Delivery of miR-675 by stem cell-derived exosomes encapsulated in silk fibroin hydrogel prevents aging-induced vascular dysfunction in mouse hindlimb
Delivery of miR-675 by stem cell-derived exosomes encapsulated in silk fibroin hydrogel prevents aging-induced vascular dysfunction in mouse hindlimb
复制标题
通过封装在丝素蛋白水凝胶中的干细胞衍生的外泌体递送 miR-675 可预防衰老引起的小鼠后肢血管功能障碍
DOI：
10.1016/j.msec.2019.01.122
复制
发表时间：
2019-06-01
影响因子：
7.9
通讯作者：
Li, Yangxin
中科院分区：
文献类型：
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作者：
Han, Chaoshan;Zhou, Jin;Li, Yangxin
血管疾病是衰老的主要并发症，但衰老诱导的血管功能障碍的分子机制尚不清楚，并且没有有效的治疗方法来预防衰老诱导的疾病。本研究的目的是确定介导衰老诱导的血管功能障碍的信号通路，并开发基于外泌体的治疗方法来抑制衰老过程。我们使用11月龄的C57 BL 6小鼠作为预衰老动物模型，并使用H2 O2处理的H9 C2细胞作为体外衰老模型来检查miR-675的治疗效果。我们发现衰老肌肉中潜在的衰老调节剂miR-675的表达降低，H2 O2处理降低了miR-675的表达，上调了衰老标记物β-gal和TGF-β 1的表达。我们还发现miR-675模拟物减少H2 O2处理的H9 C2细胞中的β-gal染色。双荧光素酶报告基因分析证实TGF-β 1是miR-675的靶基因。此外，与从用miR-675模拟物转染的UMSC中分离的外来体一起孵育的衰老H9 C2细胞显示miR-675的表达增加、衰老标记物β-gal的活性降低和TGF-β 1的蛋白水平降低。为了延长exosome在体内的半衰期，我们采用丝素蛋白水凝胶包裹exosome。傅里叶变换红外光谱（FTIR）显示，外泌体成功地被水凝胶包裹。激光多普勒灌注成像显示，包裹在丝素蛋白水凝胶中的miR-675外泌体促进缺血后肢的血液灌注。我们证明了包裹在丝素蛋白水凝胶中的miR-675外泌体在体外提供了外泌体的持续释放，并增加了红色荧光PKH 26-外泌体在组织中的保留时间。综上所述，这项研究确定了miR-675作为细胞衰老的重要调节因子，并提供了一种新的策略，通过丝素蛋白水凝胶递送强大的外泌体来治疗衰老诱导的血管功能障碍。
Vascular disease is a major complication of aging, but the molecular mechanisms underlying the aging-induced vascular dysfunction remain unclear, and there is no effective treatment to prevent aging induced diseases. The objectives of the present study are to identify the signaling pathway mediating aging-induced vascular dysfunction and to develop an exosome based therapy to inhibit aging process. We used 11-month-old C57BL6 mice as pre-aging animal model and H2O2 treated H9C2 cells as an in vitro aging model to examine the therapeutic effect of miR-675. We found decreased expression of the potential aging modulator miR-675 in aging muscle, and H2O2 treatment decreased the expression of miR-675 and upregulated the expression of the aging marker beta-gal and TGF-beta 1. We also found that miR-675 mimic decreased beta-gal staining in H2O2 treated H9C2 cells. Dual-luciferase reporter assays verified TGF-beta 1 as the target gene of miR-675. Moreover, senescent H9C2 cells incubated with exosomes isolated from UMSCs transfected with the miR-675 mimic showed increased expression of miR-675, reduced activity of the aging marker beta-gal and reduced protein levels of TGF-beta 1. We employed silk fibroin hydrogel to encapsulate exosomes in order to prolong the half-life of exosome in vivo. Fourier transform infrared spectroscopy (FTIR) revealed that exosomes were successfully encapsulated by the hydrogel. Laser Doppler perfusion imaging showed that the miR-675 exosomes encapsulated in silk fibroin hydrogel promote blood perfusion in ischemic hindlimbs. We demonstrated that miR-675 exosomes encapsulated in silk fibroin hydrogel provided sustained release of exosomes in vitro, and increased the retention time of red fluorescent PKH26-exosome in the tissue. Taken together, this study identified miR-675 as an important regulator of cell senescence and provided a novel strategy to deliver powerful exosomes by silk fibroin hydrogel to treat aging-induced vascular dysfunction.