Endothelial focal adhesions in microvascular barrier dysfunction during ischemia-

描述(申请人提供)：缺血/再灌注(I/R)损伤在微循环中触发一系列炎症反应，以血浆渗漏和白细胞渗出为特征。微血管病变很大程度上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了解。新的证据表明，微血管壁的屏障特性受内皮细胞-细胞连接和细胞-基质局灶性粘连的动态相互作用控制，后者主要由跨膜整合素受体及其相关蛋白如Kindlins和粘着斑激酶(FAK)组成。这些分子不仅为内皮屏障的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。的目标是 本研究旨在阐明FAK介导的微血管高通透性在I/R损伤中的分子机制。我们推测，I/R诱导的氧化应激上调微血管内皮细胞中的粘着斑激酶信号，诱导整合素(�5�1，�v�3)内化和点燃靶向的�-连环蛋白核转位和连接解离，导致细胞-基质和细胞-细胞黏附减弱，从而损害屏障的完整性。这一新途径将在一系列机制研究中进行测试，这些研究的中心是对肠系膜上动脉闭塞后再灌注的小鼠的肠系膜微循环、白细胞动力学和液体/蛋白质通透性进行活体显微量化。一种新开发的内皮特异性条件性FAK基因敲除的小鼠模型将用于与FAK的药物抑制进行比较。体内研究将与暴露在缺氧/复氧或氧化应激下的微血管内皮细胞的成像分析和分子分析相补充。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。从这个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意义。

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.