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(Circulation. 1999;100:5-8.)
© 1999 American Heart Association, Inc.

Brief Rapid Communication

Endothelin-1 Potentiates Human Smooth Muscle Cell Growth to PDGF

Effects of ET_A and ET_B Receptor Blockade

Zhihong Yang, MD; Nazmi Krasnici, BSc; Thomas F. Lüscher, MD

From Cardiovascular Research (Z.Y., N.K., T.F.L.), Institute of Physiology, University Zürich-Irchel and Cardiology (Z.Y., T.F.L.), University Hospital Zürich, Switzerland.

Correspondence to Thomas F. Lüscher, MD, FRCP, FACC, Professor and Head of Cardiology, University Hospital, CH-8091 Zürich, Switzerland. E-mail cardiotfl{at}compuserve.com

	Abstract

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Background—Endothelin-1 (ET-1) is a potent vasoconstrictor. However, its mitogenic effects on vascular smooth muscle cells (SMCs) remain controversial. We investigated the role of ET-1 in human SMC growth and its synergistic effect with platelet-derived growth factor (PDGF).

Methods and Results—Human aortic SMCs were cultured and cell proliferation was assayed by [³H]thymidine incorporation. PDGF receptor expression, activation of mitogen-activated protein kinase (MAPK), cell cycle regulators such as cyclin-dependent kinase 2 (Cdk2), Cdk inhibitor (p27^Kip1), and retinoblastoma protein (pRb) were analyzed by immunoblotting. ET-1 on its own was unable to stimulate [³H]thymidine incorporation but dramatically potentiated the effect of PDGF-BB up to 6-fold (P<0.001). Most of the potentiating effects (88%) were blocked by the ET_A receptor antagonist LU135252 and slightly further blocked by the ET_A/B receptor antagonist bosentan (P<0.05). ET-1 stimulated MAPK, but it neither potentiated PDGF-induced MAPK activation nor overexpressed PDGF receptors. In contrast to PDGF-BB, ET-1 had no regulatory effects on Cdk2, p27^Kip1, and pRb.

Conclusions—In human SMCs, ET-1 activates MAPK but has no mitogenic effects on its own. However, ET-1 markedly potentiates proliferation to PDGF, mainly via ET_A receptors. This may represent an important function of ET-1 for vascular structural changes in patients and provide new therapeutic opportunities for ET-1 receptor antagonists.

Key Words: cells • growth substances • kinases • platelet-derived factors

	Introduction

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Endothelin-1 (ET-1) is a potent vasoconstrictor via activation of ET_A and ET_B receptors.^{1 2} Although the role of ET-1 in cardiovascular disorders with dysregulation of vascular tone in such disease states as congestive heart failure, pulmonary and systemic hypertension, and cerebrovascular spasm after subarachnoid hemorrhage is well documented,¹ the involvement of ET-1 in vascular structural changes remains controversial. Indeed, in cultured smooth muscle cells (SMCs) obtained mainly from rat aorta, some studies demonstrated potent mitogenic effects of ET-1,^{3 4} and others showed no proliferative effects.^{5 6}

In contrast to ET-1, platelet-derived growth factor (PDGF) is a well-established mitogen for SMCs and contributes to the pathogenesis of proliferative vascular disease.⁷ Although the mechanisms of cell growth by PDGF are not yet completely understood, much of the evidence indicates that the c-Raf/MEK/mitogen-activated protein kinase (MAPK) cascade activated by PDGF tyrosine kinase receptors is important for transmitting extracellular growth signals into the cell nucleus, thereby regulating downstream gene expression and cell cycle progression.⁸

Cell cycle progression is positively regulated by the orderly activation of cyclin-dependent kinases (Cdks) and negatively regulated by several cell-cycle inhibitors known as cyclin-dependent kinase inhibitors.⁹ Our previous study showed that Cdk inhibitor (p27^Kip1) seems most likely involved in cell cycle control in human SMCs.¹⁰ Cyclin D–Cdk4/Cdk6 regulates G₁ progression, and cyclin E–Cdk2 is essential for G_1/S transition by phosphorylating and inactivating the tumor suppressor gene retinoblastoma protein (pRB),⁹ which causes release and activation of E2F transcription factor, and in turn regulating several proteins required for cell proliferation.¹¹

In this study, we clarified the proliferative role of ET-1 in human SMCs and demonstrated that a marked growth-potentiating effect of the peptide to PDGF represents an important mechanism of ET-1–induced human SMC proliferation.

	Methods

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Chemicals and Materials
Monoclonal antibody against -smooth muscle actin and all chemicals for immunoblotting were purchased from Sigma Chemical Co; all tissue culture materials were from Gibco; [³H]methylthymidine was from Amersham; recombinant human PDGF-BB was from R&D Systems GmbH; rabbit polyclonal anti-human p42^mapk (C14), p27^Kip1 (C19), Cdk2 (M2), and PDGF - (C20) and ß- (958) receptors were from Santa Cruz Biotechnology Inc; and the mouse monoclonal anti-human pRB (G3-245) was from PharMingen. Anti–phospho-p44/42^mapk was from New England BioLabs Inc; LU135252 was supplied by Knoll AG; and bosentan was supplied by Hoffman La Roche.

