Atherosclerosis 146 (1999) 351 – 359 Endothelin-1 stimulates proliferation of human coronary smooth muscle cells via the ET A receptor and is co-mitogenic with growth factors Sassan Hafizi, Sean P. Allen, Andrew T. Goodwin, Adrian H. Chester, Magdi H. Yacoub * Department of Cardiothoracic Surgery, National Heart and Lung Institute, Imperial College of Science, Technology and Medicine, Heart Science Centre, Royal Brompton and Harefield NHS Trust, Harefield, Middlesex, UB96JH, UK Received 22 June 1998; received in revised form 5 March 1999; accepted 21 April 1999 Abstract We investigated the effects of endothelin-1 (ET-1) on growth of cultured human coronary artery smooth muscle cells (cSMC). ET-1 alone stimulated DNA synthesis in growth-arrested cSMC as measured by [ 3 H]thymidine incorporation, with a maximum 63 23% increase above control by 10 -7 M(P 0.05). ET-1 (10 -7 M) also stimulated increases in cyclin D1 protein levels after 24 h, and in absolute cell number after 4 days. Furthermore, ET-1 stimulated protein synthesis (maximum 73 32% increase in [ 3 H]leucine incorporation by 10 -7 M(P 0.05)), as well as triggering intracellular calcium transients in human cSMC, as visualised under fura-2 fluorescence microscopy. The selective ET A receptor antagonist BQ123 inhibited the increases in DNA synthesis, cell number, protein synthesis and intracellular calcium concentration in response to ET-1, whereas the ET B receptor antagonist BQ788 had no such effects. Furthermore, the ET B agonist sarafotoxin 6c had no effect on cSMC DNA synthesis. In addition, co-incubation of ET-1 with threshold concentrations of the growth factors, platelet-derived growth factor-BB (PDGF-BB), basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF), resulted in pronounced synergistic increases in DNA synthesis over that observed with the factors alone. In conclusion, we have shown that ET-1 stimulates proliferation of human cSMC via the ET A receptor and is also a co-mitogen with the growth factors tested. These findings indicate a role for ET-1 in the development of coronary intimal hyperplasia in man. © 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Cell; Coronary; Endothelins; Growth factors; Receptors; Smooth muscle www.elsevier.com/locate/atherosclerosis 1. Introduction Vascular smooth muscle cell (VSMC) proliferation is a key feature in the development of atherosclerosis [1], and can occur in response to many different humoral and mechanical stimuli. The VSMC growth-promoting properties of polypeptide growth factors, e.g. platelet- derived growth factor (PDGF), basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF), in intimal thickening are well-recognised [2]. However, more recently a number of vasoactive agents with proven abilities to modulate vascular tone have also been considered as possible promoters of VSMC growth. One such agent is endothelin-1 (ET-1), a 21- amino acid peptide that is the most potent natural vasoconstrictor known [3]. The endothelium is the ma- jor source of ET-1, although secretion of the peptide has also been shown to occur in cultured human VSMC, notably at increased levels in VSMC from atherosclerotic human coronary arteries [4]. The potential of ET-1 to induce VSMC growth in addition to contraction has come to light from several studies. In vivo, ET-1 has been shown to augment the neointimal formation response after balloon injury of the rat carotid artery [5]. However, the possibility of a mitogenic effect of ET-1 and its potency in cultured VSMC in vitro has varied between different studies * Corresponding author. Tel.: +44-1895-828893; fax: +44-1895- 828902. 0021-9150/99/$ - see front matter © 1999 Elsevier Science Ireland Ltd. All rights reserved. PII:S0021-9150(99)00178-1