The Role And Mechanism Of Exosomes Derived From Human EPCs In Healing Of Cutaneous Wounds In Diabetic Rats

Background and Objective:Impaired skin wound healing is one of the most common and disabling complications of diabetes. Angiogenesis is thought to be important for wound healing, as it plays a crucial role in delivery of oxygen and nutrients to the wound sites for sustaining cell proliferation, collagen synthesis, and re-epithelialization. However, angiogenesis usually gets compromised in patients with diabetes, which consequently leads to delayed wound healing. Accumulating studies showed that the transplantation of endothelial progenitor cells(EPCs), the nature precursors of mature endothelial cells, can stimulate new capillaries formation and wound closure in diabetic animal models. 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 bioactive agents for regenerative medicine. However, application of exosomes in diabetic wound repair has been rarely reported. As EPCs exhibit robust pro-angiogenesis and wound healing potential, we hypothesized that the exosomes derived from EPCs(EPC-Exos) can induce angiogenesis and diabetic wound repair. Herein, we investigated the wound healing effects of EPC-Exos in diabetic rat models and explored the underlying mechanism. We aimed to provide a novel strategy for treating non-healing diabetic wounds. Methods: 1. Isolation and identification of EPCs derived from human umbilical cord blood(UCB)1.1 Isolation and culture of EPCs: Mononuclear cells were isolated from human UCB by density gradient centrifugation and EGM-2MV media was used to culture EPCs.1.2 EPCs identification: EPCs were detected under a microscope; Immunostaining and flow cytometry analyses were performed to assess cell-surface markers on EPCs; In vitro tube formation was evaluated on Matrigel; Uptake of Di L-labeled ac-LDL and binding of FITC-conjugated UEA-1 were assessed by fluorescent staining. 2. Collection and identification of EPC-Exos2.1 EPC-Exos collection: EPC-Exos were isolated from the conditioned medium of EPCs by ultracentrifugation and ultrafiltration.2.2 EPC-Exos identification: The morphologies of EPC-Exos were observed by a transmission electron microscopy(TEM); Tunable resistive pulse sensing(TRPS) analysis was used to measure the size distribution and concentration of EPC-Exos; Western Blot was performed to detect the surface markers on EPC-Exos. 3. The effects of EPC-Exos on wound healing in diabetic rats3.1 Established diabetic skin wound rat model and multi-point injection of EPC-Exos(2 × 1010 or 1 × 1011 particles, dissolved in 200 μL of PBS) or an equal volume of PBS around the wounds.3.2 Evaluated the wound healing effects of EPC-Exos and calculated the rate of wound-size reduction at day 4, 7, and 14 post-wounding.3.3 At 14 post-wounding, the extent of re-epithelialization and scar formation was assessed by hematoxylin and eosin(H&E) staining; Masson’s trichrome staining was adopted to determine the degree of collagen regeneration. 4. The effects of EPC-Exos on angiogenesis in the wound sites of diabetic rats4.1 Inverted the wound sites and evaluated the extent of blood vessels formation in the wound sites at 14 post-wounding.4.2 Blood vessels were perfused with microfil and reconstructed by micro-CT.4.3 Immunostaining for CD31 and α-SMA was used to evaluate the extent of new blood vessels formation and maturation. 