You are on page 1of 7

Reduced Myocardial Ischemia-Reperfusion Injury in

Toll-Like Receptor 4 Deficient Mice

Jun-ichi Oyama, MD; Charles Blais, Jr, PhD; Xiaoli Liu, MD; Minying Pu, MD;
Lester Kobzik, MD; Ralph A. Kelly, MD; Todd Bourcier, PhD
BackgroundMyocardial ischemia and reperfusion-induced tissue injury involve a robust inflammatory response, but the
proximal events in reperfusion injury remain incompletely defined. Toll-like receptor 4 (TLR4) is a proximal signaling
receptor in innate immune responses to lipopolysaccharide of Gram-negative pathogens. TLR4 is also expressed in the
heart and vasculature, but a role for TLR4 in the myocardial response to injury separate from microbial pathogens has
not been examined. This study assessed the role of TLR4 in myocardial infarction and inflammation in a murine model
of ischemia-reperfusion injury.
Methods and ResultsMyocardial ischemia-reperfusion (MIR) was performed on 2 strains of TLR4-deficient mice
(C57/BL10 ScCr and C3H/HeJ) and controls (C57/BL10 ScSn and C3H/OuJ). Mice were subjected to 1 hour of
coronary ligation, followed by 24 hours of reperfusion. TLR4-deficient mice sustained significantly smaller infarctions
compared with control mice given similar areas at risk. Fewer neutrophils infiltrated the myocardium of TLR4-deficient
Cr mice after MIR, indicated by less myeloperoxidase activity and fewer CD45/GR1-positive cells. The myocardium
of TLR4-deficient Cr mice contained fewer lipid peroxides and less complement deposition compared with control mice
after MIR. Serum levels of interleukin-12, interferon-, and endotoxin were not increased after ischemia-reperfusion.
Neutrophil trafficking in the peritoneum was similar in all strains after injection of thioglycollate.
ConclusionsTLR4-deficient mice sustain smaller infarctions and exhibit less inflammation after myocardial ischemiareperfusion injury. The data suggest that in addition to its role in innate immune responses, TLR4 serves a
proinflammatory role in murine myocardial ischemia-reperfusion injury. (Circulation. 2004;109:784-789.)
Key Words: inflammation myocardial infarction ischemia

eperfusion therapies have substantially impacted the

effective treatment of ischemic heart diseases.1,2 Therapeutic progress has been tempered, however, by a host of
events termed postischemic myocardial reperfusion injury
(myocardial ischemia-reperfusion [MIR]). A robust local and
systemic inflammatory response characterizes MIR that may
expand tissue injury and adversely affect left ventricular (LV)
recovery.3,4 Disrupting inflammatory processes such as recruitment of neutrophils, production of radical oxygen species, and activation of complement have all modulated MIR
injury.4 However, the proximal events that initiate these
proinflammatory pathways are not fully elucidated. Indeed,
such antiinflammatory strategies have thus far proven disappointing in clinical practice,5,6 emphasizing the importance of
additionally understanding the basic mechanisms of MIRinduced inflammation and injury.
The Toll-like receptors (TLRs) serve as pattern-recognition
receptors at a proximal step in the innate immune response to
microbial pathogens.7 Among these receptors, TLR4 specif-

ically signals cellular responses to bacterial lipopolysaccharide (LPS) in conjunction with accessory molecules.8 Activation of TLR4 is linked to expression of proinflammatory
cytokines and activation of nuclear factor-B signaling pathways in several cell types.7,9 TLR4 is expressed by cells of
myeloid lineage, which are central to innate immune responses, but TLR4 is also expressed in tissues without a
recognized immune function, notably the heart and vasculature.10,11 Consistent with its role as a receptor for LPS, cardiac
expression of TLR4 is essential for LPS-induced LV dysfunction and myocardial expression of tumor necrosis factor ,
interleukin (IL)-1, and inducible NO synthase.12,13 Expression of tumor necrosis factor , IL-1, and adhesion molecules
vascular cellular adhesion molecule 1 and intracellular adhesion molecule 1 on coronary endothelial cells exposed to LPS
is similarly dependent on TLR4.14 These studies support a
role for TLR4 in cardiovascular inflammation and dysfunction during bacterial sepsis, but there is no evidence that
TLR4 participates in myocardial inflammatory responses

