Activation of endothelial ras-related C3 botulinum toxin substrate 1 (Rac1) improves post-stroke recovery and angiogenesis via activating Pak1 in mice.

[BACKGROUND AND PURPOSE] Long-term disability after stroke is common yet the mechanisms of post-stroke recovery are far from clear. It has been suggested that Ras-related C3 botulinum toxin substrate 1 (Rac1) contributes to functional recovery after ischemic stroke in mice. As Rac1 activation plays diverse roles in multiple cell types after central nervous system (CNS) injury, we herein examined the functional role of endothelial Rac1 in post-stroke recovery and angiogenesis.

[METHODS] Transient middle cerebral artery occlusion (MCAO) in mice and oxygen-glucose deprivation (OGD) in human brain endothelial cell line-5i (HEBC 5i) were performed to mimic ischemic stroke. Lentivirus vectors encoding Rac1 with GFP and endothelial promotor ENG were injected into the animal's brain after stroke to overexpress Rac1. After injection, stroke recovery was tested by multiple behavioral tests including novel object recognition, adhesive removal and single pellet reaching tests. Endothelial regeneration in the peri-infarct zone was detected by immunohistochemistry (IHC). In the vitro model, the effect of Rac1 and Pak1 inhibitors to cell proliferation and migration was examined by CCK-8 and wound healing assays after OGD. The cellular protein level of brain-derived neurotrophic factor (BDNF), phosphorylated cAMP response element-binding protein (CREB), extracellular signal-regulated kinase (ERK) 1/2 and mitogen-activated protein kinase kinase (MEK) 1/2 were detected by western blots.

[RESULTS] Delayed overexpression of endothelial Rac1 after MCAO improved cognitive and sensorimotor recovery from day 14 to 21 after stroke, increased vascular density and the protein level of pericytes in the peri-infarct zone without altering tissue loss in mice. Consistently, inhibition of Rac1 prevented endothelial proliferation and migration after OGD. Pak1 inhibition exerted a similar effect on endothelial cells. However, co-incubation of Rac1 and Pak1 inhibitors with cells did not lead to additive effects when compared with either inhibitor alone. Moreover, individual inhibition of Rac1 or Pak1 suppressed OGD-induced activation of pro-regenerative molecules, including CREB, MEK1/2 and ERK1/2, as well as the production of BDNF in vitro. The level of these proteins did not further decrease if both Rac1 and Pak1 were simultaneously inhibited.

[CONCLUSIONS] We conclude that activation of endothelial Rac1 improves functional recovery and angiogenesis after stroke, and this process is mediated by Pak1 signaling. This study provides novel insight for Rac1 in the mechanism of long-term stroke recovery.