SHEAR STRESS EFFECTS ON ENDOTHELIAL CELL FOCAL CONTACTS

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The hemodynamic environment of endothelial cells (EC) is thought to play an important role in the pathogenesis of vascular diseases including atherosclerosis and hypertension. Flow-related shear stress has been shown to alter various aspects of EC structure and function including cell morphology and associated cytoskeletal organization. Focal contacts are dynamic structures where cellular morphological changes may be modulated by modifying contacts with the extracellular matrix (ECM). EC signaling mechanisms involving the focal contact proteins including vinculin, talin, alpha-actinin and the fibronectin receptor and vitronectin receptor will be examined using an in vitro system designed to provide controlled levels of shear stress to EC monolayers. Shear stress-induced alterations in the levels, cellular localization, and interactions of these proteins will be determined in response to various levels of shear stress and at various time points using immunofluorescent localization and western blotting procedures. Shear stress-related cell signaling mechanisms involving phosphorylation of focal contact proteins by protein kinase C will be examined by immunoprecipitation of specific focal contact proteins following 32P labeling of the cells. Flow-induced alterations in the localization and levels of tyrosine kinases and tyrosine-phosphorylated proteins will also be determined using specific antibodies. The possible modulation by ECM proteins of shear stress- related cellular responses underlying changes in cell morphology will be examined using EC grown on different matrices. The effect of flow on the composition of the ECM on which the EC reside will be investigated to measure specific ECM components including collagen IV, laminin, and vitronectin. Another mechanism by which EC may modify their associations with the substratum is by selective, regulated proteolysis of a focal contact protein or a component of the ECM. Shear stress-induced alterations in the levels and activity of urokinase-type plaminogen activator and metalloproteinases including collagenases will be examined using immunological methods and SDS-substrate gel zymography.