(Hypertension. 1999;33:1379-1384.)
© 1999 American Heart Association, Inc.
Scientific Contributions |
Therapeutic Angiogenesis Induced by Human Recombinant Hepatocyte Growth Factor in Rabbit Hind Limb Ischemia Model as Cytokine Supplement Therapy
From the Department of Geriatric Medicine, Osaka University Medical School, Suita 565, Japan (R.M., S.N., S.-i.H., Y.T., A.M., J.H., T.O.); Sumitomo Pharmaceutical Co, Osaka, Japan (T. Nagano, M.T., H.N.); the Second Department of Internal Medicine, Teikyo University (S.T.); and the Division of Biochemistry, Department of Oncology, Biomedical Research Center, Osaka (Japan) University Medical School (K.M., T. Nakamura).
Correspondence to Ryuichi Morishita, MD, PhD, 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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Abstract—Hepatocyte growth factor (HGF) exclusively stimulates the growth of endothelial cells without replication of vascular smooth muscle cells and acts as a survival factor against endothelial cell death. Therefore we hypothesized that a decrease in local vascular HGF might be related to the pathogenesis of peripheral arterial disease. We initially evaluated vascular HGF concentration in the vessels of patients with arteriosclerosis obliterans. Consistent with in vitro findings that hypoxia downregulated vascular HGF production, vascular HGF concentration in the diseased segments of vessels from patients with arteriosclerosis obliterans was significantly decreased as compared with disease-free segments from the same patients (P<0.05), accompanied by a marked reduction in HGF mRNA. On the other hand, a novel therapeutic strategy for ischemic diseases that uses angiogenic growth factors to expedite and/or augment collateral artery development has recently been proposed. Thus in view of the decreased endogenous vascular HGF, rhHGF (500 µg/animal) was intra-arterially administered through the internal iliac artery of rabbits in which the femoral artery was excised to induce unilateral hind limb ischemia, to evaluate the angiogenic activity of HGF, which could potentially have a beneficial effect in hypoxia. Administration of rhHGF twice on days 10 and 12 after surgery produced significant augmentation of collateral vessel development on day 30 in the ischemic model as assessed by angiography (P<0.01). Serial angiograms revealed progressive linear extension of collateral arteries from the origin stem artery to the distal point of the reconstituted parent vessel in HGF-treated animals. In addition, we examined the feasibility of intravenous administration of rhHGF in a moderate ischemia model. Importantly, intravenous administration of rhHGF also resulted in a significant increase in angiographic score as compared with vehicle (P<0.01). Overall, a decrease in vascular HGF might be related to the pathogenesis of peripheral arterial disease. In the presence of decreased endogenous HGF, administration of rhHGF induced therapeutic angiogenesis in the rabbit ischemic hind limb model, as potential cytokine supplement therapy for peripheral arterial disease.
Key Words: arterial occlusive diseases • cell, endothelial • angiogenesis • cell hypoxia • hepatocyte growth factor
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Introduction |
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Critical limb ischemia is estimated to develop in 500 to 1000 individuals per million per year.1 In a large proportion of these patients, the anatomic extent and the distribution of arterial occlusive disease make the patients unsuitable for operative or percutaneous revascularization. Thus the disease frequently follows an inexorable downhill course.2 3 Of importance, there is no optimal medical therapy for critical limb ischemia, as the Consensus Document of the European Working Group on Critical Limb Ischemia concluded.1 Therefore novel therapeutics are required to treat these patients. In the pathophysiology of the disease, in the presence of obstruction of a major artery, blood flow to the ischemic tissue is often dependent on collateral vessels. When spontaneous development of collateral vessels is insufficient to allow normal perfusion of the tissue at risk, residual ischemia occurs. Preclinical studies have demonstrated that angiogenic growth factors can stimulate the development of collateral arteries in animal models of peripheral and myocardial ischemia,4 5 6 7 8 9 a concept called therapeutic angiogenesis. Most of the studies have used vascular endothelial growth factor (VEGF), also known as vascular permeability factor as well as a secreted endothelial-cell mitogen. The endothelial cell specificity of VEGF has been considered to be an important advantage for therapeutic angiogenesis, as endothelial cells represent the critical cellular element responsible for new vessel formation.10 11 More recently, the efficacy of therapeutic angiogenesis with VEGF gene transfer has been reported in human patients with critical limb ischemia.12 13 Thus the strategy for therapeutic angiogenesis with angiogenic growth factors should be considered for the treatment of patients with critical limb ischemia.
