FAK SIGNALING IN VASCULAR INJURY

FAK(Pp125FAK)是参与细胞-基质黏附的主要信号蛋白。机械损伤和感染产生的炎性细胞因子改变内皮细胞与细胞外基质的相互作用，激活外周血单核细胞。细胞因子促进液体和炎症细胞向血管外移动，并调节血管壁上的伤口愈合。这项拟议的研究考察了FAK在细胞对血管损伤的反应中的作用。第一个具体目的是明确FAK对内皮细胞运动的调节机制。视频显微镜和FAK-绿色荧光蛋白构建将用于跟踪FAK的动力学和内皮细胞的迁移。FAK过表达、显性阴性FAK以及与巴西林、他林和Src家族激酶相互作用缺陷的突变体的影响将在运动性分析中确定。第二个具体目的是阐明FAK信号通路在调控内皮细胞增殖中的作用。FAK信号将通过外源FAK变异体的表达和细胞因子诱导的溴脱氧尿嘧啶核苷掺入和细胞周期蛋白D1表达的变化来操纵。第三个具体目的是检测FAK在内皮细胞屏障功能中的作用。FAK的表达、活性和信号将在炎症模型中进行研究，包括细胞因子处理的人内皮细胞单层和动脉粥样硬化血管的内膜表面。第四个具体目标是：确定FAK在单核细胞来源的巨噬细胞功能中的作用。在分化的单核细胞来源的巨噬细胞中，FAK的功能将因负载显性阴性形式的FAK而受到干扰。将评估黏附、趋化和细胞凋亡的影响。建议项目中的体外建模将使我们能够确定FAK信号的分子操作对炎性血管损伤的上述细胞反应的每个组成部分的影响。

FAK(pp125FAK) is a major signaling protein involved in cell-matrix adhesion. Inflammatory cytokines generated by a mechanical injury and infection alter endothelial cell interactions with extracellular matrix and activate peripheral blood monocytes. Cytokines enhance the movement of fluid and inflammatory cells out of the vascular space, and modulate wound healing in the vascular wall. The proposed studies examine the role of FAK in cellular responses to vascular injury. The first specific aim is to define the mechanisms of FAK regulation of endothelial cell motility. Video microscopy and a FAK-green fluorescent protein construct will be used to track FAK dynamics and endothelial cell migration. The effects of FAK over-expression , dominant negative FAK, and mutants defective for interactions with paxillin, talin, and Src-family kinases will be defined in motility assays. The second specific aim is to elucidate pathways of FAK signaling to the nucleus in the regulation of endothelial cell proliferation. FAK signaling will be manipulated by expression of exogenous FAK variants and cells evaluated for cytokine- induced changes in bromodeoxyuridine incorporation and cyclin D1 expression. The third specific aim is to examine the role of FAK in endothelial cell barrier function. FAK expression, activity, and signaling will be studied in models of inflammation including cytokine- treated human endothelial cell monolayers and the intimal surfaces of atherosclerotic vessels. The fourth specific aim is: To define the role of FAK in the function of monocyte-derived macrophages. FAK function will be perturbed in differentiated monocyte-derived macrophages by loading with dominant negative forms of FAK. The effects of adhesion, chemotaxis, and apoptosis will be assessed. The in vitro modeling in the proposed project will allow us to define the consequences of the molecular manipulation of FAK signaling for each of the above components of cellular response to inflammatory vascular injury.