Received January 14, 2003; de novo received July 14, 2003; revision received October 2, 2003; accepted October 6, 2003.
From the Department of Cardiovascular Medicine (J.O., C.B., M.P.); Pulmonary and Critical Care Division (X.L.); Anesthesiology, Perioperative and
Pain Medicine (T.B.), Center for Experimental Therapeutics and Reperfusion Injury, Brigham and Womens Hospital, Boston; Department of Pathology
(L.K.), Harvard Medical School, Boston; and Genzyme Corporation (R.A.K.), Framingham, Mass.
Correspondence to Todd Bourcier, PhD, Department of Anesthesiology, Perioperative and Pain Medicine, Center for Experimental Therapeutics and
Reperfusion Injury, Brigham & Womens Hospital, Medical Research Building #502, Boston, MA 02115. E-mail
2004 American Heart Association, Inc.
Circulation is available at

DOI: 10.1161/01.CIR.0000112575.66565.84

Downloaded from
by guest on May 4, 2015

Oyama et al

Reduced Myocardial IR Injury in TLR4-Deficient Mice

with a nonmicrobial etiology, like the inflammatory response

to ischemia-reperfusion injury. Such a role for TLR4 is
suggested by several observations. First, the Toll/IL-1 signaling pathway, specifically TLR2, has been implicated in the
proinflammatory response to necrotic cell death.15 Second,
endogenous factors associated with tissue injury, such as
heat-shock protein 60, may act as activating ligands for
TLR4.7,16 Lastly, constitutive expression of TLR4 in the heart
increases markedly after experimental ischemic injury.10 On
the basis of these considerations, we investigated whether
myocardial infarction and inflammation produced by ischemia-reperfusion differed in mice that lack a functional TLR4
signaling pathway.

Experimental Animals
The murine strains C57 BL/10 ScCr and C3H/HeJ do not express
functional TLR4 because of naturally occurring mutations in the
TLR4 gene.8 C57 BL/10 ScCr mice were obtained from Dr Peter
Tobias of Scripps Research Institute, La Jolla, Calif. The C57 BL/10
ScSn, C3H/HeJ, and C3H/OuJ mice were purchased from Jackson
Laboratories, Bar Harbor, Maine. Animals were housed in accordance with the Harvard Medical Area Standing Committee on
Animals in a specific pathogen-free environment.

Murine Myocardial Ischemia and Reperfusion

Mice (12 to 20 weeks old, 22 to 30 g) were subjected to 1 hour of
myocardial ischemia and 24 hours of reperfusion, as described
previously.17 Briefly, mice were anesthetized with pentobarbital
sodium (60 mg/kg body wt). Additional doses were given as needed
to maintain anesthesia. Mice were intubated and ventilated with
100% oxygen. Ischemia was achieved by ligating the left anterior
descending coronary artery (LAD) using an 8-0 silk suture with a
section of PE-10 tubing placed over the LAD, 1 mm from the tip of
the normally positioned left atrium. After occlusion for 1 hour,
reperfusion was initiated by releasing the ligature and removing the
PE-10 tubing. The chest wall was closed, the animal extubated, and
body temperature maintained by use of a 37C warm plate. Hearts
were harvested 24 hours later. The loosened suture was left in place
and then retied for the purpose of evaluating the ischemic area.

Assessment of Area at Risk and Infarct Size

To assess the ischemic area at risk, 1% Evans blue was infused into
the aorta and coronary arteries in retrograde fashion. Hearts were
excised and sliced into 5 1-mm cross sections with the aid of an
acrylic matrix (Alto Inc). The heart sections were incubated with 1%
triphenyltetrazolium chloride solution (Sigma-Aldrich) at 37C for
15 minutes. Viable myocardium stained red, and infarcted tissue
appeared white. The infarct area (I, white), the area at risk (AAR, red
and white), and the total LV area from each section were measured
using NIH Image (version 1.62) and Spot software. Ratios of
AAR/LV and of I/AAR were calculated and expressed as a

Lipid Peroxidation Assay

Hearts were collected 24 hours after reperfusion and disrupted by
Dounce homogenization in 1.0 mL of 20 mmol/L Tris-HCl, pH 7.4,
containing 5 mmol/L butylated hydroxytoluene. Lipid peroxides
(malondialdehyde and 4-hydroxynonenal) were measured as an
index of oxidative stress. Aliquots were assayed using a commercially available kit (catalog No. 437634, Calbiochem).

