Endothelial cells promote smooth muscle cell resilience to H O -induced cell death in mouse cerebral arteries.

Aim: Brain injury produces reactive oxygen species (ROS). However, little is known of how acute oxidative stress affects cell survival in the cerebral vascular supply. We hypothesized that endothelial cells (ECs) are more resilient to H O and protect vascular smooth muscle cells (SMCs) during acute oxidative stress.

Methods: Mouse posterior cerebral arteries (PCAs; diameter, ~80 µm) were exposed to H O (200 µM, 50 min, 37°C). Nuclear staining identified dead and live cells of intact and endothelium-disrupted vessels. SMC [Ca ] was assessed with Fura-2 fluorescence, and superoxide production was assessed by dihydroethidium and MitoSOX fluorescence.

Results: In response to H O : SMC death (21%) exceeded EC death (5%) and increased following endothelial disruption (to 48%) with a corresponding increase in SMC Ca entry through transient receptor potential (TRP) channels. Whereas pharmacological inhibition of TRPV4 channels prevented SMC death and reduced Ca entry for intact vessels, both remained elevated following endothelial disruption. In contrast, pharmacological inhibition or genetic deletion of TRPC3 prevented SMC death and attenuated Ca entry for both intact and endothelium-disrupted vessels. Inhibiting gap junctions increased EC death (to 22%) while SMC death and [Ca ] responses were attenuated by inhibiting nitric oxide synthesis or scavenging superoxide/peroxynitrite. Inhibiting NADPH oxidases also prevented SMC Ca entry and death. H O increased mitochondrial ROS production while scavenging mitochondria-derived superoxide prevented SMC death but not Ca entry.

Conclusions: During acute exposure of cerebral arteries to acute oxidative stress, ECs are more resilient than SMCs and the endothelium may protect SMCs by reducing Ca entry through TRPC3 channels.