Abstract DP394: Complement C3a receptor (C3aR) inhibition modulates brain endothelial cell dysfunction following OGD/reperfusion

Background: Ageing enhances chronic vascular inflammation, which promotes an increased risk of ischemic stroke, the third leading cause of death and disability worldwide. We have shown that complement C3a receptor (C3aR) activates pro-inflammatory pathways in murine stroke models as well as in brain endothelial cells subjected to Oxygen-glucose deprivation (OGD). Recent studies have suggested that ageing increases endothelial C3aR expression which promotes vascular inflammation as well as blood brain-barrier dysfunction. In this study, we investigated the extent to which OGD triggers endothelial dysfunction in aged cells, and whether C3aR deletion/inhibition is protective in this setting. Hypothesis: C3aR inhibition is protective in aged ischemic endothelial cells. Methods: Aged murine primary microvascular endothelial cells (derived from 15–18-month-old mice, Cell biologics, IL) subjected to OGD. C3aR gene knockdown was achieved using C3aR siRNA, and four groups were compared (Normoxia, OGD, OGD+Scramble siRNA and OGD+siRNA). We assessed blood-brain-barrier (BBB) integrity, release of inflammatory mediators, and oxidative stress using immunofluorescence, Western blotting and ELISA. P<0.05 was considered statistically significant. Results: In aged primary brain endothelial cells, OGD resulted in increased C3aR expression compared with Normoxia and C3aR deletion enhanced the tight junction proteins, occludin and claudin-5 expression. siRNA inhibition of C3aR also suppressed pro-inflammatory cytokines and chemokines expression, increased eNOS phosphorylation and reduced oxidative stress. Conclusion: Overall, these data suggest that C3aR inhibition in aged brain endothelial cells protects BBB integrity and vascular inflammation following OGD. Further work is warranted to explore underlying mechanisms of C3aR-mediated brain injury in ageing and stroke.