INTRODUCTION

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ENDOTHELIAL INJURY plays an important role in the pathogenesis of coronary atherosclerosis. Recent studies have demonstrated that hepatocyte growth factor (HGF) is a potent angiogenic factor that stimulates endothelial cell growth and motility (3, 25). HGF is a heparin-binding glycoprotein composed of a large -subunit and a small -subunit and was originally isolated from the sera of partially hepatectomized rats (23, 24). HGF is synthesized in various cells, including the endothelium, and exerts its mitogenic and motogenic effects on epithelial and endothelial cells by stimulating the tyrosine kinase activity of its specific receptor, c-met (3, 15, 27, 36). The HGF receptor is a heterodimeric protein consisting of an - and a -subunit and is encoded by the c-met protooncogene (2, 8, 27). Because HGF has been shown to act as an endothelial cell regeneration factor without affecting vascular smooth muscle cell growth (3, 25), HGF might have a protective effect in the complex process of atherogenesis. In the present study, we investigated the influence of native and oxidatively modified low-density lipoproteins (LDL) on HGF synthesis and c-met mRNA expression in cultured human coronary artery endothelial cells (HCAEC).

The important pathophysiological role of LDL in the development of atherosclerotic lesions has been confirmed by several studies. Hypercholesterolemia has been shown to induce endothelial injury and endothelial dysfunction (19, 29), and chronic endothelial injury is associated with an enhanced deposition of LDL in endothelial cells and the subendothelial space. LDL, when trapped in the vessel wall, can undergo progressive oxidation involving several enzyme systems and various cell types, including endothelial cells, macrophages, and smooth muscle cells (30). Oxidized LDL is no longer a ligand for the native LDL (nLDL) receptor but can be bound and internalized by means of scavenger receptors (7, 16). Deposition of LDL (oxLDL) in the vessel wall and its oxidative modification seem to initiate, or at least accelerate, the atherosclerotic process by several mechanisms, including a direct injuring effect on endothelium, increased adherence and migration of monocytes and lymphocytes into the subendothelial space, promotion of foam cell formation, and mitogenic effects on macrophages and smooth muscle cells (4, 28, 29, 37). Endothelin-1 (ET-1) seems to be one of the growth factors mediating the enhanced smooth muscle cell proliferation in atherosclerotic lesions. ET-1 is a potent vasoconstrictive peptide that acts as a mitogen and comitogen on vascular smooth muscle cells (10, 34, 35). Previous studies have demonstrated an upregulation of ET-1 expression in coronary plaque tissue (38) and a stimulatory effect of LDL on ET-1 synthesis in endothelial cells (5, 11, 33). To elucidate the potential pathophysiological role of HGF in atherogenesis, we also investigated the effect of HGF on the basal and LDL-modulated ET-1 release in HCAEC.

	METHODS

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Isolation and modification of LDL. LDL were isolated from blood samples of healthy volunteers by sequential ultracentrifugation with density adjustment by potassium bromide. For preparation of nLDL, plasma samples (4 µmol EDTA/ml blood) were supplemented with butylated hydroxytoluene (BHT; 20 µM) to prevent oxidation. LDL, isolated from serum samples of the same donors, were oxidized (oxLDL) by exposure to 5 µM CuSO₄ and O₂ for 4 or 24 h at 37°C, respectively (4-h oxLDL, 24-h oxLDL). After oxidation, LDL were dialyzed against phosphate-buffered saline in the presence of EDTA (200 µM) and BHT (20 µM) to remove CuSO₄ and prevent further oxidation. Lipopolysaccharide content of isolated LDL, assessed by the Limulus amebocyte lysate assay (BioWhittaker, Verviers, Belgium), was <0.05 EU/ml. Lipoprotein concentrations are expressed in terms of their protein content as determined by a modified Lowry protein assay (Bio-Rad DC Protein Assay, Bio-Rad, Munich, Germany). The degree of oxidation was quantified by four different methods: 1) absorption at a wavelength of 234 nm, indicating conjugated diene formation of fatty acids; 2) fluorescence, measured at 430 nm with excitation at 360 nm, attributed to the derivatization of apoB-100 lysine residues by reactive aldehydes (6); 3) relative mobility on agarose gel, indicating an enhanced negative charge of oxidatively modified LDL; and 4) capillary isotachophoresis (39). Oxidative modification for 4 h resulted in a 1.6 ± 0.3-fold increase of fluorescence emission at 430 nm (means ± SE, n = 5) without a relevant change of absorption at 234 nm and only a minor change in electrophoretic mobility, whereas oxidative modification for 24 h resulted in a 16.6 ± 2.2-fold increase of fluorescence emission at 430 nm, a 3.1 ± 0.3-fold increase of absorption at 234 nm, and an increased electrophoretic mobility, as demonstrated by agarose gel electrophoresis and capillary isotachophoresis (Fig. 1). LDL preparations were stored at 4°C and used within 1-2 days. To evaluate further LDL oxidation by the cells, LDL preparations were incubated for 24 h at 37°C either with or without HCAEC. A slight increase of fluorescence emission [1.14-, 1.09-, and 1.16-fold for nLDL, 4-h oxLDL, and 24-h oxLDL (100 µg/ml), respectively; means, n = 2] and a slight increase of absorption at 234 nm [1.12-, 1.15-, and 1.13-fold for nLDL, 4-h oxLDL, and 24-h oxLDL (100 µg/ml), respectively] were observed after incubation with HCAEC, whereas no increase was observed after incubation without cells. Electrophoretic mobility, detected by capillary isotachophoresis, was not enhanced after incubation at 37°C.