Myocardial Myeloperoxidase Activity Assay

Myeloperoxidase activity was measured as a marker of myocardial
neutrophil infiltration. Frozen myocardial tissues were placed in 0.5
mL of a 50-mmol/L potassium PBS (pH 6.0) containing 0.5%


hexadecyltrimethyl ammonium bromide, homogenized, and centrifuged for 15 minutes at 14 000 rpm at 4C. The supernatants were
mixed 1:9 (vol/vol) with 50 mmol/L PBS (pH 6.0) containing 0.2
mg/mL o-dianisidine and 0.0006% hydrogen peroxide; the absorbance change was measured at 30 and 90 seconds at a wavelength of
460 nm. Murine myocardial myeloperoxidase (MPO) activity was
normalized using human MPO as a standard.

Hearts were fixed in methyl Carnoys solution at 4C for 5 hours and
then placed in 70% ethanol overnight. Hearts were paraffin embedded and cut into 10-m sections. Sections were stained with
hematoxylin and eosin to assess morphology and evidence of injury.
Histological stains included anti-mouse CD45 antibody (Ly-5;
1:1000, Pharmingen), the neutrophil-specific marker GR-1 (Ly-6g;
1:1000, Pharmingen), and goat anti-rat complement-3 (C3, 1:500,
Cappel, ICN Pharmaceuticals). Secondary antibodies consisted of
biotinylated anti-mouse or anti-goat antibodies (diluted 1:200, Vector Laboratories), followed by incubation with horseradish peroxidase coupled streptavidin (ABC reagent, Vector Laboratories) and
development with 3,3-diaminobenzidine substrate. Specificity of
the primary antibodies was checked by using species and isotypematched nonimmune antibodies. Sections were counterstained with
hematoxylin and mounted for light microscopy. Histological analysis
was performed on hearts from 3 mice from each strain.

Serum Concentrations of Interleukin-12

and Interferon-
Blood was collected at the time of heart harvest, and serum was
prepared and assayed for interleukin-12 and for interferon- by
Quantikine sandwich ELISA (R&D Systems).

Thioglycollate-Induced Peritonitis
Mice were given a 2-mL intraperitoneal injection of 4% Brewers
thioglycollate medium (Difco No. 0236-17-7). After 3 hours, mice
were anesthetized with pentobarbital, and abdominal infiltrates were
collected with 3 washes with PBS. Cells were centrifuged and
resuspended in 1.0 mL PBS, and total cell numbers were determined
with a hemocytometer. Ratios of granulocyte to monocyte populations were determined from the forward and side-scatter characteristics on flow cytometry.

Serum Endotoxin
Levels of serum endotoxin were measured with a chromogenic
Limulus amebocyte lysate assay according to the manufacturers
directions (BioWhittaker, QCL-1000). Sensitivity of the assay is 0.1

Data are expressed as meanSEM of n observations. Comparisons
of data between Sn versus Cr and between HeJ versus OuJ were
made using a 2-sample t test assuming equal variances. Differences
were considered statistically significant when P0.05.

Reduced Infarct Size in TLR4-Deficient Mice
After Myocardial Ischemia-Reperfusion
Myocardial ischemia-reperfusion was performed on C57/
BL10 ScCr mice, which lack expression of TLR4, and on
C57/BL10 ScSn (Sn) mice, which express TLR4 normally.
After 1 hour of ligation of the LAD and 24 hours of
reperfusion, the extent of myocardial infarction was measured. The LV area affected by LAD ligation, referred to as
the area at risk (AAR), was similar between Sn and Cr mice
(515% versus 535%, respectively; PNS; Figure 1).
Despite sustaining equal areas at risk, TLR4-deficient Cr

