,
Indianapolis, IN; Mercedes Balcells, Elazer R Edelman; MIT, Cambridge, MA
Hemodynamic forces are powerful regulators of vascular endothelial cell (EC) and smooth
muscle cell (SMC) biology and phenotype. Endovascular stents, in particular drug-eluting, are
routinely used for atherosclerotic obstructive disease and yet the biology of local delivery has
not been fully elucidated. We hypothesized that stent deployment alters local hemodynamics
eliciting profound effects on the response of underlying SMC and recovery of damaged
endothelium. Single cultures of EC or SMC, and sequentially layered SMC/EC co-cultures
seeded on silicone tubes were exposed to coronary artery-like flow for 20 min or 24 h.
Perfusate consisted of cytokine-containing media (VEGF, FGF-2, IGF-1 and EGF), in the
presence and absence of the rapamycin analogue CCI-779 (10 nM). After flow exposure, cells
were immediately harvested and fixed for analysis. mTOR signalling was evaluated by flow
cytometric detection of phosphorylated S6 ribosomal protein (P-S6RP). Bare metal stents were
expanded within tubes before exposure to flow. Endothelial recovery after stenting was
assessed using microscopic examinations. Experiments were carried out in triplicate. P-S6RP
expression in EC increased 2-fold by flow exposure, but not in SMC. Mean intensity
fluorescence (MIF) of isolated EC cultures under static conditions was 535.0 21, and after
flow exposure increased to 1054 42 (p0.001). Interestingly, no differences in mTOR
activation were observed for SMC. In the sequentially layered SMC/EC co-cultures, the
expression of P-S6RP markedly increased in CD31 EC under static (MIF 980 14 p0.001
vs single cultures of EC) and flow (1578 66, p0.002 vs single culture of EC). CCI-779,
abrogated the effect of flow on mTOR signalling reducing P-S6RP to below basal levels both
with (160 16, p0.001 vs flow basal level), and without flow (151 8, p0.001 vs static
basal level). Stent endothelialization under flow in stents with inhibitor was 4.6-fold lower
(4.8 0.9 cells 10
3
/m
2
) than in stents without inhibitor (22.2 6.7 cells 10
3
/m
2
,
p0.05). Our findings suggest that activation of the mTOR pathway on EC and SMC is
differentially regulated by flow, extending our understanding of the integration of flow and
autocrine/paracrine regulation of vascular repair.
P545
In Vivo Human Lower-extremity Saphenous Vein Bypass Grafts Manifest
Flow-mediated Vasodilation
Christopher D Owens, Nicole Wake, Michael S Conte, Andres Schanzer, Marie D
Gerhard-Herman, Mark A Creager, Joshua A Beckman; BWH, Boston, MA
Objective: Physiological changes in lower extremity saphenous vein bypass grafts (SVG) used
for arterial reconstruction are undefined. The effect of arterial pressure and flow on vein graft
endothelial and vascular SMC function are unknown. We hypothesized that in patent vein
grafts, endothelial function regulates graft homeostasis and that SVGs would exhibit flow-
mediated, endothelium-dependent (ED) vasodilation. Methods: Patients with femoral to
popliteal artery SVGs without revision were enrolled. High-resolution, B-mode ultrasound was
used to measure SVG diameter before and 1 min after reactive hyperemia (RH) to determine
flow-mediated vasodilation (FMD). RH was created by application of 220 mm Hg to the calf for
5 min with a BP cuff. After a 10 min rebaseline period, TNG-mediated, endothelium-
independent (EI) vasodilation was measured 3 min after TNG 0.4 mg SL. Brachial artery FMD
was determined by standardized and validated techniques. Results: Sixteen subjects were
enrolled. The mean age was 64.79.2 years. The median time from bypass to graft study was
34.6 (21.049.7) months. Following RH, time-averaged, volumetric flow increased 219.5%
40.5%, p.0001, corresponding to an increase in shear stress of 178.9 36.7%, p.0007.
SVG flow-mediated, ED dilation was 4.8% 0.6%, p.0001. TNG-mediated, EI vasodilation
was 4.6% 0.9%, P.02. Brachial artery FMD was 9.6%. Conclusions: SVGs manifest
flow-mediated, ED and TNG-mediated vasodilation. Vein graft endothelial function likely
participates in the maintenance of vessel homeostasis. Further investigation will be required to
determine the role of endothelial function in vein graft patency.
P547
WITHDRAWN
P548
Receptor-dependent Activation of Inducible Nitric Oxide Synthase in
Endothelial and Transfected Cells: Critical Role of iNOS Phosphorylation
Yongkang Zhang, Victor Brovkovych, Fulong Tan, Svitlana Brovkovych, Tiffany Sharma,
Randal A Skidgel; Univ of Illinois College of Medicine, Chicago, IL
Endothelial-derived NO is an important mediator of vascular function. Under normal conditions,
eNOS generates low levels of NO and is strictly regulated by a variety of mechanisms. Exposure
of endothelial cells to inflammatory mediators induces expression of iNOS and the kinin B1
receptor (B1R). Once expressed, iNOS is thought to constitutively produce high output NO until
it is degraded. Here we show that iNOS activity can be acutely upregulated by activation of
B1Rs in human endothelial cells or transfected HEK293 cells to generate 2.5 to 3-fold higher
NO. To test the hypothesis that B1R agonists induce activation of extracellular signal-regulated
kinase (ERK) which then phosphorylates and activates iNOS, we cotransfected HEK cells with
B1R and iNOS. B1R agonist des-Arg
10
-kallidin (DAKD; 100 nM) stimulated ERK activation,
phosphorylation of iNOS on serine and generation of super-high output NO. ERK activation
inhibitor PD98059 greatly diminished NO production and also inhibited phosphorylation of iNOS.
Ser
745
in iNOS was identified as the critical residue phosphorylated by ERK using an in vitro
kinase assay and MALDI-TOF mass spectrometry of tryptic peptides from iNOS immunopre-
cipitated from transfected HEK293 cells. A peak corresponding to the phosphorylated form of
the tryptic peptide QNLQS
745
PTSSR at m/z 1197.526 was detected in iNOS treated in vitro with
activated ERK or iNOS from cells stimulated with 100 nM DAKD, but not in iNOS from untreated
cells or treated cells transfected with iNOS mutated at Ser
745
. iNOS and ERK were colocalized
in subcellular domains as determined by confocal imaging and also co-immunoprecipitated,
indicating that they interact. Transfection of cells with S745A mutant iNOS eliminated
phosphorylation and abolished the ability of B1R signaling to generate iNOS-dependent NO, but
did not inhibit basal iNOS activity. In contrast, the S745D mutant (mimics phosphorylation) had
much higher basal activity, but was not activated by DAKD. This is the first demonstration that
iNOS can be acutely activated by receptor-dependent ERK signaling and reveals a previously
unappreciated level of complexity in its regulation. This novel pathway may play an important
role in inflammatory vascular disease.
