血管生成素-2经肌内皮缝隙连接调节血管低反应性

Angiopoietin-2 regulates vascular hyporeactivity through myoendothelial gap junction

目的 观察肌内皮缝隙连接（MEGJ）对血管生成素-2（Ang2）调节缺氧大鼠血管平滑肌细胞（VSMCs）低反应性的介导作用,并明确参与的缝隙连接蛋白（Cx）亚型.方法 建立血管内皮细胞（VECs）和VSMCs双面共培养模型,检测异硫氰酸荧光素-牛血清白蛋白（FITC-BSA）透过率反映VSMCs收缩反应性,观察鬼笔环肽荧光反映MEGJ形成,观察磺酰罗丹明B细胞培养试剂染料转运反映MEGJ通讯功能.结果 （1）在双面共培养模型,缺氧4h后VECs和VSMCs中iNOS表达显著增高,且VSMCs中的显著强于VECs中（3.6倍,P=0.003）,此增高的VSMCs中iNOS表达可被Ang2 siRNA抑制约60％ （P =0.003）.（2） MEGJ抑制剂18α-GA、Cx43 siRNA（50 nmol/L）和促血管生成素受体2（Tie2） siRNA（50 nmol/L）可降低外源性Ang2作用下缺氧VSMCs的iNOS表达（P值分别为0.001,0.001和0.001）,改善VSMCs收缩反应性（P值分别为0.004,0.009和0.001）.（3）Cx43 siRNA和Tie2 siRNA可抑制缺氧及Ang2处理后上调的MEGJ形成（P值分别为0.005和0.005）,并抑制上调的MEGJ通讯增强（P值分别为0.003和0.004）.结论 MEGJ介导Ang2对缺氧VSMCs低反应性的调节,Cx43是参与的蛋白亚型.

Objective To observe the mediation role of myoendothelial gap junction （MEGJ） in the regulation of angiopoietin-2 （Ang2） on vascular hyporeactivity after hypoxia,and figure out the responsible Cx.Methods The double-sided cells co-culture model of vascular endothelial cells （VECs） and vascular smooth muscle cells （VSMCs） were set up,the VSMCs contractile was detected via the leakage of fluoresceine isothiocyanate-bovine serum albumin （FITC-BSA）,MEGJ formation was observed by immunocytochemistry of phalloidin,and MEGJ communication was reflected by the Sulforhodamine B transfer.Results （1） In the double-sided cells co-culture model,protein expression of iNOS after hypoxia increased more in VSMCs than that in VECs （3.6 times,P =0.003）,and the increased iNOS expression in VSMCs could be decreased by Ang2 siRNA （by 60%,P =0.003）.（2） The MEGJ non-specific inhibitor 18α-GA,the specific Cx43 siRNA,or angiopoietin receptor 2 （Tie2） siRNA could suppress the iNOS expression in VSMCs （P-0.001,0.001,and 0.001,respectively）,and improved the VSMCs contractile reactivity after hypoxia following Ang2 treatment （P =0.004,0.009,and 0.001,respectively）.（3） Cx43 siRNA or Tie2 siRNA could inhibit the increased MEGJ formation （P =0.005 and 0.005,respectively）,and inhibit the increased MEGJ communication after hypoxia following Ang2 treatment （P =0.003 and 0.004,respectively）.Conclusion MEGJ mediated VECs-dependent regulation of Ang2 on vascular hyporeactivity after hypoxia,Cx43 was the responsible Cx isoform.