Delivery of miR-675 by stem cell-derived exosomes encapsulated in silk fibroin hydrogel prevents aging-induced vascular dysfunction in mouse hindlimb

Abstract

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 β-gal and TGF-β1. We also found that miR-675 mimic decreased β-gal staining in H₂O₂ 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.

Keywords: Aging; Exosome; Ischemic hindlimb; Silk fibroin hydrogel; TGF-β1; miR-675.