(Circulation. 1999;100:II-301.)
© 1999 American Heart Association, Inc.
Aortic and Peripheral Vascular Surgery |
Potential Role of Hepatocyte Growth Factor, a Novel Angiogenic Growth Factor, in Peripheral Arterial Disease
Downregulation of HGF in Response to Hypoxia in Vascular Cells
From the Department of Geriatric Medicine, Osaka University Medical School; Sumitomo Pharmaceutical Company (T. Nagano, M.T., H.N.), Osaka, Japan; and the Division of Biochemistry (K.M., T. Nakamura), Department of Oncology, Biomedical Research Center, Osaka University Medical School, Japan.
Correspondence to Ryuichi Morishita, MD, PhD, Associate Professor, Department of Geriatric Medicine, Osaka University Medical School, 2-2 Yamada-oka, Suita 565, Japan. E-mail morishit{at}geriat.med.osaka-u.ac.jp
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Abstract |
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Background—Although hepatocyte growth factor (HGF), a novel angiogenic growth factor, plays an important role in angiogenesis, regulation of local HGF production under hypoxia has not yet been clarified in vascular smooth muscle cells (VSMC) and endothelial cells (EC). Thus, we have studied the role of HGF in hypoxia-induced endothelial injury and the regulation of local vascular HGF expression in response to hypoxia.
Methods and Results—HGF attenuated hypoxia-induced endothelial cell death. Importantly, hypoxic treatment of EC resulted in a significant decrease in local HGF production according to the severity of hypoxia and increased VEGF. Similarly, hypoxia significantly decreased in mRNA and protein of HGF and increased VEGF production in VSMC. In organ culture system, local HGF production was also significantly decreased by hypoxia (P<0.01). Downregulation of HGF by hypoxia is due to a significant decrease in cAMP, as hypoxic treatment decreased cAMP, a stimulator of HGF. Although active TGF-ß, a suppressor of HGF, was increased at 72 hours after hypoxic treatment, treatment of anti-TGF-ß antibody did not attenuate decreased HGF production. Finally, rHGF was intra-arterially administered into unilateral hind limb ischemia rabbits, to evaluate in vivo angiogenic activity. Of importance, a single intra-arterial administration of rHGF reduced severe necrosis due to ischemia in rabbit muscle, accompanied by a significant increase in angiographic score (P<0.01).
Conclusions—Overall, these data demonstrated that hypoxic treatment of vascular cells significantly downregulated HGF production due to decreased cAMP, suggesting their potential roles in the pathophysiology of ischemic diseases. Moreover, administration of rHGF induced therapeutic angiogenesis, accompanied by improvement of necrotic changes in the ischemic hind limb model, as cytokine supplement therapy for peripheral arterial disease.
Key Words: endothelium • peripheral vascular disease • angiogenesis • myocardial infarction • remodeling
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Introduction |
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The development of new blood vessels, or angiogenesis, begins with activation of parent vessel endothelial cells.1 Growth factors shown to be mitogenic for endothelial cells in vitro, as well as stimulating angiogenesis in vivo, have been referred to as angiogenic growth factors. These include vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF). In addition, recent studies identified that hepatocyte growth factor (HGF) is also a member of angiogenic growth factors.2 3 HGF is a mesenchyme-derived pleiotropic factor which regulates cell growth, cell motility, and morphogenesis of various types of cells and is thus considered a humoral mediator of epithelial-mesenchymal interactions responsible for morphogenic tissue interactions during embryonic development and organogenesis.4 Moreover, we found that HGF fulfills the characteristics of an endothelium-specific growth factor, similar to VEGF.5 6 VEGF and HGF are secreted by intact cells and are mitogenic exclusively for endothelial cells.7 8 In contrast, bFGF is neither secreted by cells nor is its mitogenic activity limited to endothelial cells only; it is also a potent mitogen for vascular smooth muscle cells (VSMC). VEGF increases vascular permeability and stimulates monocyte migration through endothelial layers, whereas HGF seems not to do so.
Capillary growth is usually limited in adult tissues, but it can resume under hypoxic conditions and pathological conditions that are associated with a decreased oxygen supply such as ischemia.9 Because inefficient vascular supply and the resultant reduction in tissue oxygen tension often lead to neovascularization in order to satisfy the needs of the tissues, knowing the triggers of natural neovascularization is important. Hypoxia is generally considered to represent a fundamental stimulus for angiogenesis, although the mechanisms responsible for its angiogenic activity remain enigmatic. Numerous studies have indicated that VEGF is upregulated by hypoxic treatment in various cells, thereby indicating its pathophysiological role in the pathogenesis of ischemic diseases.10 11 12 In contrast, no report has described the regulation of HGF in response to hypoxia, although HGF is a potent angiogenic growth factor. Therefore, in this study we hypothesized that HGF may also modulate the ischemic condition in cardiovascular disease. To understand the molecular mechanisms of neovascularization, we have addressed the following issues: (1) the role of HGF in hypoxia-induced endothelial injury, and (2) the regulation of local HGF expression in response to hypoxia. In addition, we tested whether it is possible to promote therapeutic angiogenesis by means of HGF in the rabbit ischemia model, to examine the feasibility of therapy for critical limb ischemia.