P549
Enhanced Injury Response of Murine Aortas from Mice Exposed to
Cigarette Smoke
Jason A Meyers, Amy E Hackmann, Batool Arif, Terri L Ennis, Robert W Thompson, John A
Curci; Washington Univ Sch of Medicine, St. Louis, MO
Cigarette smoke exposure leads to the enhanced development AAA in humans and murine
models. We hypothesized that aortas taken from mice exposed to tobacco smoke would have
an enhanced cytokine response to matrix injury. Methods: Male C57 mice were subjected to
smoke from 3 filterless research cigarettes a day for 6 days per week for 4 (N5) or 6 weeks
2007 ATVB Poster Presentations e-133
by on March 25, 2011 atvb.ahajournals.org Downloaded from
(N5). Littermate mice were not exposed to smoke for either 4 (N5) or 6 weeks (N4). At
sacrifice, 2 mm rings were prepared from infrarenal aorta and placed in serum free culture
media. After 4 hours, the media (CM) was removed, stored and replaced with media containing
elastase (0.16 U/mL)for another 4 hours. All CM was assayed for 13 cytokines on a bead array
apparatus. The ratio between the stimulated and unstimulated CM was calculated. Results: The
production of cytokines from the unstimulated aortic explants was similar for all mice.
Stimulation of aortas from control animals showed significant increases in the production of
IFN-, IL-10, IL-12 and MCP-1. After 6 weeks of smoke exposure, elastase stimulation
significantly increased production of the same cytokines as well as MIP-1. By comparing the
stimulated cytokine production between aortas taken from the smoke-exposed and control
animals, we found all 5 cytokines were released in significantly higher concentrations by aortas
from smoke exposed mice. The IL-1 response to stimulation was reduced by smoking,
although the change was not significant. There appeared to be a dose-dependent trend
between the duration of smoke exposure in vivo and the cytokine response. Conclusions:
Cytokine response of the aorta to matrix injury is significantly greater after in vivo tobacco
smoke exposure.
P550
Temperature and Blood Pressure Following Amlodipine Overdose: A Test of
the Thermoregulatory-Vascular Remodeling Hypothesis
Imran H Iftikhar, Fairview Hosp, Cleveland Clinic, Cleveland, OH; Robert P Blankfield, Case
Sch of Medicine, Cleveland, OH; Stuart Harris; Massachusetts General Hosp, Harvard Med
Sch, Boston, MA
BACKGROUND: The cardiovascular system participates in both blood pressure (BP) and
temperature regulation. The thermoregulatory-vascular remodeling (TVR) hypothesis postulates
that temperature homeostasis has precedence over BP homeostasis. As a result, salt ingestion
creates conflict between BP and temperature homeostasis, for prompt vasodilatation to lower
the BP would increase cutaneous blood flow, thereby accelerating heat loss and lowering the
core body temperature. In order to avoid heat loss, salt ingestion elevates the BP until the
kidneys excrete the excess salt. During these transient elevations in BP, peripheral resistance
increases due to vascular remodeling. Since vascular remodeling is irreversible, the baseline
peripheral resistance increases incrementally following an ingestion of salt. After numerous
episodes of salt ingestion, the baseline BP also rises. OBJECTIVE: A case of amlodipine
overdose offered an opportunity to test one of the predictions of the TVR hypothesis: that
vasodilators increase cutaneous blood flow, thereby accelerating heat loss. Consequently, one
would expect either a drop in body temperature and/or an increase in the metabolic rate.
METHODS: Following the ingestion of 1000 mg of amlodipine, the temperature and BP of a
single patient were monitored following presentation for emergency care, during the initial 24
hours of hospitalization, and during the final 24 hours of hospitalization. Temperature readings
were available beginning 7 hours post ingestion, and then only intermittently, varying from once
an hour to once every four hours. RESULTS: The BP dropped markedly between the 5
th
and 7
th
hours post ingestion, but then the BP rose steadily and normalized by 28 hours post ingestion.
The temperature was normal at 7 hours post ingestion, declined gradually between the 7
th
and
26
th
hours post ingestion, stabilized between the 26
th
and 31
st
hours post ingestion, then began
to rise. CONCLUSIONS: During this single case of amlodipine overdose, a modest temperature
decline lagged behind a marked BP decline. The BP normalized following medical therapy. As
the BP rose, the temperature also rose, but lagged behind the BP increases. These results are
consistent with the predictions of the TVR hypothesis.
P552
A Novel Small Molecule Inhibitor Targeted to a Ubiquitin-Ligation Enzyme
Slows Down Formation of Abdominal Aortic Aneurysms in a Rat
Experimental Model
Yoshiki Kyo, Hongxia Liu, Wayne State Univ, Detroit, MI; Brenda S Cho, Univ of Michigan,
Ann Arbor, MI; Lakshmi Padmavathi, Univ of Osnabru ck, Osnabru ck, Germany; Karen
Roelofs, John W Ford, Indranil Sinha, Univ of Michigan, Ann Arbor, MI; Guy M Lenk, Gerard
Tromp, Wayne State Univ, Detroit, MI; Gilbert R Upchurch, Jr, Univ of Michigan, Ann Arbor,
MI; Helena Kuivaniemi, Amit Banerjee; Wayne State Univ, Detroit, MI
The objective of the present study was to identify new therapeutics for abdominal aortic
aneurysms (AAA). We used microarray expression profiling to identify biological pathways
relevant to AAA pathology and to identify potential targets in these pathways against which to
develop novel small molecule inhibitors. We identified the ubiquitin-proteasome pathway and
targeted the ubiquitin ligation enzymes. Our approach is structural chemical-genetic ligand
design to identify small molecule inhibitors of selected ubiquitin-ligation enzymes. The
compounds were tested in the elastase-perfusion model for AAA in rats. Animals receiving only
the vehicle (dimethylsulfoxide, DMSO; n5) developed AAAs, while rats treated with
doxycycline (n19) and our novel inhibitor called 0464 (n9) had smaller increases in aortic
diameter 14 days following elastase-perfusion (comparison to DMSO group: p0.05 for
doxycycline group and p0.05 for 0464 group). Histological analysis showed that elastin in the
medial wall (Verhoeffs van Gieson stain) was degraded in DMSO aortas, whereas it remained
intact in the aortas of doxycycline and 0464-treated rats. The inflammatory cells were reduced
in doxycycline and 0464 groups compared to controls. The detailed mechanistic target
validation of the small molecule inhibitor 0464 is being undertaken. Together these results
demonstrate that the novel small molecule inhibitor 0464 targeted against ubiquitin- ligation
enzyme was effective in slowing down the growth of AAA in the rat elastase-perfusion model
to the same extent as doxycycline.
