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 determine the development of atherosclerosis and restenosis following intracoronary therapeutical interventions. Apoptosis was studied in cultured human umbilical vein endothelial cells (HUVEC) to investigate the regulation of endothelial cell death following serum/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-triphosphate nick end-labeling (TUNEL). An ELISA was used for detecting intracytoplasmatic nucleosomes. Untreated HUVEC showed 16+/-6% TUNEL positive cells after 24 hours as analyzed by FACS. Serum/growth factor depletion increased apoptosis by 79+/-7%, while 50 ng/ml of the pro-apoptotic drug actinomycin-D induced comparable effects (72+/-11%). Apoptosis by serum/ growth factor depletion could be blocked completely by the anti-apoptotic agent cycloheximide (2 microg/ml), but was ineffective in blocking actinomycin-D-induced 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.
Gräfe
M
M
Department of Medicine, Cardiology and Angiology, German Heart Institute and Virchow Klinikum, Humboldt University, Berlin. graefe@dhzb.de
Steinheider
G
G
Desaga
U
U
Warnecke
C
C
Lehmkuhl
H B
HB
Regitz-Zagrosek
V
V
Hildebrandt
A G
AG
Fleck
E
E
D000975:Antioxidants / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D002469:Cell Separation; D002478:Cells, Cultured; D003513:Cycloheximide / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004730:Endothelium, Vascular / Q000187:drug effects / Q000502:physiology*; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D006801:Humans; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D011759:Pyrrolidines; D013859:Thiocarbamates / Q000494:pharmacology
D000975:Antioxidants; D011500:Protein Synthesis Inhibitors; D011759:Pyrrolidines; D013859:Thiocarbamates; C020972:pyrrolidine dithiocarbamic acid; D003513:Cycloheximide