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Impaired Vitamin D Signaling in Endothelial Cell Leads to an Enhanced Leukocyte-Endothelium Interplay: Implications for Atherosclerosis Development.

Bozic M, Álvarez Á, de Pablo C, Sanchez-Niño MD, Ortiz A, Dolcet X, Encinas M, Fernandez E, Valdivielso JM - PLoS ONE (2015)

Bottom Line: Knockdown of VDR in endothelial cells (shVDR) led to endothelial cell activation, characterized by upregulation of VCAM-1, ICAM-1 and IL-6, decreased peripheral blood mononuclear cell (PBMC) rolling velocity and increased PBMC rolling flux and adhesion to the endothelium. shVDR cells showed decreased IκBα levels and accumulation of p65 in the nucleus compared to shRNA controls.In vivo, deletion of VDR led to significantly larger aortic arch and aortic root lesions in apoE-/- mice, with higher macrophage content. apoE-/-VDR-/-mice showed higher aortic expression of VCAM-1, ICAM-1 and IL-6 when compared to apoE-/-VDR+/+ mice.Our data demonstrate that lack of VDR signaling in endothelial cells leads to a state of endothelial activation with increased leukocyte-endothelial cell interactions that may contribute to the more severe plaque accumulation observed in apoE-/-VDR-/- mice.

View Article: PubMed Central - PubMed

Affiliation: Nephrology Research Department, IRB Lleida, Lleida, Spain.

ABSTRACT
Endothelial cell activation leading to leukocyte recruitment and adhesion plays an essential role in the initiation and progression of atherosclerosis. Vitamin D has cardioprotective actions, while its deficiency is a risk factor for the progression of cardiovascular damage. Our aim was to assess the role of basal levels of vitamin D receptor (VDR) on the early leukocyte recruitment and related endothelial cell-adhesion-molecule expression, as essential prerequisites for the onset of atherosclerosis. Knockdown of VDR in endothelial cells (shVDR) led to endothelial cell activation, characterized by upregulation of VCAM-1, ICAM-1 and IL-6, decreased peripheral blood mononuclear cell (PBMC) rolling velocity and increased PBMC rolling flux and adhesion to the endothelium. shVDR cells showed decreased IκBα levels and accumulation of p65 in the nucleus compared to shRNA controls. Inhibition of NF-κB activation with super-repressor IκBα blunted all signs of endothelial cell activation caused by downregulation of VDR in endothelial cells. In vivo, deletion of VDR led to significantly larger aortic arch and aortic root lesions in apoE-/- mice, with higher macrophage content. apoE-/-VDR-/-mice showed higher aortic expression of VCAM-1, ICAM-1 and IL-6 when compared to apoE-/-VDR+/+ mice. Our data demonstrate that lack of VDR signaling in endothelial cells leads to a state of endothelial activation with increased leukocyte-endothelial cell interactions that may contribute to the more severe plaque accumulation observed in apoE-/-VDR-/- mice. The results reveal an important role for basal levels of endothelial VDR in limiting endothelial cell inflammation and atherosclerosis.

No MeSH data available.


Related in: MedlinePlus

VDR knockdown-induced endothelial cell activation is mediated via NF-κB signaling.EA.hy926 cells (shc002 or shVDR) were untreated (A, B, C) or treated with IκB kinase (IKK) inhibitor PS-1145 (D) for 24 hours. (A, B, D) Whole cell lysates were immunoblotted with antibodies against VDR, IκB-α, VCAM-1 and ICAM-1. The same samples were reprobed with tubulin to ensure equal loading. (C) Western blot analysis of p65 levels in cytosolic and nuclear extracts isolated from shc002 and shVDR endothelial cells. The nuclear protein Histone 1, which is absent in the cytosolic fraction, served as a nuclear protein loading control. (D) Representative Western blot and quantitative analysis (G, H), *p<0.05 vs. shc002 or shVDR, respectivey (E) Real time PCR analysis of IL-6 mRNA in shc002 and shVDR endothelial cells. Data presented are mean ± SEM from 3 independent experiments. **p<0.01 vs. shc002. (F) Determination of IL-6 secretion into the medium by ELISA. Endothelial cells were grown in normal growth media for 72 hours. IL-6 production was determined using a human IL-6 HS ELISA kit. Data presented are mean ± SEM from 3 independent experiments. *p<0.05 vs. shc002.
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pone.0136863.g002: VDR knockdown-induced endothelial cell activation is mediated via NF-κB signaling.EA.hy926 cells (shc002 or shVDR) were untreated (A, B, C) or treated with IκB kinase (IKK) inhibitor PS-1145 (D) for 24 hours. (A, B, D) Whole cell lysates were immunoblotted with antibodies against VDR, IκB-α, VCAM-1 and ICAM-1. The same samples were reprobed with tubulin to ensure equal loading. (C) Western blot analysis of p65 levels in cytosolic and nuclear extracts isolated from shc002 and shVDR endothelial cells. The nuclear protein Histone 1, which is absent in the cytosolic fraction, served as a nuclear protein loading control. (D) Representative Western blot and quantitative analysis (G, H), *p<0.05 vs. shc002 or shVDR, respectivey (E) Real time PCR analysis of IL-6 mRNA in shc002 and shVDR endothelial cells. Data presented are mean ± SEM from 3 independent experiments. **p<0.01 vs. shc002. (F) Determination of IL-6 secretion into the medium by ELISA. Endothelial cells were grown in normal growth media for 72 hours. IL-6 production was determined using a human IL-6 HS ELISA kit. Data presented are mean ± SEM from 3 independent experiments. *p<0.05 vs. shc002.

