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(Hypertension. 1996;28:409-413.)
© 1996 American Heart Association, Inc.

Articles

A Vascular Modulator, Hepatocyte Growth Factor, Is Associated With Systolic Pressure

Yoshio Nakamura; Ryuichi Morishita; Shigefumi Nakamura; Motokuni Aoki; Atsushi Moriguchi; Kunio Matsumoto; Toshikazu Nakamura; Jitsuo Higaki; Toshio Ogihara

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

Correspondence to Toshio Ogihara, MD, PhD, Department of Geriatric Medicine, Osaka University Medical School, 2-2 Yamada-oka, Suita 565, Japan.

	Abstract

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Endothelial cells are known to secrete various antiproliferative and vasodilating factors, such as nitric oxide and natriuretic peptides. The presence of endothelial dysfunction, well known in hypertensive individuals, potentially results in the development and progression of atherosclerosis. Therefore, it is important to know the factors that might influence endothelial cell growth. We examined the mitogenic actions of hepatocyte growth factor (HGF) on human endothelial and vascular smooth muscle cells. Exogenously added human recombinant HGF stimulated endothelial but not vascular smooth muscle cell growth in a dose-dependent manner. We also compared the mitogenic action of HGF with that of basic fibroblast growth factor and vascular endothelial growth factor. Interestingly, the mitogenic action of HGF on endothelial cells was greater than the actions of basic fibroblast growth factor and vascular endothelial growth factor, whereas basic fibroblast growth factor but not HGF and vascular endothelial growth factor stimulated vascular smooth muscle cell growth. Given the characteristics of HGF as an endothelium-specific growth factor, we evaluated the relationship of circulating HGF and blood pressure in normotensive and hypertensive subjects. Serum HGF concentration has been reported to be elevated in response to organ damage, such as in hepatitis and nephritis, and recent findings show that HGF may play an important role in tissue regeneration. We hypothesized that HGF might contribute to the protection or repair of vascular endothelial cells. If so, serum HGF level might be elevated in response to endothelial cell damage induced by hypertension. To test this hypothesis, we measured serum levels of HGF, lipoprotein(a), plasminogen activator inhibitor-1, tissue plasminogen activator, total cholesterol, and blood pressure in 41 normotensive and hypertensive subjects without liver, kidney, or lung damage. Serum HGF concentration was significantly correlated with systolic pressure (P<.01, r=.43) but not diastolic pressure. Serum HGF concentration in hypertensive subjects was significantly higher than in normotensive subjects. None of the other factors showed any correlation with blood pressure. We have demonstrated that HGF is an endothelium-specific growth factor whose serum concentration is significantly associated with systolic pressure. These results suggest that HGF secretion might be elevated in response to high blood pressure as a counterregulatory system against endothelial dysfunction.

Key Words: atherosclerosis • growth substances • vascular remodeling • growth factors, endothelial

	Introduction

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Endothelial cells are known to secrete various vasoactive substances. Recently, it has been hypothesized that endothelial cells may also modulate vascular growth because many antiproliferative factors such as nitric oxide and vascular natriuretic peptides are secreted from endothelial cells.^{1 2 3} Therefore, endothelial cell dysfunction might promote abnormal vascular growth, such as in atherosclerosis. In hypertensive individuals, endothelial cell dysfunction is well known.^{4 5 6 7} The loss of substances from endothelial cells might be related to the development and progression of atherosclerosis/arteriosclerosis in hypertensive patients.^{8 9} On the other hand, VSMC growth is controlled by a balance of growth inhibitors and growth promoters, and in the normal adult vessel, this balance results in a very low smooth muscle growth rate. However, after endothelial dysfunction, this balance is shifted such that smooth muscle cell proliferation occurs.^{8 9 10} Given the importance of endothelial cells, we have hypothesized that rapid regeneration of endothelial cells not accompanied by VSMC growth may have a therapeutic potential in preventing abnormal vascular growth, such as neointimal formation after angioplasty. Therefore, we sought an endothelium-specific growth factor. Our results demonstrated the presence of a local HGF system (HGF and its receptor, c-met) in endothelial cells and VSMCs in vitro as well as in vivo.¹¹ Moreover, its regulation is controlled by the balance of TGF-ß and HGF itself, since TGF-ß is a strong suppressor and HGF a positive regulator of local HGF production (unpublished observations, 1995, and References 12 through 15). In contrast, serum HGF concentration has been reported to be elevated in response to organ damage, such as in hepatitis and nephritis.^{16 17 18 19} Recent findings show that HGF may play an important role in tissue regeneration.^{20 21 22 23} In this study, we hypothesized that HGF might contribute to the protection or repair of vascular endothelial cells. If so, serum HGF level might be elevated in response to hypertension-induced endothelial cell damage. To test this hypothesis, we evaluated the potential role of serum HGF in normotensive and hypertensive subjects without liver, kidney, or lung damage.

