The focal development of atherosclerosis involves the complex interplay of mechanical forces & chemical mediators impinging on endothelial (ECs) & smooth muscle cells (SMC) & their interactions with extracellular matrix (ECM). This program is designed to explore how these factors interact, playing specific attention to two issues; the modulation of signalling responses to humoral mediators by mechanical forces & the cell to regional heterogeneity of such interactions in the vasculature. Several humoral factors influence the adhesive interactions between endothelial & circulating cells, so characteristic of the early stages of activated receptors; PARs) G protein coupled receptors; tyrosine kinase receptors (Eph kinases) & integrins (alphaIIbbeta3 & alphaVbeta3), platelets with ECs, & cholesterol export, properties that may limit atherogenesis. Cyclooxygenase (COX)-2 is a major source of these observations will be addressed in Proj. 1, using selective inhibitors of COX-2 in humans, in conventional & novel mouse models of atherosclerosis & in genetic crosses of these models with mice lacking COX-2 or the PGI2 receptor. We shall investigate specifically how mechanical forces modulate expression of the biosynthetic & signaling pathways of PGI2 in vascular cells. Thrombin, by contrast, enhances adhesive interactions & stimulates cellular proliferation. It is a recognized ligand for some PARs but little is known of the diversity of PARs & their distinct functions in vascular cells. In Proj. 2, the mechanisms of activation, processing & regulation of PARs & the functional importance, distribution & signaling pathways activated by the noel Eph kinases will be investigated. The structural motifs which discriminate affinity for osteopontin amongst beta3 integrins will be examined in Proj. 3. We shall also determine the structural basis for selection by this adhesive ligand amongst its several receptors on platelets, examine the role of mechanical shear on the interaction, & examine the interactions or other signaling pathways outlined above (PGI2, thrombin) on regulating the activation of beta3 integrins. Since the ECM is likely to play a major role in conditioning the response of VSMCs to humoral and mechanical factors, Proj. 4 will explore the role of distinct ECM proteins interacting with mechanical forces on these interactions will also be examine din this project. Finally, Proj. 5 will address the hypothesis that differences in surface geometry amongst apparently similar endothelial cells results in differences in regulated expression of elements of the signaling pathways explored in Projects 1- 4 in response to mechanical forces. These observations will be integrated with comparisons of data derived from regions of flow separation, both in vitro & ex vivo, using mouse models & human atherosclerotic tissue. This program will take an integrated approach to the study of humoral and mechanical signaling in vascular cells. Factors which underlie the cell to cell heterogeneity of this interaction are likely to contribute to the focal nature of atherogenesis.
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