From www.bloodjournal.org by guest on September 11, 2016. For personal use only.

Plenary paper

Bifunctional role for VEGF-induced heme oxygenase-1 in vivo: induction

of angiogenesis and inhibition of leukocytic infiltration
Benedetta Bussolati, Asif Ahmed, Helen Pemberton, R. Clive Landis, Francesco Di Carlo, Dorian O. Haskard, and Justin C. Mason

Heme-oxygenases (HOs) catalyze the con-
version of heme into carbon monoxide
and biliverdin. HO-1 is induced during
hypoxia, ischemia/reperfusion, and in-
flammation, providing cytoprotection and
inhibiting leukocyte migration to inflam-
matory sites. Although in vitro studies
have suggested an additional role for
HO-1 in angiogenesis, the relevance of
this in vivo remains unknown. We investi-
gated the involvement of HO-1 in angio-
genesis in vitro and in vivo. Vascular
endothelial growth factor (VEGF) induced
prolonged HO-1 expression and activity
in human endothelial cells and HO-1 inhi-
bition abrogated VEGF-driven angiogen-
esis. Two murine models of angiogenesis
were used: (1) angiogenesis initiated by
addition of VEGF to Matrigel and (2) a
lipopolysaccharide (LPS)–induced model
of inflammatory angiogenesis in which
angiogenesis is secondary to leukocyte
invasion. Pharmacologic inhibition of
HO-1 induced marked leukocytic infiltra-
tion that enhanced VEGF-induced angio-
genesis. However, in the presence of an
anti-CD18 monoclonal antibody (mAb) to
block leukocyte migration, VEGF-induced
angiogenesis was significantly inhibited
by HO-1 antagonists. Furthermore, in the
LPS-induced model of inflammatory an-
giogenesis, induction of HO-1 with cobalt
protoporphyrin significantly inhibited leu-
kocyte invasion into LPS-conditioned Ma-
trigel and thus prevented the subsequent
angiogenesis. We therefore propose that
during chronic inflammation HO-1 has 2
roles: first, an anti-inflammatory action
inhibiting leukocyte infiltration; and sec-
ond, promotion of VEGF-driven nonin-
flammatory angiogenesis that facilitates
tissue repair. (Blood. 2004;103:761-766)
© 2004 by The American Society of Hematology

Introduction
An increasing body of evidence indicates that cells from the innate
immune system such as monocytes/macrophages are important
regulators of angiogenesis.1,2 Granulocytes, whose role is fre-
quently underestimated, also appear to play a primary role in the
initial phases of the angiogenic process.3,4 Angiogenesis is closely
associated with inflammation in several diseases including rheuma-
toid arthritis, atherosclerosis, carcinoma, and hematologic malignan-
cies.5,6 Moreover, several angiogenic growth factors are not
endothelial cell (EC) specific. In particular, vascular endothelial
growth factor (VEGF), recognized to be fundamental to the process of
angiogenesis,7 may also activate and recruit monocytes8 and has been
directly implicated in the inflammatory angiogenesis associated with
diseases such as atherosclerosis and rheumatoid arthritis.9-12
Recent studies have shown that heme oxygenase-1 (HO-1) may
exert anti-inflammatory effects including prolongation of cardiac xeno-
graft graft survival13 and inhibition of leukocyte transendothelial migra-
tion during complement-dependent inflammation14 and in response to
low-density lipoprotein (LDL) oxidation.15 Heme oxygenases are rate-
limiting enzymes that catalyze the conversion of heme into carbon
monoxide (CO) and biliverdin. Human HO exists in 2 main isoforms,
HO-1 (inducible) and HO-2 (constitutive). HO-2 is present in high
concentration in the brain, testis, and vascular EC, while HO-1 is more
widely distributed.16 HO-1 is rapidly induced during hypoxia, ischemia/
reperfusion, hyperthermia, and endotoxic shock, providing cytoprotec-
tion during the resolution of stress-induced inflammatory injury.17 The
importance of this is demonstrated by the severe and persistent
endothelial damage observed in human HO-1 deficiency18 and in
gene-targeted mice deficient in HO-1.19 Furthermore, induction of
HO-1 may directly regulate EC activation, preventing adhesion
molecule expression and chronic graft rejection.20-22 In vitro, HO-1
protects ECs from hydrogen peroxide–mediated cell death23 and
from tumor necrosis factor ␣ (TNF␣) cytotoxicity.24 In addition to
its role in vascular cytoprotection, it has been suggested that HO-1
may play a role in angiogenesis as induction of HO-1 increases EC
VEGF synthesis25 and overexpression of HO-1 induces prolifera-
tion and formation of capillary-like structures.26 However, the
importance of HO-1 in VEGF-driven angiogenesis in vivo is
currently unknown.
In the present study, we have investigated both in vitro and in
vivo the involvement of HO-1 in VEGF-dependent angiogenesis
and specifically the effect of HO-1 activation on inflammatory
angiogenesis in vivo.

