Characterization of two distinct mechanisms for induction of apoptosis in human vascular endothelial cells

Tissue homeostasis is fundamentally influenced by the functional integrity and state of endothelial cells. Survival and death of endothelial cells are encountered in cardiovascular disease and may, moreover, affect and determ ine the development of atherosclerosis and restenosis following intracorona ry therapeutical interventions. Apoptosis was studied in cultured human umbilical vein endothelial cells (H UVEC) to investigate the regulation of endothelial cell death following ser um/growth factor depletion as well as incubation with actinomycin-D. Apoptosis was verified by DNA fragmentation and quantified by fluorescence activated cell sorting (FACS) analysis after TdT-mediated deoxyuridine-trip hosphate nick end-labeling (TUNEL). An ELISA was used for detecting intracy toplasmatic nucleosomes. Untreated HUVEC showed 16 +/- 6 % TUNEL positive c ells after 24 hours as analyzed by FACS. Serum/growth factor depletion incr eased apoptosis by 79 +/- 7%, while 50 ng/ml of the pro-apoptotic drug acti nomycin-D induced comparable effects (72 +/- 11%). Apoptosis by serum/ grow th factor depletion could be blocked completely by the anti-apoptotic agent cycloheximide (2 mu g/ml), but was ineffective in blocking actinomycin-D-i nduced apoptosis. Pyrrolidine dithiocarbamate (PDTC) also acted as an anti- apoptotic agent by blocking apoptosis induced by actinomycin-D, but had no effect on apoptosis induced by factor depletion. Thus, two independent mechanisms for regulation of apoptosis are suggested to be present in human vascular endothelial cells.