Downloaded from by guest on May 4, 2015



February 17, 2004

Figure 1. Myocardial infarct size after
1-hour/24-hour MIR in mice that lack functional TLR4 signaling (Cr and HeJ) versus
wild-type controls (Sn and OuJ, respectively).
Top, Representative sections distal to the
site of ligation. Blue area depicts perfused
tissue; red plus white, at-risk tissue; white
(arrows), infarcted tissue. Lower bar graphs
from left to right depict the AAR as a percent
of the LV area, infarct size (I) as a percent of
the AAR, and infarct size as a percent of the
LV area (I/LV). Results are meanSEM from
Cr (n7), Sn (n8), HeJ (n4), and OuJ
(n5). *Significant difference between Sn
and Cr; #Significant difference between OuJ
and HeJ. Comparisons made by Students t
test at P0.05.

mice had significantly smaller myocardial infarctions compared with Sn mice, whether normalized to the area at risk or
to the LV area (Figure 1).
In addition to a lack of TLR4 expression, Cr mice harbor a
point mutation in the IL-12 receptor resulting in impaired
microbial-induced production of interferon-, a characteristic
not shared by other TLR4-deficient murine strains.18 Serum
levels of IL-12 are also reported to increase after myocardial
infarction in humans.19 Thus, part of the myocardial protection observed in Cr hearts may be linked to defective IL-12 as
well as TLR4 signaling. We determined that serum IL-12
levels in Cr mice were similar before and after 1 hour and 24
hours of MIR (Cr before MIR, 19.611 pg/mL; Cr after
MIR, 23.91.9 pg/mL). In addition, IFN-, the primary
response gene induced by IL-12, was not detected in serum
from Cr or Sn mice after MIR (2 pg/mL). Although not
exclusionary, these data suggest that IL-12/IL-12R signaling
is not activated during the course of our experiments. To
additionally address this concern, we performed MIR on
C3H/HeJ (HeJ) mice, which harbor a distinct point mutation
in the conserved TIR signaling domain of TLR4. As shown in
Figure 1, HeJ mice also sustained smaller myocardial infarctions compared with the control strain C3H/OuJ (OuJ). Thus,
both Cr and HeJ mice that harbor distinct TLR4 mutations
sustained less myocardial damage after ischemia reperfusion,
as measured by infarct size, than did mice expressing functional TLR4.
As depicted in the Table, body weights did not differ
significantly between the murine strains tested. The TLR4defective Cr mice showed an increased survival rate compared with the Sn control mice of 71.4% versus 56.8%,
respectively. However, there was not a similar increase in
survival of TLR4-defective HeJ compared with OuJ control
Body Weight and Survival Rate for Experimental Mice After 1
h/24 h Myocardial Ischemia/Reperfusion
Murine Strain


Body Weight, g

% Survival

















Part of the response to MIR may be related to postoperative

bacterial infections that flourish during the 24-hour period of
reperfusion, despite observing sterile surgical techniques.
However, differences in serum endotoxin were not detected
between unoperated versus 1-hour/24-hour MIR mice
(0.120.05 versus 0.110.06 EU/mL, P0.90), nor between
operated Sn versus Cr mice (0.110.013 versus 0.100.05,
P0.95). To ensure validity of the assay, 1.0 EU/mL LPS
was added to murine serum, which was detected by the LAL
assay (1.30.21 EU/mL).

Reduced Myocardial Inflammation in Cr Mice

After MIR
We next examined whether smaller infarctions in Cr mice
corresponded with less myocardial inflammation, defined by
neutrophil infiltration, lipid peroxidation, and complement
deposition. Hearts from Sn mice showed evidence of leukocyte infiltration at sites of tissue injury after 1-hour/24-hour
MIR, as shown by CD45-positive immunostaining (Figure
2A). The pattern of CD45-positive staining overlapped the
pattern of GR-1positive staining, the latter a specific marker
for neutrophils. Fewer CD45/GR-1 infiltrating leukocytes
were observed on sections of injured myocardium from Cr
mice compared with Sn mice, suggesting less neutrophil
trafficking (Figure 2A). Results from histological stains were
supported by quantitatively less MPO activity in hearts from
Cr compared with Sn mice after 1-hour/24-hour MIR (Cr,
0.600.05 U/100 mg; Sn, 0.940.07 U/100 mg; Figure 2B).
Reactive oxidative species are generated after MIR from
intracellular oxidases present in the myocardium and from
infiltrating leukocytes.20 Consistent with smaller infarctions
and fewer neutrophils, hearts from Cr mice contained fewer
lipid peroxides (malondialdehyde and 4-hydroxyalkenal)
compared with Sn hearts (Figure 3), suggesting a lesser
degree of oxidative stress in Cr hearts after MIR.
Neutrophils express TLR4 and respond to LPS.21 To
determine whether the TLR4 mutation in Cr mice caused a
general defect in neutrophil trafficking, we examined the
recruitment of neutrophils in response to thioglycollate, a
complement and leukotriene-dependent response.22 The number of peritoneal leukocytes 3 hours after intraperitoneal
injection of thioglycollate increased to a similar extent in Sn
and Cr mice (Figure 4), indicating a normal neutrophil
response in Cr mice.

