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(Stroke. 2001;32:561.)
© 2001 American Heart Association, Inc.

Original Contributions

Caspase Inhibitors Attenuate Oxyhemoglobin-Induced Apoptosis in Endothelial Cells

Toshinari Meguro, MD; Betty Chen, BS; Andrew D. Parent, MD John H. Zhang, MD, PhD

From the Department of Neurosurgery, University of Mississippi Medical Center, Jackson.

Correspondence to John H. Zhang, MD, PhD, Department of Neurosurgery, University of Mississippi Medical Center, 2500 N State St, Jackson, MS 39216. E-mail jzhang{at}neurosurgery.umsmed.edu

Background and Purpose—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 µmol/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 µmol/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.

Key Words: apoptosis • caspase • cerebral ischemia, transient • endothelium • oxyhemoglobins

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