From the Department of Biology and Clinical Science, University of Torino, and
Research Center for Experimental Medicine (CeRMS), Ospedale S. Giovanni
Battista, Torino, Italy; Department of Reproductive and Vascular Biology, The
Medical School, University of Birmingham, Edgbaston, Birmingham, United
Kingdom; and British Heart Foundation Cardiovascular Medicine Unit, The Eric
Bywaters Center, Imperial College London, Hammersmith Hospital, London,
United Kingdom.
Submitted June 18, 2003; accepted September 22, 2003. Prepublished online as
Blood First Edition Paper, October 2, 2003; DOI 10.1182/blood-2003-06-1974.
Supported by grants from Arthritis Research Campaign grant M0620, British
Heart Foundation Programme grant RG/98/0003, and the Italian Ministry of
University and Research (MIUR) ex 60%, and by the Special Project
“Oncology,” Compagnia San Paolo, Torino, Italy. J.C.M. is an Arthritis Research
Campaign Senior Fellow.
Reprints: Asif Ahmed, Department of Reproductive and Vascular Biology, The
Medical School, University of Birmingham, Edgbaston, Birmingham, B12 2TG,
United Kingdom; e-mail: a.s.ahmed@bham.ac.uk; and Benedetta Bussolati,
Department of Biology and Clinical Science, University of Torino, Az
Ospedaliera San Luigi, Regione Gonzole 10-10043 Orbassano, Torino, Italy;
e-mail: benedetta.bussolati@unito.it.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
© 2004 by The American Society of Hematology

