MiR-195-5p and miR-205-5p in extracellular vesicles isolated from diabetic foot ulcer wound fluid decrease angiogenesis by inhibiting VEGFA expression

Figure 1. Characteristics of EVs derived from different wound fluids. (A) TEM analysis and NTA analysis of EVs. Scale bar: 200 nm. (B) EVs-related markers were detected by Western blotting. (C) EVs were labeled with PKH26 and co-incubated with HUVECs, and the representative images photographed by confocal microscope are showed above. Scale bar: 10 μm. At least three replicates of each experiment were performed.

Figure 2. DF-EVs inhibited HUVEC angiogenesis in vitro and in vivo. (A) Transwell assay of HUVECs treated with 5 μg/ml DF-EVs or 5 μg/ml Control-EVs. Scale bar: 100 μm. (B) In vitro tube formation assay of HUVECs treated with 5 μg/ml DF-EVs or 5 μg/ml Control-EVs. (C, D) Representative images of cutaneous wounds with 200 μg Control-EVs and 200 μg DF-EVs treatment at day 0, 4 and 7 post-wounding. (E, F) HE and CD31 staining of wound section with different treatments at day 7 post-wounding. (G) Microangiography analysis of local wound treated with DF-EVs or Control-EVs. *P < 0.05, ***P < 0.001. At least three replicates of each experiment were performed.

Figure 3. MiRNA expression of DF-EVs. (A) Differential expression levels of miRNAs between Control-EVs and DF-EVs were presented in a heatmap. (B) MiRNA network predicted to target VEGFA using FunRich software. (C) Expression levels of candidate miRNAs in DF-EVs were quantitated by qRT-PCR. (D) Candidate miRNAs were detected in sera of nondiabetic patients and diabetic patients with or without foot ulcers. *P < 0.05, **P < 0.01, ***P < 0.001. At least three replicates of each experiment were performed.

Figure 4. MiR-195-5p and miR-205-5p inhibited angiogenesis through reducing VEGFA. (A) MiR-195-5p and miR-205-5p expression level in HUVECs transfected with miRNA mimics was determined by qRT-PCR. (B, C) Transwell assay of HUVECs transfected with either or both of miR-195-5p and miR-205-5p mimics. (D, E) In vitro tube formation assay of HUVECs transfected with either or both of miR-195-5p and miR-205-5p mimics. (F, G) VEGFA-3′-UTR luciferase reporter assays of HUVECs in the presence of miR-195-5p and miR-205-5p mimics separately. (H, I) The expression level of VEGFA in HUVECs overexpressing either or both of the miRNA mimics were detected by qRT-PCR and western blot separately. (J) The concentration of VEGFA in culture medium of HUVECs overexpressing either or both of the miRNA mimics were detected by Elisa. *P < 0.05, **P < 0.01, ***P < 0.001. At least three replicates of each experiment were performed.

Figure 5. DF-EVs regulate VEGFA expression through miR-195-5p and miR-205-5p. (A) Expression level of MiR-195-5p and miR-205-5p in HUVECs treated with DF-EVs or Control-EVs. (B, C) Expression level of VEGFA in HUVECs treated with DF-EVs or Control-EVs were detected by qRT-PCR and western blot separately. (D) The secretion of VEGFA in culture medium of HUVECs treated with DF-EVs or Control-EVs were detected by Elisa. *P < 0.05, **P < 0.01.