| Diabetic microangiopathy is the pathological basis of many diabetic complications,which serves as one of the major factors that contribute to diabetic morbidity and mortality.The vascular hyperpermeability and abnormal angiogenesis contribute to the development of diabetic microvascular complications.Advanced glycation end products(AGEs)are the irreversible covalent adduct formed by enzyme-free glycosylation reaction between proteins or lipids and sugars.Hyperglycemia and oxidative stress in diabetic patients accelerate the formation of AGEs,causing its excessive accumulation.It has been reported that the combination of AGEs and RAGE triggers the disruption of cell-cell adherence junctions and actin remolding through certain signal pathways,which lead to the imbalance of intercellular adhesive force and intracellular contraction force,which contributes to endothelial hyperpermeability.In addition,AGEs have been proved to induce endothelial proliferation,migration and tube formation,which are fundamental for angiogenesis.Despite many researches aim to uncover the mechanism under AGE-induced vascular hyperpermeability and angiogenesis,yet the precise mechanisms underlying the disruption of adherence junctions and actin remolding as well as vascular angiogenesis remain further investigated.?-catenin is a dual-function protein that serves as an adhesive component as well as a signal transduction component.Most of ?-catenin in endothelial cells acts as adhesive one which anchored on the membrane through VE-cadherin,while cytoplasmic-free ?-catenin can be immediately recognized and degradated by its destruction complex.When Wnt exists,the function of the destruction complex would be inhibited,which leads to ?-catenin accumulation and nuclear translocation.The nuclear-located ?-catenin combines to TCF/LEF and contributes to the transcription of Wnt target genes.As one of the adhesive components,?-catenin interacts with the cytoplasmic domain of cadherin,protecting it from degradation and hence stabilizing the adherence junctions between cells.At the same time,?-catenin combines with ?-catenin,preventing its communication with actin filaments,and hence indirectly participates in actin remolding.It has been reported that,in epithelial cells,the phosphorylation of ?-catenin Y654 attenuates E-cadherin/?-catenin association,impairing intercellular adherence junctions.In other cases,?-catenin Y142 phosphorylation has been reported to prevent its association with a-catenin and favors its nuclear tranlocation,leading to the transcription of Wnt target genes,such as VEGF,which has been detected in AGE-stimulated cell model.Those information point to a role of ?-catenin in AGE-induced angiogenesisObjective:Our research highlights effect of ?-catenin in AGE-induced endothelial hyperpermeability and angiogenesis,in which ?-catenin Y654 and Y142 phosphorylation is investigatedMethods:A series of experiments were accomplished in cell or mice by using cell culture,western blot,fluorescent staining,siRNA interference,transfected plasmids,adenovirus.Co-immunoprecipitation and immunofluorescence were performed to investigate the disruption of adherence junctions;cytoplasmic/nuclear extraction kit was used to measure ?-catenin nuclear translocation;FITC-dextran transendothelial flux and transendothelial resistance(TER)were performed to investigate HUVECs monolayer permeability and barrier function;cell proliferation was detected through CCK-8;cell migration was detected through wound scratch assay and transwell assay;Matrigel was used to investigate tube formation.Src specific inhibitor PP2,Src siRNA as well as Src wild type,kinase-deficient and kinase-mimic vectors were used to investigate whether Src is responsible for AGE-induced ?-catenin Y654 phosphorylation.FAK inhibitor was used to investigate whether FAK is indispensible for AGE-induced ?-catenin Y142 phosphorylation.?-catenin Y654 phospho-mimic(Y654E)and phosphor-deficient(Y654F)as well as ?-catenin Y142 phospho-mimic(Y142E)and phosphor-deficient(Y142F)vectors were used to investigate whether its phosphorylation is involved in AGE-induced vascular hyperpermeability and angiogenesis.For in-vivo assay,Y654F and Y142F overexpression adenovirus were administered in mice before AGEs injection,dextran flux across the mesenteric microvessels was measured to investigate microvascular permeability.Results:1.Role of ?-catenin in AGE-induced vascular hyperpermeability.1.1 AGEs mediated endothelial barrier dysfunction in vitro.Our results showed that AGEs time-dependently induced endothelial monolayer hyperpermeability,indicated by decreased TER and increased permeability coefficient for dextran(Pa).When HUVECs were incubated with AGEs for 1 h,the dissociation of VE-cadherin/?-catenin and ?-catenin/?