Blood flow exerts both shear stress and cyclic strain on vessel walls. The mechanism by which physical forces cause changes in cell function is poorly understood. The investigator previously demonstrated that cyclic strain activates several second messenger systems. His hypothesis is that application of repetitive physical strain to endothelial cells results in engagement of specific integrins on the endothelial cell (EC) surface which initiate a signal transduction cascade via modulation of membrane- or cytoskeletally-associated tyrosine kinases. The tyrosine phosphorylation events result in a cascade of biochemical events which lead to the cell responses to cyclic strain, specifically alterations in cell shape and motility. The first Specific Aim is to characterize the effect of applied strain on EC tyrosine kinase activity. The relationship between different strain parameters and tyrosine kinase activity will be studied. In addition, the effect of strain on the activity of tyrosine kinases associated with membranes and the cytoskeleton will be compared to activity of cytosolic tyrosine kinases. The second Specific Aim is to determine the mechanism by which the integrin-associated tyrosine kinase, focal adhesion kinase (FAK), is phosphorylated. Modulation of FAK activity, reorganization of FAK, and the function of reorganized FAK during strain will be evaluated. In addition, the specific integrins involved will be assessed by plating EC on tissue culture plastic coated with different matrix proteins. The role of PKC and calcium will also be studied. The third Specific Aim is to assess the biochemical consequences of phosphorylation of FAK. Tyrosine phosphoproteins will be co-precipitated with FAK or changes in tyrosine phosphorylation in cell lysates will be identified. The downstream activation of paxillin will be studied. In addition, the involvement of the GTP-binding protein, rho, will be identified. Finally, the reorganization of phosphorylated tyrosine phosphoproteins during strain will be investigated. The fourth Specific Aim is to determine the physiological consequences of phosphorylation of FAK. The effect of a panel of tyrosine kinase inhibitors or potentiators on EC morphology (as determined by measuring length to width ratios) and migration will be investigated. In addition, the effect of EC transfection with FAK cDNA mutants (leading to over-expression of FAK or expression of mutant FAK) will be performed to determine the role of FAK in changes in EC morphology and migration. These studies will identify the role of FAK in transmission of hemodynamic forces to the interior of the cell. This will aid in understanding the mechanism by which cyclic strain causes specific morphologic and functional changes in the cell. The clinical relevance of this proposal is the potential to understand the mechanism by which a hemodynamic force alters EC function.
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