MicroRNAs in BM-MSC-Derived Extracellular Vesicles Promote Angiogenesis: An in Vitro Model Study
Tomomi Kusakabe1, Yoshiki Wada1,2, Tomohiro Umezu3
1Center for Cell Therapy and Regenerative Medicine, Tokyo Medical University, Tokyo 160-0023, Japan.
Abstract:
Background/Objectives: 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. Methods: 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. Results: 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. Conclusions: 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.
Insights
Mesenchymal stromal cell (MSC)-derived extracellular vesicles (EVs) deliver microRNAs (miRNAs) that promote angiogenesis, offering a novel therapeutic approach for critical limb ischemia (CLI). This study identifies specific miRNAs within EVs that enhance blood vessel formation, supporting EV-based treatments for ischemic diseases.
Area of Science:
- Regenerative Medicine
- Molecular Biology
- Vascular Biology
Background:
- Critical limb ischemia (CLI) is a severe peripheral arterial disease with limited therapeutic options.
- Mesenchymal stromal cell (MSC) therapy shows promise, but efficacy varies, suggesting paracrine mechanisms involving extracellular vesicles (EVs) and microRNAs (miRNAs) are key.
- Angiogenesis, the formation of new blood vessels, is crucial for treating ischemic conditions.
Purpose of the Study:
- To investigate the role of specific angiogenesis-related microRNAs (miRNAs) packaged within bone marrow-derived MSC (BM-MSC) extracellular vesicles (EVs).
- To determine if these EV-associated miRNAs can enhance angiogenesis and to explore their therapeutic potential for critical limb ischemia (CLI).
Main Methods:
- Bone marrow-derived MSCs (BM-MSCs) and their EVs were analyzed for the presence of five angiogenesis-related miRNAs (miR-126, miR-135b, miR-210 were detected in EVs).
- Co-culture experiments with BM-MSCs and human umbilical vein endothelial cells (HUVECs) assessed angiogenesis via tube formation assays.
- Synthetic miRNAs were transfected into HUVECs to evaluate their individual and combined effects on angiogenesis and protein expression (VEGF, FGF, Endoglin).
Main Results:
- BM-MSC and HUVEC co-culture significantly enhanced angiogenesis in a dose-dependent manner.
- EVs selectively packaged specific angiogenic miRNAs (miR-126, miR-135b, miR-210), with expression levels influenced by donor age and individual variability.
- Combinations of miR-126 and miR-135b, and all three tested miRNAs, showed the strongest enhancement of angiogenesis and upregulated key angiogenic proteins.
Conclusions:
- BM-MSC-derived EV miRNAs promote angiogenesis through combinatorial mechanisms, providing a mechanistic basis for MSC therapy in CLI.
- These findings underscore the translational potential of EV-based nucleic acid therapeutics for treating ischemic diseases.
- The study highlights the importance of specific miRNA cargo within EVs for therapeutic efficacy.
