The long term goal of this grant application Is to understand the mechanism by which cells respond to mechanical signals which are a part of their immediate in vivo environment. We have developed an apparatus which permits us to apply precise levels of biaxial deformation to cells in culture and to assess the effects of this mechanical strain morphologically, biochemically and physiologically. We propose that mechanical strain modifies cell function and that the cell's ability to acclimate to its mechanical environment may compromise cell function and ultimately cell survival. Using this apparatus, we propose to: 1) determine If matrix protein synthesis and matrix gene expression are altered as a result of applied biaxial strain and 2), to determine a mechanism of mechanochemical transduction in endothelial cells In response to biaxial elongation. To accomplish aim 1, we will analyze matrix synthesis at the protein and RNA levels with particular emphasis on the fibronectin and the multiple forms which arise from splicing of the primary transcript. To accomplish the second aim, we will measure the Intracellular transients associated with mechanical stimulation and develop an analytical model for the calcium kinetics with emphasis on calcium accumulation as a result of injury. We will also determine if phosphorylation events are involved with the mechano-transduction process in endothelial cells. Changes in matrix protein synthesis will be assessed by radioisotope incorporation studies, polyacrylamide gel electrophoresis of isotopically labeled proteins, protease digestion of labeled proteins, Western blotting and ELISAs. Effects on matrix gene expression will be evaluated using specific matrix protein cDNA probes for collagens and fibronectin isoforms. These probes will be used in Northern hybridization and slot blot analysis of RNAs extracted from cell cultures which have been biaxially strained for different periods of time. In addition, RNAs from biaxially strained cultures will amplified and quantitated using polymerase chain reaction technology. Phosphorylation events which result from biaxial deformation of cells in culture will be characterized with a view toward understanding the mechanism of cellular response to applied mechanical forces. We will also attempt to measure the Intracellular ion transients associated with mechanical stimulation using specific fluorescent dyes which bind intracellular free Call stoichiometrically. The goal of these studies is to define the mechanotransduction system present in vascular cells and to define the molecular mechanism of cellular response to applied mechanical forces.
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