BLOOD, 1 FEBRUARY 2004 䡠 VOLUME 103, NUMBER 3 761

 From www.bloodjournal.org by guest on September 11, 2016. For personal use only.
762 BUSSOLATI et al
Materials and methods
Reagents
The HO-1 inhibitors tin (IV) protoporphyrin IX dichloride (SnPP) and zinc
(II) protoporphyrin IX (ZnPP) and the HO-1 activator cobalt (III) protopor-
phyrin IX chloride (CoPP) were purchased from Porphyrin Products
(Logan, UT). Metalloporphyrins were dissolved in 0.2 M NaOH and the pH
adjusted to 7.4. Acidic fibroblast growth factor (aFGF) and VEGF were
from PeproTech EC (London, United Kingdom). The rat antimouse ␤2
integrin (CD18) monoclonal antibody (mAb) M18/2 (immunoglobulin G 2a
[IgG2a]) was obtained from the American Type Culture Collection (Manas-
sas, VA). Lipopolysaccharide (LPS) from Escherichia coli (0111:B4) was
purchased from Sigma-Aldrich (St Louis, MO) and stock solutions were
prepared by suspending 10 mg in 2 mL 20 mM ethylenediaminetetraacetic
acid (EDTA), sonicating until clarified, and freezing at ⫺20°C. LPS
working dilutions were prepared in 10 mM HEPES (N-2-hydroxyethylpipera-
zine-N⬘-2-ethanesulfonic acid; Gibco Laboratories, Grand Island, NY).
Matrigel basement membrane matrix was obtained from Becton Dickinson
Labware (Bedford, MA).
Endothelial cells
Human umbilical vein ECs (HUVECs) and the human microvascular
endothelial cell 1 (HMEC-1) dermal microvascular line27 (a kind gift from
Dr E Ades, Centers for Disease Control, Atlanta, GA) were cultured as
previously described in detail.28,29
Western blotting analysis
HMECs or HUVECs, treated for up to 48 hours with VEGF or aFGF, were
lysed (4 mM EDTA, 50 mM Tris (tris[hydroxymethyl]aminomethane)–
HCL, pH7.4 in 150 mM NaCl with 25 mM sodium deoxycholic acid, 200
␮M sodium orthovanadate, 10 mM sodium pyrophosphate, 100 mM
sodium fluoride, 1% Triton X-100, 1 mM phenylmethysulfonyl fluoride,
and 5% protease inhibitor cocktail) and the protein content determined
using the Bio-Rad Dc protein assay (Bio-Rad, Hercules, CA). Cell lysates
were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophore-
sis (SDS-PAGE) on 12.5% gels and separated proteins transferred to
Immobilon-P transfer membranes (Millipore Corporation, Bedford, MA).
HO-1 was detected with polyclonal anti–HO-1 (Stressgen Biotechnologies,
Victoria, BC, Canada) and actin was detected as a loading control with mAb
C-2 (Santa Cruz Biotechnology, Santa Cruz, CA). The blots were developed
with an enhanced chemiluminescence substrate (Amersham Pharmacia
Biotech UK, Little Chalfont, United Kingdom).
Heme oxygenase activity assay
HMECs were harvested by scraping, centrifuged at 2000g for 10 minutes,
and stored at ⫺80°C until required. Following 3 freeze/thaw cycles the
protein concentration was determined using the Bio-Rad Dc protein assay. A
chromatographic assay was then performed30 in which the samples were
added to a reagent mix containing 0.2 mM MgCl2, 100 mM phosphate-
buffered saline (PBS) pH7.4, 0.5 mg/mL of liver cytosol, 1.1 mM
glucose-6-phosphate, 0.84 U/mL glucose-6-phosphate dehydrogenase, 28
␮M hemin, and 40 mM nicotinamide adenine dinucleotide phosphate
(NADPH). Following vortexing, the reagents were left to react at 37°C for 1
hour. The reaction was terminated with chloroform and the samples
vortexed and centrifuged at 900g for 5 minutes. After vortexing and further
centrifugation at 1100g the extracted bilirubin was quantified by calculating
the difference in absorbance at 464 nm and 530 nm (extinction coefficient
for bilirubin 40 mM⫺1cm⫺1). HO-1 activity was expressed as pmol of
bilirubin formed per mg of protein per hour.
Endothelial cell proliferation and tube formation assays
Cell proliferation was measured as previously described.31 HUVECs
(5 ⫻ 103 cells/well) were cultured in M199/10% fetal bovine serum (FBS)
in the absence of growth factor for 24 hours in 96-well plates. VEGF (25
BLOOD, 1 FEBRUARY 2004 䡠 VOLUME 103, NUMBER 3
ng/mL) was added in the absence or presence of SnPP at a range of
concentrations and ECs were cultured for 72 hours with 3H-labeled
thymidine added 16 hours prior to the end of the assay. ECs were harvested
and proliferation quantified with an automated Betaplate 96-well harvester
(Wallac Oy, Turku, Finland). EC viability was assessed by trypan blue
