Abstract
BACKGROUND: Regenerative therapy using extracellular vesicles (EVs) is a promising approach for the supportive treatment of chronic limb-threatening ischaemia. Herein, we examined the angiogenic potential of EVs derived from genetically modified mesenchymal stromal cells (MSCs), focusing on the angio-micro RNAs (miRNAs) in EVs. METHODS: Bone marrow-derived MSCs (BM-MSCs) were transfected with lentiviral vectors containing specific angio-miRNAs (miRNA-126, -135b, or -210), and miRNA overexpression was confirmed using quantitative polymerase chain reaction (qPCR). EVs were isolated from the BM-MSC culture medium and characterised using fluorometry, nanoparticle tracking analysis, and ExoScreen assays. In vitro, human umbilical vein endothelial cells (HUVECs) were used to evaluate the angiogenic potential of the EVs. In vivo, EVs were injected into the ischaemic hindlimb muscles of mice, and limb ischaemia severity, blood perfusion, and histological analysis of muscle tissue were performed. RESULTS: qPCR analysis confirmed the overexpression of angio-miRNAs in MSCs transfected with lentiviral vectors. Isolated EVs expressed CD63 and had consistent protein-to-particle ratios. Tube formation was significantly enhanced when HUVECs were cultured with EV126, EV135b, or their combination (EV126 + EV135b) (p < 0.05), compared to BM-MSC co-culture. In vivo, only the double and triple EV groups significantly improved limb perfusion compared to the EVcontrol (p < 0.05); single EVs showed no significant difference. Histological analysis showed increased capillary density in ischaemic muscles following injection of combined EVs. CONCLUSION: EVs derived from genetically modified MSCs promoted angiogenesis both in vitro and in vivo, with a combination of modified EVs demonstrating significantly superior therapeutic effects than single or native EVs.