The use of cell secreted factors in clinical settings could be an alternative to conventional cell therapy, with the advantage of limiting concerns generally associated with traditional cell transplantation, such as tumorigenicity, immunoreactivity, and carrying of infections. Based on our published data, we predict a potential role for extracellular vesicles (EVs) in contributing to the proangiogenic activity of human fetal dermal cell secretome. Depletion of nanosized EVs from secretome significantly impaired its ability to induce formation of mesh-like structures in vitro. The isolated EVs were characterized for size and concentration by nanoparticle tracking analysis, and for protein markers (Rab5⁺, Alix⁺, CD63⁺, and calnexin^-). The microRNA profile of EVs revealed 87 microRNAs significantly upregulated (≥15-fold increase) in fetal compared to adult dermal cell-derived EVs. Interestingly, these upregulated microRNAs included microRNAs with a validated role in angiogenesis according to literature. Moreover, the DIANA-TarBase v7.0 analysis confirmed enrichment in the KEGG signaling pathways associated with angiogenesis and wound healing, with the identification of putative target genes including thrombospondin 1. To validate the in silico data, EVs were also characterized for total protein contents. When tested in in vitro angiogenesis, fetal dermal cell-derived EVs were more effective than their adult counterpart in inducing formation of complete mesh-like structures. Furthermore, treatment of fibroblasts with fetal dermal-derived EVs determined a 4-fold increase of thrombospondin 1 protein amounts compared with the untreated fibroblasts. Finally, visualization of CSFE-labeled EVs in the cytosol of target cells suggested a successful uptake of these particles at 4-8 hours of incubation. We conclude that EVs are important contributors of the proangiogenic effect of fetal dermal cell secretome. Hence, EVs could also serve as vehicle for a successful delivery of microRNAs or other molecules of therapeutic interest to target cells.

中文翻译：

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来自人类胎儿皮肤细胞的小细胞外囊泡及其MicroRNA货物：与血管生成和伤口愈合相关的KEGG信号通路。

在临床环境中使用细胞分泌因子可能是常规细胞治疗的替代方法，其优点是限制了通常与传统细胞移植相关的问题，例如致瘤性，免疫反应性和感染携带。根据我们的公开数据，我们预测细胞外囊泡（EV）在促进人类胎儿真皮细胞分泌组促血管生成活性中的潜在作用。分泌组中纳米级EV的耗竭显着削弱了其在体外诱导网状结构形成的能力。通过纳米颗粒跟踪分析对分离出的电动汽车的大小和浓度进行表征，并对蛋白质标记（Rab5 ⁺，Alix ⁺，CD63 ⁺和钙粘蛋白^-）。EV的microRNA图谱显示，与成人真皮细胞衍生的EV相比，胎儿的87 microRNA显着上调（增加了15倍以上）。有趣的是，根据文献，这些上调的微RNA包括在血管生​​成中具有有效作用的微RNA。此外，DIANA-TarBase v7.0分析证实了与血管生成和伤口愈合相关的KEGG信号通路的富集，并鉴定了包括血小板反应蛋白1在内的假定靶基因。为了验证计算机模拟数据，还对电动汽车的总蛋白含量进行了表征。体外测试时血管生成方面，胎儿真皮细胞衍生的电动汽车在诱导完整网状结构形成方面比其成年电动汽车更有效。此外，与未经处理的成纤维细胞相比，用胎儿真皮来源的电动汽车对成纤维细胞的治疗可以使血小板反应蛋白1的蛋白质含量增加4倍。最后，在目标细胞的细胞质中可视化CSFE标记的EV提示在孵育4-8小时后成功摄取了这些颗粒。我们得出结论，电动汽车是胎儿真皮细胞分泌组促血管生成作用的重要贡献者。因此，电动汽车也可以作为将微RNA或其他治疗性分子成功递送至靶细胞的载体。