Xu Jing, Yang Guangming, Li Tao, Liu Liangming
State Key Laboratory of Trauma, Burns and Combined Injury, Second Department of Research Institute of Surgery, Daping Hospital, Third Military Medical University, Chongqing, People's Republic of China.
State Key Laboratory of Trauma, Burns and Combined Injury, Second Department of Research Institute of Surgery, Daping Hospital, Third Military Medical University, Chongqing, People's Republic of China
Am J Physiol Cell Physiol. 2017 Sep 1;313(3):C262-C273. doi: 10.1152/ajpcell.00369.2016. Epub 2017 Jun 21.
Angiopoietin-2 (Ang-2) contributes to vascular hyporeactivity after hemorrhagic shock and hypoxia through upregulation of inducible nitric oxide synthase (iNOS) in a vascular endothelial cell (VEC)-specific and Ang-2/Tie2 receptor-dependent manner. While iNOS is primarily expressed in vascular smooth muscle cells (VSMCs), the mechanisms of signal transfer from VECs to VSMCs are unknown. A double-sided coculture model with VECs and VSMCs from Sprague-Dawley rats was used to investigate the role of myoendothelial gap junctions (MEGJs), the connexin (Cx) isoforms involved, and other relevant mechanisms. After hypoxia, VSMCs treated with exogenous Ang-2 showed increased iNOS expression and hyporeactivity, as well as MEGJ formation and communication. These Ang-2 effects were suppressed by the MEGJ inhibitor 18α-glycyrrhetic acid (18-GA), siRNA, or siRNA. Reagents antagonizing cAMP or protein kinase A (PKA) in VECs inhibited Cx43 expression in MEGJs, decreasing MEGJ formation and associated communication, after hypoxia following Ang-2 treatment. The increased cAMP levels in VSMCs and transfer of Alexa Fluor 488-labeled cAMP from VECs to VSMCs, after hypoxia following Ang-2 treatment, was antagonized by siRNA. A cAMP antagonist added to VECs or VSMCs inhibited both increased iNOS expression and hyporeactivity in VSMCs subjected to hypoxia following Ang-2 treatment. Based on these findings, we propose that Cx43 was the Cx isoform involved in MEGJ-mediated VEC-dependent regulation of Ang-2, which induces iNOS protein expression and vascular hyporeactivity after hypoxia. Cx43 was upregulated by cAMP and PKA, permitting cAMP transfer between cells.
血管生成素-2（Ang-2）通过以血管内皮细胞（VEC）特异性和Ang-2/Tie2受体依赖性方式上调诱导型一氧化氮合酶（iNOS），导致失血性休克和缺氧后的血管反应性降低。虽然iNOS主要在血管平滑肌细胞（VSMC）中表达，但从VEC向VSMC信号传递的机制尚不清楚。采用来自Sprague-Dawley大鼠的VEC和VSMC的双面共培养模型，研究肌内皮间隙连接（MEGJ）的作用、涉及的连接蛋白（Cx）亚型以及其他相关机制。缺氧后，用外源性Ang-2处理的VSMC显示iNOS表达增加和反应性降低，以及MEGJ形成和通讯。这些Ang-2效应被MEGJ抑制剂18α-甘草次酸（18-GA）、小干扰RNA（siRNA）或短发夹RNA（shRNA）抑制。在Ang-2处理后的缺氧状态下，VEC中拮抗环磷酸腺苷（cAMP）或蛋白激酶A（PKA）的试剂抑制了MEGJ中Cx43的表达，减少了MEGJ的形成和相关通讯。在Ang-2处理后的缺氧状态下，VSMC中cAMP水平升高以及Alexa Fluor 488标记的cAMP从VEC向VSMC的转移，被siRNA拮抗。添加到VEC或VSMC中的cAMP拮抗剂抑制了Ang-2处理后的缺氧状态下VSMC中iNOS表达增加和反应性降低。基于这些发现，我们提出Cx43是参与MEGJ介导的Ang-2的VEC依赖性调节的Cx亚型，其在缺氧后诱导iNOS蛋白表达和血管反应性降低。Cx43被cAMP和PKA上调，允许cAMP在细胞间转移。