	Methods

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Cell Culture
Human aortic endothelial cells (passage 5) and human aortic VSMCs (passage 5) were obtained from Clonetech Corp and cultured in modified MCDB131 medium supplemented with 5% fetal calf serum, 100 U/mL penicillin, 100 mg/mL streptomycin, 10 ng/mL epidermal growth factor, 2 ng/mL bFGF, and 1 µmol/L dexamethasone in the standard fashion. 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 the characteristics of endothelial cells and VSMCs. All cells were used within passages 5 to 6.

Determination of DNA Synthesis
Endothelial cells and VSMCs were seeded onto uncoated 24-well tissue culture plates (Corning). At confluence, endothelial cells were rendered quiescent by incubation for 48 hours in Dulbecco's modified Eagle's medium (DMEM) with 0.5% fetal bovine serum; VSMCs were rendered quiescent by incubation for 48 hours in defined serum-free (DSF) medium supplemented with insulin (5x10^-7 mol/L), transferrin (5 mg/mL), and ascorbate (0.2 mmol/L).²⁴ Relative rates of DNA synthesis were assessed by determination of [³H]thymidine incorporation into trichloroacetic acid (TCA)–precipitable material over the next 24 hours. HGF, bFGF, VEGF, or vehicle (fresh serum-free DMEM containing 0.1% bovine serum albumin) was added 12 hours before the addition of [³H]thymidine. Twenty-four hours after the addition of [³H]thymidine, cells were washed twice with cold phosphate-buffered saline solution and twice with 10% (wt/vol) cold TCA and were incubated with 10% TCA at 4°C for 30 minutes. Cells were rinsed in ethanol (95%), dissolved in 0.25N NaOH at 4°C for 3 hours, and neutralized, and the radioactivity was determined by liquid scintillation spectrometry.³

Cell Counting Assay
In the preparation of experiments for cell counting, cells were grown to 70% confluence in 96-well culture plates (Corning). Then, the medium was changed to fresh DSF containing HGF (10 ng/mL), bFGF (10 ng/mL), VEGF (10 ng/mL), or vehicle. The cells were then incubated overnight. On day 1, the medium was again changed to fresh DSF with growth factor or vehicle. On day 4, an index of cell proliferation was determined with a WST (2-[4-lodophenyl]-3-[4-nitrophenyl]-5-[2,4-disulfophenyl]-2-tetrazolium monosodium salt) cell counting kit that is similar to the MTT (3-[4,5-dimethyl-2-thiazol]-2,5-diphenyltetrazolium bromide) assay (Wako).^{25 26} We measured the WST activity of these cells as a marker of cell number. We confirmed that a serum-stimulated increase in cell number is associated with increased absorbance at 450 nm (data not shown).

Clinical Data
For the study of serum HGF concentration, 41 age-matched subjects were studied (21 never-treated essential hypertensive subjects [10 men and 11 women] and 20 normotensive subjects [11 men and 9 women]) (normotensive subjects, 64.7±1.7 years versus hypertensive subjects, 63.5±2.0 years; P=NS). Secondary hypertension was excluded by clinical and laboratory findings. Subjects with the following disorders were excluded from the study: cardiac valvular disease; congestive heart failure; arrhythmia; diabetes mellitus; and liver, kidney, or pulmonary dysfunction. BP was measured by nurses in a standardized clinical setting in the morning (between 8:30 and 10:30 AM) before the subjects had taken any drugs. BP was taken with the subject lying down and with a standard sphygmomanometer; it was measured to the nearest 2 mm Hg in the right arm, with Korotkoff phases I and V being taken as systolic BP and diastolic BP, respectively. BP measurements were repeated at least three times in a blind fashion, and the mean values of repeated measurements represented the BP value. Subjects with greater than 140 mm Hg systolic BP and 90 mm Hg diastolic BP were defined as hypertensive.