Mentions: To assess the role of VDR in leukocyte-endothelial interplay in basal conditions, the expression of VDR in endothelial cells was disrupted by shRNA delivery. Fig 1D shows an evident decrease of VDR expression in EA.hy926 cells infected with pLKO.1-VDR (shVDR) compared with pLKO.1-SHC002 (shc002 control). Leukocyte-endothelial interactions were evaluated using a well-established in vitro dynamic flow chamber system designed to visualize and analyze multistep recruitment of leukocytes in diseases such as atherosclerotic vascular disease [40]. Knockdown of VDR in endothelial cells (shVDR) led to endothelial cell activation, seen as a decrease in the rolling velocity of human PBMC over the endothelium (shc002: 704.2±66.54, shVDR: 438.04±55.29 μm/sec) (Fig 1A) and an increase in PBMC rolling flux (shc002: 83.25±5.74, shVDR: 150.3±16.59 cells/min) (Fig 1B) and adhesion (shc002: 3.78±1.28, shVDR: 10.23±1.11 cells/mm2) (Fig 1C) (S1 and S2 Videos). To further assess the role of VDR in endothelial cell activation, we analyzed the expression of vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) in the same groups of cells. Knockdown of VDR in endothelial cells (shVDR) (Fig 1D and 1E) led to a significant increase of VCAM-1 (Fig 1D and 1F) and ICAM-1 (Fig 1D and 1G), compared with shc002 control. Furthermore, in our experimental model, downregulation of VDR led to a significant elevation of IL-6 mRNA, as well as of IL-6 secretion in shVDR cells compared with the corresponding controls (Fig 2E and 2F, respectively).


Impaired Vitamin D Signaling in Endothelial Cell Leads to an Enhanced Leukocyte-Endothelium Interplay: Implications for Atherosclerosis Development.

Bozic M, Álvarez Á, de Pablo C, Sanchez-Niño MD, Ortiz A, Dolcet X, Encinas M, Fernandez E, Valdivielso JM - PLoS ONE (2015)

VDR knockdown-induced endothelial cell activation is mediated via NF-κB signaling.EA.hy926 cells (shc002 or shVDR) were untreated (A, B, C) or treated with IκB kinase (IKK) inhibitor PS-1145 (D) for 24 hours. (A, B, D) Whole cell lysates were immunoblotted with antibodies against VDR, IκB-α, VCAM-1 and ICAM-1. The same samples were reprobed with tubulin to ensure equal loading. (C) Western blot analysis of p65 levels in cytosolic and nuclear extracts isolated from shc002 and shVDR endothelial cells. The nuclear protein Histone 1, which is absent in the cytosolic fraction, served as a nuclear protein loading control. (D) Representative Western blot and quantitative analysis (G, H), *p<0.05 vs. shc002 or shVDR, respectivey (E) Real time PCR analysis of IL-6 mRNA in shc002 and shVDR endothelial cells. Data presented are mean ± SEM from 3 independent experiments. **p<0.01 vs. shc002. (F) Determination of IL-6 secretion into the medium by ELISA. Endothelial cells were grown in normal growth media for 72 hours. IL-6 production was determined using a human IL-6 HS ELISA kit. Data presented are mean ± SEM from 3 independent experiments. *p<0.05 vs. shc002.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4556440&req=5

