Vanin-1 in Vascular Remodeling Disease Figure 2. Vanin-1 and cysteamine modulate SMC superoxide production and GSH content in response to diamide and PDGF. A, SMCs isolated from Vnn12/2 and WT mouse aortae were growth-arrested in 0.1% calf serum for 24 h, and exposed to dihydroethidium (DHE) (10 mM). Images were captured 30 min after stimulation with diamide (5 mM) and PDGF (10 ng/ml). B, Superoxide production was quantified by flow cytometry (excitation and emission wavelengths 488 nm and 610 nm, respectively). C, WT and Vnn12/2 SMCs were treated with PDGF (10 ng/ml) for 24 h, and pantetheinase activity measured (*WT+PDGF vs. Vnn12/2 +PDGFand WT control). D, In SMCs treated with cysteamine (500 ng/ml) or BSO (1 mM) for 48 h, DHE fluorescence was measured. E, WT and Vnn12/2 SMCs were treated with PDGF (10 ng/ml) for 24 h, and GSH content measured via enzymatic recycling assay. Data are mean 6 SD of 3 independent experiments. *P,0.05 vs. control; **control vs. PDGF in WT, # control vs. diamide in WT and Vnn12/2. F, SMC superoxide production in response to cysteamine and BSO treatment was quantified by flow cytometry (excitation and emission wavelengths 488 nm and 610 nm, respectively). Data are mean 6 SD of 3 independent experiments. doi:10.1371/journal.pone.0039106.g002 limits of in vitro experiments in SMCs. It remains to be determined what effects vanin-1 has in endothelial cells, macrophages, and in other resident and infiltrating cell types in the artery wall that modulate vascular remodeling. Vanin-1 pantetheinase effects on individual elements in ROS generation, such as NADPH oxidase activity and mitochondrial activities, merit further investigation. 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 [26–30]. Moreover, PPARc activity has been difficult to target for vascular and other disease in clinical medicine, since PPARc activating thiazolidine- diones (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. Methods Materials Diamide (1,1’-azobis(N,N-dimethylformamide)), PDGF-BB, and all chemical reagents were obtained from Sigma-Aldrich (St. Louis, MO), unless otherwise indicated. Antibodies against b-actin and Vanin-1 were from Cell Signaling (Boston, MA) and Santa Cruz Biotechnology, Inc (Santa Cruz, CA) respectively. b-alanine 7-amido-4-methylcoumarin trifluoroacetic acid was from Chem- Impex International Inc (Wood Dale, IL). The GSH assay kit was from Cayman Chemicals (Ann Arbor, MI) and the cell prolifer- ation assay kit was from Promega (Madison, WI). DHE was from Molecular Probes (Grand Island, NY). Anti-Ki-67 antibody was from Abcam (Kendall Square, Cambridge, MA). 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 Vnn12/2 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% parafor- maldehyde. 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 Vnn12/2 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 Biotech- nology (Santa Cruz Biotechnology, Inc. Santa Cruz, CA). Transfection was done by manufacturer recommended protocol. Vnn12/2 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. Quantitative Real-time RT-PCR Total RNA was isolated using Trizol (Invitrogen, San Diego, CA), and cDNA was transcribed from 1 mg of RNA using Superscript III enzyme (Invitrogen), and random primers. Reverse transcription and quantitative real-time RT-PCR were performed using the LightCycler 2.0 (Roche Diagnostics, San Francisco, CA), as described [38]. Oligonucleotides were designed for mouse vanin-1, using Roche Primer Express software: vanin-1, forward 5’-TGGTAGTTCAGTGGACACG-3’, and reverse 5’- AGG- GAAGACATACCGGG-3’, and mouse vanin-3, 5- CCGTTTGGGAAGTTTGGC-3’ and reverse 5’-CGAATG- GAATGGAACTGCTGA-3’. Following amplification, a mono- color relative quantification of the target gene and reference (glyceraldehyde-3- phosphate dehydrogenase; (GAPDH, F 59- CATCCCAGAGCTGAACG-39, R 59- CTGGTCCTCAGTG- TAGCC-39) was normalized for target gene to GAPDH mRNA (glyceraldehyde-3-phosphate) copy ratios using the manufacturer’s LightCycler software (version 4.0). PLoS ONE | www.plosone.org 5 June 2012 | Volume 7 | Issue 6 | e39106