Abstract WP155: Inhibition of Ras-Related C3 Botulinum Toxin Substrate 1 (Rac1) Exacerbates Endothelial Viability and Regenerative Responses After Stroke in vitro

Abstract

Brain endothelial cells play an essential role in BBB integrity and vascular recovery after injury. Accumulating evidence shows that Rac1, a Rho-related GTPase, are central in promoting endothelial vascular development. However, the contribution of endothelial Rac1 in ischemic stroke has not been directly investigated. We hypothesized that Rac1 signaling protects endothelial cells and promotes angiogenesis after stroke. Immortalized human brain endothelial cells (HBEC-5i) were challenged by oxygen-glucose deprivation (OGD) for 18 hours followed by re-oxygenation. For cell viability test, NSC 23766 (NSC, 3, 15 and 30 μM), a specific Rac1 inhibitor, was added at re-oxygenation (immediately after the18 hours ODG). The viability of endothelial cells was assessed by CCK-8 (% normoxia control) 24 hours after the18 hours OGD. For migration and tube formation assays, NSC at 30 μM was added 24 hours after the 18 hours OGD to avoid potential effects on cell survival. Cell migration using scratch assay (wound area compared to initial time %) was examined 72 hours after the 18 hours OGD. Tube formation, the ability of cells to align into tube-like structures (number of junctions), was measured 28 hours after the 18 hours OGD. Data are presented as mean±SEM. OGD reduced endothelial cell viability (control 100±3.23 vs. OGD 69.97±1.11, n=6-7, p<0.001). Acute Rac1 inhibition with NSC led to a worsening of the endothelial viability in a concentration dependent manner (3 μM: OGD+vehicle 69.97±1.11 vs. OGD+NSC 63.98±1.53, n=6, p>0.05; 15 μM: OGD+vehicle 69.97±1.11 vs. OGD+NSC 60.77, n=6, p<0.05; 30 μM: OGD+vehicle 69.97±1.11 vs. OGD+NSC 43.92±3.23, n=6, p<0.001). Using delayed treatment regimen, Rac1 inhibition reduced the endothelial regenerative responses including the cell migration assessed by the scratch assay (OGD+vehicle 9.95±2.31% vs. OGD+NSC 32.85±2.69%, n=6, p<0.001) and cell re-organization assessed by tube formation assay (OGD+vehicle 66.55±5.52 vs. OGD+NSC 33.63±6.73, n=8-11, p<0.05). In conclusion, acute inhibition of Rac1 signaling worsened endothelial cell survival and delayed inhibition of Rac1 reduced angiogenic responses after OGD. Activation of endothelial Rac1 signaling may protect BBB and promote vascular recovery after stroke.