Inhibition of NF-kappaB Activation Increases Oxygen-Glucose Deprivation-Induced Cerebral Endothelial Cell Death

Increasing evidences suggest that ischemia-induced vascular damage is an integral step in the cascade of the cellular and molecular events initiated by cerebral ischemia. In the present study, employing a mouse brain endothelioma-derived cell line, bEnd.3, and oxygen-glucose deprivation (OGD) as an in vitro stroke model, the role of nuclear factor kappa B (NF-kappaB) activation during ischemic injury was investigated. OGD was found to activate NF-kappaB and to induce bEnd.3 cell death in a time-dependent manner. OGD phosphorylated neither 32 Ser nor 42 Tyr of IkappaBalpha. OGD did not change the amount of IkappaB alpha. The extents of OGD-induced cell death after 8 h, 10 h, 12 h and 14 h of OGD were 10%, 35%, 60% and 85%, respectively. Reperfusion following OGD did not cause additional cell death, indicating no reperfusion injury after ischemic insult in cerebral endothelial cells. Three known as NF-kappaB inhibitors, including pyrrolidine dithiocarbamate (PDTC) plus zinc, aspirin and caffeic acid phenethyl ester (CAPE), inhibited OGD-induced NF-kappaB activation and increased OGD-induced bEnd.3 cell death in a dose dependent manner. There were no changes in the protein levels of bcl-2, bax and p53 which are modulated by NF-kappaB activity. These results suggest that NF-kappaB activation might be a protective mechanism for OGD-induced cell death in bEnd.3.

Role of protein kinases on NF- kappaB activation and cell death in bovine cerebral endothelial cells

Nuclear factor kappaB (NF- kappaB) activation is modulated by various protein kinases. Activation of NF- kappaB is known to be important in the regulation of cell viability. The present study investigated the effect of inhibitors of protein tyrosine kinase (PTK), protein kinase C (PKC) and protein kinase A (PKA) on NF- kappaB activity and the viability of bovine cerebral endothelial cells (BCECs). In serum-deprivation-induced BCEC death, low doses of TNF alpha showed a protective effect. TNF alpha induced NF- kappaB activation within 4 h in serum-deprivation. PTK inhibitors (herbimycin A and genistein) and PKC inhibitor (calphostin C) prevented NF- kappaB activation stimulated by TNF alpha. Likewise, these inhibitors prevented the protective effect of TNF alpha. In contrast to TNF alpha-stimulated NF- kappaB activity, basal NF- kappaB activity of BCECs in media containing serum was suppressed only by calphostin C, but not by herbimycin A. As well BCEC death was also induced only by calphostin C in serum-condition. H 89, a PKA inhibitor, did not affect the basal and TNF alpha-stimulated NF- kappaB activities and the protective effect of TNF alpha on cell death. These data suggest that modulation of NF- kappaB activation could be a possible mechanism for regulating cell viability by protein kinases in BCECs.