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(Circulation. 2003;107:1411.)
© 2003 American Heart Association, Inc.

Basic Science Reports

Angiogenic Property of Hepatocyte Growth Factor Is Dependent on Upregulation of Essential Transcription Factor for Angiogenesis, ets-1

Naruya Tomita, MD, PhD; Ryuichi Morishita, MD, PhD; Yoshiaki Taniyama, MD; Hiromi Koike; Motokuni Aoki, MD, PhD; Hideo Shimizu, MD; Kunio Matsumoto, PhD; Toshikazu Nakamura, PhD; Yasufumi Kaneda, MD, PhD; Toshio Ogihara, MD, PhD

From the Department of Geriatric Medicine (N.T., R.M., Y.T., M.A., H.S., T.O.), Division of Gene Therapy Science (R.M., H.K., Y.K.), and Division of Biochemistry, Department of Oncology, Biomedical Research Center (K.M., T.N.), Osaka University Medical School, Japan.

Correspondence to Ryuichi Morishita, MD, PhD, Associate Professor, Division of Gene Therapy Science, Osaka University Medical School, 2-2 Yamada-oka, Suita 565-0871, Japan. E-mail morishit{at}gts.med.osaka-u.ac.jp

	Abstract

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Background— Although hepatocyte growth factor (HGF) is an angiogenic growth factor, it is still unclear how it exerts its angiogenic effects. Thus, we focused on the role of an essential transcription factor for angiogenesis, ets-1. In this study, we addressed the following specific questions: (1) what genes responsible for angiogenesis can be regulated by HGF and (2) whether upregulation of gene expression for angiogenesis is dependent on ets-1.

Methods and Results— In human endothelial cells, HGF significantly stimulated the matrix-degrading pathway, such as the production of matrix metalloprotease-1 (MMP-1) through its specific receptor, c-met. In addition, HGF also significantly increased HGF itself and its specific receptor, c-met. Moreover, HGF significantly increased the transcription activity and mRNA expression of ets-1 in a time-dependent manner. Importantly, transfection of antisense ets-1 oligodeoxynucleotides (ODN) resulted in a significant reduction in MMP-1, HGF and c-met. Interestingly, HGF also stimulated ets-1 mRNA in vascular smooth muscle cells, similar to endothelial cells. Of importance, transfection of antisense ets-1 ODN resulted in a significant decrease in vascular endothelial growth factor (VEGF) and HGF expression, whereas HGF stimulated both HGF and VEGF expression. Moreover, in vivo transfection of ets-1 antisense ODN resulted in an inhibition of angiogenesis induced by the HGF gene in a rat ischemic hindlimb model.

Conclusions— Here, we demonstrated that HGF stimulated the expression of MMP-1, VEGF, HGF itself, and c-met in human endothelial cells and vascular smooth muscle cells. Upregulation of angiogenesis-related genes was largely dependent on the induction of ets, especially ets-1. These data provide new information about the mechanisms of angiogenesis.

Key Words: angiogenesis • metalloproteinases • cells • growth substances

	Introduction

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Recently, the clinical usefulness of gene therapy using the vascular endothelial growth factor (VEGF) gene has been reported for the treatment of critical limb ischemia and myocardial infarction.^1–4 These studies raise the possibility of a new strategy, therapeutic angiogenesis using angiogenic growth factors such as VEGF, for the treatment of patients with ischemic disease. Nevertheless, it is still unclear how angiogenic growth factors can stimulate angiogenesis. To answer this question, we studied the molecular mechanisms by which hepatocyte growth factor (HGF) exerts its angiogenic properties.⁵ Recently, HGF has been reported as a mitogen exclusively for endothelial cells without the replication of vascular smooth muscle cells (VSMCs).⁶ Moreover, HGF and its specific receptor, c-met, have been expressed in the blood vessels, including endothelial cells and VSMCs.⁶ Indeed, HGF is a novel angiogenic growth factor in models of hindlimb ischemia and myocardial infarction.^7–11

