Extracellular Vesicles Derived From Human Adipose-derived Stem Cells Promotes The Angiogenesis After Fat Grafting Via The Let-7/AGO1/VEGF Signaling Pathway

Background: Adipose-derived stem cells(ADSCs)have been reported to exert therapeutic effects on fat grafting by promoting angiogenesis.Increasing evidence demonstrates that extracellular vesicles(EVs)derived from ADSCs could contribute to these effects and are considered as a potential alternative for stem cell-based therapies.However,the efficacy and underlying mechanisms of EV-based treatment in fat grafting remain unclear.Thus,this study will evaluate the therapeutic effect of hADSC-EVs on fat grafting and reveal the underlying mechanism of hADSC-EVs promoting angiogenesis in hypoxic condition.Methods: Currently,the experimental protocols we designed includes:(1)in vivo,hADSC-EVs and hypoxic hADSC-EVs(EVs released by hADSCs incubated under hypoxic conditions)were employed to supplement fat grafting.The tissues were harvested at post-grafted week 2,4 and 12,and then stained with HE and CD31-perilipin double fluorescent.(2)In vitro,the proliferation,migration,tube formation and VEGF secretion ability of vascular endothelial cells co-cultured with two kinds of EVs above in hypoxic condition were analyzed.(3)microRNA sequencing was performed to reveal the species and content of microRNAs in hADSC-EVs.(4)Silencing the key microRNAs and test their effect in promoting angiogenesis via above protocols as a reverse proving.Results: Here,the results demonstrate that hADSC-EVs could improve the survival of fat grafts by promoting angiogenesis,in vivo.Meanwhile,hADSC-EVs enhanced the proliferation,migration,tube formation abilities and VEGF secretion of vascular endothelial cells in hypoxic condition,in vitro.In addition,the pro-angiogenic effect of hADSC-EVs,in vivo and vitro,could be enhanced by hypoxic treatment.Interestingly,we found that let-7 family,a kind of hypoxic-related microRNA,is enriched in hADSC-EVs.Furthermore,the pro-angiogenic effect of hADSC-EVs could be enhanced by hypoxic treatment or inhibited by down-regulating of let-7 family via the let-7/AGO1/VEGF signaling pathway.Lin28 B vector was employed to block let-7 family in vascular endothelial cells.We found that the proliferation,migration,tube formation,VEGF secreation and AGO1/VEGF signaling pathway was also blocked while the expression of let-7 was downregulated.Overall,let-7 delivering from hADSC-EVs to vascular endothelial cells promoted proliferation,migration,tube formation and VEGF secreation by targeting AGO1/VEGF signaling pathway.Conclusions: In summary,our findings suggest that the survival of fat grafts can be improved by co-transplantation of hADSC-EVs or hypoxic hADSC-EVs via the enhancing angiogenesis.The mechanism of angiogenesis may derive from let-7 delivering from hADSC-EVs to vascular endothelial cells targeting the let-7/AGO1/ VEGF signaling pathway.