Introduction: Therapeutic angiogenesis for patients with no option critical limb ischemia has not yet been a standard option, and cell therapy carries the potential risk of occult tumors. Extracellular vesicles (EV) contain bioactive agents like DNA, and micro RNA (miRNA), which are released from one cell and transported to others via the paracrine effect. They exert metabolic effects on other cells. Some miRNAs regulate angiogenic signals that promote angiogenesis. We focus on three angio-miRNAs, 126, 135b, and 210, and examine the angiogenic function of EVs in vitro.

Methods: Bone marrow mesenchymal stem cells (MSC) were obtained and transfected with lentivirus vector to overexpress specific angio-miRNAs. EVs were isolated from MSC medium using differential ultracentrifugation. The quality and quantity of EVs were assessed using nanoparticle tracking analysis and a fluorometer. Total RNA was extracted from MSCs and EVs by phenol-chloroform method. cDNA synthesis and quantitative PCR were performed. Human umbilical vein endothelial cells (HUVEC) were seeded in Matrigel-coated 24-well plates containing MSC derived EVs or PBS. After a 24-h incubation period, images were taken and we analyzed the tube formation by image analysis software.

Results: MSCs transfected with lentivirus vectors showed higher expression of miRNA 126, 135b, and 210 compared to the control vector (P< 0.05). EVs collected from MSCs transfected with lentivirus vectors showed elevated expression of these miRNAs compared to those transfected with the control vector (P< 0.05).

Tube formation of HUVECs was significantly increased in the native EV group compared to the PBS group (P< 0.05). Tube formation was also increased in EVs from transfected MSCs compared to MSCs with the control vector (P< 0.05).

Conclusion: EVs derived from MSCs overexpressing specific miRNAs demonstrated angiogenesis-promoting effects on ECs. We propose that EVs could serve as a source of cell-free therapy for lower extremity arterial disease.