Downloaded from by guest on May 4, 2015

Oyama et al

Reduced Myocardial IR Injury in TLR4-Deficient Mice


Figure 3. Lipid peroxidation products malondialdehyde and

4-hydroxynonenal (MDA4-HNE) in hearts from TLR4-deficient
(Cr) or wild-type control (Sn) mice after 1-hour/24-hour MIR.
Units are nanomole per milligram of protein and represent the
meanSEM from 6 observations per group. *Significant difference by Students t test at P0.05.


Figure 2. A, Histochemical sections of hearts from TLR4deficient (Cr) or wild-type control (Sn) mice after 1-hour/24-hour
MIR. Sections were stained with hematoxylin and eosin (A and
B), CD45 Ab (brown, C through F), or neutrophil-specific marker
GR-1 Ab (G and H). Results are representative of 3 mice from
each strain. B, MPO activity in heart homogenates from Cr
(n6) and Sn (n6) mice after 1-hour/24-hour MIR. MPO values
for sham-operated hearts were Cr, 0.370.12; and Sn,
0.520.085 U/100 mg (P0.38, NS). Units of myocardial MPO
activity are expressed as units per 100 mg tissue weight and
are meanSEM. *Significant difference by Students t test at

Complement is activated and component C3 is deposited

on tissues in response to IR injury of several organs, including the heart. After 1-hour/24-hour MIR, a distinct pattern of
C3 deposition was observed in areas of the left ventricle that
also showed evidence of injury and neutrophil infiltration
(Figure 5). Deposition of C3 appeared primarily on the
myocardium and was not observed on leukocytes. Quantification of LV C3 deposition showed a significantly greater
area of C3 staining on hearts from Sn mice compared with Cr
mice after 1-hour/24-hour MIR (Sn, 12.71.8% versus Cr,

This study demonstrates that a deficiency of TLR4 signaling

reduces myocardial infarctions after a period of ischemiareperfusion in mice. The data additionally demonstrate that
TLR4 deficiency reduces inflammatory pathways linked to
expansion of myocardial injury in MIR, including neutrophil
accumulation, oxidative stress, and deposition of activated
complement. Moreover, this study suggests that TLR4 is
involved in inflammatory responses to ischemic tissue injury
in addition to its role in inflammation triggered by microbial
pathogens. Thus, these data establish an important role for
TLR4 signaling in MIR-induced inflammation and injury in
Myocardial ischemia-reperfusion elicits an intense inflammatory response that involves production of oxidants, activation of complement, and infiltration by polymorphonuclear
neutrophils (PMNs).4 Experimental interruption of these
pathways individually is reported to reduce MIR-induced
infarctions, suggesting that the inflammatory response to
MIR contributes cooperatively with ischemia to cause myocardial damage. Among these pathways, PMN infiltration is a
prominent event after MIR. PMN infiltration was signifi-

Figure 4. Thioglycollate-induced peritonitis in TLR4-deficient

(Cr) or wild-type control (Sn) mice. Animals were injected intraperitoneally with PBS (control, n4 for Sn and n4 for Cr) or
with thioglycollate (n6 for Sn and n8 for Cr). The number of
peritoneal leukocytes was determined 3 hours later. Granulocytes and macrophages were distinguished by FACS. Values
represent mean cell numberSEM in millions; the error bars
refer to the standard error of the total leukocyte count. No significant differences from single-factor ANOVA; P0.24.

