Apelin-13 protects neurovascular unit against ischemic injuries through the effects of vascular endothelial growth factor.

Apelin-13 has protective effects on many neurological diseases, including cerebral ischemia. Here, we aimed to test Apelin-13's effects on ischemic neurovascular unit (NVU) injuries and investigate whether the effects were dependent on vascular endothelial growth factor (VEGF). We detected the expression of VEGF and its receptors (VEGFRs) induced by Apelin-13 injection at 1d, 3d, 7d and 14d after middle cerebral artery occlusion (MCAO). Meanwhile, we examined the effects of Apelin-13 on NVU in both in vivo and in vitro experiments as well as whether the effects were VEGF dependent by using VEGF antibody. We also assessed the related signal transduction pathways via multiple inhibitors. We demonstrated Apelin-13 highly increased VEGF and VEGFR-2 expression, not VEGFR-1. Importantly, Apelin-13 led to neurological functions improvement by associating with promotion of angiogenesis as well as reduction of neuronal death and astrocyte activation, which was markedly blocked by VEGF antibody. In cell cultures, Apelin-13 protected neurons, astrocytes and endothelial cells against oxygen-glucose deprivation (OGD) injuries. Moreover, the effect of Apelin-13 to up-regulate VEGF was suppressed by extracellular signal-regulated kinase (ERK) inhibitor U0126 and phosphatidylinositol 3'-kinase (PI3K) inhibitor LY294002. Our data suggest protective effects of Apelin-13 on ischemic NVU injuries are highly associated with the increase of VEGF binding to VEGFR-2, possibly acting through activation of ERK and PI3K/Akt pathways.

Huang C ，Dai C ，Gong K ，Zuo H ，Chu H ... -  《-》

Apelin-13 Protects against Ischemic Blood-Brain Barrier Damage through the Effects of Aquaporin-4.

Apelin-13 has been found to have protective effects on many neurological diseases, including cerebral ischemia. However, whether Apelin-13 acts on blood-brain barrier (BBB) disruption following cerebral ischemia is largely unknown. Aquaporin-4 (AQP4) has a close link with BBB due to the high concentration in astrocyte foot processes and regulation of astrocytes function. Here, we aimed to test Apelin-13's effects on ischemic BBB injury and examine whether the effects were dependent on AQP4. We detected the expression of AQP4 induced by Apelin-13 injection at 1, 3, and 7 days after middle cerebral artery occlusion. Meanwhile, we examined the effects of Apelin-13 on neurological function, infarct volume, and BBB disruption owing to cerebral ischemia in wild type mice, and tested whether such effects were AQP4 dependent by using AQP4 knock-out mice. Furthermore, we assessed the possible signal transduction pathways activated by Apelin-13 to regulate AQP4 expression via astrocyte cultures. It was found that Apelin-13 highly increased AQP4 expression as well as reduced neurological scores and infarct volume. Importantly, Apelin-13 played a role of BBB protection in both types of mice by reducing BBB permeability, increased vascular endothelial growth factor, upregulated endothelial nitric oxide synthase, and downregulated inducible NOS. In morphology, we demonstrated Apelin-13 suppressed tight junction opening and endothelial cell swelling via electron microscopy detection. Meanwhile, Apelin-13 also alleviated apoptosis of astrocytes and promoted angiogenesis. Interestingly, effects of AQP4 on neurological function and infarct volume varied with time course, while AQP4 elicited protective effects on BBB at all time points. Statistical analysis of 2-way analysis of variance with replication indicated that AQP4 was required for these effects. In addition, Apelin-13 upregulated phosphorylation of extracellular signal-regulated kinase (ERK) and Akt as well as AQP4 protein in cultured astrocytes. The latter was inhibited by ERK and phosphatidylinositol 3'-kinase (PI3K) inhibitors. Our data suggest that Apelin-13 protects BBB from disruption after cerebral ischemia both morphologically and functionally, which is highly associated with the increased levels of AQP4, possibly through the activation of ERK and PI3K/Akt pathways. This study provides double targets to protection of ischemic BBB damage, which can present new insights to drugs development.

