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. 2016 Nov 25;12(12):1472-1487.
doi: 10.7150/ijbs.15514. eCollection 2016.

Exosomes Derived from Human Endothelial Progenitor Cells Accelerate Cutaneous Wound Healing by Promoting Angiogenesis Through Erk1/2 Signaling

Jieyuan Zhang  1 , Chunyuan Chen  2 , Bin Hu  3 , Xin Niu  3 , Xiaolin Liu  1 , Guowei Zhang  3 , Changqing Zhang  1 , Qing Li  3 , Yang Wang  3
Affiliations

Exosomes Derived from Human Endothelial Progenitor Cells Accelerate Cutaneous Wound Healing by Promoting Angiogenesis Through Erk1/2 Signaling

Jieyuan Zhang et al. Int J Biol Sci. .

Abstract

Chronic skin wounds represent one of the most common and disabling complications of diabetes. Endothelial progenitor cells (EPCs) are precursors of endothelial cells and can enhance diabetic wound repair by facilitating neovascularization. Recent studies indicate that the transplanted cells exert therapeutic effects primarily via a paracrine mechanism and exosomes are an important paracrine factor that can be directly used as therapeutic agents for regenerative medicine. However, application of exosomes in diabetic wound repair has been rarely reported. In this study, we demonstrated that the exosomes derived from human umbilical cord blood-derived EPCs (EPC-Exos) possessed robust pro-angiogenic and wound healing effects in streptozotocin-induced diabetic rats. By using a series of in vitro functional assays, we found that EPC-Exos could be incorporated into endothelial cells and significantly enhance endothelial cells' proliferation, migration, and angiogenic tubule formation. Moreover, microarray analyses indicated that exosomes treatment markedly altered the expression of a class of genes involved in Erk1/2 signaling pathway. It was further confirmed with functional study that this signaling process was the critical mediator during the exosomes-induced angiogenic responses of endothelial cells. Therefore, EPC-Exos are able to stimulate angiogenic activities of endothelial cells by activating Erk1/2 signaling, which finally facilitates cutaneous wound repair and regeneration.

Keywords: Angiogenesis; Erk1/2.; Exosomes; Microarray; Wound healing.

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Conflict of interest statement

The authors have declared that no competing interest exists.