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Methods |
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Experiment 1
Cell Culture
Human aortic endothelial cells (passage 3) and human aortic VSMC (passage 3) were obtained from Clonetics Corp, and cultured in modified MCDB131 medium supplemented with 5% fetal calf serum, 10 ng/mL epidermal growth factor, 2 ng/mL bFGF, and 1 mmol/L dexamethasone.13 Cells were incubated at 37°C in a humidified atmosphere of 95% air/5% CO2 with medium changes every 2 days. Immunohistochemical examination and morphological observation showed these cells exhibiting specific characteristics of endothelial cells and VSMC. Briefly, human aortic endothelial cells tested positive for factor VIII antigen and for uptake of diacetylated LDL. In contrast, human aortic VSMC tested positive for
-actin and negative for expression
of factor VIII antigen. All the cells were used within passage 3-5.
Hypoxic Treatment
Hypoxia was induced with an anaerobic
device. Briefly, a hypoxic condition was achieved using BBL GasPak
(Becton Dickson, Cockeysville, Md), which catalytically reduces
O2 to undetectable levels in the severe condition
by GasPak plus or to 10% in the mild condition by Campy Pak within 90
minutes, as assessed by an oxygen electrode.14 In the
preparation of experiments for determination of cell death,
endothelial cells were grown to confluence. After
reaching confluence, the medium was changed to fresh DSF (defined serum
free) medium containing HGF, bFGF, VEGF, or vehicle. DSF medium was
supplemented with insulin (5x10-7 M),
transferrin (5 mg/mL), and ascorbate (0.2 mmol/L), as previously
described.15 The cells were then incubated. On days 1, 2,
and 3, an index of cell proliferation was determined, as described
below.
Cell Counting Assay
An index of cell proliferation was determined using WST
(sulfonated tetrazolium salt,
4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene
disulfonate)-cell counting kit, because this compound produces a highly
water-soluble formazan dye which makes the assay procedure easier to
perform.5 16 Tetrazolium salt has been used to develop a
quantitative colorimetric assay for cell growth. We
confirmed that serum-stimulated increase in cell number is associated
with increased absorbance at 450 nm (data not shown). Briefly, 50 000
cells/well reflects an absorbance of 1 under the manufacturer’s
recommended conditions. The sensitivity of WST assay is double that of
MTT assay. In our experimental conditions, an increase in
absorbance of 0.2 reflects an increase in cell number to 20 000
cells/well.
Transfection of Human HGF Vector into Endothelial
Cells Using HVJ-Liposomes
To produce an HGF expression vector, human HGF cDNA (2.2 kb) was
inserted into the Not I sites of pUC-SR expression vector plasmid.
In this plasmid, transcription of the HGF cDNA was under the control of
the SR promoter.17 As the control, we
constructed a control expression vector without HGF gene. We have
previously reported the high efficiency of transfection of cells in
culture using Hemagglutinating Virus of Japan (HVJ)-coated
liposomes17 18 ; in this study, we used the HVJ-liposome
method. Endothelial cells
(1x106) were seeded onto 6-well plates (Corning)
and grown to 80% confluence. Cells were washed 3 times with BSS
containing 2 mmol/L CaCl2 and then incubated
with 1 mL HVJ-liposomes-DNA complex (2.5 mg lipid and 10 µg
encapsulated DNA) at 4°C for 5 minutes followed by 37°C for 30
minutes. To study the release of HGF, transfected cells (48 hours
posttransfection) were washed and fed with 1 mL DSF medium. Twenty-four
hours later, conditioned medium was collected, centrifuged at
600g for 10 minutes and stored at -20°C. The
concentration of HGF in the medium was determined by enzyme immunoassay
using anti-human HGF antibody, as described below. On day 4, an index
of cell proliferation was also determined using WST-cell counting kit
(Wako).