Blood was drawn after subjects had fasted overnight, and plasma and serum were collected after centrifugation. Lp(a) concentration was determined by Sanwa Kagaku Kenkyusho Co Ltd by their developed enzyme-linked immunosorbent assay (ELISA).²⁷ PAI-1 and TPA were determined by ELISAs (TintElizae PAI-1 and TintElizae tPA, Biopool). Serum HGF concentration was assayed with a recently developed ELISA for use in humans.²⁸ Total cholesterol was measured in the standard manner.

Materials
Human and rat recombinant HGFs were purified from the culture medium of Chinese hamster ovary cells or C-127 cells, respectively, transfected with an expression plasmid containing human or rat HGF cDNA.^{29 30 31} bFGF was obtained from Sigma Chemical Co. VEGF was obtained from Biosource.

Statistical Analysis
All values are expressed as mean±SE. Experiments were repeated at least three times. ANOVA with subsequent Bonferroni's test was used for determination of the significance of differences in multiple comparisons. Multiple regression analyses were used for assessment of the relation between BP and other parameters. Values of P<.05 were considered statistically significant.

	Results

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Effect of HGF on Growth of Human Endothelial Cells
Initially, we examined the effect of exogenously added recombinant HGF on human endothelial cell growth. As shown in Fig 1 (top), HGF stimulated DNA synthesis in a dose-dependent manner. Similarly, bFGF and VEGF also stimulated DNA synthesis in endothelial cells. However, DNA synthesis stimulated by HGF was greater than that stimulated by bFGF or VEGF at the same dose. Endothelial cell numbers were increased by the addition of HGF, bFGF, and VEGF (Fig 1, middle). HGF was more efficacious in stimulating endothelial cell growth than bFGF and VEGF. HGF and VEGF did not stimulate DNA synthesis in VSMCs (Fig 1, bottom). However, the addition of exogenous bFGF resulted in a significant increase in DNA synthesis.

View larger version (86K): [in this window] [in a new window]   Figure 1. Top, Stimulatory effects of HGF, VEGF, and bFGF on [3H]thymidine incorporation in human aortic endothelial cells. Dotted line shows basal level of [3H]thymidine incorporation. **P<.01, *P<.05 vs vehicle-treated cells; n=8 per group. Middle, Stimulatory effects of HGF, VEGF, and bFGF on human aortic endothelial cell growth. DSF indicates defined serum-free medium (vehicle)–treated cells; FBS, fetal bovine serum. **P<.01 vs DSF; #P<.01 vs bFGF; +P<.01 vs VEGF; n=8 per group. Bottom, Stimulatory effect of bFGF but not VEGF and HGF on [3H]thymidine incorporation in human VSMCs. **P<.01 vs DSF; n=8 per group.

Serum HGF Concentration in Normotensive and Hypertensive Subjects
We evaluated the relationship between serum HGF concentration and BP. As shown in Fig 2 (top), serum HGF concentration was significantly correlated with systolic BP (r=.430, P<.01). Serum HGF concentration and diastolic BP showed a tendency to be related (r=.324, P<.1; Fig 2, bottom), but not significantly. Interestingly, serum HGF concentration in hypertensive subjects was significantly higher than in normotensive subjects (0.476±0.029 versus 0.381±0.020 ng/mL, respectively; P<.05).

View larger version (32K): [in this window] [in a new window]   Figure 2. Relation between serum HGF concentration and systolic BP (top) and diastolic BP (bottom).

