The objective is to elucidate the molecular and micromechanical bases of endothelial cell (EC) turnover which has been found in studies under this grant to be a key factor in causing the focal increase of LDL permeability, and hence regional susceptibility to atherosclerosis, in bifurcations and curved areas of the arterial tree. Our hypothesis is that cellular-level complex flow patterns in these lesion-prone regions induce mechanochemical transduction in the EC to modify the regulation mitosis and apoptosis, leading to the acceleration of both processes. The end result is the preservation of confluency of the EC monolayer at the expense of an accelerated turnover and the consequent increase in LDL permeability. Experiments will be conducted by using two newly designed flow devices to generatecomplex flow patterns, with an emphasis on shear stress gradient: The step flow channel generates a recirculating flow with an unsteady reattachment region that oscillates back and forth over the lenght scale of a few cells; the T-slit flow channel generates large spatial gradients in shear stress on the length scale of an individual cell.
Three specific aims are proposed to test our hypothesis. (1) To determine the influence of complex flow patterns on the molecular events in EC mitosis and apoptosis, we will use the step flow channel to study the EC expression of cyclins A,B, and D1 as a function of the flow regime and the effects of overexpressing the dominant negative mutants of cyclin dependent kinases (Cdc2, Cdk4, and Cdk6) on EC mitosis and apoptosis. (2) To decipher the signal transduction pathways underlying EC mitosis and apoptosis in response to complex flow patterns, we will use similar strategies as in specific aim 1 to test the hypothesis that the Raf-MEK-ERK and MEKK-JNKK-JNK pathways are crucial in mediating mitosis and apoptosis. (3) To elucidate the micromechanical mechanism, at individual cell level, by which hemodynamic forces lead to EC mitosis and apoptosis, we will use the T-slit flow channel t investigate the roles of integrins and focal adhesion kinase on the basal membrane of the EC, in addition to the proteins on the luminal membrane, in the signal transduction involving these proteins. This interdisciplinary research will generate new insights into the molecular mechanisms by which complex flow patterns lead to accelerated mitoxis and apoptosis of individual Ecs and enhance our understanding of the pathophysiological basis of the focal nature of lipid accumulation and atherogenesis.
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