The initiation and progression of focal atherosclerotic lesions has long been associated with regions of disturbed blood flow, yet the relationships linking hemodynamics to vessel wall biology are poorly understood. Recent in vitro and in vivo studies from this laboratory have demonstrated major variations in the lumenal 3-dimensional surface geometry from cell to cell within the nominally homogeneous endothelial monolayer. Thus the detailed distribution of hemodynamic shear stresses over the lumenal cell surface varies significantly. The predicted consequences of these differences is the hypothesis to be addressed in this proposal: that throughout the arterial circulation there is significant heterogeneity of endothelial gene expression regulated by differential shear stress as a consequence of topographic differences from cell to cell. Both single cell and regional heterogeneities will be addressed. The hypothesis will be tested by analysis of gene expression in single cells and groups of cells, removed from the precise hemodynamic locations in vitro and in vivo and analyzed by single cell amplified antisense RNA technology. Disturbed flow regions will be identified in the mouse arterial circulation and in cultures of human endothelial cells in vitro. Topographic measurements of luminal endothelial surfaces will be performed by atomic force microscopy enabling force concentrations to be mapped for individual cells. Micro- manipulation/dissection will isolate single cells (and small groups of cells) for mRNA amplification. Microarray hybridization technology will be used to determine the expression profile of known stress-responsive genes and other known and unknown human and mouse genes resulting in cell-specific mRNA expression """"""""fingerprints"""""""" related to the local hemodynamic forces. The arteries of atherosclerosis-susceptible LDL Receptor-Edit transgenic mice will be probed by in situ hybridization and immunocytochemistry (after antibody generation) to evaluate protein expression. Genes uniquely and prominently associated with specific hemodynamic stress profiles will be identified, a selective number of which will be studied by the generation of transgenic mice. In vitro flow systems will be manipulated to study the complex spatial and temporal characteristics of gene expression in controlled conditions of disturbed laminar flow. In a larger sense, expression profiles from disturbed flow regions in vivo will integrate gene discovery, hemodynamics, and focal atherosclerosis at the sites at which lesions develop.
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