To clarify the mechanisms of angiogenesis induced by HGF, we focused on the ets family, which is believed to play a pivotal role in angiogenesis. Members of the ets family play important roles in regulating gene expression in response to multiple developmental and mitogenic signals.^12,13 The ets family of transcription factors has a common DNA-binding domain that binds to a core GGA(A/T) DNA sequence.¹⁴ In situ hybridization studies have revealed that ets-1 is expressed in endothelial cells at the beginning of blood vessel formation under normal and pathological conditions.¹⁵ Thus, the ets family activated the transcription of genes encoding collagenase 1, stromelysin 1, and urokinase plasminogen activator, which are proteases involved in extracellular matrix degradation.^16–18 It is believed that the ets family takes part in regulating angiogenesis by controlling the transcription of these genes whose activity is necessary for the migration of endothelial cells from preexisting capillaries. In this study, we addressed 2 specific issues: (1) the molecular mechanisms involved in angiogenesis induced by HGF and (2) the role of ets-1 in angiogenesis induced by HGF.

	Methods

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Transfection of Antisense ets-1 ODN
Human aortic endothelial cells and VSMCs (passage 3) were obtained from Clonetics Corp and cultured in modified MCDB131 medium supplemented with 5% FCS, 10 ng/mL epidermal growth factor, 2 ng/mL basic fibroblast growth factor, and 1 mmol/L dexamethasone.⁵ VSMCs were maintained in Waymouth media with 5% fetal calf serum. Endothelial cells and VSMCs were seeded in 6-well plates and transfected with antisense ets-1 ODN using LipofectAMINE PLUS (Gibco-BRL).¹⁹ Briefly, 2 µg ODN was mixed with 10 µg liposomes for 30 minutes at room temperature. Then, liposome complex was added to the medium, which was maintained at 37°C for 4 hours. To demonstrate successful transfection, we used FITC-labeled antisense ODN. The 3' and 5' ends of the ODN were labeled with FITC by Nihon Seifun. Cells were harvested 2 and 24 hours after transfection and fixed with paraformaldehyde. Cells were examined by fluorescence microscopy. Fluorescence was readily distinguishable from the specific FITC-labeled ODN.²⁰ The sequences of antisense and sense ets-1 ODN were as follows: antisense, 5'-AGATCGACGGCCGCCTTCAT-3'; sense, 5'-ATGAAGGCG-GCCGTCGATCT-3'.¹³

Measurement of MMP-1, HGF, and VEGF in Conditioned Medium
Endothelial cells and VSMCs were seeded at a density of 5x10⁴ cells/cm² and cultured for 24 hours. After the medium was replaced with fresh defined serum free (DSF) medium and after culture for 24 hours, the concentrations of matrix metalloprotease-1 (MMP-1) and HGF in the medium were determined by enzyme immunoassays (EIAs) (MMP-1 Biotrack, Amersham; HGF, Tokushu-Meneki Co Ltd). The concentration of HGF in the medium was determined by EIA using anti-human HGF antibody.^5,6 This EIA specifically detects only human HGF, because of lack of cross-reactivity of antibodies.^5,6 To study the effects of HGF on endogenous HGF production, we used rat recombinant HGF (rHGF) to stimulate endogenous human HGF. The concentration of VEGF in the medium was determined by EIAs (VEGF; R&D systems). DSF medium did not contain immunoreactive HGF or VEGF assessed by EIA.

Western Blot for Analysis of c-met Protein
Endothelial cells and VSMCs were grown to confluence and made quiescent by incubation in DSF medium. After 24 hours of rHGF treatment, the cells were fixed with 10% trichloroacetic acid in saline. Samples containing 100 µg protein were incubated with a monoclonal antibody to c-met (1:500; Pharmingen) at 4°C overnight. Amounts of loaded proteins were confirmed to be equal by staining with Coomassie brilliant blue R (Sigma). Staining with Coomassie brilliant blue revealed identical amounts of protein in all Western blotting samples. Western blotting of tubulin using anti-tubulin antibody (anti-human mouse IgG, 1:100; Oncogene) was also performed to confirm equal amounts of loaded proteins.