Figures

Figure 1
Figure 1
Isolation of endothelial progenitor cells (EPCs) from human umbilical cord blood (UCB). (A) EPCs showed a typical cobblestone-like morphology. Scale bar: 100μm. (B) Immunostaining of cell-specific antigens on EPCs. Scale bar: 50μm. (C) Flow cytometry analysis of the cell surface markers on EPCs. The isotype controls were illustrated as blank curves and the test samples were illustrated as solid gray curves. (D) EPCs formed capillary-like networks on Matrigel surfaces. Scale bar: 100μm. (E) EPCs displayed ability to bind FITC-UEA-l and uptake Dil-ac-LDL. Scale bar: 50μm.
Figure 2
Figure 2
Characterization of exosomes released by human UCB-derived EPCs (EPC-Exos). (A) Particle size distribution and concentration of EPC-Exos measured by TRPS. (B) Morphology of EPC-Exos under a transmission electron microscopy. Scale bar: 50nm. (C) Western blotting analysis of exosomal surface marker proteins (including CD9, CD63, and CD81) and endothelial lineage cell marker (CD31) in EPC-Exos.
Figure 3
Figure 3
EPC-Exos transplantation accelerates cutaneous wound healing in diabetic rats. (A) Gross view of wounds treated with PBS or different concentration of EPC-Exos at day 4, 7, and 14 post-wounding. (B) The rate of wound-closure on different days in wounds receiving different treatments. (C) H&E staining of wound sections after treatment with PBS or different concentration of EPC-Exos at 14 days post-wounding. The double-headed arrows indicate the edges of the scar. Scale bar: 2mm. The extent of re-epithelialization (D) and widths of the scars (E) was evaluated in PBS or EPC-Exos-treated wounds at 14 days post-wounding. Ep, Epithelium. (F) Evaluation of collagen maturity by Masson's trichrome staining. Blue Scale bar: 100 μm. Yellow scale bar: 50μm. (*P < 0.05 compared with the PBS group (control), # P < 0.05 compared with the EPC-Exos 2 × 1010 particles group.)
Figure 4
Figure 4
EPC-Exos transplantation promotes new blood vessels formation in the wound sites of diabetic rats. (A) Gross view of blood vessels in wounds treated with PBS or different concentration of EPC-Exos at day 14 post-wounding. Blood vessels density was determined by microfil perfusion. The reconstructed 3D images by micro-CT were illustrated in (B). (C) Quantitative analysis of the vascularized area in wounds receiving different treatments. (D) Immunofluorescence staining for CD31 in wounds after treatment with PBS or EPC-Exos at day 14 post-wounding. Scale bar: 50μm. (E) Quantitative analysis of the number of total blood vessels in (D). (F) Double immunofluorescent staining for CD31 and SMA in wounds receiving different treatments at day 14 post-wounding. Scale bar: 50μm. (G) Quantitative analysis of the number of mature blood vessels in (F). (H) Double immunofluorescent staining for CD31 and Ki67 in wounds to identify the proliferating cells. (*P < 0.05 compared with the PBS group (control), # P < 0.05 compared with the EPC-Exos 2 × 1010 particles group.)
Figure 5
Figure 5
Internalization of EPC-Exos into endothelial cells and their pro-angiogenic effects on recipient endothelial cells. (A) Fluorescent microscopy analysis of DiO-labeled EPC-Exos internalization by human microvascular endothelial cells (HMECs). The green-labeled exosomes were visible in the perinuclear region of HMECs. Scale bar: 50μm. (B) EPC-Exos promoted HMECs proliferation as analyzed by Cell Counting Kit-8 assay, and HMECs treated with a higher dose of exosomes showed much higher proliferative capability. (C) The tube formation capability was increased in HMECs stimulated with EPC-Exos, and better tube formation was observed with the increase of exosomes concentration. Scale bar: 100μm. (D) The total branching points, total tube length, cell covered area, and total loops at the indicated time were measured to quantify the ability of HMECs to form tubes. (E) EPC-Exos augmented the motility of HMECs, and the pro-migratory effect of EPC-Exos on HMECs was enhanced with a higher concentration of exosomes. Scale bar: 250μm. (F) Quantitative analysis of the migration rates in (E). (*P < 0.05 compared with the PBS group (control), # P < 0.05 compared with the EPC-Exos 2 × 1010 particles/mL group.)
Figure 6
Figure 6
Erk1/2 signaling was activated in HMECs after EPC-Exos treatment. (A) The differentially expressed genes (DEGs) in HMECs in response to EPC-Exos stimulation were illustrated as a heat map. A p-value cut-off of 0.05 and a fold value change of ≥1.5 were used as a filter to identify the DEGs. (B) The altered expression of Erk1/2 signaling-related genes was confirmed by qRT-PCR analyses. (C) EPC-Exos enhanced the phosphorylation of Erk1/2 and the protein levels of a class of angiogenesis-related molecules downstream of Erk1/2 pathway. (*P < 0.05 compared with the control group)
Figure 7
Figure 7
Erk1/2 signaling mediated the EPC-Exos-induced pro-angiogenic effects on HMECs. EPC-Exos increased the mRNA (A) and protein levels (B) of Erk1/2 signaling-related molecules, and their up-regulation induced by EPC-Exos was abolished by a specific Erk1/2 signaling inhibitor (U0126; 10 μM). (C) The proliferation of HMECs was remarkably enhanced after EPC-Exos treatment, whereas this effect was blocked by U0126. (D) EPC-Exos increased the tube formation ability of HMECs, but this effect was inhibited by U0126. Scale bar: 100μm. (E) The total branching points, total tube length, cell covered area, and total loops at the indicated time were assessed to quantify the ability of HMECs to form tubes at each time point. (F) EPC-Exos induced a remarkable increase in the motility of HMECs, but the pro-migratory effect was dramatically decreased by U0126. Scale bar: 250μm. (G) Quantitative analysis of the migration rates in (F). (*P < 0.05 compared with the control group, # P < 0.05 compared with the EPC-Exos+U0126 group.)
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