Bone Marrow-derived Mesenchymal Stem Cells Promoted Cutaneous Wound Healing By Regulating Keratinocyte Migration Via The β₂-adrenergic Receptor Signaling

Objective（1）To explore the role of epithelial-mesenchymal transition in cutaneous wound healing.（2）To explore the role ofβ₂-AR signaling pathway in the process of skin wound healing and provide new ideas for studying the molecular mechanism of wound healing.（3）To explore the possible mechanism of bone marrow-derived mesenchymal stem cells（BMSCs）in promoting skin wound healing,and provide the solid theoretical basis for the clinical application of mesenchymal stem cells.（4）To verify the role of BMSCs in promoting skin wound healing by in vivo wound healing model.Materials and methods（1）The human normal skin and wound edge tissue samples were obtained from the patients with trauma.Immunofluorescence staining was used to detect the difference in expression and distribution of CK19,CK14,E-cadherin andα-SMA.（2）The HEK and HaCaT cells were used for cell experiments in vitro.We performed RT-qPCR and western blot analysis in human keratinocytes to detect the expression of E-cadherin,α-SMA and Snail after treatment of isoproterenol with or without ICI-118,551,a selectiveβ₂-AR antagonist.We also silencedβ₂-AR expression in epidermal keratinocytes usingβ₂-AR siRNAs in the absence or presence of isoproterenol,and detected the expression of E-cadherin andα-SMA at protein level.Forthermore,we performed scratch wound healing assays to explore the effect of isoproterenol on the migration of epidermal keratinocytes.（3）We carried out the MTT and scratch wound healing assays for keratinocytes treated with BMSC-CM to examine whether BMSCs promoted wound closure.Then isoproterenol,ICI-118551,and siRNA were used to activate or inhibit the expression ofβ₂-AR in vitro,and the possible mechanism of EMT-like changes in keratinocytes induced by BMSC and its role in the skin wound healing were detected by RT-qPCR and western blot analysis.（4）The in vivo wound healing model was established and treated with human BMSCs or vehicle（saline solution）.The wound areas were analyzed in a timely manner on days 0,3,6,9 and 12.Furthermore,the expression levels ofβ₂-AR,E-cadherin andα-SMA during skin wound healing were analysised by western blot.To further evaluate the EMT-like alterations during skin re-epithelialization after BMSCs treatment,we analyzed the expression of CK14,E-cadherin andα-SMA in keratinocytes at the wound edge by Immunofluorescence staining.Results（1）The epidermal keratinocytes proliferation abilitiy in normal skin and wound edge tissue was significantly different.Compared with normal skin,CK19 and CK14positive expression cells were widely distributed in wound edge tissue,and the epidermal keratinocytes in wound edge tissue expressed both E-cadherin andα-SMA,which suggested that EMT-like process played an important role in wound healing.（2）Our results showed that isoproterenol administration downregulated the epithelial marker E-cadherin while upregulating the expression ofɑ-SMA and Snail at mRNA and protein levels in both HEK and HaCaT cells.ICI-118,551 treatment,however,reversed the effect of isoproterenol on the expression of EMT-associated proteins in the human keratinocyte cell lines.And,the HEKs and HaCaT cells transfected withβ₂-AR siRNAs showed reduced expression ofα-SMA as well as a restoration of E-cadherin levels irrespective of isoproterenol stimulation.β₂-AR signaling also affected keratinocyte cell migration,resulting in faster closure on the wound gap as measured by in vitro scratch wound healing assay.Taken together,these results indicated thatβ₂-AR signaling was involved in the acquisition of EMT-like phenotype in human keratinocytes in vitro.（3）We investigated the effect of BMSC-CM on the biological behavior changes of human keratinocytes,and surprisingly found that BMSC-CM administration recapitulated the effect of isoproterenol on cell migration and induced the expression ofβ₂-AR and a panel of proteins associated with mesenchymal phenotype in both HEKs and HaCaT cells.Meanwhile,our results indicated that BMSC-CM contributed to the activation of PKA,whereas theβ₂-AR inhibitor ICI-118,551 could block the effects of BMSC-CM on PKA phosphorylation.Given these findings,it therefore seemed that BMSC-CM promoted the in vitro cell migration and induced the EMT-like changes in human epidermal keratinocytes by triggering activation ofβ₂-AR/PKA signaling.（4）Our data from the in vivo wound healing model revealed that exogenous application of BMSCs led to an acceleration of wound healing associated with faster wound closure and re-epithelization.BMSCs application also resulted in increased levels ofβ₂-AR andα-SMA expression,and decreased levels of E-cadherin expression in the post-injured skin tissues compared with vehicle controls.Immunofluorescent staining results indicated that some keratinocytes in the upper part of the outer root sheath of hair follicles were double staining of E-cadherin andα-SMA during the process of wound healing.It therefore makes sense that MSCs was associated with the acquisition of mesenchymal phenotype in skin tissues during wound healing and contributed to the recovery of injured skin.β₂-AR signaling,however,played potential roles in the MSCs-induced EMT-like changes during the re-epithelialization of skin tissues following injury.ConclusionThis study was performed to clarify the involvement of EMT in the BMSC-mediated re-epithelization of skin wound healing with regard toβ₂-AR activation.Mechanically,MSCs in the wound area secreted soluble factors.These factors recognized and activatedβ₂-ARs,which triggered down-stream targets,such as PKA,and subsequently promoted keratinocyte movement via inducing these cells to undergo EMT-like phenotypic changes.Our findings provided a biochemical mechanism underlying the functions of MSCs in wound re-epithelization,which provide a reliable theoretical basis for the wide application of MSCs in the treatment of chronic wounds.