Endothelial focal adhesions in microvascular barrier dysfunction during ischemia-

描述（由申请人提供）：缺血/再灌注（I/R）损伤触发微循环中的一系列炎症反应，其特征为血浆渗漏和白细胞渗出。微血管病理学主要源于内皮屏障功能障碍，这是一个复杂的细胞过程，在分子水平上尚未完全理解。新的证据表明，微血管壁的屏障特性是由内皮细胞-细胞连接和细胞-基质粘着斑的动态相互作用控制的;后者主要由跨膜整合素受体和相关蛋白如Kindlin和粘着斑激酶（FAK）组成。这些分子不仅为内皮屏障完整性提供结构支持，而且还传递调节屏障功能的生化信号。的目标 本课题旨在阐明缺血再灌注损伤中FAK介导的微血管通透性增高的分子机制。我们假设I/R引起的氧化应激上调微血管内皮中的FAK信号传导，诱导整合素（β 5 β 1，β v β 3）内化和kindlin靶向的β-连环蛋白核转位和连接解离，导致细胞-基质和细胞-细胞粘附减弱，从而损害屏障完整性。将在一系列机制研究中对这种新途径进行测试，这些研究集中在上级肠系膜动脉闭塞后再灌注的小鼠中肠系膜微循环、白细胞动力学和液体/蛋白渗透性的活体显微镜定量。将使用新开发的内皮特异性条件性FAK敲除小鼠模型与FAK的药理学抑制进行比较。体内研究将补充暴露于缺氧/复氧或氧化应激的微血管内皮细胞的成像分析和分子测定。这些研究将为I/R诱导的微血管损伤提供新的机制见解，有助于未来靶向治疗的发展，以防止复苏或再灌注后的组织损伤。从该项目中获得的知识可能对其他与微血管屏障损伤相关的疾病具有广泛的影响。

DESCRIPTION (provided by applicant): Ischemia/reperfusion (I/R) injury triggers a series of inflammatory responses in the microcirculation characterized by plasma leakage and leukocyte diapedesis. The microvascular pathology largely stems from endothelial barrier dysfunction, a complex cellular process that has not been fully understood at the molecular level. Emerging evidence indicates that the barrier property of the microvascular wall is controlled by dynamic interactions of endothelial cell-cell junctions and cell-matrix focal adhesions; the latter are maily composed of transmembrane integrin receptors and associated proteins such as kindlins and focal adhesion kinase (FAK). These molecules not only provide structural support for endothelial barrier integrity but also transmit biochemical signals that regulate barrier function. The goal of this project is to elucidate the molecular mechanisms underlying FAK-mediated microvascular hyperpermeability during I/R injury. We hypothesize that I/R-elicited oxidative stress upregulates FAK signaling in the microvascular endothelium, inducing integrin (�5�1, �v�3) internalization and kindlin- targeted �-catenin nuclear translocation and junction dissociation, leading to weakened cell-matrix and cell-cell adhesions thereby impairing barrier integrity. This novel pathway will be tested in a series of mechanistic studies centered on intravital microscopic quantification of mesenteric microcirculation, leukocyte dynamics, and fluid/protein permeability in mice subjected to superior mesenteric artery occlusion followed by reperfusion. A newly developed mouse model of endothelial-specific conditional FAK knockout will be used in comparison with pharmacological inhibition of FAK. The in vivo studies will be complemented with imaging analyses and molecular assays in microvascular endothelial cells exposed to hypoxia/reoxygenation or oxidative stress. The studies will provide new mechanistic insights into I/R-induced microvascular injury contributing to the future development of targeted therapies to prevent tissue damage following resuscitation or reperfusion. Knowledge gained from this project may have broad implications in other diseases associated with microvascular barrier injury.