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Nitric Oxide Increases Arterial Endotheial Permeability through Mediating VE-Cadherin Expression during Arteriogenesis.

Yang B, Cai B, Deng P, Wu X, Guan Y, Zhang B, Cai W, Schaper J, Schaper W - PLoS ONE (2015)

Bottom Line: We found that: 1) in normal arteriolar vessels (NAV), eNOS was moderately expressed in endothelial cells (EC) and iNOS was rarely detected.In contrast, in collateral vessels (CVs) induced by simple femoral artery ligation, both eNOS and iNOS were significantly upregulated (P<0.05).In CVs, VE-cadherin was significantly downregulated, with a discontinuous and punctate pattern.

View Article: PubMed Central - PubMed

Affiliation: Department of Histology & Embryology, School of Basic Medicine, Central South Univ., Changsha, 410078, Hunan, P.R. China; Department of Anatomy, School of Basic Medicine, Nanchang Univ., Nanchang, 330006, Jiangxi, P.R. China.

ABSTRACT
Macrophage invasion is an important event during arteriogenesis, but the underlying mechanism is still only partially understood. The present study tested the hypothesis that nitric oxide (NO) and VE-cadherin, two key mediators for vascular permeability, contribute to this event in a rat ischemic hindlimb model. In addition, the effect of NO on expression of VE-caherin and endothelial permeability was also studied in cultured HUVECs. We found that: 1) in normal arteriolar vessels (NAV), eNOS was moderately expressed in endothelial cells (EC) and iNOS was rarely detected. In contrast, in collateral vessels (CVs) induced by simple femoral artery ligation, both eNOS and iNOS were significantly upregulated (P<0.05). Induced iNOS was found mainly in smooth muscle cells, but also in other vascular cells and macrophages; 2) in NAV VE-cadherin was strongly expressed in EC. In CVs, VE-cadherin was significantly downregulated, with a discontinuous and punctate pattern. Administration of nitric oxide donor DETA NONOate (NONOate) further reduced the amounts of Ve-cadherin in CVs, whereas NO synthase inhibitor L-NAME inhibited downregulation of VE-cadherin in CVs; 3) in normal rats Evans blue extravasation (EBE) was low in the musculus gracilis, FITC-dextron leakage was not detected in the vascular wall and few macrophages were observed in perivascular space. In contrast, EBE was significantly increased in femoral artery ligation rats, FITC-dextron leakage and increased amounts of macrophages were detected in CVs, which were further enhanced by administration of NONOate, but inhibited by L-NAME supplement; 4) in vitro experiments confirmed that an increase in NO production reduced VE-cadherin expression, correlated with increases in the permeability of HUVECs. In conclusion, our data for the first time reveal the expression profile of VE-cadherin and alterations of vascular permeability in CVs, suggesting that NO-mediated VE-cadherin pathway may be one important mechanism responsible, at least in part, for macrophage invasion during arteriogenesis.

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Expression of VE-cadherin in HUVECs.A-C: VE-cadherin immunostaining. A: control, B: NONOate treated, C: L-NAME treated. Specific fluorescence: green for VE-cadherin, red for nuclei. Note that in control and L-NAME treated HUVECs VE-cadherin staining was strong and continuous and distributed around the entire periphery of cells, in NONOate treated HUVECs, VE-cadherin staining was weak and intermittent (B, arrowheads). D: quantitative analysis of immunofluorescence intensity of VE-cadherin in HUVECs (*P < 0.05 vs control, #P < 0.05 vs NONOate treated or control). E: immunoblotting of VE-Cadherin.
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pone.0127931.g008: Expression of VE-cadherin in HUVECs.A-C: VE-cadherin immunostaining. A: control, B: NONOate treated, C: L-NAME treated. Specific fluorescence: green for VE-cadherin, red for nuclei. Note that in control and L-NAME treated HUVECs VE-cadherin staining was strong and continuous and distributed around the entire periphery of cells, in NONOate treated HUVECs, VE-cadherin staining was weak and intermittent (B, arrowheads). D: quantitative analysis of immunofluorescence intensity of VE-cadherin in HUVECs (*P < 0.05 vs control, #P < 0.05 vs NONOate treated or control). E: immunoblotting of VE-Cadherin.

Mentions: To verify the effect of NONOate or L-NAME on expression of VE-cadherin and vascular permeability observed during arteriogenesis, we investigated the effects of NONOate or L-NAME on the expression of VE-cadherin and the changes of EC permeability in cultured HUVECs. The VE-cadherin was determined by immunofluorescence and immunoblotting. EC permeability was checked by FITC-dextan leakage in transwell inserts system. In untreated HUVECs, VE-cadherin staining was continuous and distributed around the entire periphery of cells (Fig 8A). Exposed to NONOate (100 μmol/L) for 24 h, the VE-cadherin staining became intermittent, showing frequent gaps (Fig 8B), the amount of VE-cadherin protein was significantly decreased (Fig 8D), which was further confirmed by immunoblotting (Fig 8E). In contrast, exposed to L-NAME (1000 μmol/L) for 24 h, VE-cadherin staining was strong, continuous and distributed around the entire periphery of cells (Fig 8C). The amount of VE-cadherin protein was even significantly higher than that in controls (Fig 8D and 8F). FITC-dextran leakage was low in untreated HUVECs, but significantly increased after exposed to NONOate, which was dose-dependent (Fig 9A). In contrast, FITC-dextran leakage was significantly reduced in L-NAME treated HUVECs (Fig 9B). Colorimetric Assay confirmed that NO production was significantly increased in HUVECs exposed to NONOate, but significantly reduced in L-NAME treated HUVECs (Fig 10).