exclusion and found to be more than 90%. EC tube formation was assessed
by adaptation of a previously described assay.32 Segments (5 mm ⫻ 5 mm)
of C57BL/6 murine abdominal wall muscle were placed in Matrigel in
24-well plates. Dulbecco modified Eagle medium (DMEM) supplemented
with 10% FBS, heparin (10 U/mL), and VEGF in the absence and presence
of SnPP (20 ␮M) was added and changed every 2 days. After 9 days EC
tube length was measured from the edge of the embedded tissue using
Optimas 6.1 Software (Optimas UK, West Malling, United Kingdom). In
individual experiments each treatment was assessed in triplicate and the
length of 10 EC tubes for each tissue segment was measured. The
endothelial nature of the tubes was confirmed by staining with fluorescein
isothiocyanate (FITC) anti–von Willebrand factor (anti-VWF; Serotec,
Oxford, United Kingdom) and the lectin Griffonia simplicifolia isolectin B4
(Vector Laboratories, Peterborough, United Kingdom). In all experiments
appropriate carrier controls were included.
In vivo murine angiogenesis assay
Angiogenesis was quantified by the Matrigel plug assay as previously
described.33 Matrigel (8.13 mg/mL), in liquid form at 4°C, was mixed with
heparin (64 U/mL, Sigma) and the experimental substances and injected
(0.25 mL) into the abdominal subcutaneous tissue of mice, along the
peritoneal midline. To evaluate the effect of HO-1 blockade or activation,
SnPP (20 ␮M), ZnPP (20 ␮M), or CoPP (25 ␮M) was added to Matrigel.
CoPP was also injected intraperitoneally (5 mg/kg) at day 0. To inhibit ␤2
integrins, mAb M18/2 or isotype-matched control mAbs were added to
Matrigel at a final concentration of 20 ␮g/mL and in addition 100 ␮g of
mAb M18/2 or isotype-matched control mAb was injected intraperitoneally
on days 0, 3, and 5. At day 6, mice were killed and gels were processed for
light microscopy and immunohistochemistry, as described.34 Rabbit anti-
VWF Ab, as well as control rabbit and mice IgG (Sigma), were used as
primary antibodies for indirect immunofluorescence. FITC-conjugated
antirabbit IgG and antirat IgG were used as secondary antibodies (all from
Sigma). Direct immunofluorescence staining was performed with antimy-
eloperoxidase (anti-MPO) phycoerythrin (PE)–conjugated monoclonal anti-
bodies (Cedarlane, Hornby, ON, Canada). The total Matrigel area and the
area occupied by vessels were planimetrically assessed from cross-sections
of Matrigel plugs observed by light microscopy as previously described.33
Results were expressed as percentage ⫾ SE of the vessel area with respect
to the total Matrigel area.
Statistics
All data are expressed as mean ⫾ SD. Nonparametric statistical analysis
was performed by the Kruskal-Wallis test for analysis of variance (ANOVA).
Results
VEGF, but not aFGF, induces HO-1 expression
The expression of HO-1 by ECs following treatment with VEGF
and aFGF was assessed by Western blotting. An increase in HO-1
expression following exposure to VEGF was seen at 24 hours and
was maximal at 48 hours in HMECs (Figure 1A). Similar results
were obtained with HUVECs (Figure 1B) and bovine aortic ECs
(not shown). In contrast aFGF, at a concentration sufficient to
induce EC proliferation, was incapable of inducing a significant
change in HO-1 expression after 48 hours (Figure 1C). Endothelial
expression of HO-1 after treatment with VEGF was coupled with
an increase in HO-1 activity (Figure 1D).
 From www.bloodjournal.org by guest on September 11, 2016. For personal use only.
BLOOD, 1 FEBRUARY 2004 䡠 VOLUME 103, NUMBER 3
Figure 1. Expression and activity of HO-1 induced by VEGF in human ECs. Lysates were prepared from HMECs and HUVECs treated with VEGF (25 ng/mL) or aFGF (100
ng/mL) for up to 48 hours and proteins were separated by SDS-PAGE and transblotted on to nitrocellulose membranes. Immunoblots were probed with a polyclonal Ab against
HO-1 and mAb C-2 against actin as a loading control. (A) HMECs and (B) HUVECs. Lane 1, unstimulated ECs; 2, VEGF 24 hours; 3, VEGF 48 hours. (C) HMECs. Lane 1,
unstimulated ECs; 2, aFGF 24 hours; 3, aFGF 48 hours. (D) Heme oxygenase activity was measured in lysates of HMECs cultured in the presence or absence of VEGF (25
ng/mL) for 24 hours. Data presented as mean ⫾ SEM.
Blockade of HO-1 inhibits VEGF-induced in vitro angiogenesis
