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

Activation of MMP-9 and migration are inhibited in <i>Vnn1</i><i>^−/−</i><b>SMCs. </b>

<p><b><i>A</i></b><i>,</i> Serum-starved SMCs were treated with PDGF (10 ng/ml), cysteamine (500 ng/ml) or diamide (5 µM) for 48 h, and conditioned media analyzed for MMP-9 activity by gelatin zymography. <b><i>B</i></b><i>,</i> Densitometric analysis of enhanced MMP-9 activity in WT compared to <i>Vnn1^−/−</i> SMCs. Data pooled from 3 independent experiments for densitometry. <b><i>C</i></b><i>,</i> We assayed migration in SMCs treated with diamide (5 µM) or PDGF (10 ng/ml), as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039106#s4" target="_blank">Methods</a>. Total number of SMCs migrated/well after 48 h treatment with diamide or PDGF are shown. Data are mean ± SD of 3 independent experiments. *P<0.05 vs. control.</p

Relationships between PPARγ expression, GSH content, and vanin-1 in arteries and cultured SMCs.

<p><i>A–B</i><i>,</i> We treated cultured aortic SMCs with diamide (5 µM) and/or PDGF (10 ng/ml) for 48 h and PPARγ expression was analyzed by Western blot, and densitometry. P<0.05 for ^#<i>Vnn1^−/−</i> control vs. WT control SMCs; *<i>Vnn1^−/−</i> control vs. diamide treated SMCs; **WT SMCs vs. PDGF and diamide treated SMCs, respectively. <b><i>C–D</i></b><i>,</i> SMCs were transfected with PPARγ siRNA, and then PPARγ expression analyzed by Western blot. *P<0.05 for <i>Vnn1^−/−</i> vs. WT SMCs, # <i>Vnn1^−/−</i> control vs. PPARγ siRNA, **PPARγ siRNA WT SMCs. <b><i>E</i></b><i>,</i> WT and <i>Vnn1^−/−</i> SMCs were treated with cysteamine (500 ng/ml) or BSO (1 µM), and GSH content determined after deproteinization<b>.</b> Data pooled from 3 experiments done in triplicate. ^# P<0.05 <i>Vnn1^−/−</i> vs. WT SMCs, *control vs. PPARγ siRNA,**control vs. cysteamine in both <i>Vnn1^−/−</i> and WT SMCs, ^Λ control vs. BSO treatment in both <i>Vnn1^−/−</i> and WT SMCs. In Panel <b><i>F</i></b>, WT and <i>Vnn1^−/−</i> SMCs were treated with diamide (5 µM) or PDGF 10 ng/ml) for 24 h, and cell proliferation compared. *P<0.05 vs. control. <b><i>G</i></b><i>,</i> SMC proliferation using PPARγ siRNA knockdown. Data are mean ± SD of 3 independent experiments. *P<0.05 control vs. PPARγ siRNA, **PPARγ siRNA vs. PPARγ siRNA + PDGF in WT and <i>Vnn1^−/−</i> SMCs.</p

Constitutive vanin-1 expression in aorta and pantetheinase activity in cultured mouse aortic SMCs.

<p><i>A</i><i>,</i> Histologic sections of aortae from WT and <i>Vnn1^−/−</i> mice were immunohistochemically stained for vanin-1 (brown positive staining). <b><i>B</i></b>, Vanin-1 analyzed by aortic tissue Western blotting. <b><i>C</i></b><i>,</i> 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 <i>Vnn1^−/−</i> mice. *P<0.05, WT vs. <i>Vnn1^−/−</i> control. <b><i>D</i></b><i>,</i> Aortic sections were incubated with the substrate pantothenate–AMC and constitutive vanin-1 immunofluorescence (green) is shown in carotid sections of WT and <i>Vnn1^−/−</i> mice. <b><i>E</i></b><i>,</i> 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 ± SD of 3 independent experiments. *P<0.05 for WT vs. <i>Vnn1^−/−.</i></p

Immunohistochemical staining for PPARγ and the cell proliferation marker Ki-67 in uninjured and injured carotid arteries.

<p><i>A</i><i>,</i> Paraffin-embedded, uninjured and injured aortae of the indicated genotypes were cut into 6 µm sections for immunohistochemistry, with PPARγ positive media and neointimal cells staining dark brown (arrowheads) in methyl green counterstained sections. <b><i>B</i></b><i>,</i> Percentage of arterial media and neointimal PPARγ positive cells, determined as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039106#s4" target="_blank">Methods</a>. <b><i>C</i></b><i>,</i> Immunohistochemical detection of Ki-67 in representative WT and <i>Vnn1^−/−</i> carotid sections. <b><i>D</i></b><i>,</i> Percentage of arterial media and neointimal Ki-67 positive cells. Data are mean ± SD of 3 independent experiments. *P<0.05 WT vs. <i>Vnn1^−/−</i>.</p

Vanin-1 mediates SMC proliferation in response to PDGF.

<p>Vanin-1 was expressed in <i>Vnn1^−/−</i> SMCs by transfection using pCMV-VNN1 expression vector. <b><i>A</i></b><i>,</i> Pantetheinase activity was measured in <i>Vnn1^−/−</i> SMCs after transfection and <b><i>B</i></b><i>,</i> Western blot analysis confirmed vanin-1 expression after transfection. <b><i>C</i></b><i>,</i> Following pCMV-VNN1 or empty vector transfection, SMCs were treated with PDGF (10 ng/ml) for 24 h, and proliferation measured. Data are mean ± SD of 3 independent experiments. *P<0.05 <i>Vnn1^−/−</i> SMCs control vs. <i>Vnn1^−/−</i> + pCMV-VNN1+ PDGF.</p

Vanin-1 and cysteamine modulate SMC superoxide production and GSH content in response to diamide and PDGF.

<p><i>A</i>, SMCs isolated from <i>Vnn1^−/−</i> and WT mouse aortae were growth-arrested in 0.1% calf serum for 24 h, and exposed to dihydroethidium (DHE) (10 µM). Images were captured 30 min after stimulation with diamide (5 µM) and PDGF (10 ng/ml). <b><i>B</i></b><i>,</i> Superoxide production was quantified by flow cytometry (excitation and emission wavelengths 488 nm and 610 nm, respectively). <b><i>C</i></b><i>,</i> WT and <i>Vnn1^−/−</i> SMCs were treated with PDGF (10 ng/ml) for 24 h, and pantetheinase activity measured (*WT+PDGF vs. <i>Vnn1^−/−</i> +PDGFand WT control). <b><i>D</i></b><i>,</i> In SMCs treated with cysteamine (500 ng/ml) or BSO (1 µM) for 48 h, DHE fluorescence was measured. <b><i>E</i></b><i>,</i> WT and <i>Vnn1^−/−</i> SMCs were treated with PDGF (10 ng/ml) for 24 h, and GSH content measured via enzymatic recycling assay. Data are mean ± SD of 3 independent experiments. *P<0.05 vs. control; **control vs. PDGF in WT, # control vs. diamide in WT and <i>Vnn1^−/−</i>. <b><i>F</i></b><i>,</i> 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 ± SD of 3 independent experiments.</p