Differential mechano-signaling in vascular endothelium by varying degrees of mechanical stretch - Resubmission 01

不同程度的机械牵张对血管内皮细胞力学信号的影响 施加在微血管内皮上的过度机械力导致微血管内皮细胞增加。 伴随危及生命的疾病（如中风、肺动脉高压或呼吸机）的渗透性 导致肺损伤等等我们先前已经描述了在血管内皮细胞中诱导的信号通路， 内皮细胞的高幅度周期性拉伸（CS），并描述了Rho GT3在高CS- 诱导内皮屏障功能障碍。然而，细胞机械转导复合物， 细胞反应的机械信号仍有待表征。我们未发表的初步研究表明 Rap 1信号被生理相关的低幅度（5%）CS激活，并促进重组 在病理相关的高幅度（18%）CS时，细胞预处理破坏了紧密连接。的 初步数据还表明，低CS诱导的EC屏障恢复与累积 衔接蛋白cingulin在紧密连接和cingulin-GEF-H1复合物的形成。中央 在本申请中测试假设是在病理性机械损伤后血管内皮屏障的恢复 应激可通过细胞暴露于生理CS水平而加速，并涉及Rap 1依赖性重组 内皮细胞紧密连接的形成、扣带蛋白向紧密连接的募集以及扣带蛋白- GEF-H1的刺激 互动这些事件导致GEF-H1核苷酸交换活性的抑制，Rho- 依赖性屏障破坏机制，以及血管内皮屏障的加速恢复。的 本研究将探讨以下问题：Aim-1将研究内皮屏障的扣带蛋白依赖性机制 通过生理机械拉伸调节。目的-2将研究扣带蛋白在下调Rho中的作用。 机械刺激微血管内皮从病理性转变为生理性后的通路 CS振幅。目的-3将评估扣带蛋白依赖的血管保护机制，在小鼠模型， 机械通气

Differential mechano-signaling in vascular endothelium by varying degrees of mechanical stretch Excessive mechanical forces imposed on the microvascular endothelium lead to increased microvascular permeability accompanying life-threatening conditions such as stroke, pulmonary hypertension, or ventilator induced lung injury, to name a few. We have previously characterized signaling pathways induced in vascular endothelium by high magnitude cyclic stretch (CS) and described a key role of Rho GTPase in high CS- induced endothelial barrier dysfunction. However, cellular mechanotransduction complexes which transform mechanical signals to cellular responses remain to be characterized. Our unpublished pilot studies indicate that Rap1 signaling is activated by physiologically relevant low magnitude (5%) CS and promotes re-assembly of tight junctions disrupted by cell preconditioning at pathologically relevant high magnitude (18%) CS. The preliminary data also suggest that low CS-induced EC barrier restoration is associated with accumulation of adaptor protein cingulin at the tight junctions and formation of cingulin-GEF-H1 complex. The central hypothesis tested in this application is that recovery of vascular endothelial barrier after pathologic mechanical stress may be accelerated by cell exposure to physiologic CS levels and involves Rap1-dependent reassembly of endothelial tight junctions, recruitment of cingulin to the tight junctions, and stimulation of cingulin - GEF-H1 interaction. These events lead to inhibition of GEF-H1 nucleotide exchange activity, suppression of Rho- dependent barrier disruptive mechanisms, and accelerated recovery of the vascular endothelial barrier. The following questions will be addressed: Aim-1 will study cingulin-dependent mechanisms of endothelial barrier regulation by physiologic mechanical stretch. Aim-2 will study the role of cingulin in downregulation of the Rho pathway in mechanically stimulated microvascular endothelium after a switch from pathologic to physiologic CS amplitude. Aim-3 will evaluate cingulin-dependent vascular protective mechanisms in a mouse model of mechanical ventilation.