The role of HO-1 induction in VEGF-driven angiogenesis was first
assessed in vitro. As seen in Figure 2A, the induction of HO-1 with
CoPP alone resulted in EC proliferation comparable to that seen with
VEGF. The presence of the HO-1 inhibitors SnPP or ZnPP (not shown)
significantly inhibited VEGF-induced EC proliferation in a dose-
Figure 2. Role of HO-1 in VEGF-induced angiogenesis in vitro. (A) HUVECs were
cultured in M199 with 10% FBS for 24 hours prior to the addition of VEGF (25 ng/mL)
and CoPP (12.5 and 25 ␮M) both for 72 hours. In test wells, ECs were preincubated
with SnPP at the concentrations shown for 60 minutes prior to the addition of VEGF.
3H-thymidine (0.037 MBq/mL [1 ␮Ci/mL]) was added 16 hours before the end of the
assay and the plates were read on an automated plate reader. Bars show the mean ⫹
SD uptake of 3H-thymidine (n ⫽ 4). (B-F) Murine abdominal muscle was implanted in
Matrigel and cultured in DMEM supplemented with 10% FBS and heparin (10 U/mL).
VEGF (25 ng/mL) was added to the medium in the absence and presence of SnPP
(20 ␮M). (B) After 9 days EC tube length was measured by image analysis. Data
presented as mean ⫹ SD tube length (n ⫽ 3). Panels C-F show representative
micrographs of the effect of SnPP on neovessels formed in the presence of VEGF.
Panels C and E show VEGF alone and panels D and F show VEGF ⫹ SnPP. Original
magnification: low power ⫻ 20 (C-D) and high power ⫻ 40 (E-F).
DUAL ROLE OF VEGF-INDUCED HO-1 IN ANGIOGENESIS 763
dependent manner. To investigate this further, murine abdominal muscle
was implanted in Matrigel and capillary sprouting and neovessel
development in response to VEGF was analyzed. As seen in Figure
2B-C, inhibition of HO-1 with both SnPP and ZnPP (not shown)
completely inhibited the development of VEGF-induced neovessels.
HO-1 blockade promotes VEGF-induced inflammation
and angiogenesis
The murine Matrigel model was used to investigate the role of
HO-1 in VEGF-induced angiogenesis in vivo. Surprisingly, HO-1
inactivation by SnPP (20 ␮M) increased the angiogenic effect
induced by VEGF (Figure 3A). In contrast, no such effect was
observed when SnPP was added to Matrigel plugs containing aFGF
(Figure 3A). Further experiments with ZnPP (20 ␮M) confirmed
these results (Figure 3B). Histologic examination of the implants
containing VEGF plus SnPP or ZnPP showed the formation of
enlarged hemorrhagic vessels when compared with the neovessels
seen in plugs containing VEGF alone (Figure 4A-B). In addition,
the presence of a dense inflammatory infiltrate was noted within
implants treated with VEGF ⫹ SnPP or ZnPP (Figure 4C) and was
confirmed by immunofluorescence staining for myeloperoxidase
(Figure 4D). Quantification demonstrated a significant leukocytic
infiltrate in plugs treated with a combination of VEGF and SnPP or
ZnPP when compared with VEGF alone (P ⬍ .05) and a smaller
but significant infiltrate was also seen with SnPP or ZnPP alone
(P ⬍ .05; Figure 3C). Interestingly, in a recent study of wound
healing, heme-induced influx of leukocytes was also significantly
elevated following pharmacologic inhibition of HO-1.35
In order to investigate further the influence of the migrating
leukocytes on angiogenesis the inhibitory anti-␤2 mAb M18/2 was
used. Treatment of mice with M18/2 did not affect VEGF-induced
angiogenesis per se but abrogated the previously observed enhance-
ment of VEGF-induced angiogenesis in the presence of SnPP
(P ⬍ .05; Figure 5A). This was related to a specific suppression of
leukocytic infiltration into Matrigel plugs by M18/2, as an isotype-
matched control mAb did not affect the proinflammatory effects of
SnPP (Figure 5B) and no effect of mAb M18/2 was seen on
VEGF-induced angiogenesis (Figure 5A). Furthermore, in the
absence of leukocyte infiltration, SnPP completely inhibited VEGF-
induced angiogenesis (Figure 5A) supporting a role for HO-1 in
angiogenesis directly driven by VEGF.
HO-1 activation inhibits leukocyte recruitment and
inflammation-related angiogenesis
To explore these findings further, lipopolysaccharide was added to
Matrigel plugs as a representative model of inflammatory angiogen-
esis in vivo. This model is characterized by an intense leukocytic
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764 BUSSOLATI et al BLOOD, 1 FEBRUARY 2004 䡠 VOLUME 103, NUMBER 3

