Oxyhemoglobin induces caspase‐mediated cell death in cerebral endothelial cells

• Published in: Journal of neurochemistry 2001-05, Vol.77 (4), p.1128-1135
• Main Authors: Meguro, Toshinari, Chen, Betty, Lancon, John, Zhang, John H.
• Format: Article
• Language: English
• Subjects: Animals; apoptosis; Apoptosis - drug effects; Biological and medical sciences; Caspase 8; Caspase 9; caspases; Caspases - metabolism; Cattle; Cell Death - drug effects; Cell Line; cerebral vasospasm; Cerebrovascular Circulation; Cysteine Proteinase Inhibitors - pharmacology; DNA Fragmentation; endothelium; Endothelium, Vascular - cytology; Endothelium, Vascular - drug effects; Endothelium, Vascular - physiology; Kinetics; Medical sciences; Microcirculation; Neurology; Oligopeptides - pharmacology; oxyhemoglobin; Oxyhemoglobins - pharmacology; Poly(ADP-ribose) Polymerases - metabolism; Vascular diseases and vascular malformations of the nervous system
• ISSN: 0022-3042; 1471-4159
• DOI: 10.1046/j.1471-4159.2001.00313.x

Damaged endothelium is one of the pathological changes of the cerebral vasospastic vessels following subarachnoid hemorrhage. Our recent study shows that oxyhemoglobin (OxyHb) induces apoptosis in vascular endothelial cells. Apoptosis generally 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 activation of caspases and the efficacy of caspase inhibitors, z‐IETD‐fmk and z‐LEHD‐fmk, for oxyhemoglobin‐induced apoptosis in vascular endothelial cells. Cultured bovine brain microvascular endothelial cells (passages 5–9) were used for this study. OxyHb (10 µmol/L) was added during the 24–72 h incubation with and without caspase‐8 or − 9 inhibitors (z‐IETD‐fmk and z‐LEHD‐fmk). Counting surviving cells, DNA laddering, western blotting of poly(ADP‐ribose) polymerase, and measurement of caspase activities were employed to confirm the cytotoxic effects of OxyHb and the protective effects of the caspase inhibitors. OxyHb produced cell detachment in a time‐dependent manner and increased caspase‐8 and ‐9 activities in the cells. z‐IETD‐fmk and z‐LEHD‐fmk (100 µmol/L) attenuated OxyHb‐induced cell loss, DNA laddering, and proteolytic cleavage of PARP, although a lower concentration (10 µmol/L) of caspase inhibitors showed partial effects. OxyHb activates caspase‐8 and ‐9 in cultured vascular endothelial cells, and blocking the action of the caspases with the inhibitors efficiently prevents loss of vascular endothelial cells from OxyHb‐induced apoptosis in vitro. These results suggest that the caspase cascade participates in OxyHb‐induced apoptosis.