P553
Angiotensin II Infusion Results in Region-specific Aortic Hypertrophy and
Hyperplasia Requiring AT1a Receptor Activation of p47
phox
and ID3 in a
Pressure-independent Manner
A P Owens III, Jessica J Moorleghen, Univ of Kentucky, Lexington, KY; Coleen A McNamara,
Univ of Virginia, Charlottesville, VA; Alan Daugherty; Univ of Kentucky, Lexington, KY
Objective: We previously demonstrated that angiotensin II (AngII) infusion leads to an AT1a
receptor (AT1aR) dependent medial arch hyperplasia as well as a medial hypertrophy in the
thoracic and abdominal regions; independent of blood pressure. The objective of this study was
to determine the mediators of this AT1aR-induced medial change. Furthermore, we assessed
the mechanism responsible for AngII-mediated aortic arch hyperplasia. Methods and Results:
P47
phox
is a critical component of the NADPH oxidase complex, which is a key mediator of
AngII-induced smooth muscle cell (SMC) hypertrophy. To determine its importance to the
development of AngII-induced aortic medial thickness, male p47
phox
mutant mice (p47/p47)
were infused with either saline, AngII (1,000 ng/kg/min), or norepinephrine (NE, 5.6 mg/kg/day)
for 28 days (n5 each). After the infusion, mice were perfusion-fixed and the ascending arch,
thorax, supra, and infra-renal abdomen were sectioned serially. The medial thickness of aortic
tissue sections ( 512 per region) was measured by image analysis. p47/p47 mice were
resistant to increased thickness during AngII-infusion, consistent with this subunit being a
critical component of AT1aR signaling. Furthermore, p47/p47 mice had greatly attenuated
increases in systolic blood pressure (SBP) during AngII-infusion. NE infused mice had
significantly increased SBP, but without medial expansion, suggesting the p47/p47 effects are
pressure independent. ID3 is a helix-loop-helix factor that induces AngII-mediated SMC
proliferation. ID3-/- male mice were infused with saline (n6) and AngII (n7) to ascertain if
this mitogenic protein leads to the aortic arch hyperplasia. ID3 deficiency did not alter
AngII-induced medial thickening, however, the AngII-induced hyperplastic response was
attenuated in the ascending arch. Conclusion: The increase in aortic medial thickness, induced
by AngII infusion, is mediated via AT1aRs leading to activation of the p47
phox
subunit of NADPH
oxidase, in a pressor independent manner. Furthermore, attenuation of the AngII-induced aortic
arch hyperplasia in ID3-/- mice suggests that this protein is responsible for the mitogenic effect
in this region.
P554
Continuous Angiotensin II Infusion Promotes Progressive Expansion and
Vascular Remodeling of Abdominal Aortic Aneurysms in Apolipoprotein
E-/- Mice
Debra L Rateri, Deborah A Howatt, Jessica J Moorleghen, Univ of Kentucky, Lexington, KY;
Chiara Barisione, Univ of Genoa, Genoa, Italy; Alan Daugherty; Univ of Kentucky, Lexington,
KY
Objective: Chronic subcutaneous angiotensin II (AngII) infusion into mice leads to rapid initiation
of abdominal aortic aneurysms (AAAs). The objective of this study was to determine whether
protracted AngII infusion led to AAA progression and changes in pathology. Methods and
Results: Male ApoE-/- mice were infused with AngII (1,000 ng/kg/min) by Alzet mini-osmotic
pump for 28 days. Lumen diameters of the abdominal aortas were measured by high frequency
(40 MHz) ultrasound. Mice with AAAs were assigned to 3 groups at 28 days. Group 1 was
terminated (n15); Group 2 was infused for a further 56 days with AngII (n15); Group 3 was
infused for a further 56 days with saline (n13). An additional Group 4, without any infusion,
was observed at 84 days. Infusion of AngII increased systolic blood pressure (SBP) by 30
mmHg throughout the infusion period in Gp 2. However, the removal of AngII led to an
immediate return to baseline SBP in Gp 3. Groups 13 exhibited increased aortic lumen
dimensions at 28 days of infusion. Lumen dimensions were further increased during continued
AngII infusion in Gp2, but remained at 28 day values in saline-infused Gp 3 mice. At 28 days
of AngII infusion, the expanded lumen was frequently associated with adventitial thickening,
thrombus and macrophage infiltration (CD68 cells). At 84 days of AngII infusion, profoundly
dilated aortas were associated with extensive wall thinning and neomedial areas composed of
CD68 cells. Thrombus was not evident in AAAs from mice infused with 84 days of AngII. Gp
3 mice infused with saline also had thinned aortic walls with reduced thrombus; however,
CD68 cells were less prevalent. No abnormal pathology was noted in abdominal aortas of
mice infused with saline for 84 days. Conclusion: Continuous AngII-infusion led to progressive
expansion of the aortic lumen. These dilated AAAs exhibited different features, including
thinned walls, and profound macrophage infiltration than normal vessels.
e-134 Arterioscler Thromb Vasc Biol June 2007
by on March 25, 2011 atvb.ahajournals.org Downloaded from
P555
Nox-1 Knockout Does Not Affect Angiotensin II-dependent Chronic
Hypertension and Cardiac Hypertrophy
Alvaro Yogi, Univ of Ottawa, Ottawa, Canada; Chantel Mercure, Joshua G Touyz, Clinical
Rsch Institute of Montreal, Montreal, Canada; Augusto C Montezano, Glaucia E Callera, Univ
of Ottawa, Ottawa, Canada; Rita C Tostes, Univ of Sao Paulo, Sao Paulo, Brazil; Rhian M
Touyz, Univ of Ottawa, Ottawa, Canada; Timothy L Reudelhuber; Clinical Rsch Institute of
Montreal, Montreal, Canada
Background: The gp91phox (Nox2)-containing NADPH oxidase is the major source of reactive
oxygen species (ROS) in the cardiovascular system. Inactivation of gp91phox blunts hyperten-
sion and cardiac hypertrophy in acute studies of Ang II-infused animals, but not in mice in
which the endogenous renin-angiotensin system (RAS) is chronically upregulated. The role of
other Nox2 homologues, such as Nox1, in chronic Ang II-dependent hypertension is unknown.