5. The mechanism by which EPC-Exos promote angiogenesis5.1 HMECs were co-cultured with green dye Di O-labeled EPC-Exos and the uptake of EPC-Exos by HMECs was analyzed with a fluorescence microscope.5.2 The effects of EPC-Exos on HMECs’ proliferation, tube formation, and migration were evaluated by CCK-8 assay, Matrigel-based tube formation assay, and scratched assay, respectively.5.3 Gene expression profiling and bioinformatics analyses were performed to detect the differentially expressed genes and altered signaling pathway in HMECs following EPC-Exos stimulation.5.4 q RT-PCR and western were used to verify the alteration of Erk1/2 signalingrelated genes and proteins in HMECs after EPC-Exos stimulation.5.5 HMECs were pre-treated with a selective Erk1/2 signaling inhibitor(U0126) and the regulatory effects of EPC-Exos on HMECs were then evaluated. Results: 1. EPCs were successfully isolated from the human UCB:EPC colonies usually appeared between 7 and 10 days of culture and EPCs exhibited typical endothelial-like cobblestone morphology; Immunostaining and flow cytometry analyses showed that EPCs were highly positive for CD31, CD34, CD133, v WF, VEGFR-2, and VE-cadherin, but negative for CD45; EPCs displayed the ability to form tube structures on Matrigel, uptake Di L-ac-LDL, and bind FITC-UEA-1. 2. EPC-Exos were successfully collected from the conditioned media of EPCsTEM showed that EPC-Exos exhibited a round- or elliptical-shaped morphology; TRPS analysis revealed that the size of EPC-Exos was approximately 50-60nm; Western blotting showed that EPC-Exos were positive for the characteristic exosomal markers(including CD9, CD63 and CD81) and the endothelial lineage marker CD31. 3. EPC-Exos transplantation accelerated wound healing in diabetic rats3.1 Wound closure in rat treated with EPC-Exos was accelerated compared to that of control group at day 4, 7, and 14 post-wounding. Rats receiving a higher dose of EPC-Exos showed a higher wound closure rate than that of low dose of EPC-Exos.3.2 H&E and Masson’s trichrome staining revealed that the extent of epithelialization and collagen regeneration in EPC-Exos-treated group was much higher than that of control group at day 14 post-wounding, whereas scar formation was inhibited. Much more neoepithelium, larger collagen deposition areas, and narrower scars were observed in wounds treated with a higher dose of EPC-Exos. 4. EPC-Exos enhanced angiogenesis in the wound sites of diabetic rats4.1 Blood vessels in control group were rarely observed. In contrast, a large amount of blood vessels was observed in the EPC-Exos-treated wounds and the number of blood vessels was enhanced with the increase of exosomes concentration.4.2 Micro CT-based microangiography revealed that EPC-Exos induced a prominent increase in blood vessels density compared to controls and higher extent of blood vessels formation was observed in wounds treated with a higher dose of exosomes.4.3 Immunostaining for CD31 and CD31/α-SMA revealed that EPC-Exos enhanced the amount of total and mature blood vessels in the wound sites compared to controls, and much more vessels were detected with the increase of exosomes concentration. 5. Erk1/2 signaling mediated the pro-angiogenic effects of EPC-Exos on HMECs5.1 The Di O-labeled EPC-Exos could be internalized by HMECs and predominantly transferred to the perinuclear region of HMECs.5.2 EPC-Exos markedly enhanced the angiogenic activities(including proliferation, tube formation, and migration) of HMECs, and better pro-angiogenic effects were observed with the increase of exosomes concentration.5.3 Results of microarray showed that the expression of a class of angiogenic genes(including FGF2, IL-6, IL-8, c-Myc, Id1, Cox-2, VEGFA, and CCND1) involved in Erk1/2 signaling was markedly enhanced in HMECs after EPC-Exos stimulation.5.4 q RT-PCR and western analyses showed that the levels of downstream targets of Erk1/2 signaling(including c-Myc, Id1, Cox-2, and VEGFA) and phosphorylation of Erk1/2 were significantly increased in HMECs after EPC-Exos stimulation.5.5 U0126 abolished the EPC-Exos-induced positive effects on gene and protein expression, proliferation, tube formation, and migration of HMECs. Conclusion:1. The local transplantation of EPC-Exos into the skin wounds of diabetic rats could markedly enhance the rate of wound closure, the formation of new blood vessels, and the regeneration of epithelium and collagen in the wound sites.2. EPC-Exos could promote the proliferation, migration, and tube formation of HMECs by activating Erk1/2 signaling.