View larger version (17K): [in this window] [in a new window]   Fig. 1.   Electrophoretic mobility of representative samples of native low-density lipoprotein (nLDL; A), 4-h oxidatively modified LDL (4-h oxLDL; B), and 24-h oxLDL (C) as investigated by capillary isotachophoresis. D: electrophoretic mobility of nLDL, 4-h oxLDL, and 24-h oxLDL (lanes 1-3, respectively) on agarose gel. LDL were isolated from human blood by sequential ultracentrifugation and oxidatively modified by exposure to CuSO4 for 4 h (4-h oxLDL) or 24 h (24-h oxLDL).

Culture of HCAEC. HCAEC (passage 3; Clonetics, San Diego, CA) were subcultured in EGM-2-MV medium (Clonetics) at 37°C and 5% CO₂. Cells (passage 6) were seeded into 6- or 24-well plates (Falcon, Franklin Lakes, NJ) that had been coated with collagen type I (Sigma, Deisenhofen, Germany). After 48 h, experiments were performed with confluent monolayers in culture medium containing 1% FCS (PAA Laboratories, Linz, Austria).

Measurement of HGF in cell culture supernatants. HCAEC, seeded in 6-well plates, were incubated for 48 h with nLDL or oxLDL at final concentrations of 0.1, 1, 10, 50, 100, and 200 µg/ml, respectively. Cell culture supernatants were concentrated with Centricon 30000 (1 h; Millipore, Eschborn, Germany), and HGF was measured by an enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems, Wiesbaden, Germany). Intra-assay coefficients of variation (n = 20) were 7.1% (425 pg/ml) and 4.4% (991 pg/ml), and interassay coefficients of variation (n = 40) were 7.2% (409 pg/ml) and 7.1% (1,058 pg/ml). Standards and samples were incubated for 2 h at room temperature in coated microtiter plates. After the plates were washed, conjugated antibody was added, followed by incubation for 1.75 h at room temperature and addition of substrate solution. After 30 min, stop solution was added, and optical density was read at 450 nm with correction at 570 nm. No immunoreactive HGF was detected in cell culture medium that had not been incubated with the cells. HGF concentrations in the cell culture supernatants were referred to the DNA content in the corresponding culture wells.