Organ Culture System
The carotid arteries of Sprague-Dawley rats (weighing 400 to
450 g) were dissected as previously reported.19 A
20-mm segment of the carotid artery was immediately transferred to DMEM
medium with 30% fetal calf serum, penicillin (100 U/mL), and
streptomycin (1000 µg/ml). Previous studies in our laboratory and
others have documented viability of the intact vessel maintained under
these conditions. After 48 hours of normoxic or hypoxic treatment,
vessels were homogenized, as previously
reported.20 HGF concentration was measured by enzyme
immunoassay using rat anti-HGF antibody, as described
below.
Measurement of HGF and VEGF in Conditioned Medium
Human endothelial cells and VSMC were seeded on
6-well plates (Corning) at a density of 5x104
cells/cm2 and cultured for 24 hours. After
replacing the medium with fresh DSF (for VSMC or DSF) with 0.5% fetal
calf serum (for endothelial cells) and following
culture under hypoxic or normoxic condition, the concentration of HGF
in the medium was determined by enzyme immunoassay using anti-human HGF
antibody, as described previously.8 20 This ELISA
specifically detects only human HGF because of lack of cross-reactivity
of antibodies.20 For the organ culture experiment, rat HGF
concentration was measured using anti-rat HGF antibody. Measurement of
VEGF was also performed using ELISA kit (R & D systems), as recommended
by the manufacturer. DSF medium did not contain immunoreactive HGF and
VEGF assessed by enzyme immunoassay (data not shown).
Northern Blot Analysis
RNA was extracted by RNAzol (Tel-Test Inc), from cells after 2,
6, and 12 hours of hypoxic or normoxic condition for Northern blot
analysis. For Northern blot analysis, 20 µg total RNA
was subjected to electrophoresis on 1.5% agarose-formaldehyde
denaturing gel and transferred to a nitrocellulose membrane (Amersham
International plc, Amersham, UK). The filter was baked, prehybridized,
and hybridized. Full-length cDNA for HGF, labeled by random-primer kit
(Amersham), were used as proves for Northern blotting. The filter was
then washed and exposed to x-ray film.
Measurement of Intracellular cAMP Level
VSMC were grown to confluence in DSF with 0.5% FCS; cells were
then exposed to hypoxia. Intracellular cAMP was measured using
an enzyme immunoassay kit from Amersham. In brief, culture medium was
removed at the indicated times, and cells were washed twice in
phosphate-buffered saline and a third time in the same buffer
containing 3-isobutyl-1-methylxanthine. Cells were then lysed by the
addition of ice-cold trichloroacetic acid (5%).21 The
trichloroacetic acid–soluble supernatant was removed from the well,
extracted 3 times with 10 mL ether, dried (SpeedVac), and resuspended
in 0.4 mL/sample of sodium acetate buffer (pH 6.2). The enzyme
immunoassay was then performed.
Effect of Neutralizing Anti-HGF Antibody
The effect of endogenously produced HGF was examined
by a neutralization procedure, using rabbit anti-human HGF
antibody.22 For the antibody, the IgG fraction (purified
with protein A-agarose) was able to neutralize a biological activity of
10 ng/mL HGF, at a concentration of 10 µg/mL. Normal rabbit serum IgG
fraction (10 µg/mL) was used as a control.
ELISA of TGF-ß
Conditioned medium was collected from VSMC, maintained in DSF
24, 48, and 72 hours later, centrifuged at 600g for
10 minutes, and stored at -20°C. ELISA for immunoreactive
TGF-ß1 in the supernatant was performed using
ELISA kit (Amersham). The antibody against
TGF-ß1 cross-reacts with rat active
TGF-ß1 but not with rat latent
TGF-ß1, TGF-ß2, or
TGF-ß3.
Effect of Neutralizing Anti-TGF-ß Antibody
Augmentation of decreased local HGF production in human
endothelial cells and VSMC was characterized as TGF-ß
specific by a neutralization procedure, using rabbit anti-human TGF-ß
(R&D Research).22 The IgG fraction (purified with protein
A-agarose) was able to neutralize a biological activity of 10 ng/mL
TGF-ß, at a concentration of 10 µg/mL23 . Normal rabbit
serum IgG fraction (10 µg/mL) was used as a control.
Experiment 2
Rabbit Ischemic Hind Limb Model
The physiological response to administration
of rHGF was investigated in the rabbit ischemic hind
limb model, described in previous reports.24 Male New
Zealand White rabbits (3.5 to 4.0 kg) (Kitayama Rabes, Nagano, Japan)
were anesthetized with a mixture of xylazine (5 mg/kg) and
ketamine (50 mg/kg). A longitudinal incision was then
performed, extending inferiorly from the inguinal ligament
to a point just proximal to the patella. Through this incision, using
surgical loupes, the operator dissected free the left femoral artery
along its entire length; all branches of the femoral artery, including
the inferior epigastric, deep femoral, lateral circumflex,
and superficial epigastric arteries, were also dissected free. After
dissection of the popliteal and saphenous arteries distally, the
external iliac artery and all of the mentioned arteries were ligated
with 4-0 silk (Ethicon). Finally, the left femoral artery was
completely excised for the ischemia limb model, from its
proximal origin as a branch of the external iliac artery to the point
distally where it bifurcates to form the saphenous and popliteal
arteries. Excision of the femoral artery results in retrograde
propagation of thrombus and occlusion of the external iliac artery.
