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

<div><p>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) <i>in vitro</i> 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.</p></div

Confocal micrographs of FITC-dextran leakage in the musculus gracilisthe.

<p>Specific fluorescence: green, red for CD31(marker of endothelial cells)., blue for nuclei. A-D: collaterals, E-H: capilaries. A and E: sham, B and F: femoral artery ligation (FAL), C and G: NONOate treated, D and H: L-NAME treated. Note that FITC-dextran was not observed in sham and L-NAME treated collaterals, but detected in FAL collaterals and enhanced in NONOate treated collaterals; in capillaries level, FITC-dextran was observed in all the groups, but more in FAL and NONOate treated ones.</p

Quantitative analysis of Evans Blue extravasation (ng/mg) in the musculus gracilis from sham, femoral artery ligation (FAL), NONOate treated (FAL + NONOate) and L-NAME treated (FAL + L-NAME) groups.

<p>*P < 0.05 vs sham., #P < 0.05 vs FAL.</p

Quantitative analysis of NO production (μM/L) in the media of cultured HUVECs treated with different doses of NONOate or L-NAME.

<p>A; NONOate treated. B: L-NAME treated. *P < 0.05 vs control., #P < 0.05 vs 1 or 100 μM group in A, vs 10 or 1000 μM group in B.</p

Quantitative analysis of fluorescence density (AU/μm2) of FITC-dextran in the collaterals and capilaries of the musculus gracilis from sham, femoral artery ligation (FAL), NONOate treated (FAL + NONOate) and L-NAME treated (FAL + L-NAME) groups.

<p>*P < 0.05 vs sham., #P < 0.05 vs FAL.</p

Quantitative analysis of fluorescence density (AU/μm2) of FITC-dextran in the media of cultured HUVECs treated with or without NONOate or L-NAME.

<p>A; NONOate treated. B: L-NAME treated. *P < 0.05 vs control., #P < 0.05 vs 1 or 100 μM group in A, vs 10 or 1000 μM group in B.</p

Quantitative analysis of NO production (μM/L) in the serum from sham, femoral artery ligation (FAL), NONOate treated (FAL + NONOate) and L-NAME treated (FAL + L-NAME) groups.

<p>*P < 0.05 vs sham., #P < 0.05 vs FAL.</p

Primary and secondary antibodies used in this study.

<p>Primary and secondary antibodies used in this study.</p

Quantitative analysis of immunofluorescence density (AU/μm2) of VE-cadherin and CD11 positive cells (%) in sham (sham), femoral artery ligation (FAL), NONOate treated (FAL + NONOate) and L-NAME treated (FAL + L-NAME) collateral vessels.

<p>*P < 0.05 vs sham., #P < 0.05 vs FLA.</p

Expression of eNOS and iNOS in collaterals of sham (Sham) and femoral artery ligation (FAL) rats.

<p>A-D: eNOS and iNOS immunostaining. E: dual immunostaining of iNOS with CD11b (marker of macrophage). Specific fluorescence: green for eNOS in A and B, for iNOS in C-E; red for F-actin in A-D, for CD11b in E; blue for nuclei. A and C: Sham; C, D and E: FAL; F: quantitative analysis of immunofluorescence intensity of eNOS in SV, and FAL. Note that in FAL, both eNOS and iNOS proteins were remarkably increased, induced iNOS was observed in all the layers of the vascular wall and macrophages (E, arrowheads). Lu: lumen; m: tunica media; ad: adventitia; *P < 0.05 vs sham.</p