Objective: In the present study we sought to determine whether Nox1 plays a role in the
activation of redox-sensitive pathways leading to development of hypertension and cardiac
hypertrophy in a model in which the RAS is chronically upregulated Methods and results:
Nox1-deficient mice and transgenic mice expressing active human renin in the liver (TTRhRen)
were crossed and four genotypes generated: control, TTRhRen transgenics (TTRhRen),
Nox1-deficient (Nox1
-
), and TTRhRen transgenic Nox1-deficient (TTRhRen/Nox1
-
). Blood
pressure, cardiac mass, and cardiac fibrosis were increased in TTRhRen versus controls. This
was associated with increased activation of NAD(P)H oxidase. Nox1 inactivation had no effect
on development of hypertension or cardiac hypertrophy in TTRhRen mice and p47phox
translocation was nor altered. Expression of Nox1 homologues, Nox2 and Nox4 was not
different between groups. Phosphorylation of redox-sensitive growth signaling molecules such
as Akt, p38MAPK, SAPK/JNK, ERK 1/2 and ERK5 was increased 23-fold in TTRhRen mice. Akt,
p38MAPK and SAPK/JNK phosphorylation was significantly reduced in TTRhRen/Nox1
-
versus
TTRhRen mice (p0.05). Activation of the non-receptor tyrosine kinase c-Src was increased
in TTRhRen mice (2.5-fold) and attenuated in TTRhRen/Nox1
-
mice. Conclusion: Our findings
demonstrate that in TTRhRen/Nox-1-deficient mice activation of NAD(P)H oxidase and
phosphorylation of redox-sensitive signaling molecules are reduced. However, these effects are
not associated with reduction of blood pressure and cardiac hypertrophy. These data suggest
that Nox1-containing NAD(P)H oxidase may not be important in hypertension and cardiac
hypertrophy in a model in which the endogenous renin-angiotensin system is chronically
upregulated.
P556
Akt Isoforms Have Distinct Roles in Photypic Modulation of VSMC and
During Vascular Injury
James E Cooper, Nihal Kaplan-Albuquerque, Mary C M Weiser-Evans, Raphael A Nemenoff;
Univ of Colorado Health Sciences Cntr, Denver, CO
Vascular smooth muscle cells (VSMC) retain the ability to de-differentiate from the contractile
to a migratory and proliferative phenotype in response to environmental cues associated with
pathophysiologic states. During this phenotypic switch, VSMC loose the expression of
contractile proteins such as smooth muscle -actin (SMA) and SM22. In cultured VSMC,
stimulation with platelet derived growth factor BB (PDGF-BB) results in a loss of smooth muscle
specific gene expression and stimulation of DNA replication, cell proliferation, and cell
migration that is characteristic of the de-differentiated state, whereas stimulation with
vasoconstrictors results in an increased expression of smooth muscle specific genes. We have
previously shown that activation of the PI3K/Akt pathway by PDGF-BB leads to transcriptional
suppression of multiple smooth muscle markers through pathways which act on serum
response factor (SRF). In mammalian cells 3 isoforms of Akt have been identified, and while
there is evidence for functional overlap of the Akt isoforms, single isoform knockout studies
have demonstrated distinct roles of Akt 1, 2, and 3 in the regulation of cell size, growth, and
metabolism. We propose that individual Akt isoforms have distinct functions in regulating
signaling pathways involved in VSMC phenotypic modulation. siRNA was used to silence
expression of either Akt1 or Akt2 in cultured rat VSMC, Silencing was verified by immuno-
blotting with isoform-specific antibodies. PDGF suppression of SMA expression was markedly
attenuated when Akt1 isoform expression was reduced. Reduction in the expression of Akt2 did
not result in a similar reversal. In wild-type VSMC, PDGF stimulation resulted in increased levels
of Akt1 in the nuclear fractions with no change in Akt2 levels, suggesting distinct nuclear
targets. Preliminary data suggest expression levels of Akt1 were also differentially regulated
after vascular injury during the formation of advanced neointima. These data imply distinct
roles for individual Akt isoforms in the regulation of VSMC, and carry implications for signaling
pathways involved in vascular remodeling.
P557
Angiotensin II Increases Vasoconstriction Through Activation of Transient
Receptor Potential Channels in Genetic Hypertension
D Y Liu, L Q Ma, Z D Luo, T B Cao, Z M Zhu; Cntr for Hypertension and Metabolic Disease,
Chongqing Institute of Hypertension, Third Military Med Univ, Chongqing, China
Transient receptor potential channels (TRPCs) mediated the changes of cytosolic free calcium
play an important role in the regulation of vascular tone. Recently, we reported an increased
TRPC3 in human hypertension ( J Hypertens, 2006). In current study, we evaluated the effect
of angiotensin II (Ang II) on TRPCs in aorta and cultured vascular smooth muscle cells (VSMCs)
from spontaneously hypertensive rats (SHR). Methods: TRPC1 and TRPC3 expression in aortic
tissues from SHR and normotensive Wistar Kyoto rats (WKY) using Western blot assay.
Measurements of cytosolic free calcium by the fluorescent dye fura2-AM technique.
Down-regulation of TRPC3 channel in VSMC using small interfering RNA (siRNA). Vasocon-
striction of aortic ring was measured. Results: The IV infusion of Ang II significantly increased
mean arterial blood pressure in WKY from 109 5 mmHg to 130 5 mmHg (p 0.01), and
in SHR from 140 9 mmHg to 192 5 mmHg (p 0.01). The increased contractile response
to Ang II in aortic rings from SHR compared to WKY (0.990.03 vs 0.760.14, P0.01). Ang
II -induced the cytosolic free calcium levels were significantly increased in VSMCs from SHR
compared to WKY (1.62 0.15 vs 1.02 0.09 P0.01). The TRPC1 and TRPC3 expressions
were significantly increased in aortic tissues from SHR compared with WKY (1.37 0.04,
1.480.05 for SHR respectively;1.000.01 and 1.000.01 for WKY respectively; p0. 01).