Detection of ET-1 release by HCAEC. Cells, seeded in 24-well plates, were incubated for 24 h with HGF (0.001-10 nM), lavendustin A (0.1-10 µM), or HGF (1 nM) combined with lavendustin A (0.1-10 µM). In another set of experiments, HCAEC were incubated for 24 h with nLDL, 4-h oxLDL, and 24-h oxLDL (0.1-200 µg/ml) alone or incubated with a combination of LDL (0.1-200 µg/ml) and HGF (1 nM). Cell culture supernatants were collected into EDTA-containing tubes, and ET-1 concentrations were measured with an ELISA kit (Biotrend, Köln, Germany; cross reactivity: ET-1, 100%; ET-2, 3.3%; ET-3, <0.1%; Big ET-1, <0.1%; Big ET-3, <0.1%). Intra-assay coefficients of variation (n = 24) were 2.9% (9.7 pg/ml) and 2.1% (56.2 pg/ml), and interassay coefficients of variation (n = 8) were 2.9% (10.0 pg/ml) and 3.3% (55.4 pg/ml). Standards and samples (100 µl) were incubated overnight in coated microtiter plates. After the plates were washed, labeled anti-ET-1 antibody was added and incubated for 30 min at 37°C. After another washing step, substrate solution was added, followed by an incubation period of 30 min at room temperature. Stop solution was added, and optical density was read at 450 nm after blanking was performed against the substrate blank. No immunoreactive ET-1 was detected in cell culture medium that had not been incubated with the cells. ET-1 concentrations in the cell culture supernatants were referred to the amount of DNA in the corresponding culture wells. Controls were performed with cell culture medium alone, with culture medium containing 5.3 mM dimethyl sulfoxide (Sigma), corresponding to the highest concentration of lavendustin A, and with culture medium supplemented with BHT-containing phosphate-buffered saline, corresponding to the highest nLDL concentration. Because ET-1 release did not differ significantly between controls with and without vehicle, results were referred to controls without vehicle.

Measurement of DNA. The DNA content of cells was measured as described previously (17) with the use of fluorescent DNA staining with bisbenzimide (Sigma) by using calf thymus DNA as a standard. Fluorescence (excitation 350 nm, emission 450 nm) was measured with a Victor 1420 Multilabel Counter (Wallac, Turku, Finland).

RNA isolation and RT-PCR of HGF, c-met, and ET-1 mRNA. For detection of HGF and c-met mRNA, HCAEC were incubated for 6, 12, and 24 h with nLDL, 4-h oxLDL (100 and 200 µg/ml), and 24-h oxLDL (10 and 100 µg/ml), respectively. To investigate the effect of HGF on ET-1 mRNA expression, cells were incubated for 3 and 5 h with HGF (1 nM). Total RNA was extracted with a High Pure RNA Isolation Kit that includes DNA digestion (Roche Diagnostics, Mannheim, Germany). The concentration and purity of RNA was determined by measuring the absorbance at 260 and 280 nm. For detection of HGF, c-met, ET-1, and -actin mRNA, 1 µg of total RNA was reverse transcribed into cDNA with 0.02 U/ml reverse transcriptase (SuperScript; GIBCO) at 42°C for 50 min. For RT-PCR, the following oligonucleotide primers (0.5 µM) were used: human HGF (24) from nucleotide +1405 to +1831 (a 427-bp fragment, accession no. M060718), sense primer 5'-ATGATGATGCTCATGGACCCT-3' and antisense primer 5'-CTGGCAAGCTTCATTAAAACC-3' (exon11-exon15); c-met (27) from nucleotide +1281 to +1955 (a 675-bp fragment, accession no. J02958), sense primer 5'-AATGGATCGATCTGCCATGT-3' and antisense primer 5'-TCCGAAATCCAAAGTCCCA-3'; preproET-1 (13) from nucleotide +415 to +662 (a 248-bp fragment, accession no. Y00749), sense primer 5'-TGCTCGTCCCTGATGGATA-3' and antisense primer 5'-TTCTCCATAATGTCTTCAGCC-3' (exon2-exon4); and -actin (21) from nucleotide +144 to +683 (a 540-bp fragment, accession no. M10277), sense primer 5'-GTGGGGCGCCCCAGGCCCA-3' and antisense primer 5'-CTCCTTAATGTCACGCACGATTTC-3' (exon2-exon4). DNA amplification was performed with 100 ng (HGF, c-met) or 60 ng (ET-1, -actin) of cDNA by using the LightCycler technology [Idaho Technology; HGF and c-met: LightCycler with FastStart DNA Master SYBR Green I (FastStart DNA Taq polymerase was activated by preincubation at 95°C for 10 min); ET-1 and -actin: LightCycler with DNA Master SYBR Green I (Roche Diagnostics)]. Reactions were cycled 34-40 times (denaturation: HGF and c-met, 95°C for 10 s, ET-1 and -actin, 95°C for 1 s; annealing: HGF and c-met, 56°C for 10 s, ET-1, 52°C for 5 s, -actin, 58°C for 7 s; and extension: HGF and c-met, 72°C for 25 s, ET-1, 72°C for 12 s, -actin, 72°C for 22 s; slopes were 20°C/s). Fluorescence was measured at the end of the extension phase. To confirm the specificity of the amplified products, melting curves were performed at the end of the amplification by cooling the sample at 20°C/s to 66°C (HGF and c-met), 62°C (ET-1), or 68°C (-actin) and then increasing the temperature to 95°C at 0.1°C/s, with fluorescence measurement every 0.1°C. For evaluation of the amplification efficiency, PCR products were purified with the QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany) and sequenced (Sequiserve, Vaterstetten, Germany). Concentrations were determined by measurement of absorbance at 260 nm, and samples were used as standard in a 10-fold serial dilution. PCR products were quantified using the LightCycler software. HGF, c-met, and ET-1 mRNA were referred to -actin mRNA in the corresponding samples. No detectable PCR products were present in water controls or in controls amplified without prior reverse transcription. For visualization, PCR products were applied to 1% agarose gel in 0.5× Tris-borate and stained with GelStar (Biozym, Hessisch Oldendorf, Germany).

