Apoptosis In Cerebral Endothelial Cells Induced By Oxygen-Glucose Deprivation Involves Bid Cleavage, Cytochrome C Release And Caspases Activation

Background: Tissue injury from ischemia and reperfusion causes significant morbidity and mortality in cerebrovascular diseases. Cerebral endothelial cells (CECs) is a primary target of hypoxic/ischemic insults in the brain. Cerebral endothelial injury following ischemia may exacerbate brain damage and contribute to the breakdown of blood-brain-barrier (BBB) leading to increased vascular permeability and vasogenic brain edema. Oxygen-glucose deprivation (OGD) presents an in vitro model of ischemic insult and CEC apoptosis has been shown in entailing OGD. However, the characteristics of cerebral endothelial cell death after hypoxia/ischemia are to be elucidated.Methods: In order to further characterize the apoptotic pathway following OGD in murine CECs in culture, we analysed cell death in CECs after different durations of OGD by MTT and LDH assay, measured the activity of caspase 3 which acts as the executioner of apoptosis and that of caspase 8 which acts as theinitiator of apoptosis by enzymatic reaction and fluorometry. We also examined the cleavage of Bid which is a proapoptotic BH3-domain-only Bcl-2 family member, the cleavage of poly-(ADP-ribose)-polymerase (PARP) which is the hallmark of apoptosis and the substrate of caspases, and the degradation of procaspase 3, 8 to activated caspase 3, 8 by Western blot. We demonstrated release of cytochrome c from mitochondria, the key event and initiator in apoptosis, by immunocytochemistry (immunostaining) and Western blot. Moreover, we investigated the effects of zVAD-fmk, a broad-spectrum caspase inhibitor, on apoptosis.Results: Murine CECs that underwent OGD for 1h showed little morphological change and sustained virtually no cell death. Changes in cell morphology occurred if OGD duration was 4h or longer Cell viability decreased with the increase of OGD duration by MTT assay and LDH release. The activity of caspase-3 increased significantly in murine CECs 1h after OGD compared with normal control and got to the highest level 4h after OGD. The activity of caspase-8 increased significantly in murine CECs 1h and 4h after OGD compared with normalcontrol but less significantly than caspase-3. Western blot analysis of cytoplasmic protein extracts showed active caspase-3 increased as early as 1h after OGD and active caspase-8 increased 4h after OGD in murine CECs. Inactive proform Bid was cleaved to truncated active form in murine CECs 4h after OGD. PARP was cleaved to inactivated 85kD fragment in murine CECs 1h after OGD. The release of cytochrome c from mitochondria to cytosol was demonstrated by immunostaining and Western blot with cytosolic extraction. In immunostaining a diffuse redistribution of cytochrome c into the cytosol after OGD compared with the punctuate patten in the absence of OGD was noted. This indicated that cytochrome c was induced by OGD to release from mitochondria to cytosol. The same phenomenon of cytochrome c release after OGD was confirmed by Western blot The activation of caspase-3 and caspase-8 in murine CECs after OGD was inhibited with treatment of zVAD-fmk (50μM). Cytochrome c release and Bid cleavage in murine CECs after OGD was also inhibited. Consistent with these was the cleavage of PARP blocked with treatment of zVAD-fmk. Conclusion: These results indicate that Bid cleavage, cytochrome c release, caspases activation and PARP cleavage participated in OGD-induced apoptosis in murine CECs, in which both Fas/TNF (death receptor) pathway and mitochondrial pathway were involved. There may exist cross-talk between these two distinct signaling pathaways in OGD-induced apoptosis in murine CECs and caspases act as both initiators and executioners of apoptosis in OGD-induced apoptosis.