MECHANISMS OF HYPOXIC/REPERFUSION INJURY IN ENDOTHELIUM

DESCRIPTION (Adapted from Investigator's Abstract): Hypoxic, anoxic and reperfusion injuries are critical processes in the pathogenesis of a broad spectrum of human disease. Because of its location between blood and tissue, the endothelium is particularly vulnerable to hypoxic/reperfusion injury. Previous work from this laboratory has demonstrated that in hepatocytes, cardiac myocytes and endothelial cells, acidosis protects potently against hypoxic, anoxic, and reperfusion injuries. The pH paradox, an acceleration of injury when intracellular pH (pHi) returns to physiologic levels during reperfusion, was documented in both tissue culture and intact organ models. The mechanisms underlying the protective effect of acidosis and the pH paradox in endothelial cells are not known but studies from this laboratory and others suggest a role for pH-dependent oxygen radical formation in mitochondria due to the oxidative stress of hypoxic and reperfusion injury, proteolysis and phospholipase A2 activation. Based upon these findings, the overall goal is to test the hypothesis that acidosis protects against endothelial cell hypoxic and reperfusion injury by inhibition of pH-dependent oxygen radical formation, proteases and phospholipases, processes that damage membrane structures in the cell. The objectives are: 1) Examine the role of the mitochondrial membrane permeability transition in endothelial cell hypoxic and reperfusion injury, 2) Determine the role of pH-dependent phospholipase A2s in endothelial cell hypoxic and reperfusion injury, and 3) Identify and determine the role of genes preferentially expressed during hypoxic and reperfusion injury in pH-dependent lethal endothelial cell injury. These studies will emphasize measurements of single living endothelial cells using a variety of novel digitized video and confocal microscopic techniques, which will be combined with cloning and antisense oligonucleotide strategies to determine the role of specific gene expression in hypoxic and reperfusion injury. Measurements of hydroperoxide formation, proteolysis, and phospholipase activity in cultures of endothelial cells as a function of pH will also be made. The proposed studies are designed to establish direct cause and effect relationships between pH-dependent mitochondrial dysfunction and oxygen radical production, proteolysis, and phospholipase activity in hypoxic and reperfusion injury. New information from these studies will better define potential sites of intervention to protect and rescue cells from hypoxic and reperfusion injury.