Caspase inhibitors attenuate oxyhemoglobin-induced apoptosis in endothelial cells.
OBJECTIVE
Our recent study showed that oxyhemoglobin (OxyHb) induces apoptosis in cultured endothelial cells. Apoptosis requires the action of various classes of proteases, including a family of cysteine proteases known collectively as the caspases. This study was undertaken to investigate the effect of 2 caspase inhibitors, Z-VDVAD-FMK and Z-DEVD-FMK, in the protection of endothelial cells from OxyHb-induced apoptosis.


METHODS
Cultured bovine brain microvascular endothelial cells (passages 5 to 9) were exposed to OxyHb (10 micromol/L) for 24 to 72 hours with and without caspase inhibitors. Cell attachment, DNA ladder, Western blotting of poly(ADP-ribose) polymerase (PARP), and caspase activities were measured to confirm the cytotoxic effect of OxyHb and the protective effect of the caspase inhibitors.


RESULTS
(1) OxyHb produced cell detachment in a time-dependent manner. (2) OxyHb increased caspase-2 and -3 activities, produced DNA ladders, and cleaved PARP in endothelial cells. (3) Z-VDVAD-FMK and Z-DEVD-FMK (100 micromol/L) attenuated OxyHb-induced cell detachment, reduced caspase-2 and -3 activities, abolished OxyHb-induced DNA ladders, and prevented OxyHb-induced cleavage of PARP.


CONCLUSIONS
OxyHb activates caspase-2 and -3 in cultured brain microvessel endothelial cells. Caspase inhibitors attenuated the cytotoxic effect of OxyHb.
Meguro
T
T
Department of Neurosurgery, University of Mississippi Medical Center, Jackson 39216, USA.
Chen
B
B
Parent
A D
AD
Zhang
J H
JH
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D001921:Brain / Q000098:blood supply / Q000166:cytology; D053143:Caspase 2; D053148:Caspase 3; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism; D002417:Cattle; D002452:Cell Count; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D002560:Cerebrovascular Circulation; D053938:DNA Fragmentation / Q000187:drug effects; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D008833:Microcirculation; D009842:Oligopeptides / Q000494:pharmacology; D010108:Oxyhemoglobins / Q000633:toxicity*; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism
D061945:Caspase Inhibitors; D004791:Enzyme Inhibitors; D009842:Oligopeptides; D010108:Oxyhemoglobins; C110772:benzoylcarbonyl-aspartyl-glutamyl-valyl-aspartyl-fluoromethyl ketone; C427308:benzoylcarbonyl-valyl-aspartyl-valyl-alanyl-aspartyl-fluoromethyl ketone; D011065:Poly(ADP-ribose) Polymerases; D053143:Caspase 2; D053148:Caspase 3; D020169:Caspases