MicroRNAs in BM-MSC-Derived Extracellular Vesicles Promote Angiogenesis: An in Vitro Model Study

<b>Background/Objectives:</b> Critical limb ischemia (CLI) is a severe manifestation of peripheral arterial disease with limited treatment options. Mesenchymal stromal cell (MSC) therapy has shown promise, but variability in efficacy suggests that paracrine mechanisms, particularly extracellular vesicle (EV)-associated microRNAs (miRNAs), may play a central role. <b>Methods:</b> We analyzed angiogenesis-related miRNAs in bone marrow-derived MSCs (BM-MSCs) and their EVs. Five angiomiRs (miR-9, miR-105, miR-126, miR-135b, miR-210) were examined; only miR-126, miR-135b, and miR-210 were consistently detected in EVs. Expression variability was assessed across donor age and individuals. Functional evaluation was performed using co-culture of BM-MSCs with human umbilical vein endothelial cells (HUVECs) and by transfecting synthetic miRNAs into HUVECs. Tube formation assays quantified angiogenesis, and angiogenesis-related protein expression (VEGF, FGF, Endoglin, uPA) was analyzed. Biological replicates (multiple donors) and technical replicates (duplicate assays) were clearly defined to ensure reproducibility. <b>Results:</b> Co-culture of BM-MSCs and HUVECs significantly enhanced angiogenesis in a dose-dependent manner. EVs selectively packaged angiogenic miRNAs, with expression levels varying according to donor age and inter-individual variability. Transfection of miR-126, miR-135b, and miR-210 individually enhanced tube formation, while the miR-126 + miR-135b combination and triple transfection elicited the strongest effects. Protein analysis confirmed upregulation of VEGF, FGF, and Endoglin. Notably, miR-210 did not further enhance angiogenesis beyond miR-126 + miR-135b but may exert context-dependent effects. <b>Conclusions:</b> This study demonstrates that BM-MSC-derived EV miRNAs promote angiogenesis via combinatorial mechanisms, providing mechanistic support for ongoing CLI therapy. Our findings highlight the translational potential of EV-based nucleic acid therapeutics for ischemic disease.