Consequently, blood flow to the ischemic limb is dependent on
collateral vessels developing from the internal iliac artery. Using
this ischemia model, we administered a single injection of 500
µg rhHGF locally (intra-arterially into the
ischemic limb) on day 10 after the operation. Animals received
the first intra-arterial bolus of rhHGF (500 µg/body) or
vehicle (3 mL saline with 0.1% rabbit serum albumin; Sigma)
administered as a bolus over 1 minute through a 3F end-hole infusion
catheter (Terumo) positioned in the internal iliac artery of the
ischemic limb.
The angiographic luminal diameter of the internal iliac artery in the ischemic limb at baseline and after drug infusion was determined on days 0, 10, and 30 by previously described techniques.24 Briefly, morphometric analysis of collateral vessel development in the ischemic limb was performed from 4-second angiograms recorded after injection of contrast medium into the internal iliac artery. A grid overlay composed of 2.5-mm (diameter) circles arranged in rows spaced 5 mm apart was placed over the angiogram in the region of the medial thigh. The number of contrast-opacified arteries crossing over circles and the total number of circles encompassing the medial thigh area were counted in a blinded fashion. The angiographic score was calculated as the ratio of overlying opacified arteries divided by the total number of circles in the ischemic thigh. This angiographic score reflects vascular density in the medial thigh.
Materials
Human recombinant HGF was purified from the culture medium of
Chinese hamster ovary cells or C-127 cells transfected with expression
plasmid containing human HGF cDNA.25 bFGF and VEGF were
obtained from Pepro Tec EC Ltd.
Statistical Analysis
All values are expressed as mean±SEM. ANOVA with subsequent
Bonferroni’s test was used to determine the significance of
differences in multiple comparisons. P<0.05 were considered
statistically significant.
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Results |
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Effect of HGF on Hypoxia-Induced Endothelial Cell Death
Initially, we tested the effect of hypoxic treatment on endothelial cell growth. After 12 hours after hypoxia, some cells started to become round and eventually detached from the plate and floated in the medium, leaving many holes in the sheet of confluent cells (data not shown). The floating cells could be recovered with the medium and neither attached onto a new plate nor proliferated. Consistent with this morphological observation, cell death rate after hypoxia was significantly increased in a time-dependent and hypoxia-dependent manner, as shown in Figure 1
. Therefore, we examined the
effects of HGF, VEGF, and bFGF, because these growth factors have been
reported to act as survival factors against endothelial
cell death. As shown in Figure 2, addition of
HGF (10 ng/mL) resulted in partial attenuation of cell death mediated
by hypoxia in a dose-dependent manner. Similarly, addition of
bFGF or VEGF attenuated endothelial cell death induced
by hypoxia in a dose-dependent manner. There was no significant
difference in the attenuation of cell death among HGF, bFGF, and VEGF.
These results suggest that endothelial cell death
induced by hypoxia may be mediated by these growth factors.
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Differential Regulation of HGF and VEGF in Vascular Cells in
Response to Hypoxia
We focused on HGF and VEGF as candidates, because they are
endothelium-specific growth factors.5 6 In
particular, VEGF has been reported to be upregulated by
hypoxia.10 11 12 Consistent with previous
reports,10 11 12 VEGF concentration in the medium of
endothelial cells was significantly higher under
hypoxic treatment than that under normoxic treatment after 24 and 72
hours of treatment (P<0.01), as shown in Figure 3. Our previous studies demonstrated that
local HGF production by endothelial cells and
VSMC regulates growth of endothelial cells in an
autocrine-paracrine manner.8 Consistent with
our previous finding,8 secretion of HGF in human
endothelial cells was also readily detected by ELISA
using specific human anti-HGF antibody. Surprisingly, HGF concentration
in the conditioned medium of endothelial cells was 4-
to 5-fold higher than VEGF concentration (Figure 3). In
addition, our previous studies demonstrated that addition of
neutralizing anti-HGF antibody to human aortic
endothelial cells resulted in a significant decrease in
number of endothelial cells,22 suggesting
that HGF secreted from endothelial cells maintain the
endothelial cell growth in the autocrine manner.
