Superoxide production by human umbilical vein endothelial cells in an anoxia-reoxygenation model

Ischaemia/reperfusion of cardiac tissue has been claimed to be associated with the production of oxygen free radicals, which can contribute to severe membrane damage and tissue injury. We investigated the effects of anoxia/reoxygenation treatment on superoxide radical production in an in vitro system consisting of preconfluent and confluent human endothelial cell monolayers. The influence of varying the anoxia and reoxygenation phases on superoxide production was studied. As a test of cytotoxicity, the release of the cytosolic enzyme lactate dehydrogenase in the culture medium was measured before and at 0, 24 and 48 h after anoxia-reoxygenation. Cellular damage was monitored by microscopic examination of the cultures during and after the experiments and by the expression of the von Willebrand protein and of the membrane glycoprotein IIa by indirect immunofluorescence with specific monoclonal antibodies. Our results show that the endothelial cells subjected to anoxia-reoxygenation release superoxide anions, as revealed by superoxide dismutase inhibitable cytochrome C reduction. Free radical production is dependent on cell confluent or preconfluent state and on both anoxia and reoxygenation duration. Free radical release does not seem to be accompanied by manifest cellular alteration.