Downloaded from by guest on May 4, 2015



February 17, 2004

Figure 5. Complement-3 (C3) deposition on representative histochemical sections of hearts from TLR4-deficient (Cr, A) or
wild-type control (Sn, B through D) after 1-hour/24-hour MIR.
Areas stained positive for C3 are depicted by arrows in A and
B. Lower bar graph depicts quantification of C3 deposition as a
percentage of the LV area from Cr and Sn mice. Values represent meanSEM from 3 observations per group. *Significant
difference by Students t test at P0.05.

cantly reduced in hearts from TLR4-deficient Cr mice.

Reduced complement activation, also observed in Cr mice,
may have contributed to fewer PMNs because of reduced
production of chemotactic anaphylotoxins, particularly C5a.23
Alternatively, a deficiency in TLR4 signaling by myocytes
may reduce expression of neutrophilic CXC-type chemokines
such as LIX and KC, which are expressed by rat myocytes in
response to MIR and to LPS, a TLR4 ligand24 (and Blais and
Bourcier, unpublished observation, 2003). It remains controversial, however, whether the presence of neutrophils in MIR
is causative or a consequence of myocardial injury.25 Thus,
fewer neutrophils in hearts from Cr mice may reflect smaller
infarct size caused by mechanisms independent of neutrophil
activity. The contribution of TLR4 to each of these pathways
is under investigation in our laboratory; however, these data
demonstrate an important role of TLR4 signaling in PMN
trafficking in response to MIR.
Reactive oxygen species are produced in MIR, and inhibiting formation of ROS can reduce infarct size in some
models.26 ROS can directly damage cells, trigger cytokine
expression, increase leukocyte chemotaxis, and initiate complement activation.26 ROS are generated in large amounts
from activated PMNs infiltrating the injured myocardium.27
Hearts from TLR4-deficient mice contained significantly

fewer lipid peroxides, major byproducts of oxidative stress. It

is possible that TLR4 activation increases ROS production in
part by upregulating expression of inducible NO synthase and
cyclooxygenase.28,29 It is more likely that reduced lipid
peroxidation in Cr mice is secondary to fewer numbers of
activated neutrophils, and thus less ROS generation, after
MIR. Our experiments, however, cannot exclude a more
direct link between TLR4 activation and generation of ROS
in MIR. Nevertheless, TLR4 appears linked to oxidative
stress in myocardial IR, which may contribute to inflammation and myocyte damage.
Inhibiting complement at the level of C3 or C5b-9 in
experimental IR reduces myocardial infarct size and postischemic inflammation.30,31 The area of C3 deposition was
significantly reduced in hearts from Cr mice compared with
Sn mice, despite identical areas at risk of infarction. Reduced
necrosis in the hearts from Cr mice might contribute to
reduced myocardial C3 deposition.32 Conversely, reduced C3
deposition predicts less generation of proinflammatory anaphylotoxins and cytotoxic complement components, which
may contribute to smaller infarct size and reduced neutrophil
trafficking in Cr mice. These data suggest that TLR4 activation may be linked and proximal to complement activation in
Reduced myocardial inflammation in Cr mice prompted us
to consider that defective TLR4 signaling may nonspecifically blunt all pathways of inflammation. However, no
difference in the number of thioglycollate (TG)-elicited
granulocytes was observed between the Sn and Cr mice. This
result suggests that neutrophil trafficking is not inherently
defective in Cr mice and that TLR4 is selectively involved in
inflammatory pathways. A key difference in TG versus
MIR-induced inflammation is the presence of tissue damage
in MIR. This is consistent with the hypothesis that endogenous TLR4 ligands are present at sites of cellular injury or
necrosis. This is supported by recent studies identifying
HSP60 as a putative ligand for TLR416 and other endogenous
host-derived molecules that appear to require TLR4 for
activity.7 Therefore, TLR4, and possibly other TLRs, would
be activated in circumstances involving tissue injury and
necrosis in addition to activation caused by recognized
microbial ligands.

In summary, 2 distinct TLR4-deficient murine strains were
protected from myocardial infarction caused by ischemiareperfusion injury. Inflammatory pathways linked to MIRinduced infarction were reduced in TLR4-deficient Cr mice,
including PMN infiltration, oxidative stress, and complement
activation. This study demonstrates that in addition to its role
in innate immune responses, TLR4 serves a proinflammatory
function in a murine model of MIR injury.

