Coronary angiogenesis is a multi-step process involving endothelial cell (EC) detachment, migration, proliferation and tube formation. The central theme of this proposal is that nitric oxide (NO) is an integrating signal molecule for regulation of coronary angiogenesis elicited by a variety of chemical and physical factors. The proposal pursues 3 broad specific aims. The first specific aim probes the direct effect of NO and the cGMP-dependent protein kinase (PKG) on activation of the MAP kinase cascade, disassembly of junctional complexes, downregulation of focal adhesions, and endothelial tube formation in vitro. We propose that NO controls the MAP kinase cascade via phosphorylation of raf-1 by PKG. The physiologic role of NO as a guanine nucleotide exchange factor capable of activating ras is also examined. The second specific aim focuses on the role of NO and PKG in mediating the pro-angiogenesis effects of vascular endothelial growth factor (VEGF), an endothelial cell specific mitogen released by many cell types during hypoxia. The role of calcium influx and membrane depolarization in VEGF signaling is considered, including the possible participation of PKG as a controller of calcium channels in the surface membrane. The role of the MAP kinase and rho-like GTPases in mediating VEGF-induced venular hyperpermeability is delineated. The requirement for upregulation of ecNOS in VEGF-induced angiogenesis is probed and the possibility that PKG modulates the cyclin/cyclin-dependent kinase cascade is explored. In addition, the role of adenosine and erythropoietin in potentiating the effects of VEGF is examined. The third specific aim focuses on the development of new intact tissue models for probing long-term (24 to 48 hrs) events in the angiogenesis process using quantitative intravital microscopy. The isolated coronary venule is maintained under culture conditions with a realistic external matrix, and a similar approach is used to develop a complete microvascular network in excised, artificially perfused papillary muscle and mesentery. To achieve the aims of the proposed work, cultured endothelial cells and isolated, perfused coronary venules are used.
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