肌内皮缝隙连接介导内皮依赖的血管生成素-2对失血性休克血管低反应性调节的作用及机制研究

There is vascular hyporeactivity after severe trauma or shock, angiopoietin-2 (Ang-2) could regulate iNOS and NO through vascular endothelial cell (VEC)-specific Tie2 receptor and induce the vascular hyporeactivity, while the iNOS is mostly produced by vascular smooth muscle cell (VSMC), how could the signal of Ang-2 transfer from VEC to VSMC is unknown. Myoendothelial gap junction (MEGJ) could induce the signal communication between VEC and VSMC, and the preliminary experiment showed the MEGJ uncoupling agent could inhibit the increasement of iNOS in VSMC and vascular hyporeactivity under Ang-2 treatment. So it is supposed that MEGJ could mediate the VEC-dependent regulation of Ang-2 on iNOS and vascular hyporeactivity after hemorrhagic shock. The present research intends to observe MEGJ via the double-sided cell culture model of VEC and VSMC, and studies on the following issue by RNAi, Western blot technique and so on: 1) If Ang-2 regulates iNOS in VSMC and vascular reactivity after hemorrhagic shock through MEGJ, and what's the relative connexin (Cx) isoforms; 2) The possible mechaniam: Ang-2 induces the activation of c-Src and PKA through Tie2 receptor, regulates the phosphorylation of Cx, the opening of MEGJ, and the transportation of cAMP/cGMP, causes the P38MAPK/ERK activation and iNOS release, then leads to the vascular hyporeactivity after hemorrhagic shock. We hope to illuminate the development of vascular hyporeactivity after hemorrhagic shock with a new view and provide experimental evidence to search for vascular hyporesponsiveness restitution agents.

严重创伤/休克晚期存在血管低反应性，血管生成素-2(Ang-2)经血管内皮细胞(VEC)特异Tie2受体调节iNOS和NO生成是其重要的发生机制，但iNOS大多由血管平滑肌细胞(VSMC)产生，Ang-2信号如何从VEC转导至VSMC尚不清楚。VEC和VSMC经肌内皮缝隙连接(MEGJ)进行信号通讯，预实验发现MEGJ解偶联剂可抑制Ang-2作用下VSMC中iNOS表达和血管低反应性，推测MEGJ可能介导VEC依赖的Ang-2对血管反应性的调节。本项目拟采用VEC和VSMC双面共培养、RNAi等技术，研究1)Ang-2是否通过MEGJ调节VSMC中iNOS和休克血管反应性，以及相关连接蛋白(Cx)亚型；2)Ang-2是否通过Tie2-c-Src/PKA-Cx，开放MEGJ传递cAMP/cGMP，调节P38MAPK/ERK-iNOS，导致血管低反应性。为寻找有效的调节靶点和恢复药物提供依据。

项目背景：严重创伤/休克晚期存在血管低反应性，血管生成素-2(Ang-2)经血管内皮细胞(VEC)特异Tie2受体调节iNOS和NO生成是其重要的发生机制，但iNOS大多由血管平滑肌细胞(VSMC)产生，Ang-2信号如何从VEC转导至VSMC尚不清楚。VEC和VSMC经肌内皮缝隙连接(MEGJ)进行信号通讯，预实验发现MEGJ解偶联剂可抑制Ang-2作用下VSMC中iNOS表达和血管低反应性，推测MEGJ可能介导VEC依赖的Ang-2对血管反应性的调节。..研究内容：1)Ang-2是否通过MEGJ调节VSMC中iNOS和休克血管反应性，以及相关连接蛋白(Cx)亚型；2)MEGJ参与的机制。..结果和数据：.1.发现缺氧后VECs和VSMCs中iNOS表达存在差异，正常对照VECs和VSMCs中的iNOS表达很低，4h缺氧后显著增高，且在VSMCs中的表达强于VECs中（约为3.6倍）；MEGJ参与了Ang-2对VSMCs中iNOS表达和缺氧血管低反应性的调节，CX43是参与的Cx亚型。.2. 研究MEGJ参与Ang-2调节VSMC中iNOS表达和血管低反应性调节的机制。.缺氧及Ang2(200ng/ml)处理后MEGJ形成增多，通讯功能增强，可被Cx43 siRNA (50 nmol/l)和Tie2 siRNA (25 nmol/l)抑制。缺氧及Ang2处理后，增高的VSMC的iNOS表达和降低的VSMC收缩可被PKA抑制剂削弱，增高的VEC中cAMP浓度和PKA活性可被Ang2进一步增强，而被Tie2 SiRNA削弱，增高的Cx43蛋白表达、MEGJ形成和通讯功能均可被cAMP和PKA抑制剂抑制。.3.研究了缺氧后通过MEGJ的信号分子。.在单独培养的VECs和VSMCs，缺氧及Ang2处理后在VECs中的cAMP浓度增高显著高于VSMCs中，但在双面共培养系统，VECs和VSMC中cAMP的浓度差异明显缩小，VSMC中增高的cAMP水平可被Cx43 siRNA抑制。缺氧及Ang2处理后，cAMP发生从VECs到VSMC的转运, 且可被Cx43 siRNA抑制。..科学意义：该研究从VECs和VSMCs间连接的角度，阐述了严重创伤/休克后的血管低反应性发生机制，为寻找有效的调节靶点和恢复药物提供理论依据。