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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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Photomicrographs of post-angioplasty swine coronary artery tissue sections.Paraffin embedded thin sections were cut, deparaffinized and histological analysis was performed using H&E, and Masson's trichrome stains and immunohistochemical analysis was done for TNF-α, VDR, and α-SMA expression. For immunohistochemistry, sections were stained with DAB as chromogen and counterstained using hematoxylin. In the H&E staining, the fracture (arrows) in the internal elastic lamina (IEL) and neointimal thickening were observed (A–B). In the Masson's trichrome staining of the tissue sections, collagen deposition in the neointimal area was prominently present(C–D). TNF-α-positive cells in post-angioplasty coronary artery are shown by arrows (E–F). The α-SMA was thoroughly expressed both in neointima and medial layer (G–H). Expression of VDR was decreased in neointima compared to media (I–J). Quantification of VDR staining in neointima and media of post-angioplasty coronary arteries was done by NIH image J (K). Two-tailed unpaired student t tests were performed to determine statistical relevance. Data are shown as mean ± SEM. ***P<0.001. Negative control is shown (L–M). (N = 6) scale bar 100 µm, magnification (100×–400×); A: adventitia L: lumen, M: medial layer, NI:neointima.
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pone-0042789-g001: Photomicrographs of post-angioplasty swine coronary artery tissue sections.Paraffin embedded thin sections were cut, deparaffinized and histological analysis was performed using H&E, and Masson's trichrome stains and immunohistochemical analysis was done for TNF-α, VDR, and α-SMA expression. For immunohistochemistry, sections were stained with DAB as chromogen and counterstained using hematoxylin. In the H&E staining, the fracture (arrows) in the internal elastic lamina (IEL) and neointimal thickening were observed (A–B). In the Masson's trichrome staining of the tissue sections, collagen deposition in the neointimal area was prominently present(C–D). TNF-α-positive cells in post-angioplasty coronary artery are shown by arrows (E–F). The α-SMA was thoroughly expressed both in neointima and medial layer (G–H). Expression of VDR was decreased in neointima compared to media (I–J). Quantification of VDR staining in neointima and media of post-angioplasty coronary arteries was done by NIH image J (K). Two-tailed unpaired student t tests were performed to determine statistical relevance. Data are shown as mean ± SEM. ***P<0.001. Negative control is shown (L–M). (N = 6) scale bar 100 µm, magnification (100×–400×); A: adventitia L: lumen, M: medial layer, NI:neointima.

Mentions: The histological examination by H&E staining revealed significant neointimal formation in both left anterior descending (LAD) and left circumflex arteries (LCX) 4 months following PTCA. Ball0on injury resulted in medial rupture and clear disruption of internal elastic lamina (IEL) in all balloon-injured coronary arteries (Fig. 1A–B). Masson's trichrome staining revealed abundant collagen in neointima and in adjoining adventitia (Fig. 1C–D). Percent stenosis for vessels with balloon angioplasty was 50.4±4.1%. Non-injured vessels had intact IEL without any neointimal development. Immunohistological analysis showed increased TNF-α expression in SMCs of neointimal area compared to normal SMCs in the media (Fig. 1 E–F). Immunohistochemistry was performed to examine the expression of smooth muscle α-actin (α-SMA) in neointimal area. Strong expression of α-SMA in these lesions confirmed that vascular SMCs were the main cellular component of neointimal proliferative lesions (Fig. 1 G–H). These findings were consistent across multiple samples from six different animals.


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)

Photomicrographs of post-angioplasty swine coronary artery tissue sections.Paraffin embedded thin sections were cut, deparaffinized and histological analysis was performed using H&E, and Masson's trichrome stains and immunohistochemical analysis was done for TNF-α, VDR, and α-SMA expression. For immunohistochemistry, sections were stained with DAB as chromogen and counterstained using hematoxylin. In the H&E staining, the fracture (arrows) in the internal elastic lamina (IEL) and neointimal thickening were observed (A–B). In the Masson's trichrome staining of the tissue sections, collagen deposition in the neointimal area was prominently present(C–D). TNF-α-positive cells in post-angioplasty coronary artery are shown by arrows (E–F). The α-SMA was thoroughly expressed both in neointima and medial layer (G–H). Expression of VDR was decreased in neointima compared to media (I–J). Quantification of VDR staining in neointima and media of post-angioplasty coronary arteries was done by NIH image J (K). Two-tailed unpaired student t tests were performed to determine statistical relevance. Data are shown as mean ± SEM. ***P<0.001. Negative control is shown (L–M). (N = 6) scale bar 100 µm, magnification (100×–400×); A: adventitia L: lumen, M: medial layer, NI:neointima.
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pone-0042789-g001: Photomicrographs of post-angioplasty swine coronary artery tissue sections.Paraffin embedded thin sections were cut, deparaffinized and histological analysis was performed using H&E, and Masson's trichrome stains and immunohistochemical analysis was done for TNF-α, VDR, and α-SMA expression. For immunohistochemistry, sections were stained with DAB as chromogen and counterstained using hematoxylin. In the H&E staining, the fracture (arrows) in the internal elastic lamina (IEL) and neointimal thickening were observed (A–B). In the Masson's trichrome staining of the tissue sections, collagen deposition in the neointimal area was prominently present(C–D). TNF-α-positive cells in post-angioplasty coronary artery are shown by arrows (E–F). The α-SMA was thoroughly expressed both in neointima and medial layer (G–H). Expression of VDR was decreased in neointima compared to media (I–J). Quantification of VDR staining in neointima and media of post-angioplasty coronary arteries was done by NIH image J (K). Two-tailed unpaired student t tests were performed to determine statistical relevance. Data are shown as mean ± SEM. ***P<0.001. Negative control is shown (L–M). (N = 6) scale bar 100 µm, magnification (100×–400×); A: adventitia L: lumen, M: medial layer, NI:neointima.
Mentions: The histological examination by H&E staining revealed significant neointimal formation in both left anterior descending (LAD) and left circumflex arteries (LCX) 4 months following PTCA. Ball0on injury resulted in medial rupture and clear disruption of internal elastic lamina (IEL) in all balloon-injured coronary arteries (Fig. 1A–B). Masson's trichrome staining revealed abundant collagen in neointima and in adjoining adventitia (Fig. 1C–D). Percent stenosis for vessels with balloon angioplasty was 50.4±4.1%. Non-injured vessels had intact IEL without any neointimal development. Immunohistological analysis showed increased TNF-α expression in SMCs of neointimal area compared to normal SMCs in the media (Fig. 1 E–F). Immunohistochemistry was performed to examine the expression of smooth muscle α-actin (α-SMA) in neointimal area. Strong expression of α-SMA in these lesions confirmed that vascular SMCs were the main cellular component of neointimal proliferative lesions (Fig. 1 G–H). These findings were consistent across multiple samples from six different animals.

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