Please cite this article in press as: Brito PM, et al. Resveratrol inhibits the mTOR mitogenic signaling evoked by oxidized LDL in smooth muscle cells. Atherosclerosis (2008), doi:10.1016/j.atherosclerosis.2008.11.011 ARTICLE IN PRESS G Model ATH-10666; No. of Pages 9 Atherosclerosis xxx (2008) xxx–xxx Contents lists available at ScienceDirect Atherosclerosis journal homepage: www.elsevier.com/locate/atherosclerosis Resveratrol inhibits the mTOR mitogenic signaling evoked by oxidized LDL in smooth muscle cells Paula M. Brito a , Raphaël Devillard b , Anne Nègre-Salvayre b,c,∗ , Leonor M. Almeida a , Teresa C.P. Dinis a , Robert Salvayre b,c , Nathalie Augé b a Laboratory of Biochemistry, Faculty of Pharmacy, and Center for Neurosciences and Cell Biology, University of Coimbra, Coimbra, Portugal b INSERM-U858, Institut Louis Bugnard CHU Rangueil, Toulouse, France c Faculté de Médecine Rangueil, Toulouse, France article info Article history: Received 19 June 2008 Received in revised form 24 October 2008 Accepted 4 November 2008 Available online xxx Keywords: Resveratrol PDK1 Akt Oxidized LDL Smooth muscle cells Proliferation abstract Objectives: Smooth muscle cell (SMC) proliferation is a major feature in atherosclerosis, since it con- tributes to the formation of the fibrous cap, thus to plaque stability, but also to arterial stenosis and post-angioplasty restenosis. Among the various mitogenic signaling pathways involved in SMC prolif- eration, the mTOR pathway regulates both the cell cycle and cell growth. Resveratrol, a polyphenolic compound from grapes and red wine, has potential anti-atherogenic and anti-cancer properties. This work was designed to investigate the activation of the mTOR pathway by the proatherogenic oxidized LDL (oxLDL) in SMC, and the potential inhibitory effect of resveratrol. Results: mTOR and its downstream target p70S6 kinase are phosphorylated and activated by mitogenic concentrations of oxLDL (50 g/ml), and are involved in SMC proliferation, as assessed by the inhibitory effect of the mTOR inhibitor rapamycin. The activation of mTOR signaling by oxLDL, requires the upstream activation of PI3K and Akt, as assessed by the inhibitory effect of the PI3K inhibitor Ly294002 on mTOR activation and DNA synthesis. Resveratrol blocked the oxLDL-induced phosphorylation and activation of the PI3K/Akt/mTOR/p70S6K pathway and strongly inhibited both the DNA synthesis and proliferation of SMC. This activity is independent of the anti-oxidant effect and of AMPK activation by resveratrol. Conclusion: These data indicate that the mTOR pathway is activated by oxLDL via PI3K/PDK1/Akt, and is required for SMC proliferation. Resveratrol blocks specifically this pathway, thereby inhibiting oxLDL- induced SMC proliferation. These data highlight a new property for resveratrol that could contribute to the general anti-atherogenic properties of this polyphenol. © 2008 Elsevier Ireland Ltd. All rights reserved. 1. Introduction Vascular smooth muscle cells proliferation is a key process in the pathogenesis of atherosclerosis as it underlies the formation of fibro-atheroma plaques, a hallmark of the disease [1]. Oxidized low density lipoproteins (oxLDL) are involved in the formation of fatty streaks and exhibit various biological properties, including vascular smooth muscle cells proliferation, that are potentially implicated in the evolution towards more advanced lesions [2]. The mito- genic effect of oxLDL depends on the activation of various signaling Abbreviations: LDL, low density lipoproteins; SMC, smooth muscle cells; oxLDL, oxidized low density lipoprotein; FCS, fetal calf serum; PI3K, phosphoinositide 3 kinase; mTOR, mammalian target of rapamycin. ∗ Corresponding author at: INSERM U858–Team 10 - IFR-31, Institut Louis Bug- nard, CHU Rangueil, 1 avenue Jean Poulhès, BP84225 - 31432 Toulouse Cedex 4, France. Tel.: +33 561 32 28 08; fax: +33 561 32 20 84. E-mail addresses: anne.negre-salvayre@inserm.fr (A. Nègre-Salvayre), nathalie.auge@inserm.fr (N. Augé). pathways including the sphingolipid pathway leading to ERK1/2 phosphorylation, and the EGF receptor/PI3K/Akt pathway involved in survival [3–5]. Beside its role in cell survival, the PI3K/Akt path- way is a key trigger of mTOR signaling. The Ser/Thr kinase mTOR is implicated in spatial and temporal aspects of cell growth, con- trolling translation and cell cycle progression by phosphorylating 4EBP1 protein family and S6 protein kinases [6,7]. The activation of mTOR through PI3K/Akt involves a direct Akt-mediated phospho- rylation of mTOR at Ser2448 and Thr2446 [8,9]. Although mTOR has been implicated in cardiovascular diseases and more particularly in cardiac hypertrophy, its role in oxLDL-induced SMC proliferation is still unknown. Resveratrol (3,4,5-trihydroxystilbene) is a natural phytoalexin found in grapes and red wine. A growing body of evidences in the literature reports a protective effect for resveratrol as anti- oxidant, anti-inflammatory, anti-thrombotic and anti-proliferative agent, thereby supporting a role for this agent in the cardiopro- tective effects observed by a moderate consumption of red wine [10–12]. Several molecular mechanisms and signaling pathways 0021-9150/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.atherosclerosis.2008.11.011