miR-709 exerts an angiogenic effect through an FGF2 upregulation induced by a GSK3B downregulation

The aim of this study was to identify angiogenic microRNAs (miRNAs) that could be used in the treatment of hindlimb ischemic tissues. miRNAs contained in extracellular vesicles (EVs) deriving from the plasma were analyzed in C57BL/6 mice, which have ischemia tolerance, and in BALB/c mice without ischemia tolerance as part of a hindlimb ischemia model; as a result 43 angiogenic miRNA candidates were identified. An aortic ring assay was employed by using femoral arteries isolated from BALC/c mice and EVs containing miRNA; as a result, the angiogenic miRNA candidates were limited to 14. The blood flow recovery was assessed after injecting EVs containing miRNA into BALB/c mice with hindlimb ischemia, and miR-709 was identified as a promising angiogenic miRNA. miR-709-encapsulating EVs were found to increase the expression levels of the fibroblast growth factor 2 (FGF2) mRNA in the thigh tissues of hindlimb ischemia model BALB/c mice. miR-709 was also found to bind to the 3’UTR of glycogen synthase kinase 3 beta (GSK3B) in three places. GSK3B-knockdown human artery-derived endothelial cells were found to express high levels of FGF2, and were characterized by increased cell proliferation. These findings indicate that miR-709 induces an upregulation of FGF2 through the downregulation of GSK3B. To compare the expression levels of miRNAs in EVs isolated from plasma between hindlimb ischemia model C57BL/6 mice exhibiting a blood flow recovery after a ligation of their femoral vessels and hindlimb ischemia model BALB/c mice not exhibiting such a blood flow recovery, the left femoral artery in mice was ligated at two points, and was severed with an electric scalpel between the ligated points. In order to collect plasma at 24 h after the ligation of the left femoral artery, blood was drawn from the abdominal vena cava by using a 26-G syringe including 0.1 mL of 1% ethylenediaminetetraacetic acid (EDTA). The collected blood was transferred to a 1.5-mL tube, and was centrifuged at 1,200 g for 20 min, at 4℃. The supernatant was placed in a new 1.5-mL tube, and was stored at −80℃. EVs were isolated from the obtained plasma by using the total exosome isolation kit (# 4484450; Thermo Fisher Scientific, Waltham, Massachusetts, USA). miRNAs in the EVs were extracted with the use of a total exosome RNA and protein isolation kit (#4478545; Thermo Fisher Scientific). The miRNA expression levels were analyzed in each of the five-sample pools (n=5 in C57BL/6, and n=5 in BALB/c)