SMCs, and in vivo after balloon injury in rat aorta [21–23]. PPARc agonists inhibit mRNA and protein expression, as well as induction of gelatinolytic activity of MMP-9 [24], an MMP implicated in SMC migration [25]. Here, we demonstrate that vanin-1 plays a major role in mediating both oxidative stress and PPARc homeostasis in SMCs, as well as migration, proliferation, and MMP-9 activity. Furthermore, vanin-1 drives neointima formation after carotid artery ligation in mice. Results Expression of Vnn1 and Vnn3 and Pantetheinase Activity in SMCs Vanin-1 was constitutively expressed in WT mouse aorta but not in Vnn12/2 mice aortae (Fig. 1A), a finding buttressed by aortic tissue Western blot analyses (Fig. 1B). The vanin enzyme subfamily includes two enzymes in mice (vanin-1, and secretory vanin- 3) [5,8]. Quantitative PCR indicated that Vnn1 deficiency was not associated with compensatory increase in the expression levels of Vnn3 normalized to GAPDH (ie, vanin-3 expression relative to GAPDH not significantly different in WT vs. vanin-1 knockout SMCs (11.0360.28 WT vs.10.4360.121 Vnn12/2 SMCs) (Fig. 1C). Last, pantetheinase activity, demonstrated by fluorescence using the substrate pantothenate–AMC, was robust in WT mouse arterial sections and SMC lysates. However, the attenuation of detectable enzyme activity in artery sections or SMC lysates from Vnn12/2 mice indicated vanin-1 expression to be required for most arterial pantetheinase activity in situ (Fig. 1 D–E). A Vanin-1 Regulatory Circuit with GSH Mediates Oxidative Stress in SMCs PDGF (10 ng/ml) and diamide (5 mM), a membrane-permeable thiol that oxidizes GSH, induced superoxide in WT SMCs; both these responses were blunted in Vnn12/2 SMCs, as assessed using the redox-sensitive dye Dihydroethidium (DHE) and by flow cytometry) (Fig. 2A–B). Next, we observed that PDGF treatment increased pantetheinase activity in WT but not in Vnn12/2 SMCs (Fig. 2C). Treatment with the vanin-1 enzymatic product cysteamine, a cGCS inhibitor, increased ROS levels in both WT and Vnn12/2 SMCs as did treatment with another GSH- depleting cGCS inhibitor buthionine sulfoximine (BSO; 1 mM) (Fig. 2 D,E). GSH levels in Vnn12/2 SMCs were significantly higher than in WT SMCs, with or without PDGF treatment (Fig. 2F). However, the GSH-oxidizing agent diamide reduced reduced GSH stores down to a comparable level in WT and Vnn12/2 SMCs (Fig. 2F). Therefore, we assessed for mechanisms beyond GSH depletion by which vanin-1 could modulate SMC function, and focused next on PPARc. Vanin-1 Modulated PPARc Expression Partly Regulates SMC Proliferation and Oxidative Stress PPARc expression was constitutively elevated in both mouse aortic sections and cultured SMCs of Vnn12/2 mice compared to WT counterparts, as confirmed by Western blotting (Fig. 3A–B). Therefore, we assessed the inter-relationships between PPARc, GSH, and vanin-1 in cultured SMCs, testing the potential contribution of vanin-1 modulation of PPARc to changes in SMC proliferation and oxidative stress mediated by vanin-1. First, Vnn12/2 SMCs were relatively resistant to the capacity of diamide to reduce PPARc expression (Fig. 3A–B). Moreover, under these conditions, PDGF treatment decreased PPARc expression in WT SMCs but not in Vnn12/2 SMCs (Fig. 3B). In contrast, diamide Vanin-1 in Vascular Remodeling Disease significantly reduced PPARc levels in WT SMCs compared to Vnn12/2 SMCs. Second, when we effectively knocked down PPARc via siRNA (Fig. 3C,D), we observed decreased GSH levels in both WT and Vnn12/2 SMCs (Fig. 3E). Though this effect of PPARc knockdown was not as extensive as the GSH depletion in response to treatment with cysteamine or BSO (Fig. 3E), it linked PPARc expression with GSH homeostasis. Third, both PDGF and diamide increased SMC proliferation in WT SMCs, but Vnn12/ 2 SMCs were resistant to induction of proliferation by PDGF and diamide (Fig. 3F). Even when PPARc was knocked down, PDGF induced proliferation more in WT than Vnn12/2 SMCs (Fig. 3G). Similarly, Vnn12/2 SMCs also were more resistant to the capacity of the PPARc inhibitor GW9662 to promote SMC proliferation (data not shown). Fourth, we expressed human vanin-1 by transfection in Vnn12/2 SMCs and linked increased pantetheinase activity and vanin-1 (Fig. 4A–B) with a permissive state for SMC proliferation to be induced by PDGF (Fig. 4C). Taken together, vanin-1 induced oxidative stress and enhanced SMC proliferation, doing so only partially by affecting PPARc expression in SMCs. Conversely, PPARc expression modulated sensitivity of SMC proliferation in response to oxidative stress. Vanin-1 Also Modulates SMC MMP Activity and Migration Diamide and PDGF, as well as cysteamine, induced MMP-9 activity more in WT than Vnn12/2 SMCs (Fig. 5A–B). In addition, vanin-1 deficiency significantly decreased both diamide- induced and PDGF-induced migration of cultured SMCs (Fig. 5C). Given the collective findings on SMC proliferation, oxidative stress, MMP activity, and migration in vanin-1 deficient SMCs, we concluded the studies by examining the role of vanin-1 in arterial remodeling and PPARc expression in response to carotid artery ligation in situ. Vanin-1 Deficiency Inhibits Post-injury Carotid Artery Neointimal Hyperplasia We observed robust development of neointima in WT mice following left carotid artery ligation, but this vascular remodeling injury response was attenuated in Vnn12/2 mice (Fig. 6A). Specifically, injured carotid arteries of Vnn12/2 mice displayed markedly decreased intima:media ratio (Fig. 6B) and cross sectional area of the neointima (Fig. 6C). There was more robust PPARc expression in injured Vnn12/2 arteries compared to WT arteries (Fig. 7A–B). Last, we observed decreased cell proliferation, assayed by Ki-67 staining, in both the media and neointima in the injured Vnn12/2 mouse arteries (Fig. 7C–D). Discussion Oxidative stress, including NADPH oxidase activity [2,26–30], and regulation of PPARc [25], are among the numerous factors implicated in activation of SMCs in vascular remodeling [21]. Given putatively redundant pathways for vascular remodeling, the net individual roles of GSH stores and PPARc in the process, let alone potential impact of their combined role, had not previously been clear. This study identified vanin-1 as a central mediator of oxidative stress and an inhibitor of constitutive PPARc expression in SMCs. Vanin-1 promoted GSH depletion and MMP activation, and vanin-1 critically mediated PDGF-induced proliferation and migration in cultured SMCs. The vanin-1 enzymatic product cysteamine also promoted oxidative stress, MMP activity, and decreased PPARc expression in SMCs. These findings were buttressed by the demonstration that vanin-1 knockout markedly limited in vivo post-injury neointima formation. Moreover, vanin-1 PLoS ONE | www.plosone.org 2 June 2012 | Volume 7 | Issue 6 | e39106