Angiogenesis is important for vascular remodeling and is strictly controlled under normal conditions. In pathological states such as cancer and diabetes, angiogenesis is altered. The process of angiogenesis requires growth and invasion/migration of endothelial cells through extracellular matrix proteins. Microvascular endothelial cells are exposed to an acid and anoxic environment, conditions that are unfavorable for growth and survival. Thus it is paradoxical that microvascular endothelial cells grow under these conditions. It is known that the extracellular environment of tumors is acidic and anoxic, yet these cells grow and survive in this hostile environment. A vacuolar type proton ATPase (pmV-ATPase) has been identified in highly invasive and metastatic tumor cells. The cells utilize pmV-ATPase to maintain an aalkaline cytosolic pH permissive for growth. Because microvascular endothelial cells also invade and migrate through extracellular matrix proteins, it is hypothesized that microvascular endothelial cells exhibit pmV-ATPase that is greater at the invading/leading edge of the cells, and that pmV-ATPase activity is exacerbated by chronic acidosis. It is also hypothesized that pmV-ATPase is decreased in microvascular endothelial cells from diabetic models where angiogenesis is impaired. To test these hypothesis, pmV-ATPase will first be analyzed in microvascular endothelial cells by evaluating the role of pmV-ATPase for pHcyt regulation using time-resolved fluorescence spectroscopy and ion substituion/pharmacological approaches. The presence of this pump's activity at the plams membrane will be analyzed by immunocytochemistry, immunogold electron microscopy and Western blot analyses of subcellular fractions. The relevance of pmV-ATPase at the leading edge of the cell will be understood by studying pHcyt regulation in single cells from leading to lagging edge using line scanning laser confocal resolution respectively. The physiological relevance of pmV-ATPase will be evaluated using in vitro invations mibration assays, in vitro angiogenesis models and the wounded monolayer model. This will be done in the presence and absence of V-ATPse inhibitors. The relevance of pmV-ATPase in angiogenesis and diabetes will be understood by using microvascular endothelial cells from a spontaneous model of diabetes with inborn defects.
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