This study was supported by grants from the National Heart, Lung,
and Blood Institute (R01 HL63927 to Dr Kelly) and the American
Heart Association (SDG 0130323N to Dr Bourcier); a Banyu
Fellowship in Cardiovascular Sciences (to Dr Oyama); and the
Medical Research Council of Canada (to Dr Blais). Special thanks to
Dr Gregory Stahl for critically reading this manuscript.

Downloaded from by guest on May 4, 2015

Oyama et al

Reduced Myocardial IR Injury in TLR4-Deficient Mice

1. Braunwald E. Personal reflections on efforts to reduce ischemic myocardial damage. Cardiovasc Res. 2002;56:332338.
2. Ribichini F, Wijns W. Acute myocardial infarction: reperfusion
treatment. Heart. 2002;88:298 305.
3. Maxwell SR, Lip GY. Reperfusion injury: a review of the pathophysiology, clinical manifestations and therapeutic options. Int J Cardiol.
4. Frangogiannis NG, Smith CW, Entman ML. The inflammatory response
in myocardial infarction. Cardiovasc Res. 2002;53:31 47.
5. Louis A, Cleland JG, Crabbe S, et al. Clinical trials update:
and RENAISSANCE and cachexia and cholesterol in heart failure. Highlights of the Scientific Sessions of the American College of Cardiology,
2001. Eur J Heart Fail. 2001;3:381387.
6. Faxon DP, Gibbons RJ, Chronos NA, et al. The effect of blockade of the
CD11/CD18 integrin receptor on infarct size in patients with acute myocardial infarction treated with direct angioplasty: the results of the
HALT-MI study. J Am Coll Cardiol. 2002;40:1199 1204.
7. Akira S, Takeda K, Kaisho T. Toll-like receptors: critical proteins linking
innate and acquired immunity. Nat Immunol. 2001;2:675 680.
8. Poltorak A, He X, Smirnova I, et al. Defective LPS signaling in C3H/HeJ
and C57BL/10ScCr mice: mutations in Tlr4 gene. Science. 1998;282:
9. Medzhitov R, Preston-Hurlburt P, Janeway CA Jr. A human homologue
of the Drosophila Toll protein signals activation of adaptive immunity.
Nature. 1997;388:394 397.
10. Frantz S, Kobzik L, Kim YD, et al. Toll4 (TLR4) expression in cardiac
myocytes in normal and failing myocardium. J Clin Invest. 1999;104:
11. Xu XH, Shah PK, Faure E, et al. Toll-like receptor-4 is expressed by
macrophages in murine and human lipid-rich atherosclerotic plaques and
upregulated by oxidized LDL. Circulation. 2001;104:31033108.
12. Baumgarten G, Knuefermann P, Nozaki N, et al. In vivo expression of
proinflammatory mediators in the adult heart after endotoxin administration: the role of toll-like receptor-4. J Infect Dis. 2001;183:16171624.
13. Nemoto S, Vallejo JG, Knuefermann P, et al. Escherichia coli LPSinduced LV dysfunction: role of toll-like receptor-4 in the adult heart.
Am J Physiol Heart Circ Physiol. 2002;282:H2316 H2323.
14. Zeuke S, Ulmer AJ, Kusumoto S, et al. TLR4-mediated inflammatory
activation of human coronary artery endothelial cells by LPS. Cardiovasc
Res. 2002;56:126 134.
15. Li M, Carpio DF, Zheng Y, et al. An essential role of the NF-B/Toll-like
receptor pathway in induction of inflammatory and tissue-repair gene
expression by necrotic cells. J Immunol. 2001;166:7128 7135.
16. Ohashi K, Burkart V, Flohe S, et al. Cutting edge: heat shock protein 60
is a putative endogenous ligand of the toll-like receptor-4 complex.
J Immunol. 2000;164:558 561.


