The purpose of the proposed study is to define completely the role of the intracellular enzyme protein kinase C (PKC) in proliferation and migration of endothelial cells, which are key processes in both the formation of new blood vessels (angiogenesis) as well as the repair of damaged endothelium. PKC, rather than being a single enzyme, is a family of related but structurally distinct isoenzymes encoded by different genes and with different cofactor requirements and substrates. Thus, it is possible that each PKC isoenzyme may selectively mediate separate effects on proliferation or migration of vascular cells, which is a principal hypothesis that will be tested in this proposal. The first specific aim is to create and characterize three classes of molecular inhibitors that selectively inhibit the function or expression of individual PKC isoenzymes normally expressed in endothelium. These three classes include pseudosubstrate, antisense, and kinase negative (dominant negative) inhibitors of each isoenzyme, and will be overexpressed to create lines of endothelial cells deficient in one or more of these isoenzymes. In the second specific aim, an isoenzyme or subset of isoenzymes of PKC that mediate migration, proliferation and vessel-forming capability of endothelium will be identified by testing the ability of endothelial cells that overexpress the PKC inhibitors created as part of Specific Aim #1 to respond to mitogens. Those lines of endothelial cells that exhibit altered proliferation or migration will be tested in a three dimensional angiogenesis assay. These functional phenotypes will be compared with phosphorylation of intracellular substrates for PKC in these cells. In the third specific aim, the mechanism by which individual PKC isoenzymes might modulate endothelial proliferation or migration will be explored by testing a model for endothelial response to mitogens. In this model, activation of individual PKC isoenzymes affects, and is affected by, the function of the endothelial integrin receptor for adhesive proteins and the activity of kinases and protooncogenes that mediate distal steps in the signal transduction pathway leading to endothelial proliferation and migration. Together, these three interrelated projects comprise a research program that should yield novel information regarding the physiologic and possible therapeutic role of the PKC gene family in endothelial and vascular cells and may provide details critical to our understanding of endothelial repair following injury and angiogenesis during disease states.
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