On the other hand, we and others have previously reported that hepatocyte growth factor (HGF) exclusively stimulated the growth of endothelial cells without replication of vascular smooth muscle cells (VSMC), thereby indicating it to be a potential angiogenic growth factor.14 15 16 17 Unexpectedly, the mitogenic activity of HGF is more potent than that of VEGF in human aortic endothelial cells.15 17 Moreover, HGF and its specific receptor, c-met, have been shown to be expressed in the heart and blood vessels including endothelial cells and VSMC.18 19 Therefore we reasoned that HGF may be a potential therapeutic angiogenic growth factor, in addition to VEGF. Indeed, activation of the HGF system promoted angiogenesis in a Matrigel system,20 but this system provides far from physiological conditions. Thus there is no direct in vivo evidence that HGF produces therapeutic angiogenesis in ischemic disease. In this study, we addressed 2 specific questions: (1) how endogenous HGF is regulated in the blood vessels of patients with ischemic limb disease and (2) 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
Subjects
For the study of serum and vascular HGF concentration, diseased segments of vessels from 7 patients with arteriosclerosis obliterans (ASO) (65.6±12.2 years old) and 6 disease-free segments from the same patients were obtained at the time of operation. Patients did not have the following disorders: cardiac valvular disease, congestive heart failure, arrhythmia, or hepatic, renal, or pulmonary dysfunction. The study protocol was approved by the Ethics Committee of Osaka University Hospital.
Measurement of HGF Concentration in Blood Vessels
Vascular HGF concentration was assayed with a recently developed
enzyme immunoassay for use in humans.18 19 21 On the day
of extraction, the tissue was thawed at 4°C, weighed, and
homogenized by polytron in assay solution. Each specimen
was centrifuged at 20 000g for 30 minutes at 4°C
to remove the lysates. The concentration of HGF in blood vessels was
determined by enzyme immunoassay with anti-human HGF antibody, as
described previously.21 The antibody against human
HGF reacts only with human HGF and not with rat HGF.21 For
the organ culture experiment, rat HGF concentration was measured with
the use of anti-rat HGF antibody.21
Reverse Transcription-Polymerase Chain Reaction
RNA was extracted from blood vessels by treatment with RNAzol
(Tel-Test Inc). Levels of HGF and G3PDH mRNA were measured by reverse
transcription–polymerase chain reaction (RT-PCR).18 19
The HGF 5' primer (nucleotides 1409 to 1426 of human
sequence) was 5'-ATG-CTC-ATG-GAC-CCT-GGT-3'; the 3' primer
(nucleotides 1797 to 1814 of human sequence) was
5'-GCC-TGG-CAA-GCT-TCA-TTA-3' (423 bp). Extreme care was taken to avoid
contamination of tissue samples with trace amounts of experimental RNA.
Aliquots of RNA (0.5 µg) derived from cultured cells were amplified
simultaneously by PCR (30 cycles), by individuals who were
blinded to the identity of the samples, and compared with a negative
control (primers without RNA). In preliminary experiments, the number
of amplification cycles for each gene was determined by performing
RT-PCR for 20, 25, 30, 35, and 40 cycles. PCR products were within
the linear logarithmic phase of the amplification curve until 40
cycles. To ensure that the RT-PCR amplified product reflects
transcribed HGF RNA without significant DNA contamination, RNA samples
treated with RNase A or amplified without RT were
simultaneously amplified as negative controls. These
samples did not result in a visual band. Moreover, PCR products
were cut by restriction enzymes, and the fragments were identical to
the theoretical bands.
