Ischemia-reperfusion injury of retinal endothelium by cyclooxygenase- and xanthine oxidase-derived superoxide.

The formation of reactive oxygen species (ROS) may be important in the pathogenesis of microvascular dysfunction and injury in ischemic retinopathies. The authors hypothesized that retinal endothelial cells can generate injurious levels of superoxide radical in response to ischemia/reperfusion, that endothelial xanthine oxidase and cyclooxygenase are important enzymatic sources of superoxide radical under these conditions, and that superoxide scavengers and inhibitors of these enzymes can protect endothelium from ischemic injury. The authors used confluent cultures of mouse retinal endothelial cells (MREC) subjected to exogenously generated superoxide or simulated ischemia-reperfusion to test these hypotheses. Cell injury was assessed biochemically by lactate dehydrogenase release into the culture medium. MREC were injured in a duration-dependent fashion by exposure to the superoxide-generating mix of hypoxanthine and xanthine oxidase. Increasing periods of oxygen and glucose deprivation (OGD) for 5-9 hr followed by replenishment of substrates for 2 hr led to progressive increases in endothelial cell injury; a significant proportion of the injury occurred during the period of substrate replenishment. Significant MREC protection was achieved by the superoxide scavengers SOD (1000 U ml(-1)) and a carboxylic acid derivative of carboxyfullerene (10 microM), the xanthine oxidase inhibitors oxypurinol (100 microM) and diphenyleneiodonium (DPI) (100 n M), and the cyclooxygenase inhibitors indomethacin (300 microM) and ibuprofen (300 microM). It is concluded that MREC are vulnerable to auto-oxidative injury by superoxide radical generated following a period of OGD. Both xanthine oxidase- and cyclooxygenase-dependent pathways are important enzymatic sources of superoxide formation in this setting. These enzymes and the ROS produced from their activity may be viable therapeutic targets to reduce microvascular dysfunction and injury in ischemic retinopathies.