HGF belongs to the family of kringle proteins, characterized by a triple disulfide loop structure (kringles), that mediate protein-protein and protein-cell interactions. Therefore, besides acting as a growth factor, HGF may have a role in the regulation of thrombosis. The kringle family contains TPA, apolipoprotein(a), plasminogen, and urokinase, etc. Therefore, we evaluated the influence on serum HGF concentration of other factors related to thrombosis and atherosclerosis. Serum HGF and total cholesterol were not significantly associated. Similarly, TPA, PAI-1, and Lp(a) did not show any correlation with serum HGF concentration (data not shown). We also examined the association of BP with TPA, PAI-1, Lp(a), and total cholesterol. None of these factors showed a significant relationship with systolic, mean, or diastolic BP values (Table).

View this table: [in this window] [in a new window]   Table 1. Correlation of Total Cholesterol, Tissue Plasminogen Activator, Plasminogen Activator Inhibitor-1, and Lipoprotein(a) With BP

	Discussion

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Locally synthesized compounds from endothelial cells and VSMCs have been postulated to control local vascular function.^{1 2 3 8 9 10} Therefore, disruption or dysfunction of endothelial cells results in the loss of multiple endothelium-derived substances (prostaglandin I₂, nitric oxide, C-type natriuretic peptide) and abnormal VSMC growth, such as in atherosclerosis. Numerous articles have reported the loss of vasodilating properties of resistance vessels in hypertensive individuals.^{4 5 6 7} These changes in vascular tone may be due to the decrease in nitric oxide content and increase in vascular hypertrophy and/or growth induced by hypertension. Given the importance of endothelial antiproliferative and vasodilating actions, it would be of interest to seek an endothelium-specific growth factor that does not stimulate VSMC growth. As shown here, HGF fulfills the above characteristics because it promotes growth of only endothelial cells and not VSMCs. HGF is a mesenchyme-derived pleiotropic factor that regulates cell growth, cell motility, and morphogenesis of various cell types and thus is considered as a humoral mediator of epithelial-mesenchymal interactions responsible for morphogenic tissue interactions during embryonic development and organogenesis.^{16 21 32} VEGF is also known to be an endothelium-specific growth factor³³ and has been reported to be secreted from VSMCs and to act on endothelial cells.³⁴ Of importance, our present study showed that HGF is more efficacious in the growth of human aortic endothelial cells than VEGF and bFGF.

Interestingly, a local HGF system (HGF and its receptor, c-met) has been identified.¹¹ The protein and mRNA expressions of HGF and c-met were detected by reverse transcription–polymerase chain reaction and ELISA in endothelial cells and VSMCs, respectively, in vitro and vascular tissues in vivo. More importantly, our preliminary data showed that TGF-ß and angiotensin II strongly inhibited HGF production, and HGF itself stimulated HGF production in both endothelial cells and VSMCs (unpublished observations, 1995). The present study demonstrated that HGF does not stimulate VSMC growth despite the presence of c-met. It is still unclear whether c-met in VSMCs is functional despite the presence of c-met mRNA. Further studies are necessary for the analysis of c-met in VSMCs. In atherosclerotic lesions, TGF-ß is upregulated, as assessed by in situ hybridization and immunohistochemistry.^{35 36} In experimental hypertensive models, activation of the vascular renin-angiotensin system and TGF-ß was also reported in the vasculature.^{37 38 39 40} Taken together, the activation of local TGF-ß and the vascular renin-angiotensin system may negatively regulate local HGF production in vascular tissues. Our preliminary data showed that vascular and cardiac HGF concentrations in spontaneously hypertensive rats were significantly lower than in Wistar-Kyoto rats in response to increased local TGF-ß and angiotensin II, and serum HGF concentration was increased in spontaneously hypertensive compared with Wistar-Kyoto rats (unpublished observations, 1995). These results may support this possibility. Interestingly, apoptosis has been recently demonstrated in vascular diseases, eg, atherosclerosis and hypertension, resulting in potential endothelial regeneration or growth.^{41 42 43} Indeed, our preliminary data showed that HGF as well as bFGF, which is known as an antiapoptotic agent in endothelial cell growth, attenuated cytokine-induced apoptosis of endothelial cells (unpublished observations, 1995). Taken together, these data show that HGF may have a role in the prevention of endothelial dysfunction, including apoptosis. Therefore, we hypothesized that HGF might contribute to the protection or repair of vascular endothelial cells. If so, serum HGF levels might be elevated in response to endothelial cell damage induced by hypertension. Indeed, our clinical data indicated that serum HGF concentration was significantly correlated with systolic BP rather than diastolic BP, since systolic BP rather than diastolic BP has been thought to be related to the arteriosclerotic changes in the vasculature.^{44 45 46} Elevation of serum HGF concentration may be considered an index of arteriosclerotic vascular changes, although further studies are needed.

