Fig 2: Identification of EPCs from human peripheral blood and exosomes from EPCs. The isolated cells were cultured and then collected for detection of CD34, CD133, and VEGFR2 expressions by immunofluorescence staining (A) and flow cytometry (B). Additionally, the phagocytic ability of the cells was examined by dual immuno-staining of Dil-Ac-LDL and FITC-UEA-1 (C). The morphology, size, and concentration of the isolated exosomes were identified by transmission electron microscope and Nanosight tracking analysis, scale bar = 100 nm (D). The expressions of exosome-associated proteins, including Alix, CD63, and TSG101 were detected by western blot (E). EPCs, endothelial progenitor cells. N = 3.

Fig 3: Effects of miR-1246 and miR-1290 on angiogenesis induced by exosomes from EPCs. The miR-375, miR-1246, and miR-1290 levels in exosomes itself or in whole EPCs were determined via real-time PCR (A). After treatment with control, CS, or Exo, HCFs were harvested to determine the expressions of miR-375, miR-1246, and miR-1290 (B). The EPCs received one of the following transfections: miR-1246 inhibitor-1, miR-1246 mimics-1, miR-1290 inhibitor-2, Mimics-2, and NCs. Then, the expressions of miR-1246 and miR-1290 in exosomes derived from the parent or transfected EPCs were determined (C). HCFs were treated with exosomes from parent EPCs (Con/Exo), exosomes from EPCs transfected with NC (NC/Exo), miR-1246 inhibitor-1 (inhibitor-1/Exo), miR-1246 mimics-1 (Mimics-1/Exo), miR-1290 inhibitor-2 (inhibitor-2/Exo), or miR-1290 mimics-2 (Mimics-2/Exo). Then, the cells were collected, expressions of miR-1246 and miR-1290 were determined by qRT-PCR (D), the level of Collagen-I (Col-I) in the medium was determined by ELISA (E), and the expressions of a-SMA, CD31, VE-Cad, and VEGFR2 were determined by western blot (F) and immunofluorescence staining (G). Additionally, the phagocytic activity of HCFs was determined by DiL-Ac-LDL staining (H), and the angiogenesis was measured by a method of tube formation assay (I). The tube length in each group was quantified according to the results of tube formation assay (J). The cells were treated with exosomes (4 µg/ml) of EPCs for 24 h. N = 3. *P < 0.05, **P < 0.01 vs. NC/Exo group. Exo, exosomes; Mimics-1, miR-1246 mimics; Mimics-2, miR-1290 mimics; Inhibitor-1, miR-1246 inhibitor; Inhibitor-2, miR-1290 inhibitor.

Fig 4: Characteristics of EPCs. (A) Fluorescence-activated cell sorting revealed that EPCs used in the present study were positive for CD34 and KDR, whereas they were negative for CD45. (B) Typical features of EPCs at day 1, 4, 7 and 14. (C) EPCs could take up ac-LDL and bind to UEA-1. Scale bar, 150 µm. ac-LDL, acetylated low-density lipoprotein; EPCs, endothelial progenitor cells; KDR, kinase insert domain receptor; UEA-1, Ulex europaeus agglutinin I.

Fig 5: Schematic illustration/of effect by exosomal mir-1246 and -1290 on myocardial infarction. MiR-1246 and -1290 in EPCs could bind to the promoter of ELF5 and SP1 in HCFs, respectively, and induce their expressions. Resultantly, increased ELF5 and SP1 promoted fibroblast-endothelial transition, demonstrated by increased expressions of endothelial markers, CD31 (by binding to the promoter), VEGFR2, and VE-Cad, and decreased expression of a fibroblast marker, a-SMA. Furthermore, they increased angiogenesis. In rats, both exosomal miR-1246 and -1290 contribute to the exosome-attenuated myocardial infarction (MI). All these indicated that both miR-1246 and -1290 in exosomes from EPCs protected the heart against MI, and this was potentially through increased expressions of ELF5 and SP1.