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. 2018 Jan 1;8(1):169-184.
doi: 10.7150/thno.21234. eCollection 2018.

Exosomes from human umbilical cord blood accelerate cutaneous wound healing through miR-21-3p-mediated promotion of angiogenesis and fibroblast function

Yin Hu  1   2 , Shan-Shan Rao  1   3 , Zhen-Xing Wang  1 , Jia Cao  1 , Yi-Juan Tan  1 , Juan Luo  1 , Hong-Ming Li  1   2 , Wei-She Zhang  4 , Chun-Yuan Chen  1 , Hui Xie  1   5   6   2   3
Affiliations

Exosomes from human umbilical cord blood accelerate cutaneous wound healing through miR-21-3p-mediated promotion of angiogenesis and fibroblast function

Yin Hu et al. Theranostics. .

Abstract

The application of blood plasma for soft tissue wound healing is receiving much more attention recently. Exosomes are critical paracrine mediators that can be obtained from biological fluids including plasma and be able to induce regenerative effects by transferring bioactive molecules such as microRNAs (miRNAs). This study aimed to investigate the effects of exosomes from human umbilical cord blood plasma (UCB-Exos) on wound healing and to elucidate the underlying mechanism. Methods: UCB-Exos were isolated by ultracentrifugation and subcutaneously injected into full-thickness skin wounds in mice. The efficacy of UCB-Exos on wound healing was evaluated by measuring wound closure rates, histological analysis and immunofluorescence examinations. In vitro, quantitative real-time PCR (qRT-PCR) analysis was performed to detect the expression levels of a class of miRNAs that have positive roles in regulating wound healing. The scratch wound assay, transwell assay and cell counting kit-8 analysis were conducted to assess the effects of UCB-Exos on migration and proliferation of human skin fibroblasts and endothelial cells. Tube formation assay was carried out to test the impact of UCB-Exos on angiogenic tube formation ability of endothelial cells. Meanwhile, by using specific RNA inhibitors or siRNAs, the roles of the candidate miRNA and its target genes in UCB-Exos-induced regulation of function of fibroblasts and endothelial cells were assessed. Results: The local transplantation of UCB-Exos into mouse skin wounds resulted in accelerated re-epithelialization, reduced scar widths, and enhanced angiogenesis. In vitro, UCB-Exos could promote the proliferation and migration of fibroblasts, and enhance the angiogenic activities of endothelial cells. Notably, miR-21-3p was found to be highly enriched in UCB-Exos and served as a critical mediator in UCB-Exos -induced regulatory effects through inhibition of phosphatase and tensin homolog (PTEN) and sprouty homolog 1 (SPRY1). Conclusion: Our results suggest that UCB-Exos are important effectors of plasma activity and can be used as a novel promising strategy for soft tissue wound healing.

Keywords: cord blood; exosomes; miR-21-3p.; wound healing.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interest exists.