Northern Blot Analysis
RNA was extracted with RNAzol (Tel-Test Inc) from cells 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). The filter was baked, prehybridized, and hybridized. Full-length cDNA for HGF, c-met, or ets-1 labeled with a random-primer kit (Amersham) was used for Northern blotting.

Gel Mobility Shift Assay
Cells were harvested at 1 day after HGF stimulation, and nuclear extracts were prepared.¹¹ ODNs containing the ets binding site (5'-GTGCCGGGGTAGGAAGTGGGCTGGG-3'; sense strand) and the mutated ets binding site were labeled as a primer at the 3' end with a 3' endo-labeling kit (Clontech Inc).¹¹ The binding mixtures (10 µL), including ³²P-labeled primers (0.5 to 1 ng, 10 000 to 15 000 cpm) and 1 µg polydeoxyinosinic-deoxycytidic acid (Sigma Co), were incubated with 10 µg nuclear extract for 30 minutes at room temperature and then loaded onto 5% polyacrylamide gel. As a control, samples were incubated with an excess (50x) of nonlabeled ets-1 ODN, which completely abolished binding. Gels were analyzed by autoradiography.

In Vivo Gene Transfer by Intramuscular Injection
A rat ischemic hindlimb model was created.^7,8 Consequently, blood flow to the ischemic limb was dependent on collateral vessels developing from the internal iliac artery. Naked human HGF vector and control vector (500 µg per body) were carefully injected directly into the ischemic limb with a 27-gauge needle (Terumo) at 10 days after surgery. Four separate injections of plasmid vectors (intramuscular into the ischemic limb near both the proximal and distal arterial stump) with or without sense or antisense ets-1 ODN (10 µmol/L) were performed. The injection volume of plasmid DNA was 100 µL. All protocols were approved by the Osaka University Institutional Animal Care and Use Committee.

Measurement of Blood Flow and Capillary Density
Measurement of blood flow with a laser Doppler imager has been performed by means of a laser Doppler blood flowmeter (Laser Doppler Imager, Moor Instruments), because laser Doppler flow velocity correlates well with capillary density.^9,11 Consecutive measurements were obtained over the same regions of interest (leg and foot). Low or no perfusion is displayed as dark blue, whereas the highest perfusion interval is displayed as white. The stored perfusion values behind the color-coded pixels remain available for data analysis. These laser images were quantitatively converted into histograms that represented the amount of blood flow on the x axis and the number of pixels on the y axis in the traced area. The average blood flow in each histogram was calculated for evaluation. Alkaline phosphatase staining was used as a specific marker of endothelial cells in paraffin-embedded sections.^9–11 Three individual sections from the middle of the transfected muscle were analyzed. The number of vessels was counted under a light microscope (magnification, x100) in a blinded manner.

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 probability value of P<0.05 were considered significant.

	Results

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Upregulation of ets-1 in Endothelial Cells
To investigate the molecular mechanisms of the angiogenic actions of HGF, we initially focused on the transcription factor ets-1. Expression of ets-1 mRNA was readily detected in human aortic endothelial cells without any stimulation (Figure 1A). Interestingly, rHGF significantly increased ets-1 mRNA expression from 1 hour, as assessed by Northern blotting (P<0.01) (Figure 1A). This increase in ets-1 mRNA by rHGF continued for at least 6 hours after treatment. Consistent with the significant increase in mRNA level, ets-1 activity was also markedly increased in endothelial cells treated with rHGF compared with vehicle, as assessed by gel mobility shift assay at 24 hours after stimulation (Figure 1B). To elucidate the role of ets-1 in the angiogenesis induced by rHGF, we used an antisense strategy. For transfection into human aortic endothelial cells, we used cationic liposomes. As shown in Figure 2, our present study using FITC-labeled ODN demonstrated the feasibility of transfecting antisense ODN into endothelial cells, because marked fluorescence could be detected in cells transfected with FITC-labeled ODN but not in untransfected cells. The average transfection rate was 80% to 90% in human endothelial cells. The fluorescence was localized primarily in cell nuclei, and some could be detected in the cytoplasm. Consistent with a previous report,¹³ we confirmed the specificity of antisense ets-1 ODN. Transfection of antisense ets-1 ODN attenuated the increase in ets-1 mRNA induced by rHGF at 24 hours, whereas sense ODN or liposomes alone did not alter ets-1 mRNA expression (P<0.01) (Figure 3). Using this specific antisense ets-1 ODN, we studied further the role of ets-1 on HGF-mediated angiogenesis-related proteins.