Ang II significantly increased TRPC1 and TRPC3 expression in VSMC from SHR compared with
WKY. Ang II-induced calcium influx was significantly increased in SHR compared to WKY
(1.62 0.15 vs 1.02 0.09, mean SEM, each n6, P0.01). Using siRNA knockdown of
TRPC3 significantly reduced TRPC3 protein expression in VSMC. Ang II -induced increased
calcium influx was decreased after specific TRPC3 knockdown in VSMCs using siRNA
(121.90 10.14% for control condition, 71.58 5.69% for transfected with siRNA against
TRPC3, P0.01).Conclusions: It concluded that increased vascular TRPC1 and TRPC3 channels
function were found in genetic hypertension, which can be modulated by Ang II. Ang II mediated
the increase blood pressure may be associated with the vascular TRPCs up-regulation in
genetic hypertension
P558
The Peroxisome Proliferator-activated Receptor /p16
INK4a
Pathway Inhibits
Telomerase Activity in Vascular Smooth Muscle Cells
Florence Gizard, Takashi Nomiyama, Elizabeth B Heywood, Karrie L Jones, Univ of
Kentucky, Lexington, KY; Bart Staels, Pasteur Institute, Lille, France; Dennis Bruemmer; Univ
of Kentucky, Lexington, KY
Proliferation of vascular smooth muscle cells (VSMC) constitutes a crucial event for the
development of occlusive cardiovascular diseases. Peroxisome Proliferator-Activated Receptor
(PPAR), a ligand-activated transcription factor belonging to the nuclear receptor family,
mediates the action of fibrates used in the treatment of dyslipidemia and atherosclerosis.
Recently, we have demonstrated that PPAR-activation inhibits VSMC proliferation by directly
targeting the cyclin-dependent kinase inhibitor p16
INK4A
. This results in an inhibition of
retinoblastoma protein (pRB) phosphorylation, the key gate-keeper for cell cycle progression.
In the present study, we hypothesized that the growth-inhibitory effects of PPAR/p16
INK4A
may
involve an inhibition of telomerase activity, which controls key cellular functions including
replicative lifespan, cell proliferation and differentiation. In human coronary VSMC, PPAR
ligands dose-dependently inhibited telomerase activity which was associated with an inhibition
of serum-induced expression of the catalytic subunit of telomerase, telomerase reverse
transcriptase (TERT), at mRNA and protein levels. Transient transfection experiments using
progressive 5-deletion series and site-directed mutagenesis of the human TERT promoter
further indicated that the transcriptional repression of the TERT promoter by PPAR is mediated
through an E2F-binding site proximal to the transcriptional start site. We further demonstrated
that the inhibitory effects of PPAR ligands on TERT expression and telomerase activity were
prevented in both PPAR- and p16
INK4a
-deficient VSMC. Altogether these findings demonstrate
that PPAR activation suppresses telomerase activity in VSMC through a p16
INK4a
-dependent
mechanism, which provide a previously unrecognized mechanism for the anti-proliferative
effects of PPAR ligands in VSMC. They support the concept that the PPAR/p16
INK4a
/
telomerase pathway may constitute a novel pharmacological target for the inhibition of VSMC
proliferation in the prevention of cardiovascular disease.
P559
Inhibition of Glycogen Synthase Kinase-3 Decreases Vascular Smooth
Muscle Cell Growth in Vitro Through Modulation of Notch Signaling
Shaunta T Guha, Ronan Murphy, Dermot Walls, DCU, Dublin, Ireland; Eileen M Redmond,
Univ of Rochester, Rochester, NY; Paul A Cahill; DCU, Dublin, Ireland
Vascular cell fate decisions are hallmarks of the vascular response to injury and play a crucial
role in the pathogenesis of vascular disease. GSK-3 and Notch signalling have been strongly
implicated in vascular morphogenesis and remodelling of the embryonic vasculature. We have
previously reported that Notch plays a major role in the regulation of adult vascular cell smooth
muscle cell (SMC) fate in vitro. Constitutive over expression of Notch 1 IC and Notch 3 IC
resulted in a significant increase in SMC proliferation. We examined the hypothesis that
GSK-3 is involved in regulation of SMC proliferation through control of Notch signalling. Using
quantitative real-time PCR, western blot analysis, luciferase reporter assays and FACS analysis,
we identified the presence of GSK- and Notch signalling components in SMC. Inhibition of
GSK-3 with the pharmacological inhibitor, SB216763 (25M) resulted in significant decrease
in cell number and proliferating cell nuclear antigen (pCNA) expression [cell counts
56.7 6.6%, pCNA 61.54 9%, FACS analysis 52.84 3.3%]. In parallel studies, SB216763
decreased the levels of Notch 1 IC and Notch 3 IC by 43.83 6.19%, and 47.37 2.9%,
respectively, concomitant with a significant decrease in Notch target gene protein (HRT-3
26.6 6.84%) and mRNA expression (HRT-1 37.11 9.6%, HRT-2 43.82 9.2%, and HRT-3
55 11.25%). Furthermore, inhibition of GSK- attenuated Notch IC-dependent CBF-1/RBP-
Jk-promoter transactivation in these cells. We conclude that GSK-3 and Notch interact to
promote SMC growth in vitro.
P560
Differential Gene Expression Profiles of Bare Metal Stents and
Paclitaxel-eluting Stents in Explanted Human Arteries
Stefan Amisten, Helen Svensson, Carl Hogberg, Atli Eyjolfsson, Ronny Gustafsson,
Anna-Karin Wihlborg, Lund Univ, Lund, Sweden; Anne Whalen, Boston Scientific Corp,
Natick, MA; David Erlinge; Lund Univ, Lund, Sweden
Implantation of coronary stents has emerged as the preferred treatment of occluded coronary
arteries. However, little is known of the molecular responses to stent implantation in human
arteries. The aim of this study was to generate gene expression profiles from intact human left
inferior mammary arteries (LIMAs) denuded and stented ex vivo with either bare metal stents
(BMS) or paclitaxel drug-eluting stent (pDES) in order to explain the molecular biology of BMS
2007 ATVB Poster Presentations e-135
by on March 25, 2011 atvb.ahajournals.org Downloaded from
and pDES implantation. Gene expression profiles were generated from five male donor LIMAs
divided into three parts: non-stented control, BMS, or paclitaxel DES. Less than 10 % of the
probe sets were differentially expressed in the BMS and pDES groups, compared to control, and
in pDES compared to the BMS. No more than 3% of all genes were differentially regulated
among the three groups. Genes involved in cell growth were up-regulated in both BMS and
pDES. However, paclitaxel DES displayed a pronounced effect on the cell cycle regulation,
down-regulating genes that promote cell cycle progression and up-regulating genes that
induce cell cycle arrest. Some smooth muscle cell (SMC) differentiation markers were
down-regulated in the pDES group (P0.005), suggesting a de-differentiation of SMC in the
vascular wall. Both pro- and anti-apoptotic genes were also regulated by pDES compared to
BMS. Expression of endothelial growth and differentiation-enhancing genes were down-
regulated in both BMS and pDES (P0.05), with no significant difference between these groups
(P0.05). In conclusion, we demonstrate a unique model for studying gene expression profiles
in stented human arteries. In comparing paclitaxel DES to BMS, gene expression patterns of
cellular growth arrest were increased in pDES, consistent with the proposed anti-restenosis
mechanism. However, no differences were observed in genes promoting endothelial cell growth
and differentiation, suggesting that BMS and paclitaxel DES promote a similar environment for
re-endothelialization and maturation within the vascular wall. These data can help guide future
work to optimize DES, by developing parameters which further promote endothelial healing.