Nitric Oxide Increases Arterial Endotheial Permeability through Mediating VE-Cadherin Expression during Arteriogenesis.

Yang B, Cai B, Deng P, Wu X, Guan Y, Zhang B, Cai W, Schaper J, Schaper W - PLoS ONE (2015)

Expression of VE-cadherin in HUVECs.A-C: VE-cadherin immunostaining. A: control, B: NONOate treated, C: L-NAME treated. Specific fluorescence: green for VE-cadherin, red for nuclei. Note that in control and L-NAME treated HUVECs VE-cadherin staining was strong and continuous and distributed around the entire periphery of cells, in NONOate treated HUVECs, VE-cadherin staining was weak and intermittent (B, arrowheads). D: quantitative analysis of immunofluorescence intensity of VE-cadherin in HUVECs (*P < 0.05 vs control, #P < 0.05 vs NONOate treated or control). E: immunoblotting of VE-Cadherin.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4489889&req=5

pone.0127931.g008: Expression of VE-cadherin in HUVECs.A-C: VE-cadherin immunostaining. A: control, B: NONOate treated, C: L-NAME treated. Specific fluorescence: green for VE-cadherin, red for nuclei. Note that in control and L-NAME treated HUVECs VE-cadherin staining was strong and continuous and distributed around the entire periphery of cells, in NONOate treated HUVECs, VE-cadherin staining was weak and intermittent (B, arrowheads). D: quantitative analysis of immunofluorescence intensity of VE-cadherin in HUVECs (*P < 0.05 vs control, #P < 0.05 vs NONOate treated or control). E: immunoblotting of VE-Cadherin.
Mentions: To verify the effect of NONOate or L-NAME on expression of VE-cadherin and vascular permeability observed during arteriogenesis, we investigated the effects of NONOate or L-NAME on the expression of VE-cadherin and the changes of EC permeability in cultured HUVECs. The VE-cadherin was determined by immunofluorescence and immunoblotting. EC permeability was checked by FITC-dextan leakage in transwell inserts system. In untreated HUVECs, VE-cadherin staining was continuous and distributed around the entire periphery of cells (Fig 8A). Exposed to NONOate (100 μmol/L) for 24 h, the VE-cadherin staining became intermittent, showing frequent gaps (Fig 8B), the amount of VE-cadherin protein was significantly decreased (Fig 8D), which was further confirmed by immunoblotting (Fig 8E). In contrast, exposed to L-NAME (1000 μmol/L) for 24 h, VE-cadherin staining was strong, continuous and distributed around the entire periphery of cells (Fig 8C). The amount of VE-cadherin protein was even significantly higher than that in controls (Fig 8D and 8F). FITC-dextran leakage was low in untreated HUVECs, but significantly increased after exposed to NONOate, which was dose-dependent (Fig 9A). In contrast, FITC-dextran leakage was significantly reduced in L-NAME treated HUVECs (Fig 9B). Colorimetric Assay confirmed that NO production was significantly increased in HUVECs exposed to NONOate, but significantly reduced in L-NAME treated HUVECs (Fig 10).

Bottom Line: We found that: 1) in normal arteriolar vessels (NAV), eNOS was moderately expressed in endothelial cells (EC) and iNOS was rarely detected.In contrast, in collateral vessels (CVs) induced by simple femoral artery ligation, both eNOS and iNOS were significantly upregulated (P<0.05).In CVs, VE-cadherin was significantly downregulated, with a discontinuous and punctate pattern.

View Article: PubMed Central - PubMed

Affiliation: Department of Histology & Embryology, School of Basic Medicine, Central South Univ., Changsha, 410078, Hunan, P.R. China; Department of Anatomy, School of Basic Medicine, Nanchang Univ., Nanchang, 330006, Jiangxi, P.R. China.

ABSTRACT
Macrophage invasion is an important event during arteriogenesis, but the underlying mechanism is still only partially understood. The present study tested the hypothesis that nitric oxide (NO) and VE-cadherin, two key mediators for vascular permeability, contribute to this event in a rat ischemic hindlimb model. In addition, the effect of NO on expression of VE-caherin and endothelial permeability was also studied in cultured HUVECs. We found that: 1) in normal arteriolar vessels (NAV), eNOS was moderately expressed in endothelial cells (EC) and iNOS was rarely detected. In contrast, in collateral vessels (CVs) induced by simple femoral artery ligation, both eNOS and iNOS were significantly upregulated (P<0.05). Induced iNOS was found mainly in smooth muscle cells, but also in other vascular cells and macrophages; 2) in NAV VE-cadherin was strongly expressed in EC. In CVs, VE-cadherin was significantly downregulated, with a discontinuous and punctate pattern. Administration of nitric oxide donor DETA NONOate (NONOate) further reduced the amounts of Ve-cadherin in CVs, whereas NO synthase inhibitor L-NAME inhibited downregulation of VE-cadherin in CVs; 3) in normal rats Evans blue extravasation (EBE) was low in the musculus gracilis, FITC-dextron leakage was not detected in the vascular wall and few macrophages were observed in perivascular space. In contrast, EBE was significantly increased in femoral artery ligation rats, FITC-dextron leakage and increased amounts of macrophages were detected in CVs, which were further enhanced by administration of NONOate, but inhibited by L-NAME supplement; 4) in vitro experiments confirmed that an increase in NO production reduced VE-cadherin expression, correlated with increases in the permeability of HUVECs. In conclusion, our data for the first time reveal the expression profile of VE-cadherin and alterations of vascular permeability in CVs, suggesting that NO-mediated VE-cadherin pathway may be one important mechanism responsible, at least in part, for macrophage invasion during arteriogenesis.

No MeSH data available.


Related in: MedlinePlus