17. Yet SF, Tian R, Layne MD, et al. Cardiac-specific expression of heme
oxygenase-1 protects against ischemia and reperfusion injury in
transgenic mice. Circ Res. 2001;89:168 173.
18. Poltorak A, Merlin T, Nielsen PJ, et al. A point mutation in the IL-12R
beta 2 gene underlies the IL-12 unresponsiveness of Lps-defective
C57BL/10ScCr mice. J Immunol. 2001;167:2106 2111.
19. Zhou RH, Shi Q, Gao HQ, et al. Changes in serum interleukin-8 and
interleukin-12 levels in patients with ischemic heart disease in a Chinese
population. J Atheroscler Thromb. 2001;8:30 32.
20. Dhalla NS, Temsah RM, Netticadan T. Role of oxidative stress in cardiovascular diseases. J Hypertens. 2000;18:655 673.
21. Sabroe I, Prince LR, Jones EC, et al. Selective roles for Toll-like receptor
(TLR)2 and TLR4 in the regulation of neutrophil activation and life span.
J Immunol. 2003;170:5268 5275.
22. Segal BH, Kuhns DB, Ding L, et al. Thioglycollate peritonitis in mice
lacking C5, 5-lipoxygenase, or p47(phox): complement, leukotrienes, and
reactive oxidants in acute inflammation. J Leukoc Biol. 2002;71:
410 416.
23. Chenoweth DE. The properties of human C5a anaphylatoxin: the significance of C5a formation during hemodialysis. Contrib Nephrol. 1987;59:
24. Chandrasekar B, Smith JB, Freeman GL. Ischemia-reperfusion of rat
myocardium activates nuclear factor-B and induces neutrophil infiltration via lipopolysaccharide-induced CXC chemokine. Circulation.
2001;103:2296 2302.
25. Baxter GF. The neutrophil as a mediator of myocardial ischemiareperfusion injury: time to move on. Basic Res Cardiol. 2002;97:
268 275.
26. Dhalla NS, Elmoselhi AB, Hata T, et al. Status of myocardial antioxidants
in ischemia-reperfusion injury. Cardiovasc Res. 2000;47:446 456.
27. Entman ML, Youker K, Shoji T, et al. Neutrophil induced oxidative
injury of cardiac myocytes: a compartmented system requiring CD11b/
CD18-ICAM-1 adherence. J Clin Invest. 1992;90:13351345.
28. Brightbill HD, Libraty DH, Krutzik SR, et al. Host defense mechanisms
triggered by microbial lipoproteins through toll-like receptors. Science.
29. Rhee SH, Hwang D. Murine TOLL-like receptor 4 confers lipopolysaccharide responsiveness as determined by activation of NF kappa B and
expression of the inducible cyclooxygenase. J Biol Chem. 2000;275:
30. Vakeva AP, Agah A, Rollins SA, et al. Myocardial infarction and apoptosis after myocardial ischemia and reperfusion: role of the terminal
complement components and inhibition by anti-C5 therapy. Circulation.
1998;97:2259 2267.
31. Weisman HF, Bartow T, Leppo MK, et al. Soluble human complement
receptor type 1: in vivo inhibitor of complement suppressing post-ischemic myocardial inflammation and necrosis. Science. 1990;249:146 151.
32. Pinckard RN, Olson MS, Giclas PC, et al. Consumption of classical
complement components by heart subcellular membranes in vitro and in
patients after acute myocardial infarction. J Clin Invest. 1975;56:
740 750.

Downloaded from by guest on May 4, 2015

Reduced Myocardial Ischemia-Reperfusion Injury in Toll-Like Receptor 4-Deficient Mice

Jun-ichi Oyama, Charles Blais, Jr, Xiaoli Liu, Minying Pu, Lester Kobzik, Ralph A. Kelly and
Todd Bourcier
Circulation. 2004;109:784-789
doi: 10.1161/01.CIR.0000112575.66565.84
Circulation is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright 2004 American Heart Association, Inc. All rights reserved.
Print ISSN: 0009-7322. Online ISSN: 1524-4539

The online version of this article, along with updated information and services, is located on the
World Wide Web at:

Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published
in Circulation can be obtained via RightsLink, a service of the Copyright Clearance Center, not the Editorial
Office. Once the online version of the published article for which permission is being requested is located,
click Request Permissions in the middle column of the Web page under Services. Further information about
this process is available in the Permissions and Rights Question and Answer document.
Reprints: Information about reprints can be found online at:
Subscriptions: Information about subscribing to Circulation is online at:

Downloaded from by guest on May 4, 2015