Abstract WP140: Activation of Endothelial Ras-Related C3 Botulinum Toxin Substrate (Rac) 1 Improves Post-Stroke Recovery and Angiogenesis via Activating P21 Activating Kinases (Pak) 1 in Mice

Abstract

Introduction: Cerebral angiogenesis is correlated with post-stroke prognosis yet its mechanism is far from clear. Accumulating evidence shows that Rac1, a Rho-related GTPase, plays a central role in promoting neovascularization via activating Pak 1 in endothelial cells. We investigated whether activating endothelial Rac1 improved functional recovery and angiogenesis after ischemic stroke. Methods and Results: Young male mice were subjected to a 60-minute middle cerebral artery (MCAO) stroke. Endothelial Rac1 was overexpressed by brain injection of lentiviral vectors encoding Rac1 with endothelial promoter ENG the day after MCAO. Treatment improved the recovery of sensorimotor and cognition function assessed by single pellet reaching (day 7: p=0.042; day 14: p=0.012, n=7/group), adhesive removal (day 7: p=0.013, n=7/group) and novel object recognition test (day 21: p=0.045, n=7/group) compared to the control vector group. Follow-up immunofluorescence revealed that ENG-Rac1 promoted angiogenesis as measured by the endothelial proliferation (CD105+BrdU, p=0.016, n=5/group) and the elevation of pericyte (NG2, p=0.008, n=5/group), which is indispensable for stable vessel structure, in the peri-infarct zone, with no effect on brain cavity size. We further explored Rac1 signaling, involving Pak1, in mediating angiogenesis in vitro. Human brain endothelial cell line-5i (HEBC 5i) was subjected to a 16-hour oxygen-glucose deprivation (OGD) followed by re-oxygenation. Rac1 (NSC23766 at 30 μM) and/or Pak1 (IPA3 at 10 μM) inhibitors were incubated with HEBC 5i 24 hours after OGD. Delayed inhibition of Rac1 reduced endothelial proliferation (CCK-8 assay) at 48 hours (p=0.039, n=4/group) and the ability of migration (scratch assay) at 72 hours (p<0.001, n=6/group) after OGD compared to the vehicle control. Similarly, Pak1 inhibition also led in the reduction in proliferation (p<0.001, n=4/group) and migration (p=0.003, n=5/group). No additional inhibitory effect if application of both inhibitors together compared with either inhibitor alone. Conclusions: Activation of endothelial Rac1 improves post-stroke recovery and angiogenesis and this pro-angiogenetic effect is mediated by Pak1.