Vanin-1 in Vascular Remodeling Disease Figure 1. Constitutive vanin-1 expression in aorta and pantetheinase activity in cultured mouse aortic SMCs. A, Histologic sections of aortae from WT and Vnn12/2 mice were immunohistochemically stained for vanin-1 (brown positive staining). B, Vanin-1 analyzed by aortic tissue Western blotting. C, In isolated aortic SMCs, vanin-1 and vanin-3 isoenzyme mRNA expression levels were compared by real-time PCR, normalized to GAPDH mRNA in samples from WT and Vnn12/2 mice. *P,0.05, WT vs. Vnn12/2 control. D, Aortic sections were incubated with the substrate pantothenate–AMC and constitutive vanin-1 immunofluorescence (green) is shown in carotid sections of WT and Vnn12/2 mice. E, Pantetheinase activity is shown from cultured SMC lysates of mice of indicated genotypes; results from SMCs of different animals are shown as distinct plots, with each result the mean 6 SD of 3 independent experiments. *P,0.05 for WT vs. Vnn12/2. doi:10.1371/journal.pone.0039106.g001 deficiency resulted in reduced lesion cell proliferation and lesion PPARc expression relative to ligated WT control carotid arteries. In our in vitro studies, SMCs from Vnn12/2 mice failed to demonstrate increased migration in response to PDGF and diamide. We also observed that regardless of whether diamide or PDGF was promoting oxidative stress, vanin-1 played a crucial role in regulating generation of ROS. Diamide acts by formation of a protein-protein internal disulfide bond, without formation of a sulfenic acid intermediate [31]. PDGF-BB-induced AP-1 activity and cell proliferation are secondary to alkylation of cysteinyl residues, essential for the catalytic activities of various enzymes, transcription factors and/or transporters [32], and PDGF effects in SMCs were blocked by addition of catalase or antioxidants in prior studies [4]. It is noteworthy that SMCs express multiple enzymes that generate ROS, including phospholipases, cytochrome P450, cyclooxygenase, lipoxygenase, xanthine oxidase, and ribonucleotide reductase, as well as a functional NADPH oxidase complex [26–30]. Moreover, vascular NADPH oxidase and ROS mediate functionally significant signal trans- duction in SMCs [26–30]. Vanin-1 is likely active in regulating SMC function because cellular GSH homeostasis modulates not only oxidative stress but also pro-inflammatory sequelae [26]. In this context, GSH depletion is one mode for enhancement of inflammation signaling pathways such as p21ras, MAP kinase activity, and NF-kB nuclear translocation [26,33]. Our demon- stration of a major role of vanin-1 in depleting SMC GSH stores identifies a novel pro-inflammatory activation switch in SMCs. In this study, siRNA-induced silencing of PPARc, by itself, depleted SMC GSH levels and modulated the threshold for SMC activation in response to oxidative stress. Our results, for SMCs, treated with PPARc siRNA and pharmacologic PPARc inhibition, indicated that vanin-1 enhanced SMC activation responses in part by modulation of PPARc expression. PPARc exerts substantial effects on SMC proliferation in vitro and in vivo [19,20,34–36]. In addition, PPARc activation is an SMC anti-inflammatory control point that inhibits activation of NF-kB, and suppresses SMC proliferation induced by PDGF and angiotensin II [19,20,34–36]. PPARc activation also inhibits SMC invasiveness, and migration mediated by MMP activation [20]. Multiple overlapping mechanisms regulate PPARc expression in SMCs [19,20,35]. Importantly, lack of vanin-1 robustly inhibited PPARc expression in SMCs, and, conversely, PPARc expression in situ was increased constitutively, and after carotid arterial injury, in Vnn12/2 mouse arteries. Limitations of the current study include confinement of the scope of in vivo analyses to carotid artery ligation, and the inherent PLoS ONE | www.plosone.org 3 June 2012 | Volume 7 | Issue 6 | e39106