P561
Aldosterone Increases Oxidant Stress to Decrease iNOS-stimulated Soluble
Guanylyl Cyclase Activity
Bradley A Maron, Shiow-Shih Tang, Joseph Loscalzo, Jane A Leopold; Brigham and
Womens Hosp, Boston, MA
Elevated levels of aldosterone (ALDO) are associated with impaired vascular reactivity. In
vascular endothelial cells, ALDO modulates this effect by increasing reactive oxygen species
(ROS) and decreasing bioavailable nitric oxide (NO); however, the effect of ALDO on vasodilator
responses in vascular smooth muscle cells (SMC) remains unknown. We hypothesized that
ALDO impairs SMC relaxation by increasing ROS to decrease soluble guanylyl cyclase (sGC)
activity. To test the effect of ALDO on ROS, bovine aortic SMC were treated with ALDO
(10
-9
-10
-7
mol/L) or a vehicle (V) control for 24 h and ROS was measured by 6-carboxy-2-
7-DCF diacetate fluorescence. Compared with V-SMC, ALDO-SMC showed a concentration
dependent increase in ROS (117.9% 5.7 vs. 124.5% 6.1 vs. 144.9% 4.6, p0.01);
treatment with apocynin (30 mol/L), a specific NADPH oxidase inhibitor, decreased
ALDO-induced ROS formation by 25.4% (p0.04). Next, to study the effect of ALDO on
endogenous NO-sGC signaling in SMC, iNOS was induced by cytokines (C-SMC) for 24 h. iNOS
protein expression was increased in C-SMC compared to V-SMC (206.38 4.89 vs.
63.7 11.1 int./mm
2
, p0.01) as was nitrite formation (0.077 0.007 vs. 0.0 0.0
mol/L/g protein, p0.01), an effect that was inhibited by L-NAME (5 mmol/L)
(0.077 0.007 vs. 0.008 0.003 mol/L/g protein, p0.001). sGC activity, assessed by
cGMP formation, was increased in C-SMC compared to V-SMC (0.014 0.0019 vs.
0.002 0.0005 pmol/g protein, p0.01). Exposure to ALDO did not increase iNOS protein
expression or nitrite formation further in C-SMC; however, ALDO decreased iNOS-generated
cGMP by 50.7% (0.014 0.0019 vs. 0.007 0.002 acetylated cGMP pmol/g protein,
p0.05). Apocynin restored iNOS stimulated cGMP production in ALDO-treated C-SMC
(0.007 0.002 vs. 0.014 0.003, pmol/g protein, p0.01) without influencing nitrite levels.
These studies demonstrate that ALDO increases ROS generation by NADPH oxidase in SMC;
when iNOS is induced, this effect is associated with decreased sGC activity and cGMP levels
without influencing NO byproduct formation. These findings suggest that ALDO-induced ROS
leads to an uncoupling of iNOS-mediated NO bioactivity and, thereby, may impair SMC
relaxation.
P562
Heme Oxygenase-1 Prevents Aldosterone-elicited Vascular Senescence
Through Mechanisms Involving SIRT1/p53/p21 Pathway
Toshisuke Morita, Eri Terada, Junko Tatebe, Gen Yoshino, Tsutomu Saji, Jun-ichi Yamasaki;
Toho Univ Sch of Medicine, Tokyo, Japan
In yeast, restriction of calorie extends life span by increasing activity of Sir2, an NAD-
dependent deacetylase. SIRT1, a human homolog of Sir2, has been reported to inhibit p53,
which is one of the key players in stimulating cellular senescence in vascular wall. Heme
oxygenase is a microsomal enzyme that catalyzes the degradation of heme into biliverdin,
which is subsequently reduced to bilirubin, free iron and carbon monoxide (CO). Induction of
heme oxygenase-1 (HO-1) has been potentially associated with cellular protection, especially
against oxidative insults, which promote age-related changes. In this study, using smooth
muscle cell-directed HO-1 over-expression mice (HO-1 Tg), we investigated anti-aging effect
of HO-1 on cardiovascular system in aldosterone/high salt (ALD)-treated mouse. Sixteen-week-
ALD promotes in vivo cellular senescence as determined by senescence-associated beta-gal
staining as well as increased expression of p53 and p21in the aorta of wild type mouse (Wt).
Consistent with these findings, expression of SIRT1 was markedly reduced, while inflammatory
cytokines were induced and eNOS was decreased, respectively, in the aorta of ALD-treated Wt.
In contrast, there are no significant changes in these senescence-related markers, and
expression of SIRT1 was not reduced in ALD-treated HO-1 Tg. Taken together, these findings
indicate that vascular HO-1 counteracts vascular senescence through restoration of SIRT1 as
well as inhibition of p53/p21 pathway in aorta. Vascular HO-1 can be a new therapeutic target
against age-related cardiovascular diseases.
P563
Enos Gene Expression by Adenovirus Prevents Angiotensin II-induced
Vascular Smooth Muscle Cell Hypertrophy Through Selective Inhibition of
the Rho/Rho-Kinase Pathway
Heigoro Shirai, Hiroyuki Suzuki, Sadaharu Higuchi, Sudhir Dhobale, Kunie Eguchi, Satoru
Eguchi; Temple Univ Sch of Medicine, Philadelphia, PA
Enhanced angiotensin II (AngII) actions are frequently associated with endothelial dysfunction,
which is characterized as decreased nitric oxide (NO) availability. Although endothelial NO
synthase (eNOS) is believed to antagonize vascular remodeling induced by the AT1 receptor,
the exact signaling mechanism remains controversial. Therefore, we investigated a possible
signal cross talk between eNOS and AT1 along with their impacts on vascular hypertrophy by
using vascular smooth muscle cells (VSMCs) infected with adenovirus encoding the eNOS gene.