Therefore, we studied the effect of hypoxic treatment on
endogenously produced HGF from vascular cells. Importantly,
hypoxic treatment of human aortic endothelial cells
resulted in a significant decrease in local HGF production
according to the severity of hypoxia (P<0.01), as
shown in Figure 3. From these results, we hypothesized that
endothelial cell death induced by hypoxia might
be mediated by decreased vascular HGF production. This
hypothesis is supported by the results of transfection of human HGF
gene. As shown in Figure 4, transfection of
human HGF gene into endothelial cells significantly
attenuated hypoxia-induced endothelial cell
death (P<0.01), accompanied by increased immunoreactive HGF
(control vector, 85±12 pg/106 cells per 24
hours; HGF vector, 203±14 pg/106 cells per 24
hours, P<0.01).
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Because VSMC also secrete HGF in addition to
endothelial cells, we also examined the effect of
hypoxia on HGF production in human aortic VSMC. As
shown in Figure 5, hypoxic treatment also
resulted in a significant decrease in local HGF
production in VSMC after 24, 48, and 72 hours of hypoxic
treatment, compared with normoxic control assessed by enzyme
immunoassay (P<0.01), whereas VEGF was significantly
increased by severe hypoxic treatment (P<0.01). Increased
severity of hypoxic condition further inhibited local HGF
production (Figure 5). Consistent with the
decreased local HGF production assessed by enzyme immunoassay,
HGF mRNA was also decreased by hypoxic treatment in VSMC
(P<0.01; Figure 6).
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Finally, we examined the effect of hypoxia on local HGF
production under more physiological
conditions: in an organ culture system. Rat carotid arteries were
exposed to normoxic and hypoxic conditions in culture medium. Similar
to cultured vascular cells, mild as well as severe hypoxic treatment
significantly decreased vascular HGF concentration according to the
severity of hypoxia (P<0.01, Figure 7). There was no significant difference in
protein contents among normoxia- and hypoxia-treated vessels
during the experimental period (48 hours; data not shown).
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Molecular Mechanisms of Downregulation of Vascular HGF in Response
to Hypoxia
Finally, we studied the molecular mechanisms of downregulation of
local HGF production in vascular cells in response to
hypoxia. Although regulation of HGF is not yet fully
understood, one of the upregulators of local HGF production is
cAMP,26 27 and TGF-ß negatively regulates local HGF
production.28 29 Thus, cAMP level and TGF-ß
concentration were measured under normoxic and hypoxic conditions.
Importantly, hypoxic treatment significantly decreased cAMP content in
VSMC and endothelial cells in a time-dependent manner,
as shown in Figure 8. During the time period
of the experiments, cAMP levels in normoxic condition were not
significantly changed in either VSMC or endothelial
cells. Moreover, the decrease in local HGF production by
hypoxic treatment was significantly attenuated by treatment with a cAMP
analogue, 8-bromo cAMP, in VSMC (Figure 9).
Of importance, addition of the cAMP analogue significantly attenuated
endothelial cell death induced by hypoxia,
whereas addition of neutralizing anti-HGF antibody attenuated the
prevention of endothelial cell death by the cAMP
analogue (% of normoxia control: severe hypoxia, 54.3±2.8%;
addition of 30 µmol/L forskolin to hypoxia,
69.9±1.2% [P<0.01 versus normoxia control]; addition of
1 mmol/L 8-bromo-cAMP to hypoxia, 73.4±3.2%
[P<0.01 versus severe hypoxia]; addition of
1 mmol/L 8-bromo-cAMP with anti-HGF antibody to hypoxia,
59.5±2.2% [P<0.01 versus 8-bromo-cAMP]). We also
measured active TGF-ß concentration under hypoxic condition. As shown
in Figure 10, hypoxic treatment increased
active TGF-ß concentration after 72 hours, but not after 24 and 48
hours, of hypoxia in VSMC. Finally, to elucidate the role of
TGF-ß activation, we examined the effects of neutralizing
anti-TGF-ß antibody (10 µg/mL). Unexpectedly, addition of
anti-TGF-ß antibody did not affect a significant decrease in local
HGF production during 48- and 96-hour incubation (48 hours,
normoxia: 0.972±0.031; hypoxia: 0.396±0.030;
hypoxia+IgG: 0.394±0.015; hypoxia+anti-TGF antibody:
0.393±0.019 ng · 24 h-1 ·
10-6 cells; 96 hours, normoxia: 1.213±0.040;
hypoxia: 0.475±0.045; hypoxia+IgG: 0.490±0.020;
hypoxia+anti-TGF antibody: 0.451±0.020 ng · 24
h-1 · 10-6 cells.