Experiment 2
Rabbit Ischemic Hind Limb Model
The physiological response to administration
of rhHGF was investigated in the rabbit ischemic hind limb
model, described in previous reports.5 6 Male New Zealand
White rabbits (3.5 to 4.0 kg) (Kitayama Rabes) were
anesthetized with a mixture of xylazine (5 mg/kg) and
ketamine (50 mg/kg). A longitudinal incision was then made,
extending inferiorly from the inguinal ligament to a point
just proximal to the patella. Through this incision, with the use of
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 to create the ischemic 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.
Excision of the femoral artery 1 cm below the peritoneum created a
moderate ischemia model; excision 1 cm above the peritoneum
created a severe ischemia model. The operative procedure was
similar in these 2 models. However, only a difference in excision
points produced a marked difference in ischemic symptoms. The
calf blood pressure ratio in the moderate ischemia model was
determined 0, 10, and 30 days after operation (0 days: 0.46±0.05, 10
days: 0.61±0.08, 30 days: 0.69±0.06). In contrast, it was impossible
to measure calf blood pressure in the severe ischemia model
both at 0 and 10 days after operation (0 days: not detected, 10 days:
not detected). In addition, probably because of some blood flow
in the moderate ischemia model, we did not find any signs of
necrosis of the nails and muscle, whereas severe necrosis was found in
all severe ischemia animals. We therefore used the relatively
moderate ischemia model in this study. Consequently, blood flow
to the ischemic limb was dependent on collateral vessels
developing from the internal iliac artery.
Design 1
To ensure the maximum effect of rhHGF, we administered 2
separate injections of 500 µg rhHGF locally
(intra-arterially into the ischemic limb): the
first dose on day 10 and the second on day 12 after the operation. The
dose of rhHGF used in the present study was chosen on the basis of
previous experiments.4 5 Ten days after surgery (day 10)
and after measurement of baseline body weight as well as baseline
noninvasive and invasive measurements of hemodynamic
parameters, the animals received the first
intra-arterial bolus of rhHGF (500 µg/animal) or vehicle
(3 mL saline with 0.1% rabbit serum albumin; Sigma)
administered over 1 minute through a 3F end-hole infusion catheter
(Terumo) positioned in the internal iliac artery of the
ischemic limb. On day 12, the same dose of drug was
administered intra-arterially.
Design 2
In design 2, rhHGF (3 mg) was intravenously
administered for 5 days from day 10 to day 14 after the operation.
Animals received rhHGF (3 mg in 6 mL vehicle/animal) or vehicle (6 mL
saline with 0.1% rabbit serum albumin)
intravenously administered over 1 hour with an infusion
pump.
Quantitative Angiography
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.5 6 Briefly, morphometric analysis of
collateral vessel development in the ischemic limb was
performed in 4-second angiograms recorded after injection of
contrast medium into the internal iliac artery. A grid with 20-mm
spaces 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 lines 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
lines in the ischemic thigh.5 6 This angiographic
score reflects vascular density in the medial
thigh.5 6
Materials
Human recombinant HGF was purified from the culture medium of
Chinese hamster ovary cells or C-127 cells transfected with an
expression plasmid containing human HGF cDNA.22
Statistical Analysis
All values are expressed as mean±SEM. ANOVA with subsequent
Duncan's test was used to determine the significance of differences in
multiple comparisons. Differences with a value of P<0.05
were considered significant.