View larger version (31K): [in this window] [in a new window]   Figure 1. A, Top, Typical example of ets-1 mRNA in human endothelial cells treated with rHGF. Bottom, Stimulatory effect of rHGF on ets-1 mRNA in human endothelial cells. 0 hour indicates Control, pretreatment; lane 2 and 1 hour, 1 hour after stimulation with rHGF (100 ng/mL); lane 3 and 3 hours, 3 hours after stimulation with rHGF (100 ng/mL); lane 4 and 6 hours, 6 hours after stimulation with rHGF (100 ng/mL). n=6 to 8 per group calculated from independent experiments. *P<0.01 vs control. B, Gel mobility shift assay for ets binding site. Lane 1, P32-labeled ODN containing ets probe without any nuclear extract; lane 2, nuclear extracts from cells treated with rHGF (100 ng/mL) incubated with P32-labeled ets probe without any competitor; lane 3, nuclear extracts from unstimulated cells incubated with P32-labeled ets probe; lane 4, nuclear extracts from cells treated with rHGF (100 ng/mL) incubated with P32-labeled ets probe with an excess amount of non–P32-labeled ets probe (competitor) (x50). These experiments were repeated at least 3 times.

View larger version (38K): [in this window] [in a new window]   Figure 2. Representative in vitro findings of fluorescence microscopy of endothelial cells transfected with FITC-labeled antisense ets-1 ODN using cationic liposomes. Untransfected indicates untransfected cells; 2 hours, 2 hours after transfection; and 24 hours, 24 hours after transfection. This experiment was performed 3 times.

View larger version (34K): [in this window] [in a new window]   Figure 3. Top, Typical example of ets-1 mRNA in human endothelial cells transfected with antisense or sense ets-1 ODN at 6 hours after transfection. Bottom, Effect of antisense ets-1 ODN on ets-1 mRNA in human endothelial cells. n=5 to 8 per group calculated from 5 to 8 independent experiments. Lane 1, UN, cells treated with vehicle; lane 2, UN+HGF, cells treated with rHGF (100 ng/mL) without ODN; lane 3, sense, cells treated with rHGF (100 ng/mL) with sense ODN; and lane 4, antisense, cells treated with rHGF (100 ng/mL) with antisense ODN.

As shown in Figure 4A, human rHGF (100 ng/mL) significantly increased the production of MMP-1 (P<0.01) and MMP-1 activity. In contrast, no detectable amount of MMP-3 and MMP-9 was observed in the conditioned medium of human endothelial cells. Of importance, transfection of antisense ets-1 ODN resulted in a significant decrease in MMP-1 production induced by rHGF (P<0.01) (Figure 4A), whereas sense ets-1 ODN or liposomes alone did not affect MMP-1 production. These findings are consistent with previous reports that the MMP-1 gene has ets binding sites in its promoter region.¹⁶ Because the promoter regions of HGF and c-met both contain ets binding sites,^21,22 we studied further the role of ets-1 in upregulation of HGF and c-met. As expected, transfection of antisense ets-1 ODN resulted in significant attenuation of endogenous human HGF expression induced by exogenously added rat rHGF (P<0.01) (Figure 4B). Similarly, the increase in c-met protein by human rHGF was also attenuated by antisense ets-1 ODN compared with vehicle and sense ODN (P<0.01) (Figure 5). The inhibition of HGF-induced c-met expression by antisense ets-1 ODN was also confirmed at the mRNA level, as assessed by Northern blotting (data not shown). These data clearly revealed that HGF activated angiogenesis through the autoinduction system of HGF and c-met mediated by ets-1.

