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Decreased expression of vitamin D receptors in neointimal lesions following coronary artery angioplasty in atherosclerotic swine.

Gupta GK, Agrawal T, Del Core MG, Hunter WJ, Agrawal DK - PLoS ONE (2012)

Bottom Line: Interestingly, there was significantly decreased expression of VDR in PCASMCs of neointimal region compared to normal media.Calcitriol has anti-proliferative properties in PCASMCs and these actions are mediated through VDR.This could be a potential mechanism for uncontrolled growth of neointimal cells in injured arteries leading to restenosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Sciences and Center for Clinical and Translational Science, Creighton University School of Medicine, Omaha, Nebraska, United States of America.

ABSTRACT

Background: Inflammatory cytokines, such as TNF-α, play a key role in the pathogenesis of occlusive vascular diseases. Activation of vitamin D receptors (VDR) elicits both growth-inhibitory and anti-inflammatory effects. Here, we investigated the expression of TNF-α and VDR in post-angioplasty coronary artery neointimal lesions of hypercholesterolemic swine and examined the effect of vitamin D deficiency on the development of coronary restenosis. We also examined the effect of calcitriol on cell proliferation and effect of TNF-α on VDR activity and expression in porcine coronary artery smooth muscle cells (PCASMCs) in-vitro.

Methodology/principal findings: Expression of VDR and TNF-α and the effect of vitamin D deficiency in post-angioplasty coronary arteries were analyzed by immunohistochemistry and histomorphometry. Cell proliferation was examined by thymidine and BrdU incorporation assays in cultured PCASMCs. Effect of TNF-α-stimulation on the activity and expression of VDR was analyzed by luciferase assay, immunoblotting and immunocytochemistry. In-vivo, morphometric analysis of the tissues revealed typical lesions with significant neointimal proliferation. Histological evaluation showed expression of smooth muscle α-actin and significantly increased expression of TNF-α in neointimal lesions. Interestingly, there was significantly decreased expression of VDR in PCASMCs of neointimal region compared to normal media. Indeed, post-balloon angioplasty restenosis was significantly higher in vitamin D-deficient hypercholesterolemic swine compared to vitamin D-sufficient group. In-vitro, calcitriol inhibited both serum- and PDGF-BB-induced proliferation in PCASMCs and TNF-α-stimulation significantly decreased the expression and activity of VDR in PCASMCs.

Conclusions/significance: These data suggest that significant downregulation of VDR in proliferating smooth muscle cells in neointimal lesions could be due to atherogenic cytokines, including TNF-α. Vitamin D deficiency potentiates the development of coronary restenosis. Calcitriol has anti-proliferative properties in PCASMCs and these actions are mediated through VDR. This could be a potential mechanism for uncontrolled growth of neointimal cells in injured arteries leading to restenosis.

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Effect of calcitriol stimulation on VDR mRNA transcript, VDR protein expression, cell proliferation in PCASMCs.Calcitriol-stimulated PCASMCs were subjected to RNA and protein isolation for qPCR (A) and Western blot (B) and data was normalized against GAPDH. Quiescent PCASMCs were incubated with calcitriol in SMCM with 10% FBS for 24 h followed by [3H] thymidine incorporation assay (C). Quiescent PCASMCs were stimulated with 20 ng/ml PDGF-BB with calcitriol for 24 h, and BrdU incorporation was analyzed (D). Representative Western blot and quantification of VDR protein in PCASMCs transfected with scrambled and VDR-specific siRNA (E). The relative expression of VDR protein was analyzed using GAPDH as a control. VDR knocked-down PCASMCs were stimulated with 20 ng/ml PDGF-BB with calcitriol for 24 h, and BrdU incorporation was analyzed (F). Data is mean± SEM (N = 3). *P<0.05, **P<0.01, ***P<0.001 all compared to control.
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pone-0042789-g004: Effect of calcitriol stimulation on VDR mRNA transcript, VDR protein expression, cell proliferation in PCASMCs.Calcitriol-stimulated PCASMCs were subjected to RNA and protein isolation for qPCR (A) and Western blot (B) and data was normalized against GAPDH. Quiescent PCASMCs were incubated with calcitriol in SMCM with 10% FBS for 24 h followed by [3H] thymidine incorporation assay (C). Quiescent PCASMCs were stimulated with 20 ng/ml PDGF-BB with calcitriol for 24 h, and BrdU incorporation was analyzed (D). Representative Western blot and quantification of VDR protein in PCASMCs transfected with scrambled and VDR-specific siRNA (E). The relative expression of VDR protein was analyzed using GAPDH as a control. VDR knocked-down PCASMCs were stimulated with 20 ng/ml PDGF-BB with calcitriol for 24 h, and BrdU incorporation was analyzed (F). Data is mean± SEM (N = 3). *P<0.05, **P<0.01, ***P<0.001 all compared to control.

Mentions: Cultured PCASMCs were incubated with various concentrations of calcitriol (0.1–100 nM). Following 24 h incubation, the mRNA transcripts and protein expression of VDR in PCASMCs was investigated by RT-PCR and western blot analysis respectively. Both RT-PCR and western blot studies showed that calcitriol stimulation significantly up-regulated the mRNA (Fig. 4A) and protein (Fig. 4B) expression of VDR gene in a dose-dependent manner.