Figure 3. Role of HO-1 during in vivo angiogenesis. (A) Quantitative evaluation of neovessels infiltrating Matrigel after injection of aFGF (50 ng/mL), VEGF (40 ng/mL), or
vehicle alone in the absence or presence of SnPP (20 ␮M). The results are expressed as percentage ⫾ SE of the vessel area to the total Matrigel area. (B) Effect of SnPP (20
␮M) and ZnPP (20 ␮M) on angiogenesis induced by VEGF (40 ng/mL). (C) Quantitative evaluation of inflammatory cells infiltrating Matrigel that were counted in sections
stained with hematoxylin and eosin. The results are expressed as the mean ⫾ SE of cells/field (⫻ 400). Six mice were used per condition in each experiment.

infiltration and an associated angiogenic response.36 As expected,
the inclusion of LPS alone induced leukocytic infiltration into the
Matrigel plug with significant angiogenesis (Figure 4E and 6). To
investigate the effect of HO-1 activation in this model of angiogen-
esis mice were treated with the CoPP. As seen in Figure 4F and
Figure 6, addition of CoPP significantly inhibited leukocyte-
induced angiogenesis, suggesting that HO-1 may act to suppress
inflammatory angiogenesis in vivo.
Discussion
One of the products of HO-1 activation, CO, is an endogenously
produced vasorelaxant molecule that activates soluble guanylate cyclase
leading to an increase in intracellular cyclic guanosine monophosphate
(cGMP).40 Recent work has demonstrated that treatment with CO
prevents arteriosclerosis in a model of aortic transplantation in which
CO exerts a marked inhibitory effect on graft leukocyte infiltration and
activation and reduces vascular smooth muscle cell proliferation via
generation of cGMP and activation of p38 mitogen-activated protein
kinase (MAPK).41 Activation of guanylate cyclase is also induced by
nitric oxide (NO), the major secondary mediator of VEGF effects.42 It is
therefore possible that HO-1 and NO synthase exert a synergistic effect

