Vanin-1 Pantetheinase Drives Smooth Muscle Cell Activation in Post-Arterial Injury Neointimal Hyperplasia

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 (PPARγ) expression. Vascular smooth muscle cells (SMCs) contribute to neointimal hyperplasia in response to injury, by multiple mechanisms including modulation of oxidative stress and PPARγ. 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. Vnn1 −/− 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 PPARγ expression. Vnn1−/− 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 PPARγ, 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.


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 promotes inflammation partly by suppression of both PPARc expression and PPARc signal transduction [15]. PPARc functions as an anti-inflammatory checkpoint in multiple inflammatory settings, and in a variety of cell types [18]. PPARcactivating 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 SMCs, and in vivo after balloon injury in rat aorta [21][22][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.

Expression of Vnn1 and Vnn3 and Pantetheinase Activity in SMCs
Vanin-1 was constitutively expressed in WT mouse aorta but not in Vnn1 2/2 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 Vnn1 2/2 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 Vnn1 2/2 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 Vnn1 2/2 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 Vnn1 2/2 SMCs (Fig. 2C). Treatment with the vanin-1 enzymatic product cysteamine, a cGCS inhibitor, increased ROS levels in both WT and Vnn1 2/2 SMCs as did treatment with another GSHdepleting cGCS inhibitor buthionine sulfoximine (BSO; 1 mM) ( Fig. 2 D,E). GSH levels in Vnn1 2/2 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 Vnn1 2/2 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 Vnn1 2/2 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, Vnn1 2/2 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 Vnn1 2/2 SMCs (Fig. 3B). In contrast, diamide significantly reduced PPARc levels in WT SMCs compared to Vnn1 2/2 SMCs.
Second, when we effectively knocked down PPARc via siRNA (Fig. 3C,D), we observed decreased GSH levels in both WT and Vnn1 2/2 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 Vnn1 2/ 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 Vnn1 2/2 SMCs (Fig. 3G). Similarly, Vnn1 2/2 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 Vnn1 2/2 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 Vnn1 2/2 SMCs ( Fig. 5A-B). In addition, vanin-1 deficiency significantly decreased both diamideinduced 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 Vnn1 2/2 mice (Fig. 6A). Specifically, injured carotid arteries of Vnn1 2/2 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 Vnn1 2/2 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 Vnn1 2/2 mouse arteries ( Fig. 7C-D).

Discussion
Oxidative stress, including NADPH oxidase activity [2,[26][27][28][29][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 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 Vnn1 2/2 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][27][28][29][30]. Moreover, vascular NADPH oxidase and ROS mediate functionally significant signal transduction in SMCs [26][27][28][29][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 demonstration 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][35][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][35][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 Vnn1 2/2 mouse arteries.
Limitations of the current study include confinement of the scope of in vivo analyses to carotid artery ligation, and the inherent We also restricted our analyses on PPAR to PPARc. Net effects of vanin-1 on individual PPARc isoforms, let alone potential effects on other PPARs that affect the artery [35], remain to be investigated. Oxidative stress is difficult to target in arteries and other tissues, since multiple mechanisms contribute to reactive oxygen species generation and elimination [26][27][28][29][30]. Moreover, PPARc activity has been difficult to target for vascular and other disease in clinical medicine, since PPARc activating thiazolidinediones (TZD) drugs can cause side effects, including severe and potentially lethal fluid retention mediated by renal effects of PPARc activation [37]. In this context, it is noteworthy that vanin-1 knockout mice are viable and grossly normal. Our study reveals vanin-1 to be a novel inflammatory switch for vascular remodeling diseases, via dual effects on PPARc and oxidative stress.

Mice Studied
All animal procedures were performed humanely and followed institutionally approved protocols, with procedures in compliance with the standards for care and use of laboratory animals of the Institute of Laboratory Animal Resource. Vnn1 +/2 mice were backcrossed for more than nine generations on a C57BL/6 background, and then interbred to generate and study Vnn1 2/2 mice and wild-type littermate progeny on the same background, as described [10,38]. All animal experimentation was assessed and approved by the IACUC (Institutional Animal Care and Use Committee) of the San Diego Veterans Affairs Medical Center.

Carotid Artery Ligation
Animals were anesthetized by intraperitoneal (i.p.) injection of 15 mg/kg Ketamine (phoenix pharmaceutical inc, ST. Joseph, MO), 1.6 mg/kg Xylazine (Akorn Inc, Decatur, IL), and 1.2 mg/ kg Acepromazine (Boehringer Ingelheim, Ridgefield, CT). In brief, the left common carotid artery, dissected from surrounding connective tissue, was ligated through a midline neck incision just proximal to its bifurcation, using 6-0 silk ligature. In control groups, dissection of the left common carotid artery from the surrounding connective tissue was performed without ligation. Twenty-one days after injury or simple dissection, animals were anaesthetized and perfused with PBS, followed by 4% paraformaldehyde. Carotid arteries were excised, and then embedded in paraffin. Cross-sections (6 microns) were taken starting at the ligation site and stained with hematoxylin and eosin.