Cell Culture
Human aortic SMCs were purchased from Clonetics Corp. The cells were cultured in DMEM containing 10% FCS supplemented with 20 mmol/L L-glutamine and HEPES buffer solution, 100 U/mL penicillin, and 100 µg/mL streptomycin in a humidified atmosphere (37°C; 95% air/5% CO₂). Culture medium was replaced every 3 days. Cells were passaged by 0.01% EDTA-trypsin.¹⁰

[³H]Thymidine Incorporation and Cell Division
SMCs in DMEM containing 10% FCS were seeded on 12-well plates (density, 2x10⁴/mL) overnight to allow attachment. Culture media were then replaced with serum-free DMEM containing all ingredients as described above and 0.2% BSA instead of FCS without any possible growth factors, such as insulin. Cells in serum-free DMEM were incubated for 48 hours to achieve a quiescent stage. Quiescent cells were stimulated with PDGF-BB or ET-1 or both together at various concentrations in the presence or absence of the ET-1 receptor antagonists LU135252 or bosentan 10^-6 to 10^-5 mol/L. [³H]thymidine incorporation was then assayed as described.¹⁰ In another set of experiments, quiescent SMCs (10⁴/well) in 12-well plates were stimulated by PDGF-BB 1 ng/mL or ET-1 10^-7 mol/L or both together over 8 days. Cell number was counted every 2 days (Coulter Counter).

MAPK Activation
p42/44^mapk activation or phosphorylation was analyzed by immunoblotting as described.¹⁰

Cell Cycle Regulatory Proteins
In another series of experiments, the cells were stimulated either with ET-1 10^-7 to 10^-6 mol/L or PDGF-BB 10 ng/mL for 24 hours. p27^Kip1, Cdk2, and pRb were then analyzed by immunoblotting on 12% or 8% SDS-PAGE.

PDGF Receptors
To study the effects of ET-1 on PDGF receptor proteins, the cells were stimulated with ET-1 10^-7 to 10^-6 mol/L for 24 hours and then harvested for PDGF - and ß-receptor protein analysis by immunoblotting on 8% SDS-PAGE.

Statistics
Data are mean±SEM. [³H]thymidine incorporation was expressed as percent increase of control. n equals the number of different independent experiments. Student's t test for paired observations and ANOVA followed by Scheffé's test with repeated measurements were used. A 2-tailed P value <0.05 was considered significant.

	Results

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ET-1 Potentiated Cell Growth to PDGF
In cultured human aortic SMCs, ET-1 10^-9 to 10^-6 mol/L on its own did not stimulate [³H]thymidine incorporation, whereas PDGF-BB 0.01 to 10 ng/mL concentration-dependently enhanced the [³H]thymidine incorporation (data not shown). The effects of PDGF-BB at low concentrations (0.1 or 1 ng/mL) were dramatically potentiated by ET-1 10^-9 to 10^-7 mol/L in a concentration-dependent manner. Similarly, the increase in cell number in response to a low concentration of PDGF-BB (1 ng/mL) was markedly potentiated by ET-1 10^-7 mol/L over 8 days (Figure 1A and 1B).

View larger version (20K): [in this window] [in a new window]   Figure 1. Potentiation of cell growth by ET-1. A, ET-1 had no effect on [3H]thymidine incorporation, but it concentration-dependently potentiated effect of PDGF-BB (n=3). B, Cell number increase stimulated by PDGF-BB 1 ng/mL was time-dependently potentiated by ET-1 10-7 mol/L. This experiment was repeated 3 times independently (C). PDGF-BB 1 ng/mL significantly enhanced [3H]thymidine incorporation, which was remarkably potentiated by ET-1 10-7 mol/L (n=3). Potentiating effect of ET-1 was almost completely inhibited by selective ETA receptor antagonist LU 135252 10-6 to 10-5 mol/L in a concentration-dependent manner and was modestly but significantly further reduced by ETA/B-receptor antagonist bosentan 10-6 to 10-5 mol/L. *P<0.05 vs control, P<0.001 vs PDGF-BB 1 ng/mL alone, §P<0.001 vs PDGF-BB plus ET-1, P<0.05 vs LU 135252.

Potentiation of Cell Growth by ET-1 Mainly Through ET_A Receptor
A low concentration of PDGF-BB (1 ng/mL) stimulated [³H]thymidine incorporation (1103±4% increase of control, n=3), which was dramatically potentiated by ET-1 10^-7 mol/L (Figure 1C; 6868±358% increase above control, corresponding to 6.2-fold; P<0.001). The potentiating effects of ET-1 were concentration-dependently prevented by the selective ET_A-receptor antagonist LU135252 10^-6 to 10^-5 mol/L (1820±24%, n=3; P<0.001 versus PDGF-BB plus ET-1) and slightly but significantly further prevented by the ET_A/B-receptor antagonist bosentan 10^-6 to 10^-5 mol/L (Figure 1C, 1595±12% increase, n=3, P<0.05 versus LU135252).

