Work

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

Year: 2025

Type: article

Abstract: Background/Objectives: Critical limb ischemia (CLI) is a severe peripheral vascular disease with limited treatment options. We are conducting a clinical trial using intramuscular transplantation of autologous bone marrow-derived mesenchymal stromal cells (BM-MSCs) in CLI patients. Our previous work showed that transplanted BM-MSCs do not differentiate into endothelial cells in vivo, suggesting that secreted factors, including microRNAs (miRNAs), may contribute to therapeutic effects through paracrine mechanisms. This study aims to elucidate the angiogenic mechanisms of miRNAs derived from BM-MSC-derived extracellular vesicles (EVs) using an in vitro model reflecting our clinical protocol. Methods: BM-MSCs were co-cultured with human umbilical vein endothelial cells (HUVECs) in a tube formation assay to model the therapeutic environment. Total RNA was extracted from both BM-MSCs and their EVs, and angiogenesis-related miRNAs were analyzed. Three miRNAs (miR-126, miR-135b, and miR-210) were selected for further evaluation based on EV expression profiles. Synthetic miRNAs were transfected into HUVECs individually and in combination. Tube formation was quantified, and angiogenesis-related protein expression (e.g., VEGF, FGF, Endoglin) was assessed using antibody arrays. Results: Co-culture of BM-MSCs and HUVECs significantly enhanced tube formation in a dose-dependent manner. EV analysis revealed selective packaging of angiogenic miRNAs into EVs. Transfection of miR-126, miR-135b, and miR-210 promoted tube formation, with the combination of miR-126 and miR-135b or the triple combination producing the strongest effect. Protein analysis showed significant upregulation of VEGF, FGF, and Endoglin, indicating the activation of multiple angiogenic pathways. Conclusions: This study provides preliminary mechanistic insights into the pro-angiogenic effects of EV-derived miRNAs in BM-MSC-based therapy for CLI. While each miRNA has been individually reported, our data highlight their synergistic effects in combination, which better reflects the clinical context. These findings support the development of EV-based nucleic acid therapeutics for ischemic disease and provide a foundation for future drug discovery efforts.

Source: Biomedicines

DOI: 10.3390/biomedicines13102353