Measurement of LDH release. Lactate dehydrogenase (LDH) release into the cell culture supernatants was determined with a coupled enzymatic assay, which results in the conversion of a tetrazolium salt into a red formazan product (CytoTox96 Non-Radioactive Cytotoxicity Assay; Promega, Madison, WI). LDH release was referred to the DNA content in the corresponding culture wells.

Statistical analysis. Results are expressed as means ± SE. Differences between the HGF or ET-1 release after stimulation with different concentrations of LDL, HGF, or lavendustin A were evaluated by one-way analysis of variance, followed by the Newman-Keuls test. Differences between ET-1 release after incubation with corresponding LDL concentrations with and without HGF were evaluated by the two-sample t-test.

	RESULTS

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Effect of LDL on HGF release by HCAEC. nLDL, 4-h oxLDL, and 24-h oxLDL, incubated for 48 h with cultured HCAEC, induced a significant concentration-dependent stimulation of HGF release (Fig. 2). Maximum stimulation of HGF release was achieved by incubation with 1 µg/ml 24-h oxLDL. With higher concentrations of 24-h oxLDL, a stepwise decrease of HGF release was observed, and after incubation with 200 µg/ml 24-h oxLDL, HGF release was even below control values. This phenomenon might be due to a toxic effect of increasing 24-h oxLDL concentrations. LDH release showed only minor changes after incubation with nLDL, 4-h ox LDL, and lower 24-h oxLDL concentrations but was significantly increased after incubation with high 24-h oxLDL concentrations [100 µg/ml: 274 ± 19%; 200 µg/ml: 539 ± 31%; n = 5; P < 0.01 and P < 0.001 vs. control (100 ± 6%), respectively].

View larger version (24K): [in this window] [in a new window]   Fig. 2.   Effect of nLDL, 4-h oxLDL, and 24-h oxLDL on hepatocyte growth factor (HGF) release by cultured human coronary artery endothelial cells (HCAEC). HCAEC were incubated for 48 h with nLDL, 4-h oxLDL, and 24-h oxLDL, and HGF concentrations were determined by ELISA. HGF release was referred to the DNA content in the corresponding culture wells. Results are presented as means ± SE (n = 5) and are expressed as relative HGF release compared with controls (culture medium containing 1% FCS). *P < 0.05; **P < 0.01; and ***P < 0.001 vs. control. Inset: HGF mRNA expression of HCAEC after incubation with nLDL (100 µg/ml), 4-h oxLDL (100 µg/ml), and 24-h oxLDL (10 µg/ml) for 12 h (lanes 2-4, respectively; lane 1: control without LDL). RT-PCR products were amplified and quantified with the LightCycler and were visualized by application to 1% agarose gel.

Stimulation of HGF and c-met mRNA expression by LDL. For investigation of HGF and c-met mRNA expression, HCAEC were incubated for 6, 12, and 24 h with LDL, and RT-PCR was performed using the LightCycler technology. The LDL-induced stimulation of HGF release was confirmed by an increase of HGF mRNA expression. Maximum stimulation of HGF mRNA expression was observed after 12 h with a 10.8-, 2.9-, 3.2-, 1.7-, 6.8-, and 1.2-fold increase after incubation with 100 and 200 µg/ml nLDL, 100 and 200 µg/ml 4-h oxLDL, and 10 and 100 µg/ml 24-h oxLDL, respectively (means, n = 2) (Fig. 2). mRNA expression of the HGF receptor c-met was also increased after incubation with nLDL and oxLDL (maximum stimulation after 12 h ) (Fig. 3).