ET-1 Had No Effects on Cell Cycle Regulators
Although both ET-1 10^-7 mol/L and PDGF-BB 10 ng/mL time-dependently stimulated p42^mapk, as demonstrated by a slower mobility on immunoblotting (data not shown), only PDGF-BB 10 ng/mL was capable of activating Cdk2, accompanied by an increase in its electrophoretic mobility on SDS-PAGE (Figure 2A) due to Cdk2 phosphorylation on Thr160,⁹ downregulating p27^Kip1 and hyperphosphorylating pRb (Figure 2B).

View larger version (37K): [in this window] [in a new window]   Figure 2. Effects of ET-1 on p42mapk and cell cycle regulators: A, PDGF-BB 10 ng/mL (24 hours) but not ET-1 10-7 to 10-6 mol/L markedly activated Cdk2, downregulated p27Kip1, and hyperphosphorylated pRb. B, Both ET-1 10-7 mol/L and PDGF-BB 10 ng/mL alone stimulated p42/44mapk phosphorylation at 2 minutes. No potentiating effect was observed when cells were stimulated with ET-1 and PDGF-BB together. C, Stimulation of cells with ET-1 10-7 or 10-6 mol/L for 24 hours had no effects on PDGF ß- and -receptor protein level. Results were repeated with 3 independent experiments.

ET-1 Had No Effects on PDGF Receptors and PDGF-Stimulated MAPK
Treatment of the cells with ET-1 10^-7 to 10^-6 mol/L for 24 hours influenced neither PDGF - nor ß-receptor protein level (Figure 2C). Furthermore, no potentiation effects of PDGF-BB–induced p42/44^mapk activation by ET-1 10^-7 mol/L were observed at 2 minutes (Figure 2B).

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The present study for the first time demonstrates a remarkable growth-potentiating effect (almost 70-fold) of ET-1 in human aortic SMCs in response to PDGF, whereas the peptide on its own did not exhibit mitogenic effects despite MAPK activation. This effect of ET-1 is mediated primarily by ET_A receptor and is not attributable to overexpression of PDGF receptors and potentiation of PDGF-induced MAPK activation.

Recent studies using animal models or human tissue indicate that ET-1 contributes to vascular structural changes in proliferative cardiovascular disease.^{12 13} However, in vitro studies with cultured SMCs did not always find such mitogenic effects of ET-1.^{5 6} It has been postulated that the putative proliferative effects of ET-1 may depend on the species differences or different culture conditions in particular ingredients of the culture medium such as serum.^{5 6} To clarify the issue, we therefore used human aortic SMCs in medium devoid of any growth factors. Under these conditions, we clearly demonstrated that ET-1 on its own was unable to stimulate DNA synthesis even in the µmol/L range, although the peptide activated MAPK. The dissociation of MAPK activation from cell growth is in line with our previous study, which suggested that MAPK activation alone is necessary but not sufficient for cell proliferation.¹⁰ The ineffectiveness of ET-1 to stimulate SMC growth was further confirmed by the fact that in contrast to PDGF, ET-1 was unable to activate Cdk2, downregulate p27^Kip1, or hyperphosphorylate pRb, which is crucial for cell cycle progression.

The most important and intriguing finding of the present study is that ET-1 dramatically potentiated [³H]thymidine incorporation in human SMCs in response to PDGF-BB, with a potency reaching almost 6-fold. The potentiating effects of ET-1 were inhibited mainly by the selective ET_A-receptor antagonist LU135252 and slightly but significantly further inhibited by the ET_A/B-receptor antagonist bosentan.¹ This indicates that activation of ET_A receptors is primarily responsible for the potentiating effects of ET-1. Furthermore, our results demonstrated that the potentiating effect of cell growth by ET-1 is due to a mechanism other than overexpression of PDGF receptors or potentiation of PDGF-induced MAPK activation by the peptide. The crosstalk of G-protein–coupled receptors, such as ET-1 receptors, with tyrosine kinase pathways has been documented.¹⁴ However, which tyrosine kinase–mediated pathways are influenced by PDGF deserves further investigation.

The data presented in this study may explain the contradictory reports on the mitogenic effects of ET-1 in several in vitro studies. The dramatic potentiation of proliferative responses to other growth factors by ET-1 may be crucial in the development of structural vascular changes in patients with atherosclerosis, restenosis, and venous bypass graft disease. The present study also provides strong support for the clinical use of ET-1 antagonists in those clinical situations.

	Acknowledgments

 
Original research reported in this article was supported by grants from the Swiss National Research Foundation (32-51069.97/1), the Swiss Cardiology Foundation, and the Swiss 3R Research Foundation.

Received November 20, 1998; revision received May 12, 1999; accepted May 12, 1999.

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