Oxyhemoglobin induces caspase‐mediated cell death in cerebral endothelial cells

Abstract

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 ef®cacy 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 mmol/L) was added during the 24±72 h incubation with and without caspase-8 or 2 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 con®rm 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-IETDfmk and z-LEHD-fmk (100 mmol/L) attenuated OxyHbinduced cell loss, DNA laddering, and proteolytic cleavage of PARP, although a lower concentration (10 mmol/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 ef®ciently prevents loss of vascular endothelial cells from OxyHb-induced apoptosis in vitro. These results suggest that the caspase cascade participates in OxyHb-induced apoptosis.

Synergistic Protection of a General Caspase Inhibitor and MK-801 in Bilirubin-Induced Cell Death in Human NT2-N Neurons

“…The execution of caspase-mediated apoptosis is thought to be the result of a cascade of events converging in the activation of the executioner caspases -3, -6, or -7, where caspase-3 has been identified as a major downstream execution caspase (15). In the present study, treatment with the apoptosisinducer STS evoked a large activation of caspase-3.…”

Induction of Caspase-Mediated Cell Death by Matrix Metalloproteinases in Cerebral Endothelial Cells after Hypoxia—Reoxygenation

“…MMP-9-mediated disruption of cellmatrix interactions promotes neuronal death (Gu et al, 2002). Here, we showed that similar mechanisms may be involved, insofar as endogenous apoptotic machinery has been previously demonstrated to be present in endothelial cells (Aoudjit and Vuori, 2001;Lin and Maiese, 2001;Meguro et al, 2001;Xu et al, 2001). After hypoxia-reoxygenation, MMP-2 and MMP-9 were upregulated in human brain endothelial cells, and subsequent degradation of extracellular matrix fibronectin appeared to trigger a form of caspase-mediated cytotoxicity.…”

Interactions Between p38 Mitogen-Activated Protein Kinase and Caspase-3 in Cerebral Endothelial Cell Death After Hypoxia-Reoxygenation

“…In cerebral endothelial cultures, oxidative stress can trigger caspase-mediated pathways of cellular demise. [7][8][9] Because oxidative stress plays a critical role in ischemia and reperfusion, it is likely that similar caspase-mediated pathways operate in the vascular compartment after stroke. Nevertheless, the signaling pathways that regulate caspase activity during cerebral endothelial cell death remain to be fully elucidated.…”