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.

MicroRNA-365 modulates astrocyte conversion into neuron in adult rat brain after stroke by targeting Pax6.

Reactive astrocytes induced by ischemia can transdifferentiate into mature neurons. This neurogenic potential of astrocytes may have therapeutic value for brain injury. Epigenetic modifications are widely known to involve in developmental and adult neurogenesis. PAX6, a neurogenic fate determinant, contributes to the astrocyte-to-neuron conversion. However, it is unclear whether microRNAs (miRs) modulate PAX6-mediated astrocyte-to-neuron conversion. In the present study we used bioinformatic approaches to predict miRs potentially targeting Pax6, and transient middle cerebral artery occlusion (MCAO) to model cerebral ischemic injury in adult rats. These rats were given striatal injection of glial fibrillary acidic protein targeted enhanced green fluorescence protein lentiviral vectors (Lv-GFAP-EGFP) to permit cell fate mapping for tracing astrocytes-derived neurons. We verified that miR-365 directly targets to the 3'-UTR of Pax6 by luciferase assay. We found that miR-365 expression was significantly increased in the ischemic brain. Intraventricular injection of miR-365 antagomir effectively increased astrocytic PAX6 expression and the number of new mature neurons derived from astrocytes in the ischemic striatum, and reduced neurological deficits as well as cerebral infarct volume. Conversely, miR-365 agomir reduced PAX6 expression and neurogenesis, and worsened brain injury. Moreover, exogenous overexpression of PAX6 enhanced the astrocyte-to-neuron conversion and abolished the effects of miR-365. Our results demonstrate that increase of miR-365 in the ischemic brain inhibits astrocyte-to-neuron conversion by targeting Pax6, whereas knockdown of miR-365 enhances PAX6-mediated neurogenesis from astrocytes and attenuates neuronal injury in the brain after ischemic stroke. Our findings provide a foundation for developing novel therapeutic strategies for brain injury.

Neurovascular Interaction Promotes the Morphological and Functional Maturation of Cortical Neurons.

Brain microvascular endothelial cells (BMEC) have been found to guide the migration, promote the survival and regulate the differentiation of neural cells. However, whether BMEC promote development and maturation of immature neurons is still unknown. Therefore, in this study, we used a direct endothelium-neuron co-culture system combined with patch clamp recordings and confocal imaging analysis, to investigate the effects of endothelial cells on neuronal morphology and function during development. We found that endothelial cells co-culture or BMEC-conditioned medium (B-CM) promoted neurite outgrowth and spine formation, accelerated electrophysiological development and enhanced synapse function. Moreover, B-CM treatment induced vascular endothelial growth factor (VEGF) expression and p38 phosphorylation in the cortical neurons. Through pharmacological analysis, we found that incubation with SU1498, an inhibitor of VEGF receptor, abolished B-CM-induced p-p38 upregulation and suppressed the enhancement of synapse formation and transmission. SB203580, an inhibitor of p38 MAPK also blocked B-CM-mediated synaptic regulation. Together these results clearly reveal that the endothelium-neuron interactions promote morphological and functional maturation of neurons. In addition, neurovascular interaction-mediated promotion of neural network maturation relies on activation of VEGF/Flk-1/p38 MAPK signaling. This study provides novel aspects of endothelium-neuron interactions and novel mechanism of neurovascular crosstalk.

VEGF Axonal Transport Dependent on Kinesin-1B and Microtubules Dynamics.

Axon-transport plays an important role in neuronal activity and survival. Reduced endogenous VEGF can cause neuronal damage and axon degeneration. It is unknown at this time if VEGF can be transported within the axon or whether it can be released by axonal depolarization. We transfected VEGF-eGFP plasmids in cultured hippocampal neurons and tracked their movement in the axons by live-cell confocal imaging. Then, we co-transfected phVEGF-eGFP and kinesin-1B-DsRed vectors into neurons and combined with immunoprecipitation and two-color imaging to study the mechanism of VEGF axon-trafficking. We found that VEGF vesicles morphologically co-localized and biochemically bounded with kinesin-1B, as well as co-trafficked with it in the axons. Moreover, the capacity for axonal trafficking of VEGF was reduced by administration of nocodazole, an inhibitor of microtubules, or kinesin-1B shRNA. In addition, we found that VEGF could release from the cultured neurons under acute depolarizing stimulation with potassium chloride. Therefore, present findings suggest that neuronal VEGF is stored in the vesicles, actively released, and transported in the axons, which depends on the presence of kinesin-1B and functional microtubules. These results further help us to understand the importance of neuronal VEGF in the maintenance of neuronal activity and survival throughout life.

Neurovascular coupling protects neurons against hypoxic injury via inhibition of potassium currents by generation of nitric oxide in direct neuron and endothelium cocultures.

This study examined the effect of neuron-endothelial coupling on the survival of neurons after ischemia and the possible mechanism underlying that effect. Whole-cell patch-clamp experiments were performed on cortical neurons cultured alone or directly cocultured with brain microvascular endothelial cells (BMEC). Propidium iodide (PI) and NeuN staining were performed to examine neuronal death following oxygen and glucose deprivation (OGD). We found that the neuronal transient outward potassium currents (IA) decreased in the coculture system, whereas the outward delayed-rectifier potassium currents (IK) did not. Sodium nitroprusside, a NO donor, enhanced BMEC-induced IA inhibition and nitro-l-arginine methylester, a NOS inhibitor, partially prevented this inhibition. Moreover, the neurons directly cocultured with BMEC showed more resistance to OGD-induced injury compared with the neurons cultured alone, and that neuroprotective effect was abolished by treatment with NS5806, an activator of the IA. These results indicate that vascular endothelial cells assist neurons to prevent hypoxic injury via inhibiting neuronal IA by production of NO in the direct neuron-BMEC coculture system. These results further provide direct evidence of functional coupling between neurons and vascular endothelial cells. This study clearly demonstrates that vascular endothelial cells play beneficial roles in the pathophysiological processes of neurons after hypoxic injury, suggesting that the improvement of neurovascular coupling or functional remodeling may become an important therapeutic target for preventing brain injury.

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.