View larger version (47K): [in this window] [in a new window]   Fig. 3.   Effect of nLDL, 4-h oxLDL, and 24-h oxLDL (µg/ml) on c-met mRNA expression in HCAEC after an incubation period of 12 h. mRNA expression was quantified by RT-PCR with the use of the LightCycler technology and is expressed as relative mRNA expression compared with control. Results are presented as means (n = 2). Inset: amplification products were visualized by application to 1% agarose gel. Lane 1: control without LDL; lanes 2-4: nLDL (100 µg/ml), 4-h oxLDL (100 µg/ml), and 24-h oxLDL (10 µg/ml), respectively.

Inhibitory action of HGF on ET-1 synthesis. Incubation of HCAEC with HGF induced a significant concentration-dependent reduction of ET-1 release (Fig. 4) and a marked decrease of ET-1 mRNA expression (71 and 59% of control after incubation with HGF 1 nM for 3 and 5 h, respectively; means, n = 2). To investigate whether the inhibitory effect of HGF on ET-1 synthesis is mediated by the tyrosine kinase activity of c-met, we incubated HCAEC with the tyrosine kinase inhibitor lavendustin A alone or in combination with HGF. Lavendustin A induced a slight increase of ET-1 release, possibly by antagonizing the effect of endogenously produced HGF. When HCAEC were incubated with lavendustin A and HGF (1 nM), the inhibitory action of HGF on ET-1 release was concentration-dependently antagonized by lavendustin A (Fig. 4).

View larger version (22K): [in this window] [in a new window]   Fig. 4.   A: effect of HGF on endothelin-1 (ET-1) release (n = 9) by cultured HCAEC. B: effect of the tyrosine kinase inhibitor lavendustin A on ET-1 release by HCAEC (n = 6). C: ET-1 release by HCAEC after incubation with HGF (1 nM) and lavendustin A (n = 6). After an incubation period of 24 h, ET-1 concentrations were measured with an ELISA, and ET-1 release was referred to the DNA content in the corresponding wells. Results are presented as means ± SE and are expressed as relative ET-1 release compared with controls (culture medium supplemented with 1% FCS). *P < 0.05 and ***P < 0.001 vs. control.

Antagonizing effect of HGF on the LDL-induced modulation of ET-1 release. nLDL, 4-h oxLDL, and lower concentrations of 24-h oxLDL exerted a stimulatory effect on ET-1 release, which was significantly antagonized by HGF (Fig. 5). Incubation with higher concentrations of 24-h oxLDL (50-200 µg/ml) induced a decrease of ET-1 release, probably due to a toxic effect of high 24-h oxLDL concentrations, as indicated by a significant increase of LDH release (100 µg/ml 24-h oxLDL: 267 ± 17%; 200 µg/ml 24-h oxLDL: 453 ± 24%; control 100 ± 3%; n = 6; P < 0.01 vs. control). Interestingly, the inhibitory effect of high 24-h oxLDL concentrations on ET-1 release was also antagonized by exogenously administered HGF. In addition, LDH release was not increased after incubation with high 24-h oxLDL concentrations in the presence of HGF.

View larger version (19K): [in this window] [in a new window]   Fig. 5.   Antagonizing effect of HGF (1 nM) on the LDL-induced modulation of ET-1 release: nLDL (A), 4-h oxLDL (B), and 24-h oxLDL (C). After an incubation period of 24 h, ET-1 concentrations were measured with an ELISA, and ET-1 release was referred to the DNA content in the corresponding wells. Results are presented as means ± SE (n = 6) and are expressed as relative ET-1 release compared with controls (culture medium containing 1% FCS: 100 ± 3%). *P < 0.05; **P < 0.01; and ***P < 0.001 vs. corresponding LDL concentration without HGF.