SMC Culture and Transfection
Mouse aortic SMCs were isolated by enzymatic digestion [39] from Vnn1 2/2 and control littermate WT mice. Cells were cultured in DMEM supplemented with 10% FBS, 100 Units/ml penicillin, 100 mg/ml streptomycin, and 2 mM L-glutamine at 37uC in a humidified 95% air and 5% CO2 incubator. The purity of each mouse SMC preparation in culture was confirmed by immunocytochemistry for a-smooth muscle actin. Cells were passaged at 1:3 ratio. Experiments were performed using cells between passage 3-8, and serum-deprived conditions were generated by incubation for 24 h in DMEM containing 0.1% FBS. The small interfering RNA (siRNA) and scrambled RNA (scRNA) employed were purchased from Santa Cruz Biotechnology (Santa Cruz Biotechnology, Inc. Santa Cruz, CA). Transfection was done by manufacturer recommended protocol. Vnn1 2/2 SMCs were transfected with pCMV-VNN1 full-length plasmid DNA using 293T transient overexpression reagent (Abnova, Walnut, CA, Catalog # H00008876-T01), as per manufacturer instructions.

Quantification of GSH
To determine GSH and oxidized glutathione levels, we used an enzymatic recycling assay (glutathione assay kit, Cayman Chemicals, Ann Arbor, MI) in the presence of glutathione reductase. We spectrophotometrically determined 5-thio-2-nitrobenzoic acid generation in deproteinated cells [38].

Measurement of Superoxide Generation
Superoxide generation in SMCs was measured, as described [41], in cells incubated with dihydroethidium (DHE) (10 mM) in PBS at 37uC in the dark for 30 min in a 5% CO2 humidified chamber. Propidium iodide (10 mg/ml) was added 1 min before flow cytometry and examined by FACS with excitation at 488 and emission at 610 nm. Flow cytometry (FACScan; BD Biosciences, San Jose, CA) was used to select a homogeneous population of 10,000 live cells. Bivariate flow cytometry was performed with a FACS scan, and the data were analyzed with Cell Quest software (Becton Dickinson, San Jose, CA, USA), in the cell population from which apoptotic cells were gated out against forward and side scatter or PI-positivity. The geometrical mean of ethidium fluorescence intensity (excitation 488 and emission at 610 nm) in thepopulation was used for analysis. Alternatively, samples were examined by fluorescence microscopy (Advanced Microscopy Group, EVOS FL).

Immunohistochemistry
Formalin-fixed and paraffin-embedded tissue sections were deparaffinized and followed by rehydration, endogenous peroxidase activity was quenched using 3% H2O2. Followed by blocking, the sections were incubated overnight at 4uC with primary antibody against vanin-1, PPARc (1:100) and Ki-67 (1:100) as a cell proliferation marker, sections were then incubated with biotinylated secondary antibody and peroxidase-labeled (Invitrogen, Carlsbad, CA). Peroxidase activity was demonstrated by exposing sections to the substrate, 3,39-diaminobenzidine tetrahydrochloride (DAB) and counterstaining with 1% methyl green. For negative control sections, PBS was substituted for the primary antibody. We calculated percentage of positive staining SMCs in media and neointima (relative to total cell number) in 5 different 20 X magnification fields in each section, studying 8 sections from each control and ligated sample from each mouse.

Assay of MMP Activity
Gelatinase activity was determined by zymography as previously described [42,43]. Equal amounts of conditioned media from identical numbers of cells, grown under serum free conditions for 48 h, were loaded onto 10% SDS-polyacrylamide gels containing 0.1% gelatin, and zymography was performed. After electrophoresis, gels were incubated for 45 min in renaturation buffer and in developing buffer (Invitrogen, Carlsbad, CA) for 24 h at 37uC. Gels were stained with Coomassie blue; clear bands indicated active enzymes (MMP-9 pro-form, 92 kDa; active form, 83 kDa). Enzymatic activity was estimated by densitometry of negative-image zymographic gels and measured in OD units.

Cell Proliferation and Migration Studies
We used the Invitrogen non-radioactive Cell Proliferation Assay kit, per manufacturer instructions, to determine cell proliferation. SMC migration was assayed using the Transwell system (Corning, NY), with a polycarbonate membrane in six-well plates. SMCs were plated at a concentration of 1.0x 10 6 cells/ml. Numbers of cells migrated per well, after 48 h treatment with PDGF or diamide, were determined by thiozolidine bluestaining. Images were analyzed using a microscope with digital camera (Advanced Microscopy Group, Bothell, WA).

Statistical Analyses
Unless otherwise indicated, data are presented as the mean 6 SD of determinations from 3 or more experiments. Results were compared by one-way ANOVA followed by Bonferroni multiplecomparison analysis. A value of p,0.05 was used to define statistical significance.