The influence of cellular hypoxia and reactive oxygen species on the development of endothelial cell edema

We investigated in vitro whether endothelial cell edema is induced by cellular hypoxia or oxygen radical formation. Measurements of relative cell volume (RCV) were made using microweight analysis, liquid scintillation spectrometry and analysis of cellular protein content. To validate this method of determining cell volume, endothelial cells were incubated in media of different osmolarities. Vascular endothelial cells reacted to osmotic stress with a volume increase or decrease. The addition of xanthine oxidase (XOD; 3 mU/ml) and hypoxanthine (1 mM) for the enzymatic production of O2- caused a reproducible and significant increase in RCV by 29 +/- 8% (from 5.5 to 7.1 microliters/10(6) cells; p < 0.001) after an incubation time of 60 min. Nonenzymatically produced H2O2 (100 microM) caused a similar increase in RCV by 35 +/- 5% (from 5.5 to 7.6 microliters/10(6) cells; p < 0.001) over the same incubation period. The addition of catalase (50 U/ml) diminished the increasing effect of XOD as well as that of H2O2 on cell volume. As assessed by the uptake of the vital dye trypan blue and the release of lactate dehydrogenase into the medium, there was no significant loss of viability during the incubation time. Lower concentrations of H2O2 as well as lower activities of XOD did not induce a significant increase in RCV. Higher H2O2 concentrations and increased XOD activities caused a considerable time- and concentration-dependent injury of endothelial cells. RCV was unchanged even after long exposure (5 h) to two different hypoxic gas mixtures (3% O2:5% CO2:92% N2; 0% O2:5% CO2:95% N2). Cell viability was not impaired under hypoxic conditions. The results suggest that reactive oxygen species play a more important role in the development of endothelial cell edema than cellular hypoxia.