Signal Transduction Pathways Involved Oxidative Stress-induced Endothelial Cell Apoptosis

Backgroud:Stroke or "brain attack" refers to an acute onset of cerebrovascular accident with long-lasting neurological deficits.The brain functions entirely depend on the continuous blood supply of oxygen and glucose.Notably,mitochondrial oxidative stress plays a criticle role in the development of ischemic stroke.We previously reported that an abnormal elevation of intracellular Ca²⁺ triggers activation of various Ca²⁺-dependent enzymes,accompanying with nitric oxide(NO) and its other reactive species during neurovascular injury.NO along with superoxide radical forms a highly reactive peroxynitrite radical(ONOO^-) leads to membrane lipid peroxidation,protein nitration, as well as endothelial injury.However,little is known about cross-talk between ONOO^-and mitochondrial oxidative stress signaling in endothelial cell death.Therefore,we asked whether cross-talk of ONOO^-/Omi/HtrA2 signaling involve in endothelial cell injury.Aim:The present study aims to investigate the mechanisms involved in oxygen-glucose deprivation(OGD)-induced endothelial cell death.We first address the morphological changes of endothelial cells following OGD.Secondly,we examine the temporal changes of Omi/HtrA2-PED/pea-15 pathway after OGD by Western blot and immunohistochemical studies.We also determine the protective effect of uric acid (ONOO^- inhibitor) and Ucf-101(Omi/HtrA2 inhibitor) on OGD-induced endothelial cell death.Methods and results:Human umbilical vein cell line EA.hy926 cell(EA strain) were used in the study,which were subject to OGD for 1 h,3 h,6 h,12 h and 24 h, respectively.The morphological changes of endothelial cells were observed by the light microscope,transmission electron microscopy,as well as JC -1 staining.Moreover, Western-blot and immunohistochemical staining were used to detect the changes in protein levels.Immunoblotting of cytosolic extracts of endothelial cells revealed that Omi/HtrA2 significantly increased 3 h after OGD induction and remained elevated at least until 24 h.By contrast,XIAP(X-linked inhibitor of apoptosis protein) in endothelial cells was observed down-regulated significantly since 3 h after OGD induction. Additionally,we also found that PED/pea15 in endothelial cells was observed down-regulated significantly 6 h after OGD induction.Consistently,the pro-caspase3 and pro-caspase8 protein levels also concomitantly decreased following OGD treatment, as did activation of gelatinase B(MMP-9).Ucf-101treatment effectively inhibited the Omi/HtrA2 release from Mitochondria to the cytosol,moderately reduced ischemia-induced XIAP degradation,whereas did not decrease the overexpression of cleaved-caspase3.Meanwhile,we observed that UA treatment significantly reduced OGD-induced cell death,along with significantly enhanced XIAP and PED/pea 15 expression in same context.By gene-chip analysis,a total of 1184 genes were differentially expressed in the 6 h after OGD induction compared to the normal ECs,Including 636 up-regulated genes and 548 down-regulated genes.These gene play important roles in the ECs apoptosis, cell cycle regulation,signal transduction and transcription. Conclusions:our results provide evidence that ONOO--mediated oxidatice stress associated with changes in mitochondrial Omi/HtrA2-XIAP-caspases and Omi/HtrA2-PED/pea15 signaling,which accounts for endothelial cell death.Targeting ONOO^- or Omi/HtrA2-PED/pea15 signaling might be a potential therapeutic strategy against neurovascular unit damage in cerebral ischemia.