Recent findings also have revealed that HGF may play an important role in tissue regeneration.^{20 21 22 23} For example, HGF mRNA and blood HGF levels increased rapidly and markedly after hepatic injury and disease,^{16 17} and intravenously injected recombinant HGF markedly enhanced liver regeneration in vivo.²² Systemic HGF may work in tissue regeneration as a humoral mediator, in addition to autocrine-paracrine local HGF production. However, these results support the hypothesis that systemic HGF is not sufficient to promote tissue regeneration caused by a decrease in local HGF production. Since serum HGF concentration is increased in hypertensive individuals, serum HGF may act protectively against endothelial dysfunction in organs such as the vasculature and kidney.

Besides acting as a growth factor, HGF may have a role in the regulation of thrombosis because it belongs to the family of kringle proteins, characterized by a triple disulfide loop structure (kringles), that mediate protein-protein and protein-cell interactions. Therefore, we evaluated the correlation of HGF with other factors related to thrombosis and atherosclerosis, eg, Lp(a), PAI-1, and TPA, which belong to kringle families. However, there was no relationship between serum HGF concentration and total cholesterol, TPA, PAI-1, and Lp(a). On the other hand, BP was also not associated with these factors. Although our present data failed to show an interaction of HGF with other members of kringle families, further study is necessary to clarify such interactions. Overall, we demonstrated that HGF is an endothelium-specific growth factor whose serum concentration is significantly associated with systolic BP. These results suggest that systemic HGF secretion might be elevated in response to high BP as a counterregulatory system against endothelial dysfunction. Serum HGF concentration may be considered as a new index of endothelial dysfunction in hypertensive individuals.

	Selected Abbreviations and Acronyms

 

bFGF = basic fibroblast growth factor BP = blood pressure HGF = hepatocyte growth factor Lp(a) = lipoprotein(a) PAI-1 = plasminogen activator inhibitor-1 TGF-ß = transforming growth factor-ß TPA = tissue plasminogen activator VEGF = vascular endothelial growth factor VSMC = vascular smooth muscle cell

	Acknowledgments

 
Dr Ryuichi Morishita is the recipient of a Japan Vascular Disease Research Foundation Award and is a Research Fellow of the Japan Society for the Promotion of Science. This work was partially supported by grants from the Japan Cardiovascular Research Foundation, Mochida Memorial Foundation for Medical and Pharmaceutical Research, Tokyo Biochemistry Foundation, Japan Society for the Promotion of Science, and Molecular Cardiology and Research Foundation for Pharmaceutical Sciences. We wish to thank Drs Hirotsugu Fukuda, Hon-zi Bai, and Yoshiki Sawa (First Department of Surgery, Osaka University Medical School) for kindly providing rat coronary endothelial cells. Neointimal VSMCs were kindly provided by Dr Yasuko Kato (Fujisawa Pharmaceutical Co Ltd). We thank Misako Mashimoto and Keiko Zaitsu for their excellent technical assistance.

Received April 11, 1996; first decision April 18, 1996; accepted April 18, 1996.

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				J.-W. Lin, W. H.-H. Sheu, W.-J. Lee, Y.-T. Chen, T.-J. Liu, C.-T. Ting, and W.-L. Lee Circulating Hepatocyte Growth Factor Level but Not Basic Fibroblast Growth Factor Level Is Elevated in Angiography-Proven Symptomatic Peripheral Artery Disease Angiology, September 1, 2007; 58(4): 420 - 428. [Abstract] [PDF]

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