Bone-derived Nestin-positive mesenchymal stem cells improve cardiac function via recruiting cardiac endothelial cells after myocardial infarction
Bone-derived Nestin-positive mesenchymal stem cells improve cardiac function via recruiting cardiac endothelial cells after myocardial infarction
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骨源性巢蛋白阳性间充质干细胞通过在心肌梗塞后招募心脏内皮细胞来改善心脏功能
DOI：
10.1186/s13287-019-1217-x
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发表时间：
2019-04-27
影响因子：
7.5
通讯作者：
He, Wen
中科院分区：
文献类型：
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作者：
Lu, Dihan;Liao, Yan;He, Wen
背景据报道，骨源间充质干细胞（BMSC）移植可有效治疗缺血性心脏病，但 BMSC 是否是最佳细胞类型仍存在争议。越来越多的研究表明，Nestin（一种中间丝蛋白）是 MSC 的潜在标记物，这提出了 BMSC 中的 Nestin+ 细胞是否可能在心肌修复中发挥更关键作用的问题。 方法通过流式细胞术，通过从 Nestin-GFP 转基因小鼠的致密骨中门控 CD45−Ter119−CD31− 细胞（由 Nestin 启动子驱动表达 GFP），分离出 Nestin+ 细胞。进行集落形成和增殖曲线测定以确定这些细胞的增殖能力，同时使用qRT-PCR分析相关趋化因子和生长因子的mRNA水平。通过 Transwell 测定评估心脏内皮细胞 (CEC) 的募集情况。此外，对左冠状动脉前降支（LAD）进行永久结扎，建立急性心肌梗死（AMI）小鼠模型。细胞移植后，在MI后1周和4周进行常规超声心动图检查，并在MI后1周收获心脏进行苏木精-伊红(HE)染色和免疫荧光染色。进一步评估MI心脏中的旁分泌因子水平和给予中和抗体（TIMP-1、TIMP-2和CXCL12）或CXCR4拮抗剂（AMD3100），以阐明Nestin+BMSCs体内趋化作用的机制。趋化因子代替生长因子。体外观察到Nestin+BMSC共培养组有更多的CEC募集，而在用针对TIMP-1、TIMP-2或CXCL12的中和抗体处理后，这种效应明显消除，更重要的是，用AMD3100阻断CXCL12/CXCR4轴显着降低了Nestin+BMSC的趋化作用。移植到暴露于心肌梗塞 (MI) 的小鼠后，与 Nestin-BMSC 治疗的小鼠相比，Nestin+BMSC 治疗的小鼠表现出显着改善的存活率和左心室功能。此外，在Nestin+BMSC治疗的梗塞边缘区，内源性CEC显着增加，趋化因子水平显着升高。同时，体内中和每种TIMP-1、TIMP-2或CXCL12可降低MI后1周和4周的左心室功能；重要的是，这三种中和抗体的联合使用可以对心脏功能产生更高的意义。最后，用AMD3100阻断CXCL12/CXCR4轴显着降低了左心室功能，并极大地抑制了Nestin+BMSC诱导的CEC体内趋化作用。结论这些结果表明，Nestin+BMSC移植可以通过CXCL12/CXCR4趋化因子途径将驻留的CEC募集到梗塞边缘区域，从而改善AMI模型中的心脏功能。我们还证明了 Nestin+BMSC 分泌的 TIMP-1/2 增强了 AMI 后缺血心脏中 CXCL12(SDF1α)/CXCR4 轴驱动的内源性 Sca-1+内皮细胞的迁移。综上所述，我们的结果表明，Nestin 是鉴定功能性 BMSC 的有用标记，并表明 Nestin+BMSC 可能是心脏修复更好的治疗候选者。
BackgroundBone-derived mesenchymal stem cell (BMSC) transplantation has been reported to be effective for the treatment of ischemic heart disease, but whether BMSCs are the optimal cell type remains under debate. Increasing numbers of studies have shown that Nestin, an intermediate filament protein, is a potential marker for MSCs, which raises the question of whether Nestin+cells in BMSCs may play a more crucial role in myocardial repair.MethodsNestin+cells were isolated using flow cytometry by gating for CD45−Ter119−CD31−cells from the compact bone of Nestin-GFP transgenic mice, expressing GFP driven by the Nestin promoter. Colony-forming and proliferative curve assays were conducted to determine the proliferative capacity of these cells, while qRT-PCR was used to analyze the mRNA levels of relative chemokines and growth factors. Cardiac endothelial cell (CEC) recruitment was assessed via a transwell assay. Moreover, permanent ligation of the left anterior descending (LAD) coronary artery was performed to establish an acute myocardial infarction (AMI) mouse model. After cell transplantation, conventional echocardiography was conducted 1 and 4 weeks post-MI, and hearts were harvested for hematoxylin-and-eosin (HE) staining and immunofluorescence staining 1 week post-MI. Further evaluation of paracrine factor levels and administration of a neutralizing antibody (TIMP-1, TIMP-2, and CXCL12) or a CXCR4 antagonist (AMD3100) in MI hearts were performed to elucidate the mechanism involved in the chemotactic effect of Nestin+BMSCs in vivo.ResultsCompared with Nestin−BMSCs, a greater proliferative capacity of Nestin+BMSCs was observed, which further exhibited moderately high expression of chemokines instead of growth factors. More CEC recruitment in the Nestin+BMSC-cocultured group was observed in vitro, while this effect was obviously abolished after treatment with neutralizing antibodies against TIMP-1, TIMP-2, or CXCL12, and more importantly, blocking the CXCL12/CXCR4 axis with a AMD3100 significantly reduced the chemotactic effect of Nestin+BMSCs. After transplantation into mice exposed to myocardial infarction (MI), Nestin+BMSC-treated mice showed significantly improved survival and left ventricular function compared with Nestin−BMSC-treated mice. Moreover, endogenous CECs were markedly increased, and chemokine levels were significantly higher, in the infarcted border zone with Nestin+BMSC treatment. Meanwhile, neutralization of each TIMP-1, TIMP-2, or CXCL12 in vivo could reduce the left ventricular function at 1 and 4 weeks post-MI; importantly, the combined use of these three neutralizing antibodies could make a higher significance on cardiac function. Finally, blocking the CXCL12/CXCR4 axis with AMD3100 significantly reduced the left ventricular function and greatly inhibited Nestin+BMSC-induced CEC chemotaxis in vivo.ConclusionsThese results suggest that Nestin+BMSC transplantation can improve cardiac function in an AMI model by recruiting resident CECs to the infarcted border region via the CXCL12/CXCR4 chemokine pathway. And we demonstrated that Nestin+BMSC-secreted TIMP-1/2 enhances CXCL12(SDF1α)/CXCR4 axis-driven migration of endogenous Sca-1+endothelial cells in ischemic heart post-AMI. Taken together, our results show that Nestin is a useful marker for the identification of functional BMSCs and indicate that Nestin+BMSCs could be a better therapeutic candidate for cardiac repair.