View larger version (44K): [in this window] [in a new window]   Figure 4. Effects of antisense ets-1 ODN on (A) MMP-1 protein and (B) endogenous HGF protein in human endothelial cells at 24 hours after transfection. n=5 to 8 per group calculated from 5 to 8 independent experiments. Control indicates cells treated with vehicle; Vehicle, cells treated with rHGF (100 ng/mL) without ODN; Sense, cells treated with rHGF (100 ng/mL) with sense ODN; and Antisense, cells treated with rHGF (100 ng/mL) with antisense ODN.

View larger version (36K): [in this window] [in a new window]   Figure 5. Top, Typical example of c-met protein in human endothelial cells transfected with antisense or sense ets-1 ODN at 48 hours after transfection as assessed by Western blotting. Bottom, Effect of antisense ets-1 ODN on c-met protein in human endothelial cells at 24 hours after transfection. n=5 to 8 per group calculated from 5 independent experiments. Lane 1, Control, cells treated with vehicle; lane 2, Vehicle, cells treated with rHGF (100 ng/mL) without ODN; lane 3, Sense, cells treated with rHGF (100 ng/mL) with sense ODN; and lane 4, Antisense, cells treated with rHGF (100 ng/mL) with antisense ODN.

Upregulation of ets-1 in VSMCs
We have previously reported that HGF stimulated endothelial cell growth exclusively without replication of VSMC growth.⁵ However, we were aware that the expression of the specific receptor of HGF, c-met, could be detected in VSMCs.⁶ Indeed, the presence of c-met was clearly demonstrated by Western blotting, although the amount of c-met protein in human VSMCs was obviously lower than that in endothelial cells (VSMCs, 100%; endothelial cells, 492±42%; P<0.01). Moreover, it has been reported that HGF induced migration of VSMCs.²³ Therefore, we elucidated the role of c-met in VSMCs. As in endothelial cells, rHGF significantly stimulated the production of MMP-1 (vehicle, 31.5±2.5 ng/mL; HGF, 47.2±2.2 ng/mL; P<0.01). Thus, HGF may stimulate the expression of angiogenesis-related genes in VSMCs. Interestingly, the expression of ets-1 mRNA was significantly increased by rHGF in a time-dependent manner, as assessed by Northern blotting (P<0.01) (Figure 6A), whereas transfection of antisense ets-1 ODN inhibited the upregulation of ets-1 mRNA induced by rHGF (data not shown). Accordingly, transfection of antisense ets-1 ODN also resulted in significant inhibition of endogenous human HGF production induced by rat HGF (P<0.01) (Figure 6B). Finally, we examined the effects of HGF on other angiogenic growth factors such as VEGF, because a previous report documented that HGF stimulated VEGF expression.⁸ In this study, we confirmed the previous observation that addition of rHGF resulted in a significant, but weak, increase in VEGF protein in human VSMCs (P<0.01) (Figure 6C). Interestingly, upregulation of VEGF by rHGF was also diminished by treatment with antisense ets-1 ODN (P<0.01) (Figure 6C). In contrast, rHGF did not affect basic fibroblast growth factor expression (data not shown). These results demonstrated that HGF upregulated the degradation pathway of extracellular matrix, such as production of MMP-1, via the induction of HGF and VEGF through the upregulation of ets-1, without affecting the growth of VSMCs.

View larger version (22K): [in this window] [in a new window]   Figure 6. A, Top, Typical example of ets-1 mRNA in human VSMCs treated with rHGF as assessed by Northern blotting. Bottom, Stimulatory effect of rHGF on ets-1 mRNA in human endothelial cells as assessed by Northern blotting. Lane 1 and 0 hour, pretreatment; lane 2 and 1 hour, 1 hour after stimulation with rHGF (100 ng/mL); lane 3 and 3 hours, 3 hours after stimulation with rHGF (100 ng/mL). n=6 to 8 per group calculated from independent experiments. *P<0.01 vs 0 hour. B and C, Effects of antisense ets-1 ODN on endogenous HGF (B) and VEGF (C) protein in human VSMCs at 24 hours after transfection. n=5 to 8 per group calculated from 5 to 8 independent experiments. Control indicates cells treated with vehicle; Vehicle, cells treated with rHGF (100 ng/mL) without ODN; Sense, cells treated with rHGF (100 ng/mL) with sense ODN; and Antisense, cells treated with rHGF (100 ng/mL) with antisense ODN.

