Anti-proliferative effect of rosiglitazone on angiotensin II-induced vascular smooth muscle cell proliferation is mediated by the mTOR pathway Jung-Sun Kim 1 *, Il-Kwon Kim 1{,{ , Se-Yeon Lee 1{,1 , Byeong-Wook Song {,1 , Min-Ji Cha {,1 , Heesang Song " , Eunmi Choi { , Soyeon Lim {,1 , Onju Ham {,1 , Yangsoo Jang *,{ and Ki-Chul Hwang 2 * ,{,{,1 * Cardiology Division, Yonsei Cardiovascular Center, Yonsei University Health System, Seoul, South Korea { Severance Biomedical Science Institute, Yonsei University College of Medicine, Seoul, South Korea { Cardiovascular Research Institute, Yonsei University College of Medicine, Seoul, South Korea 1 Brain Korea 21 Project for Medical Science, Yonsei University College of Medicine, Seoul, South Korea " Department of Biochemistry and Molecular Biology, Chosun University School of Medicine, Gwangju, South Korea Abstract VSMC (vascular smooth muscle cell) proliferation contributes significantly to intimal thickening in atherosclerosis, restenosis and venous bypass graft diseases. Ang II (angiotensin II) has been implicated in VSMC proliferation though the activation of multiple growth-promoting signals. Although TZDs (thiazolidinediones) can inhibit VSMC proliferation and reduce Ang II-induced fibrosis, the mechanism underlying the inhibition of VSMC proliferation and fibrosis needs elucidation. We have used primary cultured rat aortic VSMCs and specific antibodies to investigate the inhibitory mechanism of rosiglitazone on Ang II-induced VSMC proliferation. Rosiglitazone treatment significantly inhibited Ang II-induced rat aortic VSMC proliferation in a dose-dependent manner. Western blot analysis showed that rosiglitazone significantly lowered phosphorylated ERK1/2 (extracellular-signal-regulated kinase 1/2), Akt (also known as protein kinase B), mTOR (mammalian target of rapamycin), p70S6K (70 kDa S6 kinase) and 4EBP1 (eukaryotic initiation factor 4E-binding protein) levels in Ang II-treated VSMCs. In addition, PPAR-c (peroxisome-proliferator-activated receptor c) mRNA increased significantly and CTGF (connective tissue growth factor), Fn (fibronectin) and Col III (collagen III) levels decreased significantly. The results demonstrate that the rosiglitazone directly inhibits the pro-atherosclerotic effect of Ang II on rat aortic VSMCs. It also attenuates Ang II-induced ECM (extracellular matrix) molecules and CTGF production in rat aortic VSMCs, reducing fibrosis. Importantly, PPAR-c activation mediates these effects, in part, through the mTOR-p70S6K and -4EBP1 system. Keywords: angiotensin II; mammalian target of rapamycin (mTOR); proliferation; rosiglitazone; smooth muscle cell 1. Introduction VSMC (vascular smooth muscle cell) proliferation and migra- tion are essential features of vasculogenesis and blood vessel maturation. Furthermore, these processes clearly play a role in the pathophysiology of several prominent cardiovascular disease states, such as atherosclerosis and restenosis (Lee et al., 2004). Following migration, VSMCs proliferate in the intima and secrete matrices and proteases to form atheromatous plaques under the influence of stimulatory cytokines. Various stimuli induce VSMC proliferation and migration; Ang II (angiotensin II), the effector molecule of the renin–Ang system, has profound effects on SMC (smooth muscle cell) proliferation and migration (Perlegas et al., 2005; Zuo et al., 2005). These effects are not only haemodynamic in nature but also comprise inflammation, thrombosis and cell proliferation by stimulating cytokine and growth factor production (Horiuchi et al., 2003). ARBs [AT 1 R (Ang II type 1 receptor) blockers] (Zuo et al., 2005) and knockdown experiments have shown that intracellular signals involving Rac1 activation are important for the Ang II-mediated hypertrophy of SMCs and neointimal formation in injured arteries (Horiuchi et al., 2003). Rosiglitazone is an agonist of PPAR-c (peroxisome-proliferator-activated receptor c) and is the most potent of the TZD (thiazolidinedione) anti-diabetic agents. The FDA (Food and Drug Administration) recently approved rosiglitazone for the treatment of type II diabetes mellitus. Rosiglita- zone is thought to attenuate Ang II-induced vascular inflammation (Ji et al., 2009). In addition, PPAR-c activators decrease BP (blood pressure) and cell growth, and improve endothelial dysfunction in mesenteric resistance arteries from Ang II-infused rats. Thus, PPAR-c could contribute to the regulation of different vascular genes in hypertension (Ryan et al., 2004). Furthermore, rosiglita- zone inhibits VSMC proliferation by blocking growth factor-induced phosphorylation of retinoblastoma tumour suppressor proteins (Bruemmer and Law, 2003). However, the role of mTOR (mammalian target of rapamycin) in rosiglitazone-inhibited VSMC proliferation and fibrosis induced by Ang II is unknown. We demonstrate that rosiglitazone directly 1 These authors contributed equally to this work. 2 To whom correspondence should be addressed (email kchwang@yuhs.ac). Abbreviations: AMV, avian myeloblastosis virus; Ang II, angiotensin II; Col III, collagen III; CTGF, connective tissue growth factor; DMEM, Dulbecco’s modified Eagle’s medium; 4EBP, eukaryotic initiation factor 4E-binding protein; ECM, extracellular matrix; ERK, extracellular-signal-regulated kinase; FBS, fetal bovine serum; Fn, fibronectin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; mTOR, mammalian target of rapamycin; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H- tetrazolium bromide; PI3K, phosphoinositide 3-kinase; PPAR, peroxisome-proliferator-activated receptor; p70S6K, 70 kDa S6 kinase; RT–PCR, reverse transcription PCR; siRNA, small interfering RNA; SMC, smooth muscle cell; TBS-T, TBS-Tween 20; TZD, thiazolidinedione; VSMC, vascular smooth muscle cell. Cell Biol. Int. (2012) 36, 305–310 (Printed in Great Britain) Research Article E The Author(s) Journal compilation E 2012 Portland Press Limited Volume 36 (3) N pages 305–310 N doi:10.1042/CBI20100524 N www.cellbiolint.org 305