In VSMCs infected with eNOS adenovirus, basal G kinase activity was enhanced as detected by
enhanced VASP Ser239 phosphorylation. AngII-induced VSMC hypertrophy as judged by protein
synthesis as well as by cell volume was again markedly inhibited by eNOS adenovirus. These
effects are accompanied with selective inhibition of the Rho/Rho-kinase(ROCK) cascade. The
downstream components of Rho/ROCK, MYPT phosphorylation and myosin light chain
phosphorylation induced by AngII, were inhibited by the eNOS expression. Thus, eNOS
adenovirus inhibited AngII-induced G12/13 and RhoA activation in VSMCs. By contrast,
intracellular Ca2 elevation, EGF receptor transactivation and ERK activation induced by AngII
were not affected by the eNOS expression. From these data, we conclude that under eNOS
gene transfer, NO/cGMP production and the resultant G kinase activation appear to prevent
AngII-induced VSMC hypertrophy by selective inhibition of the Rho/ROCK pathway. These data
suggest a novel molecular mechanism of endothelial dysfunction leading to vascular
remodeling through select induction of the Rho/ROCK cascade.
P564
The Reversible Oral P2Y12 Antagonist AZD6140 Inhibits ADP-induced
Contractions in Mouse Aorta in Addition to Established Inhibitory Effects
on Platelet Aggregation
Helen Svensson, David Erlinge; Lund Univ Hosp, Lund, Sweden
Introduction. Platelet P2Y
12
receptor has long been used as a target for antithrombotic drugs
of the thienopyridine family such as clopidogrel and ticlopidine, which are prodrugs whose
bioactive metabolites bind irreversibly to P2Y
12
receptor. AZD6140, a reversible oral P2Y
12
antagonist with no requirement for metabolic activation, is being studied for its potential to
prevent thrombotic events in patients with acute coronary syndromes (ACS). Clinically,
AZD6140 showed greater, more consistent effects on inhibition of platelet aggregation than
clopidogrel, with a similar incidence of total bleeding events. P2Y
12
receptors have also been
shown on vascular smooth muscle cells (VSMC), where they mediate a contractile function after
stimulation by ADP; this could contribute to local vasospasm and poorer outcomes in ACS.
Objective. To elucidate if AZD6140, in contrast to clopidogrel, can act on VSMC and thereby
inhibit ADP-mediated contractions. Methods. Nine female mice were used: 5 were pretreated
with clopidogrel 50 mg/kg the day before, and 2 hours before the experiment; 4 were not
pretreated. Thoracic aorta sections obtained from all mice were dissected from connective
tissue and denuded. Ring segments (7 or 8 per mouse) were mounted into temperature-
controlled tissue baths with physiological Krebs buffer. AZD6140 10 M or DMSO 1:1000 as
control was added; after 20 minutes, segments were precontracted with 10 nM norepinephrine,
followed by the P2Y
12
agonist 2-MeSADP (10 M). Results. Mean 2-MeSADP-induced
contraction (% maximal contraction induced by 60 mM K

) in clopidogrel-treated and untreated


DMSO control groups was 64% and 59%, respectively; this was reduced to 32% (P0.002)
and 33% (P0.015), respectively, with AZD6140. Conclusion. AZD6140 blocked ADP-induced
vasoconstriction mediated by P2Y
12
receptors in denuded mouse aortic rings, regardless of in
vivo pretreatment with clopidogrel. This effect of AZD6140 could potentially modulate
vasoreactivity mediated by locally elevated concentrations of ADP in vivo. Further investigation
is required to examine this question.
P565
TGF- Increases Intimal Hyperplasia After Vascular Injury Through
Smad3-Mediated Stimulation of Cell Proliferation
Shirling Tsai, Scott T Hollenbeck, Evan J Ryer, Weill Med College-Cornell Univ, New York,
NY; Rishi Kundi, Beth Israel Med Cntr, New York, NY; Kentaro Kamiya, Andrew Zohlman,
Chunjie Wang, Sophia Chu, Bo Liu, K C Kent; Weill Med College-Cornell Univ, New York, NY
Introduction: Intimal hyperplasia is associated with the transformation of vascular smooth
muscle cells (VSCM) from a quiescent to a proliferative phenotype. TGF-beta has been shown
to be a critical factor in the development of intimal hyperplasia. We hypothesize that TGF-beta,
through Smad3 signaling, increases intimal hyperplasia by stimulating VSMC proliferation.
Methods: Male Spague-Dawley rats underwent left carotid balloon injury followed by
intra-arterial dwell with adenoviral vectors expressing Smad3, Smad7 or control (LacZ or empty
virus). Injured arteries were harvested at specified time points and subject to histological
analysis. Results: Endogenous Smad3 is upregulated in the rat carotid artery after balloon
injury. Further upregulation of Smad3 by adenovirus-mediated gene transfer increased intimal
hyperplasia while inhibition of Smad3 signaling through Smad7 overexpression decreased
neointimal formation. Upon immunohistochemical analysis, PCNA staining was increased in
Smad3 infected arteries and decreased in Smad7 infected arteries, thus suggesting a role for
Smad3 signaling in cell proliferation. (Figure 1) This was confirmed by an in vitro proliferation
assay using tritiated thymidine. To explore the mechanism of the observed stimulation of
proliferation by Smad3, we found by western blotting that upregulation of Smad3 results in
serine-10 phosphorylation of p27 and a subsequent decrease in total p27. Conclusions:
Upregulation of the TGF-beta signaling protein Smad3 following arterial injury causes quiescent
VSMC to re-enter the cell cycle by phosphorylation and subsequent degradation of the cyclin
dependent kinase inhibitor p27.
e-136 Arterioscler Thromb Vasc Biol June 2007
by on March 25, 2011 atvb.ahajournals.org Downloaded from
P566
81 Integrin Is Upregulated in the Neointima Concomitant with Late
Luminal Loss After Balloon Injury
Ramin Zargham, Gaetan Thibault; IRCM, Montreal, Canada
Introduction: Constrictive remodeling of the neointima results in the late lumen loss and
restenosis after balloon angioplasty. Previously we showed that 81 integrin is downregu-
lated in the early stages of postangioplasty concomitant with the loss of the contractile state
in tunica media. 81 integrin is also intensely expressed in the contractile state of vascular
smooth muscle cells (VSMCs) and in myofibroblasts. Thereby, we hypothesized that 81 is
involved in the process of constrictive remodeling of neointima and late lumen narrowing.
