Effects And Mechanism Of Liraglutide On Wound Angiogenesis Via Regulating Endothelial Colony Forming Cell Senescence Induced By High Glucose

Background and ObjectiveSkin wound is a serious and common complication of diabetes mellitus,and refractory ulcer in lower limb is an important contributor of amputation.Previous studies have shown that neovascularization impaired is a key factor in the delayed healing of diabetic wounds.Endothelial colony-forming cells(ECFCs)have attracted more attention due to their ability of clonal proliferation ability,differentiation into mature endothelial cells and promoting angiogenesis in vivo and vitro.However,ECFCs exposed to high glucose display decreased number and dysfunction.Meanwhile,our previous study found that high glucose could induce ECFCs senescence and down-regulate Sirt7.Another study also demonstrated that in senescent cells(HEK293FT)induced by camptothecin,the acetylation level of NPM1 was increased in conjunction with decreased levels of Sirt7.In addition,recent study has shown that liraglutide can significantly reduce the risk of foot ulcer-related amputation in diabeticpatients.Therefore,in this study,firstly,the appropriate intervention concentration of liraglutide was explored,then the wound model of nude mice was constructed and ECFCs transplantation was carried out to explore the effects of high glucose and liraglutide intervention on wound healing and angiogenesis.2.The expression of SMP30,Ac-NPM1 levels and p53 was detected under high glucose conditions and the intervene with liraglutide was performed.3.To examine the changes in SMP30 expression,the acetylation levels of NPM1,p53 expression,ECFCs senescence induced by Sirt7,the expression vector was transfected into ECFCs.MethodsPeripheral blood of healthy adults was collected,mononuclear cells were obtained by density gradient centrifugation,and ECFCs was cultured and induced in EGM-2 complete medium.After the optimal concentration of liraglutide was explored,the cells were randomly divided into three groups: normal glucose group(NG)(5.5mmol/L D-glucose),high glucose group(HG)(30mmol/L D-glucose)and high glucose intervention group(HG+Lira)(30mmol/LD-glucose + 100nmol/L liraglutide).After a full-thickness skin wound model established,CM-Dil-labeled ECFCs transplantation was performed to observe the wound healing rate and capillary density.In vitro,cells were randomly divided into three groups:normal glucose group(NG)(5.5mmol / L D-glucose),high glucose group(HG)(30mmol / L D-glucose),osmotic pressure group(OSM)(5.5mmol/LD-glucose+24.5mmol/L mannitol)to explore the effects of high glucose on the expression of SMP30,acetylated NPM1 levels,and p53.Two groups were added(normal glucose intervention group(NG+Lira)(5.5mmol/L D-glucose+100nmol/L liraglutide),high glucose intervention group(HG Lira)(30 mmol/LD-glucose+100 nmol/L liraglutide))to explore the effects of liraglutide on the related protein expression.After the expression of Sirt7 was up-regulated by lentiviral vector,under high glucose conditions,the expression of Sirt7,SMP30,acetylated NPM1 levels,and p53 in the overexpression group and the empty vector group were detected respectively.The intervention time of all the groups mentioned above was 6days.Results1.In the case of high glucose,the intervention of liraglutide up-regulated the expression of Sirt7,and the expression level of Sirt7 rose to the plateau at 100 nmol/L.2.After the ECFCs treated with high glucose were transplanted into mice,the wound healing rate of the mice was lower than that of the normal glucose group,and the capillary density decreased.But the intervention of liraglutide could improve the healing rate and increase the capillary density.In addition,ECFCs were labeled with CM-Dil before transplantation and showed red,which was observed at the vascular structure of the mouse,suggesting that the transplanted ECFCs were incorporated into thevasculature of the mouse.3.High glucose could down-regulate SMP30,up-regulate acetylated NPM1 levels and p53.After treatment with liraglutide and Sirt7up-regulated by lentivirus,SMP30 was up-regulated and acetylated NPM1 levels and p53 were down-regulated.ConclusionHigh glucose could induce ECFCs senescence,leading to the decreased angiogenesis in wound and delayed wound healing.The mechanism may be related to the reduction of Sirt7 expression by high glucose,which caused increased Ac-NPM1 levels,up-regulated p53 and increased ECFCs senescence.After the administration of Liraglutide,the pathways mentioned above can be reversed by upregulation of Sirt7,thereby reducing the high glucose-induced ECFCs senescence,increasing angiogenesis and promoting wound healing.