Vanin-1 Pantetheinase Drives Smooth Muscle Cell Activation in Post-Arterial Injury Neointimal Hyperplasia K. Jagadeesha Dammanahalli, Stephanie Stevens, Robert Terkeltaub* San Diego VA Healthcare System and Department of Medicine, University of California San Diego, San Diego, California, United States of America Abstract The pantetheinase vanin-1 generates cysteamine, which inhibits reduced glutathione (GSH) synthesis. Vanin-1 promotes inflammation and tissue injury partly by inducing oxidative stress, and partly by peroxisome proliferator-activated receptor gamma (PPARc) expression. Vascular smooth muscle cells (SMCs) contribute to neointimal hyperplasia in response to injury, by multiple mechanisms including modulation of oxidative stress and PPARc. Therefore, we tested the hypothesis that vanin-1 drives SMC activation and neointimal hyperplasia. We studied reactive oxygen species (ROS) generation and functional responses to platelet-derived growth factor (PDGF) and the pro-oxidant diamide in cultured mouse aortic SMCs, and also assessed neointima formation after carotid artery ligation in vanin-1 deficiency. Vnn12/2 SMCs demonstrated decreased oxidative stress, proliferation, migration, and matrix metalloproteinase 9 (MMP-9) activity in response to PDGF and/or diamide, with the effects on proliferation linked, in these studies, to both increased GSH levels and PPARc expression. Vnn12/2 mice displayed markedly decreased neointima formation in response to carotid artery ligation, including decreased intima:media ratio and cross-sectional area of the neointima. We conclude that vanin-1, via dual modulation of GSH and PPARc, critically regulates the activation of cultured SMCs and development of neointimal hyperplasia in response to carotid artery ligation. Vanin-1 is a novel potential therapeutic target for neointimal hyperplasia following revascularization. Citation: Dammanahalli KJ, Stevens S, Terkeltaub R (2012) Vanin-1 Pantetheinase Drives Smooth Muscle Cell Activation in Post-Arterial Injury Neointimal Hyperplasia. PLoS ONE 7(6): e39106. doi:10.1371/journal.pone.0039106 Editor: Rudolf Kirchmair, Medical University Innsbruck, Austria Received February 23, 2012; Accepted May 17, 2012; Published June 13, 2012 Copyright: 2012 Dammanahalli et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the Veterans Affairs Research Service and NIH (HL077360, HL087252) awards to Robert Terkeltaub. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: rterkeltaub@ucsd.edu Introduction SMC proliferation, MMP activation, and migration play pivotal roles in the progression of intimal lesions in atherosclerosis, and in arterial injury following interventional revascularization [1]. There are multiple mediators of intimal proliferation and neointima formation in model arterial injury, with some evidence suggesting that reactive oxygen species (ROS) generation and oxidative stress are a common thread [2]. In this light, reduced glutathione (GSH) is the major cellular thiol, and increasing cellular GSH levels can be protective against oxidative stress [3]. GSH homeostasis is regulated by oxidation of GSH to glutathione disulfide (GSSG), with GSH subsequently regenerated by glutathione reductase, expressed under the control of antioxidant response elements [4,5]. In addition, cell and tissue levels of GSH stores are regulated by gammaglutamylcysteine synthetase (cGCS), the rate-limiting enzyme for GSH synthesis [6]. Vanin-1 is a widely expressed glycosyl-phosphatidylinositol (GPI)-linked plasma membrane pantetheinase ectoenzyme, and nitrilase superfamily member, that contains the invariant catalytic triad residues glutamate, lysine, and cysteine [7,8]. Vanin pantetheinase activity generates the amino-thiol cysteamine, and regulates a variety of responses to stress [9,10]. Cysteamine inhibits cGCS [5]. Concordantly, GSH stores in multiple tissues are significantly increased in vanin-1 knockout mice [10]. Cysteamine also appears to decrease protective activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH- Px) against ROS toxicity, allowing free radical production to overwhelm antioxidant defense systems [11]. Conversely, vanin-1 deficiency is associated with both decreased superoxide produc- tion, and resistance to oxidative stress and tissue injury induced in vivo by paraquet or c-irradiation [5,10,12]. Several inflammatory stimuli, exemplified by PDGF, promote SMC proliferation and migration [13,14]. Vanin-1, like several other mediators of oxidative stress, promotes inflammation [15,16]. For example, in intestinal epithelial cells, vanin-1 facilitates expression of certain pro-inflammatory cytokines [9]. Conversely, vanin-1 deficiency is protective in vivo for intestinal inflammation and injury in response to both nonsteroidal anti- inflammatory drugs and Schistosomal infection in vivo [17,18]. Vanin-1 promotes inflammation partly by suppression of both PPARc expression and PPARc signal transduction [15]. PPARc functions as an anti-inflammatory checkpoint in multiple inflam- matory settings, and in a variety of cell types [18]. PPARc- activating thiazolidinedione (TZD) treatment suppresses SMC proliferation and migration in vitro, as well as in vivo in the intimal hyperplasia process after arterial injury [18]. Furthermore, PPARc is expressed by normal vascular SMCs, and arterial PPARc expression normally increases in early human atherosclerotic lesions and 1–2 weeks after arterial balloon injury in rodents [19,20]. PPARc activation inhibits SMC invasion, and suppresses induction by PDGF of the transcription factor Ets-1, a mediator of MMP expression and SMC invasion both in cultured PLoS ONE | www.plosone.org 1 June 2012 | Volume 7 | Issue 6 | e39106