Angiogenesis plays a critical role in the revascularization of ischemic organs and in the progression of cancer, atherosclerosis, rheumatoid arthritis, and diabetic retinopathy. The outcome of angiogenesis depends on neovessel survival. Developing vessels uses the angiopoietin/Tie2 system to acquire a stabilizing layer of mural cells (smooth muscle cells/pericytes). The mechanisms regulating this process are, however, poorly understood because the Tie2 receptor is reportedly expressed in endothelial cells but not in mural cells. Using the rat aorta model of angiogenesis we found that Tie2 is transiently expressed in intimal- derived mesenchymal cells that have the capacity to differently into mural cells. Tie2+ mural precursor cells migrate and secrete matrix metalloproteinases in response to Ang-1 which they produce, and Ang-2, which is produced by endothelial cells. Based on the additional observation that the intimal/subintimal layers of the rat aorta contain Flk- 1+ and Tie2+ non-endothelial mesenchymal cells and have angioinformative properties we postulate that Tie2+ mural-precursor cells arise from vascular progenitor cells capable of both endothelial and mural cell differentiation. We linked mural cell recruitment to the p38 MAPK signaling pathway by demonstrating that pharmacologic inhibition of p28, which is activated upon Tie2 stimulation, abrogates mural cell development resulting in naked neovessels. Based on these observations the specific aims of this grant focus on the following hypotheses. 1) The aortic wall. contains vascular progenitor cells capable of both endothelial and mural cell differentiation. 2. The muscular wall of blood vessels originates from Tie2+ mural precursor cells. 3. Mural cell recruitment during angiogenesis is selectively mediated by the p38 MAPK pathway. Vascular progenitor cells of neonatal, young and old rat aortas will be identified by immunohistochemistry and confocal microscopy and isolated by surface marker-based magnetic beads technology. Their angioformative and mural cell differentiation properties will be studied in models of vascular organ culture, microvessel assembly and chemotaxis. The role of Tie2 and p38 MAPK pathway in mural cell recruitment will be studied by gene transfer technology using adeno-associated viral vectors carrying wild type or dominant-negative genes Mural cell recruitment will be analyzed by Immunohistochemistry, confocal microscopy and image analysis. Gene/protein expression and function will be evaluated by Northern and Western analysis, RT-PCR, kinase assays, gel zymography, in situ hybridization, and immunohistochemistry,. These studies will define key cellular and molecular mechanisms of vessel wall development. This knowledge may lead to novel approaches for the stabilization of neovessels in ischemic conditions, the induction of vascular regression in angiogenesis-dependent disorders, and the bioengineering of blood vessels for therapeutic applications.
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