The long term goal of the proposed research is to understand the effect of flow, fluid mechanical stresses and mechanical strain on the arachidonic acid metabolism of endothelial cells and leukocytes and subsequent modulation of endothelia cell gene expression and platelet and leukocyte interactions with endothelial cells. Initial studies from our laboratory have indicated quite striking stress-metabolism coupling in each of these cell types. Proposed work will center on understanding the mechanisms of membrane signal transduction and gene regulation for these unique (and physiologically relevant) stimuli. Potential mediators include alteration of ion fluxes across cell membranes caused by mechanical perturbations (including stretch activated ion channels), with calcium, potassium and hydrogen ion regulation being most likely. Three dimensional reconstructions of intracellular calcium and pH distribution will be obtained utilizing fluorescence video reconstructions of intracellular calcium and pH distribution will be obtained utilizing fluorescence video microscopy coupled with digital image processing. We hypothesize that both the level of stress (and strain) and the ate of change of stress (and strain) acting on the cell membrane are important factors in modulation of time, stress (strain) magnitude and the time history of stress (strain) applications. Specifically designed viscometers, flow chambers, and uniaxial stretching chambers will be employed. At the gene regulation level, polymerase chain reaction methods and northern analysis will be employed to ascertain changes in steady state messenger RNA levels caused by mechanical perturbations. Altered levels of secretion of metabolites, such as tPA, PAI-1 and endothelia, in addition to expression of membrane associated adhesion receptors caused by fluid flow and strain will be quantified. Again, preliminary data from our laboratory have documented changes in secretion of many potent vasoactive compounds. The roles of flow and strain on expression of cell adhesion molecules such as ICAM-I, VCAM-1 and P- and E-selectin-- as well as modulation of subsequent neutrophil adhesion may be very important in stroke pathophysiology. We will extend our studies of human umbilical vein and bovine pulmonary and aortic endothelial cells to a newly developed bovine brain endothelium preparation. Studies of flow and arachidonic acid metabolite control of the permeability of endothelial cell monolayers from these different vascular beds will be conducted in specially designed flow chambers with nuclepore membrane inserts. Comparison of flow and strain regulation of metabolism in these different cell types will be important with respect to in vivo stroke applications. We believe our results should be of importance in understanding the role of flow in metabolic events associated with ischemia and reperfusion. Many of the metabolites produced are extremely bioactive and can lead to changes in vessel permeability, cell adhesion and smooth muscle tone-all of which may be important in the pathogenesis of stroke.
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