	DISCUSSION

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The pathogenesis of atherosclerosis is a complex process including endothelial cell injury, deposition of LDL in the vessel wall, and an enhanced proliferation of vascular smooth muscle cells. Until now, little was known about a possible protective role of HGF, a potent endothelial cell and epithelial cell regeneration factor (12, 20, 25), in the development of atherosclerotic lesions. In the present study, we demonstrated a significant concentration-dependent stimulatory effect of nLDL and oxLDL on HGF synthesis and HGF receptor (c-met) mRNA expression in HCAEC. In parallel with our findings, an upregulation of HGF and c-met expression in endothelial cells has been observed after myocardial ischemia (26). In addition, serum HGF levels have been considered as an index of endothelial dysfunction in hypertensive patients. Elevated serum HGF levels have been observed in hypertensive patients, especially in hypertensive patients with complications, and HGF concentrations correlated with systolic blood pressure (22, 25). Induction of acute renal failure also resulted in an upregulation of tissue HGF and c-met expression as well as in an increase of plasma HGF levels (9, 12, 18). These data suggest a compensatory upregulation of HGF and its receptor in endothelial cell and renal epithelial cell injury.

In the current study, HGF release was significantly stimulated by nLDL, mildly oxidized LDL, and extensively oxidized LDL. Extensively modified LDL (24-h oxLDL) exhibited the most prominent stimulatory effect on HGF release with a maximum stimulation of 423% at a 24-h oxLDL concentration of 1 µg/ml. However, the stimulatory effect decreased with higher 24-h oxLDL levels, and at the highest 24-h oxLDL concentration (200 µg/ml), HGF release was reduced to 56% of control. This phenomenon might be due to the toxic effect of high 24-h oxLDL concentrations, indicated by a marked increase in LDH release. However, these data also might suggest that, at high local concentrations of LDL, especially oxLDL, the initial compensatory rise of HGF synthesis might switch to a downregulation and thus might promote endothelial cell injury.

The facts that, in previous studies, gene transfection of HGF attenuated reperfusion injury in the heart (32) and that administration of HGF prevented tacrolimus- and cyclosporine A-induced renal injury (1, 31) suggest that HGF administered in addition to the endogenously upregulated HGF might exert additional beneficial effects.

In this study, administration of exogenous HGF concentration dependently reduced the basal ET-1 release and ET-1 mRNA expression in HCAEC. This effect seems to be mediated via the tyrosine kinase activity of the HGF receptor c-met. The tyrosine kinase inhibitor lavendustin A induced a slight increase of ET-1 release, possibly by antagonizing the action of endogenously produced HGF. When HCAEC were coincubated with lavendustin A and HGF, the inhibitory effect of HGF on ET-1 release was concentration-dependently antagonized by lavendustin A. In addition to the inhibitory action on basal ET-1 release, HGF potently antagonized the LDL-induced stimulation of ET-1 release. ET-1 is a potent vasoconstrictor peptide that acts as a mitogen and comitogen on vascular smooth muscle cells. Several studies have suggested a pathophysiological role of ET-1 in the development of atheroslerotic lesions. An upregulation of ET-1 expression has been observed in coronary and peripheral atherosclerotic lesions (14, 38), and nLDL and oxLDL have been shown to induce an increase of ET-1 synthesis in endothelial cells (5, 11, 33). The present findings of an inhibitory effect of HGF on the basal and LDL-stimulated ET-1 synthesis in coronary endothelial cells also provide support for a potential beneficial effect of HGF in the development of atherosclerotic lesions. At high 24-h oxLDL concentrations, a decrease of ET-1 synthesis was observed that seems to be due to a toxic effect of extensively modified LDL, indicated by an increased LDH release (a decrease of ET-1 synthesis in toxically damaged cells was also observed in previous studies). Interestingly, exogenously administered HGF not only antagonized the LDL-induced rise of ET-1 release but also antagonized the decrease of ET-1 release at high 24-h oxLDL concentrations. These results provide further evidence for the protective role of HGF in endothelial cell injury.

In summary, HGF synthesis in HCAEC was upregulated by LDL, and exogenously administered HGF concentration dependently reduced the basal ET-1 synthesis and antagonized the LDL-induced modulation of ET-1 release. These findings provide support for a protective role of HGF in coronary atherosclerosis because HGF promotes endothelial cell regeneration but does not affect smooth muscle cell growth, whereas ET-1 is a potent vasoconstrictor peptide that stimulates vascular smooth muscle cell proliferation. The present data might encourage further studies to elucidate the pathophysiological role of HGF in coronary atherosclerosis and to evaluate a potential beneficial effect of locally administered HGF in coronary artery disease.