Zhu J, Liu Q, Jiang Y, Wu L, Xu G, Liu X
Department of Neurology, Jinling Hospital, Medical School of Nanjing University, 305 East Zhongshan Road, Nanjing 210002, Jiangsu, China.
Neuroscience. 2015 Apr 2;290:288-99. doi: 10.1016/j.neuroscience.2015.01.038. Epub 2015 Jan 28.
Cellular therapy has provided hope for restoring neurological function post stroke through promoting endogenous neurogenesis, angiogenesis and synaptogenesis. The current study was based on the observation that transplantation of human umbilical cord mesenchymal stem cells (hUCMSCs) promoted the neurological function improvement in stroked mice and meanwhile enhanced angiogenesis in the stroked hemisphere. Grafted hUCMSCs secreted human vascular endothelial growth factor A (VEGF-A). Notch1 signaling was activated after stroke and also in the grafted hUCMSCs. To address the potential mechanism that might mediate such pro-angiogenic effect, we established a hUCMSC-neuron co-culture system. Neurons were subjected to oxygen glucose deprivation (OGD) injury before co-culturing to mimic the in vivo cell transplantation. Consistent with the in vivo data, co-culture medium claimed from hUCMSC-OGD neuron co-culture system significantly promoted the capillary-like tube formation of brain-derived endothelial cells. Moreover, coincident with our in vivo data, Notch 1 signaling activation was detected in hUCMSCs after co-cultured with OGD neurons as demonstrated by the up-regulation of key Notch1 signaling components Notch1 and Notch1 intercellular domain (NICD). In addition, OGD-neuron co-culture also increased the VEGF-A production by hUCMSCs. To verify whether Notch1 activation was involved in the pro-angiogenic effect, γ-secretase inhibitor N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) was added into the co-culture medium before co-culture. It turned out that DAPT significantly prevented the Notch1 activation in hUCMSCs after co-culture with OGD neurons. More importantly, the pro-angiogenic effect of hUCMSCs was remarkably abolished by DAPT addition as demonstrated by inhibited capillary-like tube formation and less VEGF-A production. Regarding how Notch1 signaling was linked with VEGF-A secretion, we provided some clue that Notch1 effector Hes1 mRNA expression was significantly up-regulated by OGD-neuron co-culturing and down-regulated after additional treatment of DAPT. In summary, our data provided evidence that the VEGF-A secretion from hUCMSCs after being triggered by OGD neurons is Notch1 signaling associated. This might be a possible mechanism that contributes to the angiogenic effect of hUCMSC transplantation in stroked brain.
细胞疗法为中风后恢复神经功能带来了希望，其作用机制是促进内源性神经发生、血管生成和突触形成。当前的这项研究基于以下观察结果：人脐带间充质干细胞（hUCMSCs）移植可促进中风小鼠的神经功能改善，同时增强中风半球的血管生成。移植的hUCMSCs会分泌人血管内皮生长因子A（VEGF-A）。中风后以及移植的hUCMSCs中Notch1信号通路均被激活。为了探究可能介导这种促血管生成作用的潜在机制，我们建立了hUCMSC-神经元共培养系统。在共培养之前，先对神经元进行氧糖剥夺（OGD）损伤，以模拟体内细胞移植的情况。与体内实验数据一致，来自hUCMSC-OGD神经元共培养系统的共培养基显著促进了脑源性内皮细胞形成毛细血管样管状结构。此外，与我们的体内实验数据相符，与OGD神经元共培养后，hUCMSCs中检测到Notch 1信号通路激活，关键的Notch1信号成分Notch1和Notch1细胞内结构域（NICD）上调即证明了这一点。另外，OGD-神经元共培养还增加了hUCMSCs的VEGF-A分泌量。为了验证Notch1激活是否参与了促血管生成作用，在共培养前向共培养基中添加了γ-分泌酶抑制剂N-[N-(3,5-二氟苯乙酰基)-L-丙氨酰基]-S-苯甘氨酸叔丁酯（DAPT）。结果表明，DAPT显著抑制了与OGD神经元共培养后的hUCMSCs中的Notch1激活。更重要的是，添加DAPT后，hUCMSCs促血管生成作用明显被消除，表现为毛细血管样管状结构形成受到抑制以及VEGF-A分泌减少。关于Notch1信号通路如何与VEGF-A分泌相关联，我们发现了一些线索：OGD-神经元共培养显著上调了Notch1效应分子Hes1 mRNA的表达，而在额外添加DAPT处理后其表达下调。总之，我们的数据表明，OGD神经元触发后hUCMSCs分泌VEGF-A与Notch1信号通路相关。这可能是hUCMSC移植对中风大脑产生血管生成作用的一种潜在机制。