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Results |
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Decreased Endogenous HGF Production in Ischemic Limb in Humans
As shown in Figure 1, vascular HGF concentration in the diseased segments of blood vessels from patients was significantly decreased as compared with that in disease-free segments of blood vessels from the same patients as control vessels. The decrease in vascular HGF concentration was accompanied by a decrease in HGF mRNA in the diseased segments of blood vessels (Figure 1c). In contrast, serum HGF concentration in patients with peripheral arterial disease was significantly higher than that in healthy volunteers (patients, 0.43±0.03 ng/mL vs control 0.35±0.03 ng/mL, P<0.01), probably in response to damaged vasculature. Downregulation of the vascular HGF system may be related to the pathogenesis of peripheral arterial disease, since HGF showed an antiapoptotic action on endothelial cells, in addition to its mitogenic activity.23 24 25 26 The decrease in local HGF production in the vessels of patients with ASO might be due to transforming growth factor-ß or hypoxia, which are strong suppressors of vascular HGF in vitro as well as in vivo.19 Alternatively, HGF belongs to the family of kringle proteins that mediate protein/protein and protein/cell interactions, which suggests a potential role in the regulation of thrombosis. Therefore decrease in local HGF production may also be responsible for disturbance of local microcirculation.
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Angiogenesis Induced by Intra-Arterially Injected
rhHGF
Given the significant decrease in endogenous HGF
production in the ischemic limb, we hypothesized that
administration of rhHGF into the ischemic limb might result in
a beneficial effect in hypoxia. Therefore rhHGF was
intra-arterially administered through the internal iliac
artery of rabbits in which the femoral artery was 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 (vehicle, n=7: 3.63±0.13 kg, rhHGF; n=5: 3.86±0.07 kg). Administration of rhHGF into the ischemic limb on days 10 and 12 after surgery produced significant augmentation of collateral vessel development as assessed by angiography on day 30 in the moderate ischemia model, as shown in Figure 2 (P<0.01). Serial angiograms revealed progressive linear extension of collateral arteries from the origin stem artery to the distal point of the reconstituted parent vessel in HGF-treated animals (Figure 2). Moreover, we evaluated a single administration of rhHGF in the ischemia model and found that it also caused a significant increase in angiographic score as compared with vehicle-treated rabbits.
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Angiogenesis Induced by Intravenously Injected
rhHGF
Finally, we evaluated the efficacy of intravenous
administration of rhHGF in the rabbit ischemia model to examine
the clinical feasibility of rhHGF for the treatment of ASO patients.
Consistent with the angiogenic activity of
intra-arterially injected rhHGF, repeated
intravenous administration of rhHGF (3 mg/d/animal) for 5
days from day 0 to day 4 also resulted in a significant increase in the
angiographic score as compared with vehicle treatment
(P<0.01, Figure 3).
Consistent with other experiments, there was no significant
difference in body weight between the rabbits intravenously
injected with rhHGF and vehicle on day 40 after surgery (data not
shown).
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Discussion |
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In patients with critical limb ischemia, since there is no pharmacological treatment. Amputation, despite its associated morbidity, mortality rates, and functional implications,1 27 28 is often recommended as a solution to the disabling symptoms, in particular excruciating ischemic rest pain.29 30 31 Indeed, a second major amputation is required in nearly 10% of such patients. Consequently, the need for alternative treatment strategies in patients with critical limb ischemia is compelling. A novel therapeutic strategy that uses angiogenic growth factors to expedite and/or augment collateral artery development has recently entered the realm of treatment of ischemic diseases. Indeed, the clinical utility of gene therapy with VEGF, an endothelium-specific growth factor gene, has been recently reported for the treatment of critical limb ischemia.12 13 The present study raises the possibility of a new strategy, therapeutic angiogenesis with HGF instead of VEGF, for the treatment of patients with critical limb ischemia. HGF is a mesenchyme-derived pleiotropic factor that 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.32 33 On the basis of our recent finding that HGF exclusively stimulated the growth of endothelial cells without VSMC replication, we further studied the capability of HGF to stimulate angiogenesis.
