Basal Apoptotic Rate Research Articles

HYPOXIA EXPOSURE REDUCES APOPTOSIS (PROGRAMMED CELL DEATH) IN CULTURED ENDOTHELIAL CELLS. † 281

Hypoxia/reoxygenation has been implicated as a signal for endothelial cell apoptosis. We examined the possibility that exposure of endothelial cells (EC) to varying periods of hypoxia and reoxygenation would be associated with altered basal levels of apoptosis. Cultured pulmonary artery endothelial cells(passage 18-20) were incubated in room air and 5% CO2 and grown to confluence in 6 well microtiter plates. The confluent EC monolayers were exposed to hypoxia and reoxygenation for varying periods of time (0-12-24 hrs) in a plexiglass chamber continuously purged with 5% N2 and 95% CO2. Control cells were exposed to normoxia in a similar manner and duration. Cell viability for all groups was assessed by trypan blue exclusion. Cells were trypsinized and morphology assessed using cytospin preparations, stained with DAPI and observed by UV fluorescence microscopy. Analysis of DNA fragmentation was done using 1% agarose gel electrophoresis of low molecular weight DNA stained with ethidium bromide. Viabilty for all groups was >96%. Control cells had a basal apoptotic rate of 10.5±2.0% and showed the typical morpology with characteristic chromatin condensation and margination as well as formation of apoptotic nuclear fragments. Cells exposed to hypoxia had a 50% decrease in the basal rate of apoptosis (4.5±1.5%)(p<0.01) compared to control cells and also displayed the typical morphology. The decrease in basal apoptosis with hypoxia was confirmed by DNA analysis which showed a significant reduction in internucleosomal DNA fragmentation. Reoxygenation had no effect on the rate of apoptosis. The results suggest that regulation of endothelial cell death following hypoxia and reoxygenation is more complex than previously believed.