Methods and Results: Balloon injury was used to induce neointima formation in the rat carotid
artery. Male rats were divided into 4 groups and sacrified at 7, 14, 21, and 28 days
postangioplasty(N12/time point). Immunohistochemical analysis and immunoconfocal studies
showed that late lumen narrowing at 21 d and 28 d was concomitant with the upregulation of
smooth muscle (SM) -actin and 8 integrin in the neointima. 8 integrin and SM -actin
-positive cells were localized in the medial side of the neointima at 21 d in a similar pattern
to the collagen and fibronectin accumulation, markers of constrictive remodelling. The
transforming growth factor-beta (TGF-) stimulation of either fibroblasts or VSMCs in vitro,
populated in 3D collagen matrices, led to an upregulation of 8 integrin as well as increased
fibronectin fibril-8 integrin interaction and gel contraction. TGF--induced SM contractile
features in Rat1 fibroblasts and VSMCs were impaired in cells pre-treated with a small
interference RNA targetting 8 integrin. Conclusion: The close correlation between 8 integrin
upregulation in the neointima and late luminal loss and 8 integrin being required for
contractile characteristics induced by TGF- highlight a possible role for 8 integrin in
post-angioplasty restenosis.
P567
Age-dependent Impairment of Endothelial Progenitor Cells Is Corrected by
Growth HormoneMediated Increase of Insulin-like Growth Factor-1
Thomas Thum, Sarah Hoeber, Julius-Maximilians Universitaet, Wuerzburg, Germany; Ivonne
Klink, Med Sch Hannover, Hannover, Germany; Dirk O Stichtenoth, Dimitrios Tsikas, Med
Sch, Hannover, Germany; Stefan D Anker, Philip A Poole-Wilson, Imperial College, London,
United Kingdom; Ju rgen Borlak, Fraunhofer Institute for Experimental Medicine, Hannover,
Germany; Georg Ertl, Johann Bauersachs; Julius-Maximilians Universitaet, Wuerzburg,
Germany
Aging is associated with an increased risk for atherosclerosis. A possible cause is low numbers
and dysfunction of endothelial progenitor cells (EPC) which insufficiently repair damaged
vascular walls. We hypothesized that decreased levels of IGF-1 during age contribute to
dysfunctional EPC. We measured the effect of growth hormone (GH), which increases
endogenous insulin-like growth factor-1 (IGF-1) levels, on EPC in mice and human subjects. We
compared EPC number and function in healthy middle-aged male volunteers (57.41.4 years)
before and after a ten day treatment with recombinant GH (0.4 mg/day) with that of younger
and elderly male subjects (27.50.9 and 74.10.9 years). Middle-aged and elderly subjects
had lower circulating CD133

/VEGFR-2

EPC with impaired function and increased senes-


cence. GH treatment in middle-aged subjects elevated IGF-1 levels (126.07.2ng/ml vs.
241.113.8ng/ml; p0.0001), overall NO bioavailability, increased circulating EPC with
improved colony forming and migratory capacity, enhanced incorporation into tube-like
structures, and augmented endothelial nitric oxide synthase (eNOS) expression in EPC
comparable to that of the younger group. EPC senescence was attenuated, whereas telomerase
activity was increased after GH treatment. Treatment of aged mice with GH (7 days) or IGF-1
increased IGF-1 and EPC levels and improved EPC function, whereas a two day GH treatment
did not alter IGF-1 or EPC levels. Systemic blocking of the IGF-1 receptor in vivo abolished the
beneficial GH-mediated effects on NO production and EPC. Ex vivo treatment of EPC from
elderly individuals with IGF-1 improved function and attenuated cellular senescence. IGF-1
stimulated EPC differentiation, migratory capacity and the ability to incorporate into forming
vascular networks in vitro via the IGF-1 receptor. IGF-1 increased telomerase activity, eNOS
expression, phosphorylation and activity in EPC in a phosphoinositide-3-kinase/Akt dependent
manner. Small interference RNA-mediated knockdown of eNOS in EPC abolished the IGF-1
effects. Growth hormone-mediated increase in IGF-1 reverses age-related EPC dysfunction and
may be a novel therapeutic strategy against vascular disorders with impairment of EPC.
P568
Abundance and Plasticity of Circulating Stem Cells in Patients with
Abdominal Aortic Aneurysm
Evelyn Torsney, Joseph Dawson, Jenny Tooze, Hosaam Nasr, Edward Choke, Ian Loftus,
Matt M Thompson, Gillian Cockerill; St Georges, Univ of London, London, United Kingdom
Background: Abdominal aortic aneurysms (AAAs) are characterised by progressive degener-
ative changes in the wall of the aorta, leading to a structurally weaker vessel which is at risk
of potentially fatal rupture. Recently, we reported that aneurysmal rupture correlated with
medial neo-vascularisation. As neo-vascularisation is most likely to require engraftment and
differentiation of a circulating stem cell population, we have investigated the role of stem cells
in the pathophysiology of AAA. Objectives: We aimed to evaluate the number of circulating
stem cells, and then investigate the effects of in vitro conditions on adhesion, migration and
proliferation. Methods: Venous blood samples are collected from patients undergoing
endovascular repair of AAA. CD133 cells are isolated from peripheral blood mononuclear
cells (PBMCs) using magnetic cell sorting of CD133 antibody labelled cells (Miltenyi Biotech).
CD133/34 cells are measured by flow cytometry following magnetic bead enrichment
using another epitope of CD133. CD133 enriched cells are plated onto Permanox coated
Tekslides, for subsequent analysis. PBMC-conditioned medium was HUVEC MEM harvested,
spun, filtered and aliquoted for further use. Plasma was collected and ELISA analysis performed
using an RD System Sandwich ELISA kit (Quantikine). Results: CD133/34 cell numbers
are increased in patients in AAA compared to normals (2.43 versus 1.25%, p0.008). Plasma
VEGF concentration is increased compared to normals (33.20.36 vs 18.60.14, p0.002).
GCSF demonstrated the same trend (9.70.02 vs 6.80.02, p0.03). CD133cells were
cultured in HUVEC MEM, 2 day and 5 day PBMC conditioned medium. Both 2 day and 5 day
PBMC conditioned medium preferentially support the growth of CD133-enriched cells in
comparison to standard HUVEC-MEM. Conclusions: The number of circulating CD133/
CD34stem cells are increased in patients with AAA. We have found that PBMC-conditioned
medium optimises the growth conditions of CD133enriched cells. Mediator substances such
as growth factors and cytokines secreted by the PBMCs promotes cultivation of these rare
progenitor cells in preference to endothelial cell growth factor alone.
P570
WITHDRAWN
2007 ATVB Poster Presentations e-137
by on March 25, 2011 atvb.ahajournals.org Downloaded from

You might also like