Roles And Mechanism Of Exosome Derived From Umbilical Cord MSC In Cutaneous Regeneration

Objective:Burns are a common trauma in everyday life, with high morbidity and mortality. Some reports have confirmed that human umbilical cord mesenchymal stem cells (hucMSC) can enhance cutaneous wound healing, but the problems such as the preservation, transportation and safety issues of MSC limit its clinical application. Exosome is the main component of MSC paracrine, the roles and mechanism of hucMSC-exosome (hucMSC-Ex) in cutaneous regeneration is unclear. The present study was designed to prove whether hucMSC-Ex can enhance the healing of deep II burn repair and clarify the signal transduction mechanism of hucMSC-Ex in process of promoting cutaneous regeneration.Methods:HucMSC cells were isolated by adherent method. HucMSC-Ex was isolated by sucrose density gradient centrifugation. HucMSC-Ex surface markers:CD63, CD81 and CD9 were detected by Western blot. The shape and size of hucMSC-Ex were observed by TEM. The diameter distribution of hucMSC-Ex was analyzed by nanosight. Injury condition of 80℃ hot water 8 seconds was used to make deep II burn of skin, hucMSC-Ex treated this model and analyzed the results of its repair by observing wound, HE staining, PCNA and CK19 staining. Cell apoptosis was detected by analyzing the expression of apoptosis Bcl-2, Bax by Annexin V/PI double staining. Cell count was used to detect cell proliferation, cell migration was analyzed by scratch test. The cell cycle was analyzed by flow cytometry. PI3K inhibitor LY294002 was used to prove the role of AKT pathway in hucMSC-Ex induced wound healing.β-catenin inhibitor ICG001 reversed the role of Wnt/β-catenin signal in hucMSC-Ex induced wound healing. The lentivirus-mediated shRNA was used to interfere the expression of Wnt4 in hucMSC and hucMSC-Ex. Stem cells markers CK15, CK19, Integrin a6, CD44, Oct4 and Nanog were detected by immunohistochemistry and immunofluorescence. qRT-PCR was used to detect the expression of collagen I and III. The deposition of collagen was showed by Masson staining of skin tissue. Immunofluorescence staining of a-SMA in skin tissue and dermal fibroblasts (DFL) was used to show the differentiation of myofibroblast. Immunofluorescence and nuclear/cytoplasmic protein analysis were used to detect the nuclear translocation of YAP and β-catenin protein. TopFlash assay was applied to detect the transcriptional activity of β-catenin. The transcription activity of YAP was analyzed by YAP-TEAD4 luciferase reporter. The expression of YAP target genes CTGF and Cy61 was detected by qRT-PCR. The expression and nuclear translocation of p-YAP and YAP in skin tissue was detected by immunohistochemistry. YAP Serl27 site and LATS Thr1079 site was mutated to prove founction of these two phosphorylation sites. Knockdown 14-3-3ζ,α-catenin, YAP, LATS1/ 2 and other molecules by shRNA and overexpressed YAP and 14-3-3ζ to prove their action. Co-precipitation technique(IP) was used to detect the interaction among 14-3-3ζ, YAP and p-LATS.Results:HucMSC-Ex was isolated successfully. The morphology of hucMSC-Ex was spherical. The diameter of hucMSC-Ex was about 100nm by Nanosight. CD63, CD81 and CD9 were expressed in hucMSC-Ex. After successfully establishing skin deep Ⅱ degree burn model in SD rats, hucMSC-Ex enhanced the wound healing of burned rat and promoted the proliferation of skin cells and induced the rational distribution of collagen in the skin tissue. HucMSC-Ex reversed thermal damage induced apoptosis and promoted cell proliferation in vitro. Screening the activation of AKT pathway and MAPK pathway, we found that hucMSC-Ex significantly promoted the activation of AKT signaling pathway. Luminex detection discovered that a number of possible factors could induce the activation of AKT pathway. PI3K inhibitor LY294002 could reverse the inhibition of apoptosis induced by hucMSC-Ex in cells treated with heat stress, but cuold not change the promotion of cell cycle and migration induced by hucMSC-Ex. Further studies showed that hucMSC-Ex activated Wnt/P-catenin signal and β-catenin inhibitors ICG001 reversed hucMSC-Ex induced wound healing. After interfering the expression of Wnt4 in hucMSC-Ex (Wnt4-shRNA-Ex), the promotion of cell cycle and migration induced by hucMSC-Ex in skin cells disappeared, and Wnt4-shRNA-Ex could not enhance wound healing. Longer observation was conducted to exosome repaired deep Ⅱ burn model and found that, after the first promotion, hucMSC-Ex inhibited the cell proliferation and the activation of β-catenin. HucMSC-Ex restricted the expansion of skin stem cells and collagen deposition at 4W after treating with hucMSC-Ex. In vitro studies have found that the inhibition of cell proliferation and β-catenin activation induced by hucMSC-Ex depended on the high cell density culture conditions. HucMSC-Ex inhibited the expression of α-SMA and collagen in DFL cells. Further studies revealed that hucMSC-Ex induced YAP cytoplasmic retention by promoting YAP Serl27 site phosphorylation and the inhibition of β-catenin activation induced by hucMSC-Ex depended on the phosphorylation of YAP Serl27 site. The results of LC/MS assay discovered that hucMSC-Ex have regulatory role in Hippo pathway. The knockdown of 14-3-3ζ in hucMSC-Ex reversed YAP Serl27 phosphorylation and inhibition of cell proliferation in vitro, and the overexpression of 14-3-3ζ in hucMSC-Ex got the opposite results. After interfering the expression of LATS or conducting the point mutation LATS Thr1079,the promotion role of 14-3-3ζ on YAP phosphorylation was reduced.14-3-3ζ, YAP and p-LATS could be formed binding complex, the binding of YAP and its upstream kinases p-LATS depended on 14-3-3ζ proteins. Interfering the expression of 14-3-3ζ in hucMSC-Ex (sh14-3-3ζ-Ex). Sh14-3-3ζ-Ex treated skin tissue was appeared to show stronger proliferation ability, thicker epidermis, less subsidiary structure, more myofibroblast and collagen deposition than that control.Conclusion:Our findings have clearly indicated that hucMSC-Ex enhances skin second-degree burn injury repair and Wnt4 is the key mediator delivered by hucMSC-Ex in cutaneous wound healing. HucMSC-Ex-mediated 14-3-3ζ effectively coordinates self-control of Wnt4 activity via modulation of YAP to balance growth and differentiation of stem cells during cutaneous remodeling.