P<0.01 versus normoxia for all).
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Angiogenesis Induced by Intra-Arterially Injected
rhHGF
Given the significant decrease in endogenous HGF
production in response to hypoxia, we hypothesized that
administration of rhHGF into the ischemic limb might result in
a beneficial effect in hypoxia. Therefore, rhHGF was
intra-arterially administered via the internal iliac artery
of rabbits in whom the femoral artery had been excised to induce
unilateral hind limb ischemia. There was no significant
difference in body weight between the rabbits treated with rhHGF and
vehicle on day 40 after surgery (data not shown). A single
administration of rhHGF into the ischemic limb on day 10 after
surgery produced significant augmentation of collateral vessel
development as assessed by angiography 30 days later in the critical
limb ischemia model (vehicle: 120.1±4.0%; versus rhHGF:
180±5.8%, P<0.01), as shown in Figure 11. Serial angiograms revealed progressive
linear extension of the collateral arteries of the origin stem artery
to the distal point of the parent vessel reconstitution in HGF-treated
animals (Figure 11A). Of importance, a single administration of
rHGF also reduced severe necrosis due to ischemia in rabbit
muscle, whereas vehicle-treated rabbits demonstrated severe necrosis of
muscle and the nail (Figure 11B, Table 1).
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Discussion |
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In this study, we focused on 2 principle candidates as potential mediators of endothelial cell death by hypoxia: VEGF and HGF. Both VEGF and HGF are secreted from vascular cells and act on endothelial cells.7 8 Because addition of anti-HGF antibody attenuated endothelial cell growth, local HGF production in vascular cells may have a pathophysiological role in endothelial growth in an autocrine-paracrine manner. Our study demonstrated the differential regulation of HGF and VEGF in response to hypoxia: VEGF was upregulated and HGF was downregulated by hypoxic treatment. Some stimuli capable of inducing the development of neovessels in vivo, specifically certain cytokines30 and hypoxia, fail to stimulate endothelial cell proliferation in vitro, suggesting a role for additional mediators and/or cell types. Decreased local HGF may abolish the mitogenic activity of the increased vascular VEGF expression. In the human heart, the degree of collateral growth is highly variable among individuals; in many, the functional capacity of collaterals is not sufficient to counterbalance myocardial functional derangement caused by coronary obstruction. Importantly, our study demonstrated that a single intra-arterial administration of rHGF was sufficient to induce angiogenesis in the rabbit hind limb ischemia model. Because development of collateral vessels in the ischemic porcine heart may require several weeks, neovascularization therapy using recombinant and/or gene transfer of angiogenic growth factors might be considered.
What are the molecular mechanisms of the downregulation of HGF in response to hypoxia? Upregulation of VEGF by hypoxia has been reported to be due to the presence of the homology sequence in the VEGF promoter, identified as a binding site for a hypoxia-specific transcription factor (HIF-1).12 In contrast, our previous study documented a marked reduction of local HGF production by TGF-ß in vascular cells,22 whereas accumulation of cAMP induced by 8-bromo cAMP stimulated local HGF production.27 The present study revealed a significant decrease in cAMP concentration at an early time point (from 8 hours) and an increase in active TGF-ß concentration at a later time point (72 hours). Unexpectedly, anti-TGF-ß antibody failed to alter a significant decrease in local HGF production by hypoxia, suggesting that decreased cAMP rather than TGF-ß activation may be responsible for downregulation of HGF by hypoxia. Our hypothesis is supported by the observation that addition of cAMP attenuated endothelial cell death induced by hypoxia, accompanied by increased HGF. Increased local HGF production by cAMP may explain the previous findings that a cAMP analogue preserved vascular function.31 32 However, in diseased vessels (eg, restenotic lesions), activation or upregulation of TGF-ß might play a more important role in the regulation of local vascular HGF system. Additional studies will establish whether the differential regulation of VEGF and HGF expression observed in this study in response to hypoxia might involve an HIF-1-controlled mechanism(s).
Overall, this study demonstrated that hypoxic treatment of vascular cells significantly downregulated endogenous HGF production through decreased cAMP, suggesting their potential roles in the pathophysiology of ischemic diseases.
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Acknowledgments |
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We wish to thank Chihiro Noguchi and Shiori Takase for excellent technical assistance. Dr Ryuichi Morishita is the recipient of a Harry Goldblatt Award from the Council of High Blood Pressure, the American Heart Association. This work was partially supported by grants from the Japan Heart Foundation-Pfizer Pharmaceuticals Grant for Research on Coronary Artery Disease, the Uehara Memorial Foundation, the Hoan-sya Foundation, a Japan Heart Foundation Research Grant, Grant-in-Aid from The Tokyo Biochemical Research Foundation, and Grant-in-Aid for Scientific Research (A) and (B) from The Ministry of Education, Science, Sports and Culture.
