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.
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