Limits...
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.

Show MeSH

Related in: MedlinePlus

Effect of TNF-α on VDR expression in PCASMCs.(A) PCASMCs cultured in chamber slides were quiesced before addition of 10 nM calcitriol and/or 10 ng/ml TNF-α for 24 h. PCASMCs were fixed, permeabilized and stained with mouse anti-VDR and goat anti-mouse cy3 as secondary antibody. Red color indicates VDR expression and nuclei are stained blue with DAPI. PCASMCs were stained with Alexa Fluor 488 phalloidin as green for filamentous actin. Scale Bar 50 µm, Magnification 200×. (B) PCASMCs were treated with TNF-α (10 ng/ml) and/or calcitriol (10 nM) for 24 hr. Total protein was isolated followed by Western-blot analysis. Relative expression of VDR protein was analyzed against GAPDH. (C) PCASMCs transiently transfected with pVDRE-Luc were treated with 10 ng/ml TNF-α for 24 h in the absence or presence of 1onM calcitriol and subjected to luciferase assay. Fold change in relative luciferase activity was assessed relative to control. Data is shown as mean ± SEM (N = 3). *P <0.05, **P<0.01, ***P<0.001.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3412822&req=5

pone-0042789-g006: Effect of TNF-α on VDR expression in PCASMCs.(A) PCASMCs cultured in chamber slides were quiesced before addition of 10 nM calcitriol and/or 10 ng/ml TNF-α for 24 h. PCASMCs were fixed, permeabilized and stained with mouse anti-VDR and goat anti-mouse cy3 as secondary antibody. Red color indicates VDR expression and nuclei are stained blue with DAPI. PCASMCs were stained with Alexa Fluor 488 phalloidin as green for filamentous actin. Scale Bar 50 µm, Magnification 200×. (B) PCASMCs were treated with TNF-α (10 ng/ml) and/or calcitriol (10 nM) for 24 hr. Total protein was isolated followed by Western-blot analysis. Relative expression of VDR protein was analyzed against GAPDH. (C) PCASMCs transiently transfected with pVDRE-Luc were treated with 10 ng/ml TNF-α for 24 h in the absence or presence of 1onM calcitriol and subjected to luciferase assay. Fold change in relative luciferase activity was assessed relative to control. Data is shown as mean ± SEM (N = 3). *P <0.05, **P<0.01, ***P<0.001.

Mentions: To ascertain whether the downregulation of VDR by TNF-α is maintained in vitro, PCASMCs were treated with TNF-α (10 ng/ml) and/or calcitriol (10 nM) for 24 h and protein expression of VDR was analyzed by Western blot and immunocytochemistry analysis. Calcitriol stimulation significantly upregulated VDR expression whereas TNF-α treatment significantly downregulated the protein expression of VDR. However, stimulation with calcitriol abolished the TNF-α-induced downregulation of protein expression of VDR (Fig. 6A). The findings from the immunocytochemical studies were consistent with the Western blot results (Fig. 6B).


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 TNF-α on VDR expression in PCASMCs.(A) PCASMCs cultured in chamber slides were quiesced before addition of 10 nM calcitriol and/or 10 ng/ml TNF-α for 24 h. PCASMCs were fixed, permeabilized and stained with mouse anti-VDR and goat anti-mouse cy3 as secondary antibody. Red color indicates VDR expression and nuclei are stained blue with DAPI. PCASMCs were stained with Alexa Fluor 488 phalloidin as green for filamentous actin. Scale Bar 50 µm, Magnification 200×. (B) PCASMCs were treated with TNF-α (10 ng/ml) and/or calcitriol (10 nM) for 24 hr. Total protein was isolated followed by Western-blot analysis. Relative expression of VDR protein was analyzed against GAPDH. (C) PCASMCs transiently transfected with pVDRE-Luc were treated with 10 ng/ml TNF-α for 24 h in the absence or presence of 1onM calcitriol and subjected to luciferase assay. Fold change in relative luciferase activity was assessed relative to control. Data is shown as mean ± SEM (N = 3). *P <0.05, **P<0.01, ***P<0.001.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3412822&req=5

pone-0042789-g006: Effect of TNF-α on VDR expression in PCASMCs.(A) PCASMCs cultured in chamber slides were quiesced before addition of 10 nM calcitriol and/or 10 ng/ml TNF-α for 24 h. PCASMCs were fixed, permeabilized and stained with mouse anti-VDR and goat anti-mouse cy3 as secondary antibody. Red color indicates VDR expression and nuclei are stained blue with DAPI. PCASMCs were stained with Alexa Fluor 488 phalloidin as green for filamentous actin. Scale Bar 50 µm, Magnification 200×. (B) PCASMCs were treated with TNF-α (10 ng/ml) and/or calcitriol (10 nM) for 24 hr. Total protein was isolated followed by Western-blot analysis. Relative expression of VDR protein was analyzed against GAPDH. (C) PCASMCs transiently transfected with pVDRE-Luc were treated with 10 ng/ml TNF-α for 24 h in the absence or presence of 1onM calcitriol and subjected to luciferase assay. Fold change in relative luciferase activity was assessed relative to control. Data is shown as mean ± SEM (N = 3). *P <0.05, **P<0.01, ***P<0.001.
Mentions: To ascertain whether the downregulation of VDR by TNF-α is maintained in vitro, PCASMCs were treated with TNF-α (10 ng/ml) and/or calcitriol (10 nM) for 24 h and protein expression of VDR was analyzed by Western blot and immunocytochemistry analysis. Calcitriol stimulation significantly upregulated VDR expression whereas TNF-α treatment significantly downregulated the protein expression of VDR. However, stimulation with calcitriol abolished the TNF-α-induced downregulation of protein expression of VDR (Fig. 6A). The findings from the immunocytochemical studies were consistent with the Western blot results (Fig. 6B).

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