A COMPARISON OF FIBRINOLYTIC CAPACITY AND RESPONSE TO TNF-ALPHA STIMULATION IN THE CULTURED ENDOTHELIAL-CELL LINES ECV-304 AND EA-HY-926

Experiments were carried out to investigate the ability of the inflamm atory mediator tumour necrosis factor (TNF-alpha) to modulate the fibr inolytic state of two immortalised endothelial cell lines: EA.hy 926 ( hybrid) and ECV 304 (spontaneously transformed). Results were compared and contrasted to responses of related endothelial cells from human u mbilical cord veins (HUVECs). As with HUVECs, EA.hy 926 cells secreted substantial basal quantities of plasminogen activator inhibitor (PAI- 1) protein into the cell medium. Moreover, addition of TNF-alpha (0.02 -20.0 ng/ml) to EA.hy 926 cells significantly increased levels of secr eted PAI-1 antigen up to 2-fold, in concentration-dependent fashion. I n contrast, whereas tissue-type plasminogen activator (t-PA) antigen i ncreased in EA.hy 926 cell supernatants upon stimulation with TNF-alph a (45% at 20 ng/ml TNF-alpha; p<0.001), a decrease was observed in HUV ECs (40% at 20 ng/ml TNF-alpha; p<0.001). ECV 304 cells, under normal or TNF-alpha stimulated conditions, showed a different fibrinolytic pr ofile from that of HUVECs or EA.hy 926 cells. High basal amounts of ur okinase plasminogen activator (u-PA) were secreted into the cell mediu m compared to levels of t-PA and PAI-1 antigen. Furthermore, in the pr esence of TNF-alpha, whilst PAI-1 antigen levels rose only modestly, s ecreted levels of u-PA increased substantially in a concentration-rela ted manner (approximately 3-fold at 20 ng/ml TNF-alpha; p<0.001). The use of continuous human cell lines such as EA.hy 926 and ECV 304 prese nts an opportunity to study in vitro mechanisms of fibrinolysis and it s involvement in cell function, whilst removing the requirement for pr imary endothelial cells.