Previous in vitro studies have reported a role for HO-1 in EC survival,

proliferation, and tube formation, suggesting that HO-1 induction may
promote angiogenesis and that this is mediated, at least in part, by
CO.25,26 A recent study has also reported HO-1 induction in the chicken
chorioallantoic membrane following treatment with VEGF.37 We found
that VEGF, a potent angiogenic factor, induced HO-1 expression and
increased HO-1 activity in human ECs. The potential involvement of
endothelial HO-1 in VEGF-driven angiogenesis was demonstrated by
the ability of the HO-1 antagonists SnPP and ZnPP to significantly
inhibit EC proliferation and tube formation in vitro. Furthermore, the
results presented herein demonstrate for the first time that HO-1 plays an
important role in VEGF-driven angiogenesis in vivo and acts as a key
regulator in the control of the anti-inflammatory versus proinflammatory
actions of VEGF.
VEGF is known to act on both leukocytes and ECs. However, in
vivo models have suggested that VEGF is principally an inducer of
noninflammatory angiogenesis.38 This may reflect a predominant
influence of VEGF on ECs rather than leukocytes. In our in vivo
experiments inhibition of HO-1 with SnPP shifted VEGF-driven
angiogenesis from noninflammatory to proinflammatory, as evi-
denced by the presence of a dense leukocytic infiltrate. This
suggests that induction of HO-1 by VEGF may act to both promote
endothelial proliferation and to limit proinflammatory leukocytic
infiltration. This hypothesis was confirmed in vivo by the effect of
the inhibitory anti-␤2 integrin mAb M18/2. M18/2 did not affect
VEGF-induced angiogenesis per se but completely abrogated the
VEGF-induced leukocytic infiltration observed in the presence of
HO-1 inhibitors. Moreover, when leukocyte infiltration was inhib-
ited by M18/2, the HO-1 antagonists ZnPP and SnPP inhibited Figure 4. Histologic and immunohistochemical analysis of Matrigel implants.
VEGF-induced angiogenesis confirming the role of HO-1 in this Representative hematoxylin and eosin–stained sections of Matrigel containing VEGF
(A) or VEGF plus SnPP (B) implanted in mice and excised 6 days after injection. A
process. Furthermore, experiments performed with aFGF, which dense inflammatory infiltrate around vessels was observed in plugs containing VEGF
did not induce HO-1, showed that blockade of HO-1 did not plus SnPP when compared with VEGF alone (original magnification ⫻ 160). (C-D)
influence aFGF-induced angiogenesis, thus demonstrating that the Double immunofluorescence staining for VWF (C) and MPO (D) showing the
presence of leukocytes around vessels in plugs with VEGF plus SnPP (original
HO-1 inhibitors themselves do not affect angiogenesis. These data
magnification ⫻ 250). (E-F) Representative hematoxylin and eosin–stained sections
support previous studies showing that VEGF and FGF induce of Matrigel containing LPS (E) or LPS in the presence of CoPP, which prevented
angiogenesis through distinct pathways.39 leukocyte-dependent angiogenesis (F) (original magnification ⫻ 160).
 From www.bloodjournal.org by guest on September 11, 2016. For personal use only.

BLOOD, 1 FEBRUARY 2004 䡠 VOLUME 103, NUMBER 3 DUAL ROLE OF VEGF-INDUCED HO-1 IN ANGIOGENESIS 765

Figure 5. Effect of anti–␤2-integrin Ab M18/2 on angiogen-

esis and leukocyte infiltration. M18/2 or a isotype-
matched control mAb was added to Matrigel and injected
intraperitoneally every other day, as described in “Materials
and methods.” (A) Quantitative evaluation of neovessels
infiltrating Matrigel after treatment with SnPP, VEGF, or
VEGF plus SnPP in presence of M18/2 or a control mAb. The
results are expressed as percentage ⫾ SE of the vessel area
with respect to the total Matrigel area. (B) Quantitative
evaluation of inflammatory cells infiltrating Matrigel counted
in hematoxylin and eosin–stained sections. The results are
expressed as the mean ⫾ SE of cells/field (⫻ 400). Four
mice were used per condition in each experiment. ANOVA
with Dunnett multicomparison test was performed between
control Ab and M18/2 (*P ⬍ .05).

