Study On Enhancing Angiogenesis Ability Of Adipose-derived Stem Cells By Metformin

Background and ObjectiveNon-healing cutaneous wounds are a huge medical and social burden around the world.Morbidity from diabetes is the major cause of persistent,non-healing ulcers,which put patients at danger of limb amputation.The diminished ability to rebuild microvasculature by angiogenesis is one main pathophysiological causes of non-healing wounds in diabetes and peripheral vascular disease.Angiogenesis is required for the transfer of systemic oxygen,nutrients,and other needs into proximal injured regions during the entire tissue regeneration process in all types of cutaneous wound healing.Additionally,a variety of procedures and dressings have been used to promote chronic wound healing,including growth factor delivery,gene therapy,stem cell therapies,and mechanical/pressure-based stimulatio.Previous research has shown that human adipose tissue-derived stem cells(hADSCs),a population of pluripotent mesenchymal cells.The key variables that assist hADSCs in exerting their biological effects are paracrine cytokines,exosomes,and another active compound.Interestingly,as alternatives to traditional hADSCs therapy,hADSCsconditioned medium(hADSCs-CM)containing active molecules released by hADSCs such as cytokines,exosomes,deoxyribonucleic acid(DNA),and ribonucleic acid(RNA).However,cytokine generation in hADSCs-CM is unstable,limiting its therapeutic usefulness.Some studies have shown that specific chemicals can influence the level of release of active compounds secreted by mesenchymal stem cells(MSCs).We aimed to stimulate hADSCs with a chemical that will allow for secretion of more cytokines,which is beneficial for wound vascularization.MET,a biguanide,is a regularly used oral antihyperglycemic medication for the treatment of type 2 diabetes mellitus.MET is an insulin booster that boosts insulin sensitivity.As a result,it may be beneficial for wound healing.Autophagy is a cellular protective process that degrades misfolded proteins and damaged organelles.Many studies have shown that autophagy is involved in VEGFa-mediated angiogenesis relevant to endothelial cells.However,it is unclear whether or not autophagy of hADSCs has a therapeutic effect on wound healing.Therefore,we aimed to investigate whether,by increasing autophagy,MET can promote the quality and quantity of cytokine secretion,which is crucial for wound healing in hADSCs-CM.MethodsCCK-8 assay,cell cycle assay and flow cytometry were used to detect the effects of metformin on hADSCs in vitro.The effect of conditioned medium on migration of HUVECs was studied by cell scratch assay,the effect of conditioned medium on the ability of tube formation of HUVECs was studied by tube formation assay,the expression of angiogenesisrelated proteins Ang Ⅱ and VEGFa was detected by western blot,the secretion of VEGFa was detected by ELISA,and the expression of VEGFa was detected by transmission electron microscopy(TEM)Autophagosomes in hADSCs were directly observed,autophagosomes were detected by immunofluorescence assay,and autophagy-related proteins Beclin-1,LC3Ⅱ and Atg 5 were detected by western blot.To explore the relationship between the autophagy level of hADSCs and their angiogenic ability,rapamycin and 3-methyladenine were used(3-MA)increase or inhibit the autophagy level of hADSCs,then explore the influence of conditioned medium of hADSCs under various autophagy levels on the tube formation ability of HUVECs through tube formation assay,and use western blot to verify whether there is correlation between the expression levels of autophagy-related proteins Beclin-1,LC3 Ⅱ and Atg 5 and the expression levels of angiogenesis-related proteins Ang Ⅱ and VEGFa under various autophagy levels of hADSCs.ELISA was used to verify the level of VEGFa secretion in the conditioned media of hADSCs at various levels of autophagy.Then,in order to verify that VEGFa secreted by hADSCs is a key factor for promoting angiogenesis of HUVECs,VEGFa released by hADSCs is neutralized by using a VEGFa neutralizing antibody,and then the effects of three groups of conditioned media of hADSCs with or without metformin pretreatment and conditioned media of metformin-treated hADSCs added with VEGFa neutralizing antibody on the angiogenesis ability of HUVECs are tested through a tube formation assay.Finally,the matrigel plug assay was used to examine whether metformin-treated hADSCs promoted angiogenesis in vivo.ResultsThe results showed that low concentrations of metformin had no effect on the proliferation of hADSCs.Metformin upregulates angiogenic proteins Ang Ⅱ and VEGFa and autophagy-related proteins Beclin-1,LC3 Ⅱ and Atg 5 in hADSCs.Metformin-induced autophagy of hADSCs is associated with increased hADSCs production and VEGFa release.VEGFa secreted by hADSCs plays a key role in promoting angiogenesis in vivo.The results showed that hADSCs promoted angiogenesis in vivo after metformin treatment compared with untreated hADSCs.ConclusionsIn conclusion,our research has shown that hADSCs pretreated with MET have a stronger ability to secrete VEGFa in vivo and in vitro,and this process is mediated by autophagy promoted by MET.