Although an angiogenic response to HGF has been reported in an experimental model system of implanted reconstituted basement membrane (Matrigel),20 few reports have examined the angiogenic activity of HGF in physiological conditions in normal tissue without a supplement such as Matrigel, and controversial reports have described the inability of HGF to stimulate angiogenesis.34 Here we demonstrated direct in vivo evidence of angiogenesis induced by recombinant HGF, consistent with previous findings.14 20 Notably, a single intra-arterial administration of rhHGF was sufficient to induce angiogenesis in the rabbit hind limb ischemia model. As previously reported, HGF has been postulated to promote angiogenesis as a result of a combination of direct effects on endothelial cells and indirect effects, including paracrine upregulation of VEGF, on VSMC.35 Of importance, the promotion of angiogenesis by HGF prevented necrosis of muscle and nails in a critical limb ischemia model, accompanied by a significant increase in angiographic score (data not shown). These results demonstrate the utility of therapeutic angiogenesis induced by recombinant HGF. In this study we used the moderate ischemia model because this model appears to be more similar to human ischemic disease (see Methods).
Finally, we investigated the feasibility of intravenous administration of rhHGF in the ischemia model to examine the clinical utility of the therapeutic angiogenesis induced by HGF because there is no report of the effects of intravenous administration of rhHGF. Importantly, intravenous injection of rhHGF sufficiently increased angiogenesis, as shown in Figure 3. Although further studies are necessary, the present data suggest the clinical utility of intravenous administration of rhHGF for the treatment of ischemic disease. In contrast, endogenously expressed HGF was markedly decreased in the diseased segments of blood vessels from patients with critical ischemic limb as compared with disease-free segments of vessels, which suggests an insufficient level of HGF to salvage damaged vessels. Our in vitro experiments suggest that the decrease in vascular HGF production is caused by hypoxia (unpublished observation). Therefore a sufficient supply of recombinant HGF would be expected to enhance collateral formation in patients with critical limb ischemia (see Figure 4). What is the clinical relevance of therapeutic angiogenesis induced by HGF as compared with VEGF? First, HGF may not cause edema as a side-effect, as it does not increase permeability, different from VEGF. Second, c-met (the specific receptor for HGF) has been reported to be upregulated in response to hypoxia in a myocardial ischemia model,36 probably enhancing the angiogenic activity of HGF. Third, previous reports showed elevated levels of cell-associated matrix degrading enzymes (MMP-1, and so on) and enhanced plasmin-generating ability (uPA) by HGF.37 38 39 Interestingly, uPA activates pro-HGF in vitro, and activation of pro-HGF involves the formation of a stable complex between pro-HGF and uPA,38 which suggests that the biological effects of HGF can be titrated in vivo by the level of uPA activity. Increased amounts of uPA locally induced by HGF may condition the tissue microenvironment by rendering HGF bioavailable to its target cells. Although further studies are necessary to distinguish the mechanisms of HGF and VEGF, the present study at least demonstrated the potential utility of rHGF for therapeutic angiogenesis.
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Overall, the present studies suggest a novel therapeutic strategy that might reduce the symptoms of critical limb ischemia by use of the angiogenic properties of recombinant HGF. It is noteworthy that the supply of HGF into the ischemic limb supplements the downregulated endogenous vascular HGF expression. In addition, stimulation of new vessel formation by HGF is likely to create new therapeutic options in angiogenesis-dependent conditions such as wound healing, inflammatory diseases, ischemic heart disease, myocardial infarction, and peripheral arterial disease.
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Acknowledgments |
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We wish to thank Chihiro Noguchi and Shiori Takase for their excellent technical assistance. We also wish to thank Drs Jun-ichi Kanbayashi (America Otsuka Pharmaceutical Co) and Tomio Kawasaki (First Department of Surgery, Osaka University) for providing human vessels. Dr Ryuichi Morishita is the recipient of a Harry Goldblatt Award from the Council of High Blood Pressure, 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, the Japan Cardiovascular Research Foundation, and a Japan Heart Foundation Research Grant.
Received December 9, 1998; first decision January 5, 1999; accepted February 9, 1999.
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