Vascular endothelial growth factor promotes transdifferentiation of astrocytes into neurons via activation of the MAPK/Erk-Pax6 signal pathway.

Reactive astrocytes can be transformed into new neurons. Vascular endothelial growth factor (VEGF) promotes the transformation of reactive astrocytes into neurons in ischemic brain. Therefore, in this study, the molecular mechanism of VEGF's effect on ischemia/hypoxia-induced astrocyte to neuron transformation was investigated in the models of rat middle cerebral artery occlusion (MCAO) and in astrocyte culture with oxygen and glucose deprivation (OGD). We found that VEGF enhanced ischemia-induced Pax6, a neurogenic fate determinant, expression and Erk phosphorylation in reactive astrocytes and reduced infarct volume of rat brain at 3 days after MCAO, which effects could be blocked by administration of U0126, a MAPK/Erk inhibitor. In cultured astrocytes, VEGF also enhanced OGD-induced Erk phosphorylation and Pax6 expression, which was blocked by U0126, but not wortmannin, a PI3K/Akt inhibitor, or SB203580, a MAPK/p38 inhibitor, suggesting VEGF enhanced Pax6 expression via activation of MAPK/Erk pathway. OGD induced the increase of miR365 and VEGF inhibited the increase of OGD-induced miR365 expression. However, miR365 agonists blocked VEGF-enhanced Pax6 expression in hypoxic astrocytes, but did not block VEGF-enhanced Erk phosphorylation. We further found that VEGF promoted OGD-induced astrocyte-converted to neuron. Interestingly, both U0126 and Pax6 RNAi significantly reduced enhancement of VEGF on astrocytes-to-neurons transformation, as indicated Dcx and MAP2 immunopositive signals in reactive astrocytes. Moreover, those transformed neurons become mature and functional. We concluded that VEGF enhanced astrocytic neurogenesis via the MAPK/Erk-miR-365-Pax6 signal axis. The results also indicated that astrocytes play important roles in the reconstruction of neurovascular units in brain after stroke.

Vascular endothelial growth factor increases the function of calcium-impermeable AMPA receptor GluA2 subunit in astrocytes via activation of protein kinase C signaling pathway.

Astrocytic calcium signaling plays pivotal roles in the maintenance of neural functions and neurovascular coupling in the brain. Vascular endothelial growth factor (VEGF), an original biological substance of vessels, regulates the movement of calcium and potassium ions across neuronal membrane. In this study, we investigated whether and how VEGF regulates glutamate-induced calcium influx in astrocytes. We used cultured astrocytes combined with living cell imaging to detect the calcium influx induced by glutamate. We found that VEGF quickly inhibited the glutamate/hypoxia-induced calcium influx, which was blocked by an AMPA receptor antagonist CNQX, but not D-AP5 or UBP310, NMDA and kainate receptor antagonist, respectively. VEGF increased phosphorylation of PKCα and AMPA receptor subunit GluA2 in astrocytes, and these effects were diminished by SU1498 or calphostin C, a PKC inhibitor. With the pHluorin assay, we observed that VEGF significantly increased membrane insertion and expression of GluA2, but not GluA1, in astrocytes. Moreover, siRNA-produced knockdown of GluA2 expression in astrocytes reversed the inhibitory effect of VEGF on glutamate-induced calcium influx. Together, our results suggest that VEGF reduces glutamate-induced calcium influx in astrocytes via enhancing PKCα-mediated GluA2 phosphorylation, which in turn promotes the membrane insertion and expression of GluA2 and causes AMPA receptors to switch from calcium-permeable to calcium-impermeable receptors, thereby inhibiting astrocytic calcium influx. The present study reveals that excitatory neurotransmitter glutamate-mediated astrocytic calcium influx can be regulated by vascular biological factor via activation of AMPA receptor GluA2 subunit and uncovers a novel coupling mechanism between astrocytes and endothelial cells within the neurovascular unit.

VEGF overexpression enhances the accumulation of phospho-S292 MeCP2 in reactive astrocytes in the adult rat striatum following cerebral ischemia.

PURPOSE: Astrocytes can be reactivated after cerebral ischemia by expressing nestin and other characteristic markers of neural stem cells (NSCs). However, the epigenetic features of reactive astrocytes are not well known. Methyl-CpG-binding protein 2 (MeCP2) is a vital transcriptional modulator in brain development. Although the expression and function of some phosphorylated MeCP2 isoforms have been clarified, phospho-serine 292 (pS292) MeCP2 has not yet drawn much attention. In this study, we used western blot analysis and immunohistochemical and immunofluorescent staining to reveal the expressive features of pS292 MeCP2 and MeCP2 in the adult rat striatum following transient middle cerebral artery occlusion (MCAO). RESULTS: We first discovered that the ischemia-induced expression of cytoplasmic pS292 MeCP2 is primarily accumulated in nestin-positive reactive astrocytes in the stroke-injured striatum. Moreover, the enhancement of astrocytic pS292 MeCP2 was correlated with the augmentation of VEGF in astrocytes, as determined by the substantial co-localization of pS292 MeCP2 and VEGF after stroke. Finally, the exogenous overproduction of VEGF further promoted the expression of pS292 MeCP2 in reactive astrocytes, and this effect was accompanied by a marked increase in reactive astrocytes. On the contrary, MeCP2 was predominantly expressed in the neuronal nucleus, and the level of this protein was not significantly altered after ischemic injury and VEGF overproduction. CONCLUSION: Our data provide the first demonstration that overexpression of VEGF enhances the accumulation of pS292 MeCP2 in reactive astrocytes in the ischemic-injured rat striatum, implicating a pS292 MeCP2-related epigenetic role of exogenous VEGF in reactive astrocytes following cerebral ischemia.