during VEGF-induced angiogenesis. Moreover, HO-1 may play a role
in different angiogenic settings, such as during hypoxia, where NO
production and endothelial NO synthase expression are suppressed.43
Indeed, a role for HO-1 in angiogenesis during hypoxia has been
suggested by the observation that HO-1 activation enhances VEGF
production and that VEGF induced by hypoxia was down-regulated by
HO-1 inhibitors but not by inhibitors of NO synthase.44
HO-1 breaks down heme to equimolar amounts of CO, biliver-
din, and free iron. Biliverdin is rapidly reduced to bilirubin by the
action of biliverdin reductase. Although less is known about the
anti-inflammatory and cytoprotective effects of bilirubin, these
have been clearly demonstrated.45,46 Hence, the relative contribu-
tion of bilirubin and CO in mediating the effects of VEGF-induced
HO-1 in angiogenesis merits further investigation.
An anti-inflammatory effect of HO-1 has been demonstrated in vitro
and in vivo. The induction of HO-1 decreases EC expression of the
cellular adhesion molecule ICAM-122 and inhibits monocyte chemo-
taxis.15 Pretreatment with HO-1 agonists attenuates tissue damage in a
variety of models including carrageenan-induced pleuritis, endotoxic
shock, and oxidant-induced lung injury.14,47,48 Moreover, increased
leukocyte adhesion to the vessel wall and spontaneous perivascular
infiltration of leukocytes into the liver, lungs, and kidneys was observed
in HO-1–deficient mice.19 To investigate the role of HO-1 in inflamma-
tory angiogenesis we used LPS-conditioned Matrigel. In this model,36
neutrophils invade and begin to degrade the gel creating clefts and this is
followed by migration of macrophages. Growth factors, such as VEGF
and basic FGF (bFGF), released by neutrophils and macrophages
subsequently induce EC migration and tube formation. Thus, this model
of angiogenesis is directly dependent upon leukocyte migration. Treat-
ment of mice with the HO-1 inducer CoPP significantly inhibited
LPS-induced leukocyte infiltration and the subsequent angiogenic
response. This suggests that HO-1–mediated inhibition of leukocyte
recruitment represents an important mechanism for the control of
angiogenesis associated with inflammation.
The data presented herein, and in other recent studies, suggests
that many anti-inflammatory and cytoprotective molecules act
through a mechanism dependent upon the expression of HO-1 and
that the products of HO-1 facilitate the protective effects.46 This is
supported by the demonstration that HO-1 expression, or treatment
with CO, reproduces these beneficial actions. Thus, HO-1 and CO
have been shown to mediate the protective effects of interleukin-10
in LPS-induced septic shock in mice.49 Further examples of the
pivotal role of HO-1 include its importance in the antiproliferative
effects of rapamycin,50 the anti-inflammatory actions of 15-Deoxy-
12,14-prostaglandin J2,51 and the cytoprotective effects of heat-
shock preconditioning.52
VEGF may act both as a potent proangiogenic factor and as a
leukocyte chemoattractant. We propose that HO-1 induced by
VEGF acts to promote angiogenesis while inhibiting the local
recruitment of leukocytes (Figure 7). Thus, in the setting of
inflammatory diseases associated with VEGF-driven angiogenesis,
such as rheumatoid arthritis, the action of VEGF-induced HO-1
may be to maximize angiogenesis associated with the resolution of
tissue injury while inhibiting leukocyte adhesion and transmigra-
tion. However, HO-1 induction may be impaired during inflamma-
tion, as recently reported in human chronic graft rejection.53 Thus,
therapeutic induction of HO-1, or delivery of CO or bilirubin, may
be beneficial in the treatment of chronic inflammatory diseases, not
only through their anti-inflammatory actions but also via a proan-
giogenic effect enhancing tissue repair.

Figure 6. Effect of HO-1 induction on inflammatory angiogenesis in vivo.

Matrigel containing LPS (10 ng/mL) plus CoPP (25 ␮M) was injected subcutaneously
into C57BL/6 mice. After 6 days plugs were explanted and fixed in formalin and
paraffin embedded. Quantification of neovascularization was performed on hematoxy-
lin and eosin–stained sections as described in “Materials and methods.” The results
are expressed as percentage ⫾ SE of the vessel area with respect to the total
Matrigel area. Nine mice were used per condition in each experiment. ANOVA with
Dunnett multicomparison test was performed between LPS and LPS plus CoPP
(*P ⬍ .05).
Figure 7. Proposed mechanism for the actions of HO-1 in vivo. HO-1 activation
by VEGF favors endothelial cell proliferation while inhibiting leukocyte migration and
activity, thus resulting in a predominantly noninflammatory angiogenesis. However,
when HO-1 is inhibited, there is increased leukocyte migration with subsequent local
release of growth factors and induction of inflammatory angiogenesis.
 From www.bloodjournal.org by guest on September 11, 2016. For personal use only.
766 BUSSOLATI et al
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 From www.bloodjournal.org by guest on September 11, 2016. For personal use only.

2004 103: 761-766

doi:10.1182/blood-2003-06-1974 originally published online
October 2, 2003

Bifunctional role for VEGF-induced heme oxygenase-1 in vivo: induction

of angiogenesis and inhibition of leukocytic infiltration
Benedetta Bussolati, Asif Ahmed, Helen Pemberton, R. Clive Landis, Francesco Di Carlo, Dorian O.
Haskard and Justin C. Mason

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