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References |
|---|
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
1. D’Amore PA, Thompson RW. Mechanisms of angiogenesis. Annu Rev Physiol. 1987;49:453–464.[Medline] [Order article via Infotrieve]
2.
Bussolino F, Di Renzo MF, Ziche M, Bocchietto E,
Olivero M, Naldini L, Gaudino G, Tamagnone L, Coffer A, Comoglio PM.
Hepatocyte growth factor is a potent angiogenic factor
which stimulates endothelial cell motility and growth.
J Cell Biol. 1992;119:629–641.
3. Silvagno F, Follenzi A, Arese M, Prat M, Giraudo E, Gaudino G, Camussi G, Comoglio PM, Bussolino F. In vivo activation of met tyrosine kinase by heterodimeric hepatocyte growth factor molecule promotes angiogenesis. Arterioscler Thromb Vasc Biol. 1995;5:1857–1865.
4.
Matsumoto K, Nakamura T. Emerging multipotent aspects
of hepatocyte growth factor. J Biochem. 1996;119:591–600.
5. Nakamura Y, Morishita R, Higaki J, Kida I, Aoki M, Moriguchi A, Yamada K, Hayashi S, Yo Y, Nakano H, Matsumoto K, Nakamura T, Ogihara T. Hepatocyte growth factor is a novel member of the endothelium-specific growth factors: additive stimulatory effect of hepatocyte growth factor with basic fibroblast growth factor but not with vascular endothelial growth factor. J Hypertens. 1996;14:1067–1072.[Medline] [Order article via Infotrieve]
6.
Nakamura Y, Morishita R, Nakamura S, Aoki M, Moriguchi
A, Matsumoto K, Nakamura T, Higaki J, Ogihara T. A vascular modulator,
hepatocyte growth factor, is associated with
systolic pressure. Hypertension. 1996;28:409–413.
7. Ferrara N, Winer J, Burton T. Aortic smooth muscle cells express and secrete vascular endothelial growth factor. Growth Factors. 1991;5:141–148.[Medline] [Order article via Infotrieve]
8. Nakamura Y, Morishita R, Higaki J, Kida I, Aoki M, Moriguchi A, Yamada K, Hayashi S, Yo Y, Matsumoto K, Nakamura T, Ogihara T. Expression of local hepatocyte growth factor system in vascular tissues. Biochem Biophys Res Commun. 1995;215:483–488.[Medline] [Order article via Infotrieve]
9.
Hudlicka O, Brown M, Egginton S. Angiogenesis in
skeletal and cardiac muscle. Physiol Rev. 1992;72:369–417.
10. Shweiki D, Itin A, Soffer D, Keshet E. Vascular endothelial growth factor induced by hypoxia may mediate hypoxia-initiated angiogenesis. Nature. 1992;359:843–845.[Medline] [Order article via Infotrieve]
11. Minchenko A, Bauer T, Salceda S, Caro J. Hypoxic stimulation of vascular endothelial growth factor expression in vitro and in vivo. Lab Invest. 1994;71:374–379.[Medline] [Order article via Infotrieve]
12. Forsythe JA, Jiang BH, Iyer NV, Agani F, Leung SW, Koos RD, Semenza GL. Activation of vascular endothelial growth factor gene transcription by hypoxia-inducible factor 1. Mol Cell Biol. 1996;16:4604–4613.[Abstract]
13. Bonin PD, Leadley RJ, Erickson LA. Growth factor-induced modulation of endothelin-1 binding to human smooth-muscle cells. J Cardiovasc Pharmacol. 1993;22:S125–127.
14. Shweiki D, Itin A, Soffer D, Keshet E. Vascular endothelial growth factor induced by hypoxia may mediate hypoxia-initiated angiogenesis. Nature. 1992;359:843–845.
15. Libby P, O’Brien KV. Culture of quiescent arterial smooth muscle cells in a defined serum-free medium. J Cell Physiol. 1983;115:217–223.[Medline] [Order article via Infotrieve]
16. Ishiyama M, Tominaga H, Shiga M, Sasamoto K, Ohkura Y, Ueno K. A combined assay of cell viability and in vitro cytotoxicity with a highly water-soluble tetrazolium salt, neutral red and crystal violet. Biol Pharm Bull. 1996;19:1518–1520.[Medline] [Order article via Infotrieve]
17. Hayashi S, Morishita R, Higaki J, Aoki M, Moriguchi A, Kida I, Sawa Y, Matsumoto K, Nakamura T, Kaneda Y, Ogihara T. Autocrine-paracrine effects of overexpression of hepatocyte growth factor gene on growth of endothelial cells. Biochem Biophys Res Commun. 1996;220:539–545.[Medline] [Order article via Infotrieve]
18.
