Mesenchymal stem cells (MSCs) can provide therapeutic benefits for myocardial infarction (MI) recovery; however, the molecular mechanism by which MSCs improve the heart function is unclear. Microarray analysis was performed to examine the expression profiling of human MSCs (hMSCs) grown as adherent cultures (AC-hMSCs) or nonadherent cultures on ultra-low-adherent plates (nonAC-hMSCs). Real-time quantitative polymerase chain reaction (RT-qPCR), western blotting, and enzyme-linked immunosorbent assays (ELISA) were used to assess VEGFA expression and secretion in the AC-hMSCs and nonAC-hMSCs. The paracrine effect of VEGFA-overexpressing AC-MSCs (AC-VEGFA-hMSCs) or VEGFA-knockdown nonAC-hMSCs (nonAC-shVEGFA-hMSCs) on the angiogenic ability of human umbilical vein endothelial cells (HUVECs) was evaluated using tube formation assay. AC-VEGFA-hMSCs or nonAC-shVEGFA-hMSCs were transplanted into myocardial infarction rats to investigate the therapeutic effect of AC-VEGFA-hMSCs or nonAC-shVEGFA-hMSCs. Luciferase reporter assay was used to confirm the association of VEGFA with miR-519d. Microarray analysis revealed that VEGFA is downregulated in AC-hMSCs compared to nonAC-hMSCs. Functional assays revealed that high levels of VEGFA produced from AC-VEGFA-hMSCs increased the tube formation capacity of HUVECs in vitro, improved angiogenesis and cardiac performance, and reduced infarct size in a rat MI model. Low levels of VEGFA secretion from nonAC-shVEGFA-hMSCs had the opposite effects. Mechanistically, we found that miR-519d directly targets VEGFA. High levels of VEGFA secreted from VEGFA-overexpressing nonAC-hMSCs abolished the repressive effect of miR-519d on HUVEC angiogenesis. Our findings indicate that nonadherent culture-induced secretion of VEGFA plays an important role in MSCs via the miR-519d/VEGFA pathway and may provide a novel therapeutic strategy for MI treatment.

中文翻译：

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非贴壁培养方法通过 miR-519d/VEGFA 途径促进心肌梗死中 MSC 介导的血管化。

间充质干细胞（MSC）可以为心肌梗死（MI）的恢复提供治疗益处；然而，间充质干细胞改善心脏功能的分子机制尚不清楚。进行微阵列分析以检查作为贴壁培养物（AC-hMSCs）或在超低贴壁平板上的非贴壁培养物（nonAC-hMSCs）生长的人MSC（hMSCs）的表达谱。使用实时定量聚合酶链反应（RT-qPCR）、蛋白质印迹和酶联免疫吸附测定（ELISA）来评估AC-hMSC和非AC-hMSC中的VEGFA表达和分泌。使用管形成实验评估 VEGFA 过表达 AC-MSCs (AC-VEGFA-hMSCs) 或 VEGFA 敲低非 AC-hMSCs (nonAC-shVEGFA-hMSCs) 对人脐静脉内皮细胞 (HUVEC) 血管生成能力的旁分泌作用。将AC-VEGFA-hMSCs或nonAC-shVEGFA-hMSCs移植到心肌梗死大鼠体内，研究AC-VEGFA-hMSCs或nonAC-shVEGFA-hMSCs的治疗效果。使用荧光素酶报告基因测定来确认 VEGFA 与 miR-519d 的关联。微阵列分析显示，与非 AC-hMSC 相比，AC-hMSC 中 VEGFA 下调。功能测定显示，AC-VEGFA-hMSC 产生的高水平 VEGFA 增加了体外 HUVEC 的管形成能力，改善了血管生成和心脏性能，并减少了大鼠 MI 模型中的梗塞面积。nonAC-shVEGFA-hMSC 的低水平 VEGFA 分泌具有相反的效果。从机制上讲，我们发现 miR-519d 直接靶向 VEGFA。过度表达 VEGFA 的非 AC-hMSC 分泌的高水平 VEGFA 消除了 miR-519d 对 HUVEC 血管生成的抑制作用。我们的研究结果表明，非贴壁培养诱导的 VEGFA 分泌通过 miR-519d/VEGFA 途径在 MSC 中发挥重要作用，并可能为 MI 治疗提供一种新的治疗策略。