Intracellular pH regulation in microvascular endothelial cells
Rojas, Jose D
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Many advances in vascular biology are a result of the use of cultured endothelial cells as a model. This model system has shown that endothelial cells synthesize and secrete various substances involved in regulating blood flow. There can be considerable heterogeneity in the properties and function of endothelial cells. These heterogeneities are a result of the tissue of origin and whether they are from macro- or microcirculation. Nitric oxide, a vasoactive substance synthesized by endothelial cells, has been shown to activate other second messenger systems in both a calcium-dependent and –independent fashion. There is a substantial body of work on intracellular calcium [Ca^2+] regulation in endothelial cells. Similarly, it is known that intracellular pH (pH'") is important in signal transduction following hormonal stimulation, and in regulating many physiological processes including cell growth, secretion, contraction, and invasion/migration. Significant work has been done on pH'" regulation in macrovascular endothelial cells; however, there remains a large gap in the knowledge of pH'" regulation in microvascular endothelial cells. Since there have been reported differences in [Ca^2+ ] regulation and nitric oxide production in microvascular and macrovascular endothelial cells, one specific aim of this dissertation was to perform an in-depth study of pH^in regulation in microvascular endothelial cells from different vascular beds. Microvascular endothelial cells are intimately involved in the invasive process of angiogenesis and there have been reports of plasma membrane vacuolar-type ATPase (pmV-ATPase) in several invasive cell types. These observations led to the formulation of the hypothesis that pmV-ATPase is required for invasion and was tested in this study. To our knowledge, this study is the first reported observation of pmV-ATPase as a pH^10 regulatory mechanism in microvascular endothelial cells along with evidence that the mechanism is involved in the process of invasion. Diabetes affects the cardiovascular system with complications that include acidosis, abnormalities in blood flow regulation, and altered microvascular proliferation and angiogenesis. Part of this study examined pH'" regulation in microvascular endothelial cells from a model of spontaneous diabetes. As a result of this work we determined that diabetic cells have decreased pmV-ATPase activity. Along with decreased pmV-ATPase activity diabetic cells also exhibit decreased invasive Potential and angiogenesis. Pharmacologic inhibition of pmV-ATPase activity in normal cells renders them as non-invasive as diabetic cells and unable to form capillary-like structures in an angiogenesis model. Finally, the study examines endothelial cells from lung micro vasculature. This vascular bed is known to undergo extensive vascular remodeling with prolonged hypoxia. Since prolonged hypoxia results in acidosis, it was predicted that these cells would have a dynamic pH'" regulatory that allows them to cope with this situation. Moreover, because vascular remodeling is an invasive process, it was predicted that these endothelial cells would also express pmV-ATPase. In support of the hypothesis; immunocytochemical, fluorescence spectroscopy, and pharmacologic studies reveal pmV-ATPase as a major pH'" regulatory mechanism in these cells. In summary this work demonstrates the presence of pmV-ATPase in several microvascular beds and provides functional evidence for its role in the invasive process of vascular remodeling.