Effects Of NO/NOS On Expression Of VE-cadherin And Permeability During Collateral Vessel Growth

Part I The alteration of VE-cadherin expression and vascular permeability during collateral vessel growth induced by femoral artery ligation in rat hind limbObjective:The present study was designed to investigate the changes of expression of vascular endothelial cadherin (VE-cadherin), a key molecule of adhering junction, and vascular permeability during collateral vessel growth after femoral artery ligation in rat hind limb, exploring whether VE-cadherin and vascular permeability were involved in collateral vessel growth, and enriching the theories of inflammation formation in collateral vessel growth mechanisms.Methods:Forty-five adult Sprague-Dawley (SD) rats were randomly divided into femoral artery ligation group, sham group and normal control group. Femoral artery ligation was carried out by using double knots. The surgery of sham group was same as the femoral artery ligation group but without femoral artery ligation. Animals were survival for one or four weeks, then collateral vessel growth and expression of VE-cadherin were examined by using gelatin-tetroxide lead angiography, H.E. staining and confocal immunofluorescence. The vascular permeability was detected with EB permeability experiment.Results:As compared to the sham group, at one week after femoral artery ligature angiography showed that the number of collateral vessels and Ki67positive cells (a marker of cell proliferation) were significantly increased (P<0.01), that the expression of VE-cadherin was strongly downregulated (P<0.01), and the amount of extravasated EB was significantly increased (P<0.01). At4weeks after femoral artery ligature, the expression of VE-cadherin was significantly upregulated, but the amount of extravasated EB was decreased. Ki67positive cells at this stage were visible, but decreased. Few collateral vessels and ki67positive cells were seen in the animals of the sham group. There was no significant difference regarding the above-mentioned parameters between sham and normal control group.Conclusions:①The number of collateral vessels and their lumen areas were increased after femoral artery ligation;②The VE-cadherin expression was different in course of collateral vessel growth. At the early stage of collateral vessel growth, the expression of VE-cadherin was down-regulated and the vascular permeability was increased. At the late stage of collateral vessel growth, the VE-cadherin expression was increased to normal level, but the permeability was reduced. The data suggest that VE-cadherin may play an important role in vascular permeability in collateral vessel growth. Part II Effects of NO/NOS on expression of VE-cadherin and vascular permeability during collateral vessel growthObjective:The purpose of this study was to investigate the effect of nitric oxide (NO) on collateral vessel growth, expression of VE-cadherin and vascular permeability and inflammation formation in rat femoral artery ligation induced collateral vessel growth model by administration of DETA NONOate (NO donator) and L-NAME (NOS inhibitor) to change the NO level in vivo.Methods:Seventy-two adult SD rats were divided into four groups: femoral artery ligation plus DETA NONOate (NONO group), femoral artery ligation plus L-NAME (L-NAME group), simple femoral artery ligation (SL group) and sham group (S group). The preparation of femoral artery ligation model was the same as described in Part I. The agents, DETA NONOate and L-NAME were respectively intraperitoneal injected following femoral artery ligation. After survival for one week, the expression of VE-cadherin and eNOS in collateral vessels and vascular permeability were detected by confocal immunofluorescence, EB and FITC-dextran permeability experiment respectively. In addition, the expression of the following molecules in collateral vessels were detected, including the marker of cell proliferation (Ki67), the marker of macrophage (CD11b).Results:The main findings in present study are:1. administration of DETA NONOate led to an increased ratio of Ki67and CD11b positive cells and high level of eNOS expression and the vascular permeability;2. administration of DETA NONOate led to reduced VE-cadherin expression showing a discontinuous distribution on endothelium;3. administration of L-NAME led to a reduced ratio of Ki67and CD11b positive cells, the expression of eNOS and the vascular permeability, but an increased expression of VE-cadherin with continuous distrubution on the endothelium. The ratio of CDllb positive cells, the expression of eNOS and iNOS in collateral vessels in SL group were higher than that in S group.Conclusions:①There were increased expressions of eNOS and iNOS and vascular permeability and inflammatory cell infiltration during native collateral vessels growth.②Exogenous NO donor could enhance vascular permeability and extravasation of inflammatory cell by alternating VE-cadherin expression and adhesion junctions, by which promotion of the collateral vessels growth may be achieved.③Exogenous L-NAME downregulated eNOS and iNOS expression, which may inhibit the collateral vessel growth by lowering vascular permeability and extravasation of inflammatory cell via increasing VE-cadherin expression and stablizing adherens junctions. Part Ⅲ Effects of NO/NOS on the expression of VE-cadherin, cell permeability and cell proliferation in HUVECObjective:The present study was designed to verify the findings gained in Part Ⅰ and Ⅱ by investigating the effect of NO on the proliferation of endothelial cell, expression of VE-cadherin and eNOS, and the permeability via alternating the level of NO in cultured HUVEC.Methods:Either DETA NONOate or L-NAME was added to the medium of cultured HUVEC. Griess test was used to examine the levels of NO metabolic products in the media. The HUVEC activity was assessed with MTT assay, the effect of NO donor (DETA NONOate) and NOS inhibitor (L-NAME) on HUVEC proliferation activity and the expression of eNOS and VE-cadherin were examined with immunocytochemistry and Western blot, and HUVEC permeability was detected with Transwell permeability experiment.Results:Griess and Transwell test showed that both NO product concentration and intercellular permeability were significantly increased in HUVEC treated with1,10,100μmol/L DETA NONOate, as compared to control group (P<0.01). This increase was dose-dependent. In contrast, in HUVEC treated with10,100,1000μmol/L L-NAME, both NO product concentration and intercellular permeability were lower. Treated with10μmol/L DETA NONOate, HUVEC showed increased cell activity and proliferation (Ki67) and down-regulated expression of VE-cadherin, while treatment with100μmol/L L-NAME led to an opposite reaction.Conclusions:Exogenous NO could decrease the expression of VE-cadherin and increase cellular permeability; Suppressing eNOS expression in HUVEC could increase the VE-cadherin expression and reduce the activity of the cell proliferation and cellular permeability; increasing the level of NO and eNOS could promote the activity of cell proliferation. Taken together, NO is an important factor that contributes to cell proliferation, the formation of adhesion junctions and change of permeability, thereby promoting the collateral vessel growth. Figures:37；table:0；references:92.