Decreased expression of vitamin D receptors in neointimal lesions following coronary artery angioplasty in atherosclerotic swine.

Gupta GK, Agrawal T, Del Core MG, Hunter WJ, Agrawal DK - PLoS ONE (2012)

Effect of calcitriol stimulation on VDR mRNA transcript, VDR protein expression, cell proliferation in PCASMCs.Calcitriol-stimulated PCASMCs were subjected to RNA and protein isolation for qPCR (A) and Western blot (B) and data was normalized against GAPDH. Quiescent PCASMCs were incubated with calcitriol in SMCM with 10% FBS for 24 h followed by [3H] thymidine incorporation assay (C). Quiescent PCASMCs were stimulated with 20 ng/ml PDGF-BB with calcitriol for 24 h, and BrdU incorporation was analyzed (D). Representative Western blot and quantification of VDR protein in PCASMCs transfected with scrambled and VDR-specific siRNA (E). The relative expression of VDR protein was analyzed using GAPDH as a control. VDR knocked-down PCASMCs were stimulated with 20 ng/ml PDGF-BB with calcitriol for 24 h, and BrdU incorporation was analyzed (F). Data is mean± SEM (N = 3). *P<0.05, **P<0.01, ***P<0.001 all compared to control.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3412822&req=5

pone-0042789-g004: Effect of calcitriol stimulation on VDR mRNA transcript, VDR protein expression, cell proliferation in PCASMCs.Calcitriol-stimulated PCASMCs were subjected to RNA and protein isolation for qPCR (A) and Western blot (B) and data was normalized against GAPDH. Quiescent PCASMCs were incubated with calcitriol in SMCM with 10% FBS for 24 h followed by [3H] thymidine incorporation assay (C). Quiescent PCASMCs were stimulated with 20 ng/ml PDGF-BB with calcitriol for 24 h, and BrdU incorporation was analyzed (D). Representative Western blot and quantification of VDR protein in PCASMCs transfected with scrambled and VDR-specific siRNA (E). The relative expression of VDR protein was analyzed using GAPDH as a control. VDR knocked-down PCASMCs were stimulated with 20 ng/ml PDGF-BB with calcitriol for 24 h, and BrdU incorporation was analyzed (F). Data is mean± SEM (N = 3). *P<0.05, **P<0.01, ***P<0.001 all compared to control.
Mentions: Cultured PCASMCs were incubated with various concentrations of calcitriol (0.1–100 nM). Following 24 h incubation, the mRNA transcripts and protein expression of VDR in PCASMCs was investigated by RT-PCR and western blot analysis respectively. Both RT-PCR and western blot studies showed that calcitriol stimulation significantly up-regulated the mRNA (Fig. 4A) and protein (Fig. 4B) expression of VDR gene in a dose-dependent manner.

Bottom Line: Interestingly, there was significantly decreased expression of VDR in PCASMCs of neointimal region compared to normal media.Calcitriol has anti-proliferative properties in PCASMCs and these actions are mediated through VDR.This could be a potential mechanism for uncontrolled growth of neointimal cells in injured arteries leading to restenosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Sciences and Center for Clinical and Translational Science, Creighton University School of Medicine, Omaha, Nebraska, United States of America.

ABSTRACT

Background: Inflammatory cytokines, such as TNF-α, play a key role in the pathogenesis of occlusive vascular diseases. Activation of vitamin D receptors (VDR) elicits both growth-inhibitory and anti-inflammatory effects. Here, we investigated the expression of TNF-α and VDR in post-angioplasty coronary artery neointimal lesions of hypercholesterolemic swine and examined the effect of vitamin D deficiency on the development of coronary restenosis. We also examined the effect of calcitriol on cell proliferation and effect of TNF-α on VDR activity and expression in porcine coronary artery smooth muscle cells (PCASMCs) in-vitro.

Methodology/principal findings: Expression of VDR and TNF-α and the effect of vitamin D deficiency in post-angioplasty coronary arteries were analyzed by immunohistochemistry and histomorphometry. Cell proliferation was examined by thymidine and BrdU incorporation assays in cultured PCASMCs. Effect of TNF-α-stimulation on the activity and expression of VDR was analyzed by luciferase assay, immunoblotting and immunocytochemistry. In-vivo, morphometric analysis of the tissues revealed typical lesions with significant neointimal proliferation. Histological evaluation showed expression of smooth muscle α-actin and significantly increased expression of TNF-α in neointimal lesions. Interestingly, there was significantly decreased expression of VDR in PCASMCs of neointimal region compared to normal media. Indeed, post-balloon angioplasty restenosis was significantly higher in vitamin D-deficient hypercholesterolemic swine compared to vitamin D-sufficient group. In-vitro, calcitriol inhibited both serum- and PDGF-BB-induced proliferation in PCASMCs and TNF-α-stimulation significantly decreased the expression and activity of VDR in PCASMCs.

Conclusions/significance: These data suggest that significant downregulation of VDR in proliferating smooth muscle cells in neointimal lesions could be due to atherogenic cytokines, including TNF-α. Vitamin D deficiency potentiates the development of coronary restenosis. Calcitriol has anti-proliferative properties in PCASMCs and these actions are mediated through VDR. This could be a potential mechanism for uncontrolled growth of neointimal cells in injured arteries leading to restenosis.

Show MeSH
Related in: MedlinePlus