Role of ets-1 in Angiogenesis Induced by HGF In Vivo
To clarify the role of ets-1 in angiogenesis induced by HGF, we also used a rat hindlimb ischemia model in vivo. Injection of human HGF vector into the ischemic hindlimb resulted in a significant increase in blood flow at 4 weeks after transfection, as assessed by laser Doppler imager, compared with ischemic hindlimb transfected with control vector (P<0.01), as shown in Figure 7. Moreover, transfection of human HGF vector significantly increased the number of capillary arteries in the ischemic hindlimb of rat around the injection site compared with control vector (P<0.01) (Figure 8A). Importantly, cotransfection of antisense ets-1 ODN resulted in a significant decrease in blood flow induced by the HGF gene compared with sense ets-1 ODN transfection (P<0.01). Capillary density was also significantly decreased in rats transfected with antisense ets-1 ODN compared with sense ODN (P<0.01) (Figure 8). There was no significant difference in the blood flow or the capillary density between rats transfected with sense ODN and untransfected rats.

View larger version (50K): [in this window] [in a new window]   Figure 7. A, Typical image of blood flow analyzed by laser Doppler imager at 4 weeks after transfection with ets-1 antisense or sense ODN in ischemic hindlimb. Arrows indicate operated legs. Low or no perfusion is displayed as dark blue, whereas lightest perfusion is displayed as white. B, Quantitative analysis of blood flow in percent change in ratio of blood flow in ischemic (operated) limbs to nonischemic limbs. Control indicates rats transfected with control vector; HGF, rats transfected with HGF vector and vehicle; HGF+sense, rats transfected with human HGF vector and sense ODN; and HGF+antisense, rats transfected with HGF vector and antisense ets-1 ODN. Each group contains 7 or 8 animals.

View larger version (66K): [in this window] [in a new window]   Figure 8. A, Effect of transfection of HGF vector with antisense or sense ets-1 ODN on vascular formation at 4 weeks after transfection. Representative cross sections (x200) are shown. B, Effect of transfection of human HGF vector on number of vessels. Control indicates rats transfected with control vector; HGF, rats transfected with HGF vector and vehicle; HGF+sense, rats transfected with human HGF vector and sense ODN; and HGF+antisense, rats transfected with HGF vector and antisense ets-1 ODN. Each group contains 7 or 8 animals.

	Discussion

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Members of the ets family play important roles in regulating angiogenesis for the migration of endothelial cells from preexisting capillaries. In this study, we demonstrated that HGF upregulated ets-1 mRNA and activity in human endothelial cells. With an antisense strategy, inhibition of ets-1 attenuated the increase in MMP-1 induced by HGF. These results were consistent with the previous report that endothelin-1 and phorbol 12-myristate 13-acetate induced MMP-1 through ets-1 in endothelial cells.²⁴ Interestingly, similar results were observed in another cell type, the human hepatic stellate cell line.²⁵ In addition, we also demonstrated upregulation of endogenous HGF and c-met expression by exogenously added HGF, consistent with our previous in vivo findings.²⁶ Interestingly, induction of ets activity by HGF regulated this autoloop upregulation of the HGF system. This phenomenon is consistent with the previous report that the promoter region of the HGF gene contains putative regulatory elements, such as a B-cell– and macrophage-specific transcription factor binding site (PU.1/ETS).²¹ The upregulation of c-met has been reported in the acute phase of a myocardial infarction model.²⁷ Considering the clinical usefulness of therapeutic angiogenesis by HGF, it is important to continue stimulation of the angiogenesis pathway by autoinduction of the HGF system after a single stimulation. Because our previous study clearly demonstrated induction of ets binding activity in infarcted myocardium by HGF gene transfer in vivo, an increase in ets may play a pivotal role in the regulation of angiogenesis by HGF in physiological situations.

