The broad long-term goal of the applicants is to increase knowledge of the role of phospholipases in signal transduction in endothelial cells. The importance of this information is greatly enhanced by the accumulating evidence implicating endothelial cells in the processes of coagulation, inflammation, thrombosis and atherosclerosis. The pro- and anti-thrombotic properties of endothelial cells can be altered by vasoactive and inflammatory mediators. Tumor angiogenesis and endothelial proliferation are regulated by growth factors. A very early event initiated by these mediators is activation of phospholipase C (PLC) which generates inositol 1,4,5-triphosphate and 1,2-diacylglycerol, two second messengers in signal transduction of ligand-receptor interactions. To better understand how endothelial cell and growth factor participate in normal and pathological angiogenic processes will require detailed knowledge of the intracellular signaling pathways. The studies proposed in this application shall provide information to three important aspects that have not yet been investigated. These are the mechanism of PLC- gamma activation, PLC activation in human endothelial cells and in association with vascular endothelial growth factor (VEGF). A better understanding of how endothelial cell activation in modulated may aid the development of better strategies for the prevention and treatment of cardiovascular and metastatic diseases. In this proposal, more specifically we propose the following lines of inquiry: 1. Investigate the role of PLC-gamma2 in signal transduction of epidermal growth factor (EGF) in human endothelial cells. We will determine if EGF activates PLC-gamma2, and if so, evaluate the activation mechanism of PLC-gamma2 by examining the effect of PLC-gamma2 phosphorylation on its catalytic activity and on the interaction with membrane. 2. Study the activation of PLC isozymes induced by VEGF in human endothelial cells. 3. Evaluate the modulation of the expression of PLC isozymes in in vitro endothelial cell culture. 4. Determine by in situ hybridization and immunohistochemistry the tissue specificity of PLC isozyme expression in the endothelium of normal tissues and inflammatory lesions. 5. Characterize a potentially unique gene induced by interleukin-1 in human endothelial cells.