pone.0136863.g002: VDR knockdown-induced endothelial cell activation is mediated via NF-κB signaling.EA.hy926 cells (shc002 or shVDR) were untreated (A, B, C) or treated with IκB kinase (IKK) inhibitor PS-1145 (D) for 24 hours. (A, B, D) Whole cell lysates were immunoblotted with antibodies against VDR, IκB-α, VCAM-1 and ICAM-1. The same samples were reprobed with tubulin to ensure equal loading. (C) Western blot analysis of p65 levels in cytosolic and nuclear extracts isolated from shc002 and shVDR endothelial cells. The nuclear protein Histone 1, which is absent in the cytosolic fraction, served as a nuclear protein loading control. (D) Representative Western blot and quantitative analysis (G, H), *p<0.05 vs. shc002 or shVDR, respectivey (E) Real time PCR analysis of IL-6 mRNA in shc002 and shVDR endothelial cells. Data presented are mean ± SEM from 3 independent experiments. **p<0.01 vs. shc002. (F) Determination of IL-6 secretion into the medium by ELISA. Endothelial cells were grown in normal growth media for 72 hours. IL-6 production was determined using a human IL-6 HS ELISA kit. Data presented are mean ± SEM from 3 independent experiments. *p<0.05 vs. shc002.
Mentions: To assess the role of VDR in leukocyte-endothelial interplay in basal conditions, the expression of VDR in endothelial cells was disrupted by shRNA delivery. Fig 1D shows an evident decrease of VDR expression in EA.hy926 cells infected with pLKO.1-VDR (shVDR) compared with pLKO.1-SHC002 (shc002 control). Leukocyte-endothelial interactions were evaluated using a well-established in vitro dynamic flow chamber system designed to visualize and analyze multistep recruitment of leukocytes in diseases such as atherosclerotic vascular disease [40]. Knockdown of VDR in endothelial cells (shVDR) led to endothelial cell activation, seen as a decrease in the rolling velocity of human PBMC over the endothelium (shc002: 704.2±66.54, shVDR: 438.04±55.29 μm/sec) (Fig 1A) and an increase in PBMC rolling flux (shc002: 83.25±5.74, shVDR: 150.3±16.59 cells/min) (Fig 1B) and adhesion (shc002: 3.78±1.28, shVDR: 10.23±1.11 cells/mm2) (Fig 1C) (S1 and S2 Videos). To further assess the role of VDR in endothelial cell activation, we analyzed the expression of vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) in the same groups of cells. Knockdown of VDR in endothelial cells (shVDR) (Fig 1D and 1E) led to a significant increase of VCAM-1 (Fig 1D and 1F) and ICAM-1 (Fig 1D and 1G), compared with shc002 control. Furthermore, in our experimental model, downregulation of VDR led to a significant elevation of IL-6 mRNA, as well as of IL-6 secretion in shVDR cells compared with the corresponding controls (Fig 2E and 2F, respectively).

Bottom Line: Knockdown of VDR in endothelial cells (shVDR) led to endothelial cell activation, characterized by upregulation of VCAM-1, ICAM-1 and IL-6, decreased peripheral blood mononuclear cell (PBMC) rolling velocity and increased PBMC rolling flux and adhesion to the endothelium. shVDR cells showed decreased IκBα levels and accumulation of p65 in the nucleus compared to shRNA controls.In vivo, deletion of VDR led to significantly larger aortic arch and aortic root lesions in apoE-/- mice, with higher macrophage content. apoE-/-VDR-/-mice showed higher aortic expression of VCAM-1, ICAM-1 and IL-6 when compared to apoE-/-VDR+/+ mice.Our data demonstrate that lack of VDR signaling in endothelial cells leads to a state of endothelial activation with increased leukocyte-endothelial cell interactions that may contribute to the more severe plaque accumulation observed in apoE-/-VDR-/- mice.

View Article: PubMed Central - PubMed

Affiliation: Nephrology Research Department, IRB Lleida, Lleida, Spain.

ABSTRACT
Endothelial cell activation leading to leukocyte recruitment and adhesion plays an essential role in the initiation and progression of atherosclerosis. Vitamin D has cardioprotective actions, while its deficiency is a risk factor for the progression of cardiovascular damage. Our aim was to assess the role of basal levels of vitamin D receptor (VDR) on the early leukocyte recruitment and related endothelial cell-adhesion-molecule expression, as essential prerequisites for the onset of atherosclerosis. Knockdown of VDR in endothelial cells (shVDR) led to endothelial cell activation, characterized by upregulation of VCAM-1, ICAM-1 and IL-6, decreased peripheral blood mononuclear cell (PBMC) rolling velocity and increased PBMC rolling flux and adhesion to the endothelium. shVDR cells showed decreased IκBα levels and accumulation of p65 in the nucleus compared to shRNA controls. Inhibition of NF-κB activation with super-repressor IκBα blunted all signs of endothelial cell activation caused by downregulation of VDR in endothelial cells. In vivo, deletion of VDR led to significantly larger aortic arch and aortic root lesions in apoE-/- mice, with higher macrophage content. apoE-/-VDR-/-mice showed higher aortic expression of VCAM-1, ICAM-1 and IL-6 when compared to apoE-/-VDR+/+ mice. Our data demonstrate that lack of VDR signaling in endothelial cells leads to a state of endothelial activation with increased leukocyte-endothelial cell interactions that may contribute to the more severe plaque accumulation observed in apoE-/-VDR-/- mice. The results reveal an important role for basal levels of endothelial VDR in limiting endothelial cell inflammation and atherosclerosis.

No MeSH data available.


Related in: MedlinePlus