One of the distinguishing features between HGF and VEGF is the presence of the HGF-specific receptor c-met in VSMCs, because VEGF and HGF are well-known angiogenic growth factors. Although VEGF has no effect on VSMCs because of the lack of its receptors, HGF is reported to stimulate the migration of VSMCs without replication.⁵ These different actions of HGF and VEGF in VSMCs may document their different properties in the maturation of blood vessels. Indeed, HGF does not induce edema in transfected sites, whereas VEGF is well known to induce edema. The initial event in angiogenesis induced by VEGF is the migration of endothelial cells, leading to the sprouting of blood vessels. Later, migration of VSMCs occurs because of the release of platelet-derived growth factor, followed by the migration of endothelial cells. However, a delay in the maturation of blood vessels might exist in the case of angiogenesis induced by VEGF. In contrast, HGF simultaneously stimulates the migration of both endothelial cells and VSMCs. Thus, the blood vessels may mature at an earlier time point, thereby avoiding the release of blood-derived cells into the extracellular space, although further studies are needed. The functionality of c-met in VSMCs was confirmed by the present study, which shows that HGF stimulated ets-1 mRNA and MMP-1 production.

Importantly, HGF also stimulated the production of VEGF in VSMCs, consistent with a previous report.⁸ Thus, it is believed that HGF may exert a potent combination of direct and indirect effects, including direct effects on endothelial cells and indirect effects mediated via an increase in the production of VEGF.⁸ However, no report has documented how HGF induced VEGF expression. Here, using an antisense strategy, we clearly demonstrated that the induction of VEGF by HGF was also mediated by ets-1. This finding is supported by the observation that the promoter region of VEGF contains ets binding sites.²⁸ Furthermore, HGF is known to increase the expression of the VEGF receptor (VEGFR) flk-1 in human endothelial cells.²⁹ Interestingly, both the VEGFRs Flt-1/VEGFR-1 and KDR/VEGFR-2 carry a putative ets-responsive element in their promoters.^30,31 It has also been proposed that ets-1 stimulates its own expression in vitro.^32,33 These results extend our findings, suggesting that the ability of HGF to induce angiogenesis by direct effects on the proliferation and migration of endothelial cells may be potentiated by its ability to induce angiogenesis indirectly by upregulating 1 or more cytokines, such as VEGF, through ets-1. Although the angiogenesis phenomenon is multicomplex, including migration, proliferation, and tubular morphogenesis of endothelial cells, in this study we did not perform experiments regarding proliferation and migration so as to avoid duplication of previous works. Transfection of transdominant mutant ets-1 cDNA into endothelial cells resulted in a decrease of bromodeoxyuridine incorporation and DNA synthesis, a lesser migration of endothelial cells, and reduction in tube formation in vitro.³⁴ In addition, transfection of the mutant ets-1 gene also resulted in a significant decrease in cell number and DNA synthesis in endothelial cells stimulated with basic fibroblast growth factor in vitro. The length of tube-like formation stimulated by basic fibroblast growth factor was also smaller in cells transfected with the mutant ets-1 gene.³⁵ Most importantly, the present study proved that the in vivo angiogenesis induced by HGF was inhibited by antisense, but not sense, ets-1 ODN in the ischemic hindlimb model. The present studies revealed that HGF is located upstream of the angiogenesis cascade and acts through induction of the coordinated trans-activating genes necessary for the processes for angiogenesis modulated by ets-1. The widespread activation of these genes by HGF is critical in treating ischemic disease.

	Acknowledgments

 
This work was supported in part by a Grant-in-Aid from the Ministry of Public Health and Welfare, a Grant-in-Aid for the Development of Innovative Technology, a Grant-in-Aid from Japan Promotion of Science, and Special Coordination Funds of the Ministry of Education, Culture, Sports, Science, and Technology, the Japanese Government.

	Footnotes

 
Dr Morishita works as a board member of and is a shareholder in AnGes MG, which has interests in the development of the HGF gene therapy drug.

Received October 28, 2002; accepted December 5, 2002.

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