Department of Biophysics, Federal University of São Paulo, São Paulo, Brazil.
Department of Biochemistry, Federal University of São Paulo, São Paulo, Brazil.
PLoS One. 2022 Aug 16;17(8):e0272962. doi: 10.1371/journal.pone.0272962. eCollection 2022.
Extracellular vesicles (EVs) are known as molecular carriers involved in cell communication and the regulation of (patho)physiological processes. miRNAs and growth factors are the main contents of EVs which make them a good candidate for the treatment of diseases caused by ischemia, but the low production of EVs by a cell producer and a significant variation of the molecular contents in EVs according to the cell source are the main limitations of their widespread use. Here, we show how to improve the therapeutic properties of mesenchymal stromal cell (MSC)-derived EVs (MSC-EVs) by modifying MSCs to enrich these EVs with specific angiomiRs (miR-135b or miR-210) using lentiviral vectors carrying miR-135b or miR-210. MSCs were obtained from the mouse bone marrow and transduced with a corresponding lentivector to overexpress miR-135b or miR-210. The EVs were then isolated by ultracentrifugation and characterized using a flow cytometer and a nanoparticle tracking analyzer. The levels of 20 genes in the MSCs and 12 microRNAs in both MSCs and EVs were assessed by RT‒qPCR. The proangiogenic activity of EVs was subsequently assessed in human umbilical vein endothelial cells (HUVECs). The results confirmed the overexpression of the respective microRNA in modified MSCs. Moreover, miR-135b overexpression upregulated miR-210-5p and follistatin, whereas the overexpression of miR-210 downregulated miR-221 and upregulated miR-296. The tube formation assay showed that EVs from MSCs overexpressing miR-210-5p (EVmiR210) significantly promoted tubular structure formation in HUVECs. A significant increase in angiogenic proteins (PGF, endothelin 1, and artemin) and genes (VEGF, activin A, and IGFBP1) in HUVECs treated with VEmiR210 justifies the better tubular structure formation of these cells compared with that of EVmiR135b-treated HUVECs, which showed upregulated expression of only artemin. Collectively, our results show that the EV cargo can be modified by lentiviral vectors to enrich specific miRNAs to achieve a specific angiogenic potential.
细胞外囊泡 (EVs) 是参与细胞通讯和 (病理) 生理过程调节的分子载体。miRNA 和生长因子是 EVs 的主要内容，这使它们成为治疗缺血引起的疾病的良好候选物，但细胞生产者产生的 EVs 产量低，以及根据细胞来源 EVs 中分子含量的显著变化是其广泛应用的主要限制。在这里，我们展示了如何通过修饰间充质基质细胞 (MSC) 来提高 MSC 衍生的 EVs (MSC-EVs) 的治疗特性，方法是使用携带 miR-135b 或 miR-210 的慢病毒载体将这些 EVs 富集特定的血管生成 miRNA (miR-135b 或 miR-210)。MSC 从鼠骨髓中获得，并通过相应的慢病毒载体转导以过表达 miR-135b 或 miR-210。然后通过超速离心分离 EVs，并使用流式细胞仪和纳米颗粒跟踪分析仪进行表征。通过 RT-qPCR 评估 MSC 中的 20 个基因和 MSC 和 EVs 中的 12 个 microRNAs 的水平。随后在人脐静脉内皮细胞 (HUVEC) 中评估 EVs 的促血管生成活性。结果证实了修饰后的 MSC 中相应 microRNA 的过表达。此外，miR-135b 的过表达上调了 miR-210-5p 和卵泡抑素，而 miR-210 的过表达下调了 miR-221 并上调了 miR-296。管形成试验表明，过表达 miR-210-5p 的 MSC 来源的 EV (EVmiR210) 显著促进了 HUVEC 中管状结构的形成。与 EVmiR135b 处理的 HUVEC 相比，用 VEmiR210 处理的 HUVEC 中血管生成蛋白 (PGF、内皮素 1 和 artemin) 和基因 (VEGF、激活素 A 和 IGFBP1) 的显著增加证明了这些细胞的管状结构形成更好，而 EVmiR135b 处理的 HUVEC 仅上调了 artemin 的表达。总的来说，我们的结果表明，EV 货物可以通过慢病毒载体进行修饰，以富集特定的 miRNA，从而实现特定的血管生成潜力。
