FAK SIGNALING IN VASCULAR INJURY

FAK（pp 125 FAK）是参与细胞-基质粘附的主要信号蛋白。由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并激活外周血单核细胞。细胞因子增强流体和炎性细胞移出血管空间的运动，并调节血管壁中的伤口愈合。拟议的研究检查FAK在血管损伤的细胞反应中的作用。第一个具体的目标是确定FAK调节内皮细胞运动的机制。视频显微镜和FAK-绿色荧光蛋白构建体将用于跟踪FAK动力学和内皮细胞迁移。FAK过表达、显性阴性FAK和与桩蛋白、talin和Src家族激酶相互作用缺陷的突变体的影响将在运动性测定中确定。第二个具体的目的是阐明FAK信号传导到细胞核中调节内皮细胞增殖的途径。通过表达外源性FAK变体操纵FAK信号传导，并评价细胞的溴脱氧尿苷掺入和细胞周期蛋白D1表达中细胞因子诱导的变化。第三个具体目标是研究FAK在内皮细胞屏障功能中的作用。将在炎症模型中研究FAK的表达、活性和信号传导，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在单核细胞源性巨噬细胞功能中的作用。FAK功能将在分化的单核细胞衍生的巨噬细胞中通过加载显性阴性形式的FAK而受到干扰。将评估粘附、趋化性和细胞凋亡的影响。在拟议的项目中的体外建模将使我们能够定义FAK信号传导的分子操作的后果，为炎症性血管损伤的细胞反应的上述每个组件。

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.