Fig 1: Role of ELF5 and SP1 in angiogenesis induced by exosomes from EPCs. In parent HCFs or those transfected with empty plasmids (OE-NC), SP1 overexpression plasmids (OE-SP1), ELF5 overexpression plasmids (OE-ELF5), siRNA-1 against SP1 (siSP1-1), siRNA-2 against SP1 (siSP1-2), siRNA-1 against ELF5 (siELF5-1), siRNA-2 against ELF5 (siELF5-2), or the negative control (si-NC), the expressions of ELF5, SP1, VE-Cad, VEGFR2, and ACTA2 were determined by qRT-PCR (A), the level of COL-I in medium was examined by ELISA (B), and the proteins levels of ELF5, SP1, Lamin A, a-SMA, CD31, VE-Cad, and VEGFR2 were determined by western blot (C). Additionally, the angiogenesis of the cells were determined by tube formation assay (D). N = 3. *P < 0.05, **P < 0.01 vs. OE-NC group; #P < 0.05, ##P < 0.01 vs. si-NC group. Exo, exosomes; Mimics-1, miR-1246 mimics; Mimics-2, miR-1290 mimics.
Fig 2: Interaction between miR-1246 and ELF5, miR-1290, and SP1. In HCFs treated with Con/Exo, NC/Exo, inhibitor-1/Exo, Mimics-1/Exo, inhibitor-2/Exo, or Mimics-2/Exo, the expressions of ELF5 and SP1 were determined by immunofluorescence staining (A) and by western blot together with Lamin A (B). Potential binding of miR-1246 to the promoter of ELF-5 and miR-1290 to that of SP1 and induced expressions of the genes were observed in luciferase report assay (C). N = 3. **P < 0.01 vs. NC group. Exo, exosomes; Mimics-1, miR-1246 mimics; Mimics-2, miR-1290 mimics; Inhibitor-1, miR-1246 inhibitor; Inhibitor-2, miR-1290 inhibitor.
Fig 3: Effects of miR-1246 and miR-1290 in exosomes from EPCs on angiogenesis in rats with myocardial infarction. After administration of PBS to sham and model groups, and of exosomes from EPCs transfected with NC/Exo, Mimics-1/Exo, or Mimics-2/Exo, the expressions of miR-1246, miR-1290, CD31, ACTA2, ELF5, and SP1 were examined by qRT-PCR (A), the expressions of ELF5, SP1, Lamin A, α-SMA, and CD31 were examined by western blot (B), and the expressions of ELF5, SP1, α-SMA, and CD31 were also examined by IHC (C). N = 10. **P < 0.01 as compared with the Sham group; #P < 0.05, ##P < 0.01 as compared with the Model group. Exo, exosomes; Mimics-1, miR-1246 mimics; Mimics-2, miR-1290 mimics.
Fig 4: Regulation of CD31 directly by both ELF5 and SP1. Potential binding of ELF5 and SP1 to the promoter of CD31 and induction of the expression were examined by luciferase report assay (A). In HCF cells treated with control (IgG), NC/Exo, mMimics-1/Exo or Mimics-2/Exo, the association of miR-1246 and miR-1290 with the potential binding of ELF5 and SP1 to the DNA of CD31, respectively, was examined by CHIP (B) and EMSA (C). In HCFs that received non-transfection or transfection with OE-NC, OE-ELF, or OE-SP1 (si-NC, siELF5-1, siELF5-2, siSP1-1, or siSP1-2), the expression of CD31 was examined by qRT-PCR (D). N = 3. **P < 0.01 as compared with the NC group. Exo, exosomes; Mimics-1, miR-1246 mimics; Mimics-2, miR-1290 mimics.
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.
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