Department of Cardiovascular Surgery and Institute for Cardiovascular Science, First Affiliated Hospital of Soochow University, Suzhou, Jiangsu 215123, PR China.
Department of General Surgery, the First Affiliated Hospital of Soochow University, Suzhou, Jiangsu 215123, PR China.
Mater Sci Eng C Mater Biol Appl. 2019 Jun;99:322-332. doi: 10.1016/j.msec.2019.01.122. Epub 2019 Jan 28.
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 HO 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 HO treatment decreased the expression of miR-675 and upregulated the expression of the aging marker β-gal and TGF-β1. We also found that miR-675 mimic decreased β-gal staining in HO treated H9C2 cells. Dual-luciferase reporter assays verified TGF-β1 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 β-gal and reduced protein levels of TGF-β1. 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.
血管疾病是衰老的主要并发症，但衰老引起的血管功能障碍的分子机制尚不清楚，也没有有效的治疗方法来预防衰老引起的疾病。本研究的目的是确定介导衰老引起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 个月大的 C57BL6 小鼠作为衰老前动物模型，并用 HO 处理的 H9C2 细胞作为体外衰老模型来研究 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，HO 处理降低了 miR-675 的表达，并上调了衰老标志物 β-gal 和 TGF-β1 的表达。我们还发现 miR-675 模拟物降低了 HO 处理的 H9C2 细胞中的 β-gal 染色。双荧光素酶报告基因实验验证了 TGF-β1 是 miR-675 的靶基因。此外，与转染 miR-675 模拟物的 UMSC 分离的外泌体孵育的衰老 H9C2 细胞显示出 miR-675 的表达增加，衰老标志物 β-gal 的活性降低，TGF-β1 的蛋白水平降低。我们采用丝素蛋白水凝胶来包裹外泌体，以延长外泌体在体内的半衰期。傅里叶变换红外光谱（FTIR）显示外泌体被水凝胶成功包裹。激光多普勒灌注成像显示，丝素蛋白水凝胶包裹的 miR-675 外泌体促进缺血后肢的血液灌注。我们证明，丝素蛋白水凝胶包裹的 miR-675 外泌体在体外提供了外泌体的持续释放，并增加了组织中红色荧光 PKH26-外泌体的保留时间。综上所述，本研究确定了 miR-675 是细胞衰老的重要调节因子，并提供了一种通过丝素蛋白水凝胶递送强大外泌体来治疗衰老引起的血管功能障碍的新策略。
