Signal‐specific contributions of NADPH oxidase and mitochondria to oxidative‐stress induced cerebral vascular endothelial injury.

In the cerebral circulation, oxidative stress associated with seizures, hypoxia and inflammation causes endothelial injury. The excitatory neurotransmitter, glutamate, and the inflammatory cytokine, TNF‐alpha (TNF), are major mediators of oxidative‐stress related endothelial injury. In cerebral vascular endothelial cells from newborn pigs, glutamate (2mM) and TNF (15ng/ml) increase formation of reactive oxygen species (ROS) and cause oxidative‐stress related apoptosis as indicated by caspase‐3 activation, NFkB nuclear translocation, DNA fragmentation, loss of cell‐cell contacts, and cell detachment. We investigated contributions of NADPH oxidase, NO synthase, and mitochondria to oxidative‐stress mediated endothelial death. TNF‐evoked ROS and apoptosis were greatly reduced by NADPH oxidase inhibitors (DPI and apocynin) and, to a lesser extent, by a mitochondrial complex II inhibitor, TTFA. Conversely, glutamate‐evoked ROS and apoptosis were blocked by TTFA, but not affected by NADPH oxidase inhibitors. The NO synthase inhibitor, L‐NNA, did not alter ROS responses to either glutamate or TNF. Overall, these data indicate signal‐specific activation of distinct ROS‐generating pro‐apoptotic pathways in the cerebral vasculature. TNF causes cerebrovascular endothelial cell death largely by activating NADPH oxidase, while glutamate causes apoptosis through the mitochondrial pathway.