Kaneda Y, Iwai K, Uchida T. Increased expression of DNA
co-introduced with nuclear protein in adult rat liver.
Science. 1989;243:375–378.
19.
Morishita R, Gibbons GH, Kaneda Y, Ogihara T, Dzau VJ.
Novel in vitro gene transfer method for study of local modulators in
vascular smooth muscle cells. Hypertension. 1993;21:894–899.
20. Yamada A, Matsumoto K, Iwanari H, Sekiguchi K, Kawata S, Matsuzawa Y, Nakamura T. Rapid and sensitive enzyme-linked immunosorbent assay for measurement of HGF in rat and human tissues. Biomed Res. 1995;16:105–114.
21.
Ogawa S, Koga S, Kuwabara K, Brett J, Morrow B, Morris
SA, Bilezikian JP, Silverstein SC, Stern D. Hypoxia-induced
increased permeability of endothelial monolayers occurs
through lowering of cellular cAMP levels. Am J Physiol. 1992;262:C546–554.
22.
Nakano N, Morishita R, Moriguchi A, Nakamura Y, Hayashi
S, Aoki M, Kida I, Matsumoto K, Nakamura T, Higaki J, Ogihara T.
Negative regulation of local hepatocyte growth factor (HGF)
expression by angiotensin II and transforming growth
factor-ß in blood vessels: potential role of HGF in
cardiovascular disease. Hypertension. 1998;32:444–451.
23. Gibbons GH, Pratt RE, Dzau VJ. Vascular smooth muscle cell hypertrophy vs. hyperplasia: autocrine transforming growth factor beta 1 expression determines growth response to angiotensin II. J Clin Invest. 1992;90:456–461.
24. Takeshita S, Zheng LP, Brogi E, Kearney M, Pu LQ, Bunting S, Ferrara N, Symes JF, Isner JM. Therapeutic angiogenesis. A single intra-arterial bolus of vascular endothelial growth factor augments revascularization in a rabbit ischemic hind limb model. J Clin Invest. 1994;93:662–670.
25. Nakamura T, Nishizawa T, Hagiya M, Seki T, Shimonishi M, Sugimura A, Tashiro K, Shimizu S. Molecular cloning and expression of human hepatocyte growth factor. Nature. 1989;342:440–443.[Medline] [Order article via Infotrieve]
26.
Matsumoto K, Okazaki H, Nakamura T. Novel function of
prostaglandins as inducers of gene expression of HGF and
putative mediators of tissue regeneration. J Biochem
Tokyo. 1995;117:458–464.
27. Morishita R, Higaki J, Hayashi S, Yo Y, Aoki M, Nakamura S, Moriguchi A, Matsushita H, Matsumoto K, Nakamura T, Ogihara T. Role of hepatocyte growth factor in endothelial regulation: prevention of high D-glucose-induced endothelial cell death by prostaglandins and phosphodiesterase type 3 inhibitor. Diabetologia. 1997;40:1053–1061.[Medline] [Order article via Infotrieve]
28.
Matsumoto K, Tajima H, Okazaki H, Nakamura T. Negative
regulation of hepatocyte growth factor gene expression in
human lung fibroblasts and leukemic cells by transforming growth
factor-beta 1 and glucocorticoids. J Biol Chem. 1992;267:24917–24920.
29. Yo Y, Morishita R, Yamamoto K, Tomita N, Kida I, Hayashi S, Moriguchi A, Kato S, Matsumoto K, Nakamura T, Higaki J, Ogihara T. Actions of hepatocyte growth factor as a local modulator in the kidney: potential role in pathogenesis of renal disease. Kidney Int. 1997;53:50–58.
30. Klagsbrun M, D’Amore PA. Regulators of angiogenesis. Annu Rev Physiol. 1991;53:217–39.[Medline] [Order article via Infotrieve]
31. Beavo JA, Conti M, Heaslip RJ. Multiple cyclic nucleotide phosphodiesterases. Mol Pharmacol. 1994;46:39.
32. Fujitani K, Kambayashi J, Murata K, Yano Y, Shiozaki K, Yukawa M, Sakon M, Murata T, Kawasaki T, Shiba E, Mori T. Clinical evaluation of combined administration of oral prostacyclin analogue beraprost and phosphodiesterase inhibitor cilostazol. Pharmacol Res. 1995;31:121–125.[Medline] [Order article via Infotrieve]
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