Figures

Figure 1
Figure 1
Characterization of UCB-Exos. (A) Particle size distribution of UCB-Exos measured by DLS analysis. (B) Morphology of UCB-Exos observed by TEM. Scale bar: 50 nm. (C) Representative flow cytometry histograms showing the presence of exosomal surface markers CD63 and TSG101 on UCB-Exos-bound beads. Negative controls: isotype control primary antibody + secondary antibody or secondary antibody only. The beads un-coated with exosomes but incubated with the respective primary and secondary antibodies served as blank.
Figure 2
Figure 2
UCB-Exos transplantation was beneficial for cutaneous wound healing in mice. (A) Gross view of wounds treated with UCB-Exos or PBS at day 2, 5 and 8 post-wounding. (B) The rate of wound-closure in wounds receiving different treatments. n = 10 per group. (C) H&E staining of wound sections treated with UCB-Exos or PBS at 8 days after operation. The black arrows indicate the edges of the scar. Scale bar: 500 μm. (D) Quantification of the scar widths and the extent of re-epithelialization. n = 3 per group. (E) Masson's trichrome staining of wound sections treated with UCB-Exos or PBS. *P < 0.05 compared with the PBS group (control).
Figure 3
Figure 3
UCB-Exos enhance angiogenesis in the wound sites of mice. (A) Gross view of wounds receiving different treatments at day 8 post-wounding from the undersurface. Newly formed blood vessels were detected in the wound sites. (B) CD31 immunofluorescence staining of wound sections treated with UCB-Exos or PBS. Scale bar: 50 μm. (C) Quantitative analysis of the number of blood vessels in (B). n = 3 per group. *P < 0.05 compared with the PBS group (control).
Figure 4
Figure 4
UCB-Exos deliver miR-21-3p into fibroblasts and endothelial cells. (A) Detection of the expression of the indicated miRNAs by qRT-PCR analysis. n = 3. (B) Fluorescence microscopy analysis of PKH67-labeled UCB-Exos internalization by HSFs and HMECs. The green-labeled exosomes were visible in the perinuclear region of recipient cells. Scale bar: 50 μm. (C-D) HSFs and HMECs incubated with UCB-Exos for 3 h showed higher expression levels of miR-21-3p than controls did. n = 3 per group. Incubation with UCB-Exos for 24 h reduced the expression of PTEN and SPRY1 in HSFs (E) and HMECs (F). n = 3 per group. UCB-Exos were used at a concentration of 100 μg/mL. *P < 0.05 compared with the PBS group (control).
Figure 5
Figure 5
UCB-Exos promote the proliferation and migration of fibroblasts via transferring miR-21-3p. (A) UCB-Exos promoted HSFs migration as analyzed by scratch wound assay, but this effect was reduced by miR-21-3p inhibitor. Scale bar: 250 μm. (B) Quantitative analysis of the migration rates in (A). n = 3 per group. (C) The migratory ability of HSFs receiving different treatments was further confirmed by the transwell assay. Scale bar: 100 μm. (D) Quantitative analysis of the migrated cells in (C). n = 3 per group. (E) CCK-8 assay showed that UCB-Exos promoted HSFs proliferation, whereas this effect was attenuated by miR-21-3p inhibition. n = 4 per group. (F) UCB-Exos reduced the expression of PTEN and SPRY1 in HSFs, but their down-regulation was reversed by the miR-21-3p inhibitor. n = 3 per group. (G) UCB-Exos incubation enhanced the protein levels of p-Akt and p-Erk1/2 in HSFs, whereas this effect was reduced in the miR-21-3p co-treatment group. (H) Densitometric quantification of the relative band intensity in (G). n = 4 per group. *P < 0.05 compared with the control group, # P < 0.05 compared with the UCB-Exos (100 μg/mL) group.
Figure 6
Figure 6
Inhibition of PTEN and SPRY1 induces UCB-Exos-like positive effects on fibroblast function. (A) The inhibitory efficiency of the siRNAs targeting PTEN and SPRY1 was verified by qRT-PCR. n = 3 per group. (B) The migration of HSFs in different treatment groups was tested by the scratch wound assay. (C) Quantitative analysis of the migration rates in (B). n = 3 per group. (D) CCK-8 analysis of HSFs proliferation in different treatment groups. n = 4 per group. *P < 0.05 compared with the PBS group, # P < 0.05 compared with the control (Con) siRNA group.
Figure 7
Figure 7
MiR-21-3p mediates the pro-angiogenic effects of UCB-Exos on endothelial cells. UCB-Exos induced a significant increase in the motility of HMECs, but the pro-migratory effect was decreased by the miR-21-3p inhibitor, as analyzed by the scratch wound assay (A-B) (Scale bar: 250 μm) and the transwell assay (C-D) (Scale bar: 100 μm). n = 3 per group. (E) The proliferation of HMECs receiving different treatments was assessed by CCK-8 analysis. n = 4 per group. (F) UCB-Exos increased the tube formation ability of HMECs, but this effect was decreased by miR-21-3p inhibition. Scale bar: 100 μm. (G) Quantitative analysis of the total branching points and total tube length in (F). n = 3 per group. (H) Detection of the expression of PTEN and SPRY1 by qRT-PCR analysis. n = 3 per group. (I) Detection of the phosphorylation levels of Akt and Erk1/2 by western blotting. n = 4 per group. *P < 0.05 compared with the control (Con) siRNA group, # P < 0.05 compared with the UCB-Exos (100 μg/mL) group.
Figure 8
Figure 8
Inhibition of PTEN and SPRY1 induces UCB-Exos-like pro-angiogenic effects on endothelial cells. (A) The inhibitory efficiency of the siRNAs targeting PTEN and SPRY1 was verified by qRT-PCR. n = 3 per group. (B) The migration of HMECs in different treatment groups was tested by the scratch wound assay. (C) Quantitative analysis of the migration rates in (B). n = 3 per group. (D-E) Representative images and quantification of HMECs tube formation in different treatment groups. n = 3 per group. (F) CCK-8 analysis of HMECs proliferation in different treatment groups. n = 4 per group. *P < 0.05 compared with the PBS group, # P < 0.05 compared with the Con siRNA group.
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