The phenotype of the vascular endothelium changes dramatically in the evolution of a number of human vascular diseases, recapitulating changes seen during embryonic vascular development. A switch similar to the epithelial-to-mesenchymal transition, activating migration and proliferation pathways, is seen during the angiogenic response to tumors or healing wounds and also during the restitution of vessels following inflammatory injury. This coupling of endothelial cell migration and proliferation reflects the linkage between cell fate decisions and cytoskeletal mechanics at functional, biochemical, and spatial levels. Migrating cells in particular display striking subcellular polarization of proximal signaling proteins which govern cytoskeletal dynamics as well as survival and proliferation pathways. Notably, endogenously- produced reactive oxidants have been shown to concentrate at the leading edge of migrating endothelial cells, and appear to be necessary for both locomotion and mitogenic signaling. These observations suggest that the endothelial cell oxidase may be similarly targeted to leading edge structures, and that such precise targeting may be essential to preserve the fidelity of oxidant-related signals. However, the molecular basis and biological rationale for such putative subcellular oxidase localization is virtually unknown. During the prior funding period, we identified a number of protein binding partners of the principal NADPH oxidase adapter, p47phox, and demonstrated the involvement of several protein-protein interactions in specifying oxidant-related signaling to specific signaling modules. In this application, we propose to examine in detail the role of these interactions in changing the endothelial phenotype, using a combination of microscopic, biochemical, and functional studies. Relevance to Public Health: Diseases that account for the leading causes of death in the United States, among them cancer and cardiovascular disease, involve fundamental changes in the phenotype of the vascular endothelium. We propose to investigate in detail one facet of the biochemical basis for these changes, in hopes of reversing or preventing these changes. This application is a competing renewal of R01-HL67256. ? ? ?
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