Mesenchymal Stem Cell-Derived Exosomes Promote Ischemic Hindlimb Repair By Delivering MiR-24 And CircPWWP2A

Mesenchymal stem cells(MSCs)not only have the biological characteristics of stem cells,but also protect ischemic tissue through paracrine mechanism.Many of the beneficial effects of MSCs are mediated by paracrine factors which are released through exosomes.Many studies have shown that exosomes and MSCs have similar biological functions.Compared with MSCs,exosomes have the advantages of low immunogenicity,easy to obtain,easy to store and transport,and no ethical issues.The exosomes can greatly reduce the acute immune rejection induced by allogeneic stem cell transplantation.Therefore,cell-free exosome replacement therapy is expected to become an effective strategy for clinical treatment of ischemic vascular diseases.In our study,we found that human umbilical cord mesenchymal stem cells(UMSCs)can promote the repair of injured muscles.However,mesenchymal stem cells express HLA class I antigen,so MSCs still have immunogenicity,which is a major obstacle in MSC-based therapy.We transplanted MSCs into the ischemic injury site of the hindlimbs of mice,and observed immune rejection.Deletion of B2M gene in UMSCs inhibited the expression of HLA class I antigen,and reduced immune rejection in vivo after UMSCs transplantation.Compared with regular UMSCs,B2M knockout UMSCs showed better blood flow recovery and hindlimb motor function.Subsequently,we performed high-throughput sequencing of UMSCs and exosomes,and found that both UMSCs and exosomes highly expressed miR-24.Therefore,we speculate that miR-24 may play an important role in mediating exosome’s beneficial effect.By modifying the expression level of miR-24 in exosomes,we found that exosomes overexpressing miR-24 had more significant protective effects.Dual luciferase gene reporter assay revealed that Bim is the molecular target of miR-24.Exosome-derived miR-24 inhibits apoptosis,and enhances the motor function of hind limbs by inhibiting the expression of Bim protein.It is known that in addition to miRNA,exosomes also contain circRNAs which have a stable circular structure.We found that circPWWP2A was relatively abundant in exosomes.By comparing the sequencing results of normal and ischemic muscles,we found that the expression of circPWWP2A was reduced after ischemia.We further showed that exosomes promoted the repair of hindlimb ischemia by releasing circPWWP2A which regulates the CDK6/Rb1/AMPKα2 signaling pathway by targeting miR-29b,leading to inactivation of NLRP3 inflammasome,reduced active caspase-1 and reduced levels of IL-1β,IL-18 in the ischemic environment,and enhanced recovery of acute ischemic hind limb.In summary,our study found that allogeneic MSCs transplantation is associated with the risk of acute immune rejection.Exosomes derived from UMSCs have similar biological effects as UMSCs,therefore,exosomes can be used for the treatment of ischemic diseases.Exosomes contain a large number of non-coding RNAs.We demonstrated that exosomes can inhibit apoptosis and promote tissue repair by releasing miR-24.We obtained exosomes expressing high levels of miR-24 by transfecting the exosomes with miR-24 mimics,which showed better effects.Moreover,we showed that circPWWP2A which acts as the molecular sponge of miR-29b,inhibited the activation of NLRP3 inflammasome and the release of IL-1β and IL-18 in ischemic tissues,and promoted the recovery of ischemic hindlimb.