Chu H ，Yang X ，Huang C ，Gao Z ，Tang Y ，Dong Q ... -  《-》

Vascular endothelial growth factor signaling implicated in neuroprotective effects of placental growth factor in an in vitro ischemic model.

Placental growth factor (PlGF) is involved in the angiopoiesis of the placental chorion and the maintenance of the placenta. Some additional roles of PlGF in other tissues have recently been described. Relatively little is known about PlGF expression in the CNS and the involvement of PlGF in cerebral ischemia injury. We examined the expression of PlGF in cerebral ischemia, utilizing a permanent middle cerebral artery occlusion (MCAO) model in the rat. PlGF expression and release from brain microvascular endothelial cells (BMECs) in response to oxygen and glucose deprivation (OGD) were examined in primary culture. To elucidate the effects of PlGF in cerebral ischemic injury, we investigated the effects of varying concentrations of PlGF upon neurons in an in vitro model of OGD. The effects of PlGF upon neuronal vascular endothelial growth factor receptor-1 (VEGFR-1) and vascular endothelial growth factor receptor-2 (VEGFR-2) expression were examined. We detected PlGF immunoreactivity mainly in the microvessels and interstitum of rat brain cortex after cerebral ischemic injury. In primary BMECs, PlGF expression and release were significantly higher under OGD conditions in culture. In primary cultures of rat cortical neurons, PlGF administration reduced cell death in an in vitro model of OGD. VEGFR-1 and VEGFR-2 were expressed in primary cortical neurons as measured by Western blotting. VEGFR-2 expression in primary neurons was significantly higher following PlGF administration. These data demonstrate that VEGFR-2 signaling may play a role in PlGF-mediated neuroprotection, and that PlGF may be a promising target for therapeutic intervention in ischemic injury.

Du H ，Li P ，Pan Y ，Li W ，Hou J ，Chen H ，Wang J ，Tang H ... -  《-》

Apelin-36, a potent peptide, protects against ischemic brain injury by activating the PI3K/Akt pathway.

Apelin is an endogenous ligand of G protein-coupled receptor-apelin and angiotensin-1-like receptor (APJ). The biological effects of apelin-APJ system are reported in multiple systems including cardiovascular, endocrinal, and gastrointestinal system. Previous studies had shown that apelin-13 is a potential protective agent on cardiac ischemia; however, the role of apelin in the central nervous system remained unknown. In this study, we investigated therapeutic effects of apelin-36, a long form of apelin, in ischemic brain injury models. We found that apelin-36 reduced cerebral infarct volume in the middle cerebral artery occlusion (MCAO) model and the neonatal hypoxic/ischemic (H/I) injury model. Apelin-36 improved neurological deficits in the MCAO model and promoted long-term functional recovery after H/I brain injury. We further explored the protective mechanisms of apelin-36 on H/I brain injury. We clearly demonstrated that apelin-36 significantly reduced the levels of cleaved caspase-3 and Bax, two well-established apoptotic markers after H/I injury, indicating the anti-apoptotic activity of apelin-36 in ischemic injury. Since apelin-36 increased the level of phosphorylated Akt after H/I injury, we treated neonates with a specific PI3K inhibitor LY294002. We found that LY294002 decreased the phosphorylated Akt level and attenuated protective effects of apelin-36 on apoptosis. These suggested that the PI3K/Akt pathway was at least in part involved in the anti-apoptotic mechanisms of apelin-36. Our findings demonstrated that apelin-36 was a promising therapeutic agent on the treatment of ischemic brain injury.

Gu Q ，Zhai L ，Feng X ，Chen J ，Miao Z ，Ren L ，Qian X ，Yu J ，Li Y ，Xu X ，Liu CF ... -  《-》

Protection of granulocyte-colony stimulating factor to hemorrhagic brain injuries and its involved mechanisms: effects of vascular endothelial growth factor and aquaporin-4.

Chu H ，Tang Y ，Dong Q 《-》