Abstract TP121: Inhibition of Ezh2 Simultaneously Blocks Pro-Inflammatory Polarization and Enhances Vegfa Expression in Male and Female Microglia

Abstract

Background: Microglia are the innate immune cells of the central nervous system and are poised to be the first responders to ischemic stroke by polarizing to a damaging pro-inflammatory or a reparative anti-inflammatory phenotype. Oxygen glucose deprivation (OGD) alters microglia epigenetic regulation to promote an anti-inflammatory phenotype and up-regulation of Vegfa which may enhance angiogenesis after ischemic stroke. Interestingly, OGD down-regulates Enhancer of Zeste Homologue 2 (Ezh2), a histone lysine N -methyltransferase associated with repression of anti-inflammatory genes to promote pro-inflammatory polarization. Hypothesis: We hypothesized that OGD drives microglial anti-inflammatory polarization through down-regulation of Ezh2 . Methods: We cultured male and female primary microglia from neonatal P0.5-P3 C57Bl/6 mice in phenol red-free DMEM (Gibco) and charcoal stripped FBS (Sigma). Cultures were treated with GSK343 (Cayman Chemical) or DMSO control and exposed cells to OGD (95% N 2 , 5% CO 2 ) for 8hrs. Microglia were collected for RNA (Qiagen) and analyzed using real-time PCR (Bio-Rad). Results: We found that OGD up-regulated anti-inflammatory genes, notably Gdf15 , Il1rn , Irf4 , and Vegfa while down-regulating pro-inflammatory genes including Tnfa , Il1b , Il6 , and Ccl2 (n=14, p<0.05). Ezh2 expression was down-regulated with 8hr OGD (p<0.001). Pharmacological inhibition of Ezh2 with GSK343 in OGD resulted in exaggerated down-regulation of Il1b and Il6 (p<0.01) as well as enhanced up-regulation of Vegfa (p<0.05). Notably, there were no sex differences identified in our work (n=6-7, p>0.05). Conclusion: Our data shows that OGD-induced down-regulation of Ezh2 is necessary for down-regulation of Il1b and Il6 and up-regulation of Vegfa . This may be a novel therapeutic target for ischemic stroke available to both men and women to simultaneously diminish pro-inflammatory gene expression and enhance angiogenesis and functional recovery.