We investigated the hypothesis that endothelial cells activated by erythropoietin (EPO) promote the migration of neuroblasts. This hypothesis is based on observationsin vivothat treatment of focal cerebral ischemia with EPO enhances the migration of neuroblasts to the ischemic boundary, a site containing activated endothelial cells and angiogenic microvasculature. To model the microenvironment within the ischemic boundary zone, we used a coculture system of mouse brain endothelial cells (MBECs) and neural progenitor cells derived from the subventricular zone of the adult mouse. Treatment of MBECs with recombinant human EPO (rhEPO) significantly increased secretion of matrix metalloproteinase 2 (MMP2) and MMP9. rhEPO-treated MBEC supernatant as conditioned medium significantly increased the migration of neural progenitor cells. Application of an MMP inhibitor abolished the supernatant-enhanced migration. Incubation of neurospheres alone with rhEPO failed to increase progenitor cell migration. rhEPO activated phosphatidylinositol 3-kinase/Akt (PI3K/Akt) and extracellular signal-regulated kinase (ERK1/2) in MBECs. Selective inhibition of the PI3K/Akt and ERK1/2 pathways significantly attenuated the rhEPO-induced MMP2 and MMP9, which suppressed neural progenitor cell migration promoted by the rhEPO-activated MBECs. Collectively, our data show that rhEPO-activated endothelial cells enhance neural progenitor cell migration by secreting MMP2 and MMP9 via the PI3K/Akt and ERK1/2 signaling pathways. These data demonstrate that activated endothelial cells can promote neural progenitor cell migration, and provide insight into the molecular mechanisms underlying the attraction of newly generated neurons to injured areas in brain.

Angiogenesis and neurogenesis are coupled processes. Using a coculture system, we tested the hypothesis that cerebral endothelial cells activated by ischemia enhance neural progenitor cell proliferation and differentiation, while neural progenitor cells isolated from the ischemic subventricular zone promote angiogenesis. Coculture of neural progenitor cells isolated from the subventricular zone of the adult normal rat with cerebral endothelial cells isolated from the stroke boundary substantially increased neural progenitor cell proliferation and neuronal differentiation and reduced astrocytic differentiation. Conditioned medium harvested from the stroke neural progenitor cells promoted capillary tube formation of normal cerebral endothelial cells. Blockage of vascular endothelial growth factor receptor 2 suppressed the effect of the endothelial cells activated by stroke on neurogenesis as well as the effect of the supernatant obtained from stroke neural progenitor cells on angiogenesis. These data suggest that angiogenesis couples to neurogenesis after stroke and vascular endothelial growth factor likely mediates this coupling.

MicroRNAs are small RNAs that attenuate protein expression by complementary binding to the 3′‐UTR of a target mRNA. Currently, very little is known about microRNAs after cerebral ischemia. In particular, microRNA‐21 (miR‐21) is a strong antiapoptotic factor in some biological systems. We investigated the role of miR‐21 after stroke in the rat. We employed in situ hybridization and laser capture microdissection in combination with real‐time RT‐PCR to investigate the expression of miR‐21 after stroke. In situ hybridization revealed that miR‐21 expression was upregulated in neurons of the ischemic boundary zone, and quantitative real‐time RT‐PCR analysis revealed that stroke increased mature miR‐21 levels by approximately threefold in neurons isolated from the ischemic boundary zone by laser capture microdissection as compared with homologous contralateral neurons 2 days (n = 4; P < 0.05) and 7 days (n = 3; P < 0.05) after stroke. In vitro, overexpression of miR‐21 in cultured cortical neurons substantially suppressed oxygen and glucose deprivation‐induced apoptotic cell death, whereas attenuation of endogenous miR‐21 by antisense inhibition exacerbated cell death after oxygen and glucose deprivation. Moreover, overexpression of miR‐21 in neurons significantly reduced FASLG levels, and introduction of an miR‐21 mimic into 293‐HEK cells substantially reduced luciferase activity in a reporter system containing the 3′‐UTR of Faslg. Our data indicate that overexpression of miR‐21 protects against ischemic neuronal death, and that downregulation of FASLG, a tumor necrosis factor‐α family member and an important cell death‐inducing ligand whose gene is targeted by miR‐21, probably mediates the neuroprotective effect. These novel findings suggest that miR‐21 may be an attractive therapeutic molecule for treatment of stroke.

Stroke is one of the leading causes of death and disability worldwide. Stroke recovery is orchestrated by a set of highly interactive processes that involve the neurovascular unit and neural stem cells. Emerging data suggest that exosomes play an important role in intercellular communication by transferring exosomal protein and RNA cargo between source and target cells in the brain. Here, we review these advances and their impact on promoting coupled brain remodeling processes after stroke. The use of exosomes for therapeutic applications in stroke is also highlighted.