-catenin were detected.Cells were stimulated by AGEs for 8 h and immunofluorescent was performed.Results showed impaired adherence junctions(AJs).Furthermore,we showed that AGEs induced actin remolding in endothelial cells,indicated by transferring of periphery-localized F-actin into centralized stress fibers1.2 ?-catenin Y654 phosphorylation was involved in AGE-induced endothelial hyperpermeability.HUVECs were incubated with AGEs for different times and ?-catenin Y654 phosphorylation was detected by western blotting.The resulted showed that P-catenin Y654 phosphorylation was rapidly enhanced at 10 min,reached a peak at 60 min,and finally returned to normal level at 120 min.Next,HUVECs were stimulated with different concentrations of AGEs and ?-catenin Y654 phosphorylation was determined.Results showed that AGEs concentration-dependently increase ?-catenin Y654 phosphorylation.HUVECs were pretreated with PP2,Src siRNA,Src overexpression vectors followed by incubation of AGEs for 60 min,then ?-catenin Y654 phosphorylation was detected.The results showed that either pharmological inhibition of Src activity or genetic knockdown of Src with siRNA attenuated AGE-induced ?-catenin Y654 phosphorylation.In addition,over expression of Src kinase-deficient vector prevented this effect,while Src kinase-mimic vector itself triggered ?-catenin Y654 phosphorylation.HUVECs were pretransfected with Y654F overexpression vectors followed by AGEs stimulation.The disruption of VE-cadherin/?-catenin and monolayer cell permeability were detected.CO-IP and immunofluorescent results showed that overexpression Y654F prevented dissociation of VE-cadherin/?-catenin in response to AGEs.In addition,overexpression Y654F attenuated AGE-induced endothelial hyperpermeability.Those data suggested that AGEs mediate ?-catenin Y654 phosphorylation and hence lead to VE-cadherin/?-catenin dissociation,which contributes to endothelial hyperpermeability.1.3 ?-catenin Y142 phosphorylation was involved in AGE-induced endothelial hyperpermeability.HUVECs were incubated with AGEs for 60 min and ?-catenin Y142 phosphorylation was detected by western blotting.The results showed that ?-catenin Y142 phosphorylation was increased after AGEs stimulation,indicating that AGEs induce ?-catenin Y142 phosphorylation.HUVECs were pretreated with FAK inhibitor PF573228,followed by incubation of AGEs for 60 min,then ?-catenin Y142 phosphorylation was detected.The results showed that PF573228 attenuated AGE-induced ?-catenin Y142 phosphorylation,suggesting that AGEs mediate ?-catenin Y142 phosphorylation through FAK.HUVECs were pretransfected with Y142F overexpression vectors followed by AGEs stimulation.Disruption of ?-catenin/?-catenin,formation of stress fiber and monolayer cell permeability were detected.Immunofluorescent results showed that overexpression Y142F prevented dissociation of ?-catenin/?-catenin and stress fiber formation in response to AGEs.In addition,overexpression Y142F attenuated AGE-induced endothelial hyperpermeability.1.4 ?-catenin Y654,Y142 phosphorylation was involved in AGE-induced mice mesentery microvascular hyperpermeability.Y654F and Y142F overexpression adenovirus were administered in mice before AGEs injection,dextran flux across the mesenteric micro vessels was measured.the results showed that either overexpress Y654F or Y142F attenuated AGE-induced mice mesentery microvascular hyperpermeability.2.Role of ?-catenin in AGE-induced angiogenesis.2.1 AGEs induced ?-catenin nuclear translocation.HUVECs were stimulated by AGEs,the cytoplasmic and nuclear ?-catenin was detected.The results showed that after AGEs stimulation for 60 min,the cytoplasmic?-catenin was decreased,whereas the nuclear ?-catenin was increased.As the stimulation time increase,this effect became more significant,indicating that AGEs time-dependently induce ?-catenin nuclear translocation.2.2 ?-catenin Y142 phosphorylation was indispensible for AGE-induced?-cateninin nuclear translocationHUVECs were pretransfected with Y142F overexpression vectors followed by AGEs stimulation for 2 h,cytoplasmic and nuclear ?-catenin was detected.The results showed that overexpression of Y142F prevented AGE-induced ?-catenin nuclear translocation,indicating that ?-cateninin Y142 phosphorylation is indispensible for AGE-induced ?-cateninin nuclear translocation.2.3 ?-catenin Y142 phosphorylation was involved in AGE-induced HUVEC angiogenesis.HUVECs were pretransfected with Y142F overexpression vectors followed by AGEs stimulation for 24 h,cell proliferation,cell migration and tube formation were investigated.The results suggested that overexpression of Y142F prevented AGE-induced cell proliferation,migration and tube formation,indicated respectively by decreased OD value,ameliorated migration area after scratch and decreased migration cells through transwell,decreased total branching points and total tube length.ICG-001,a specific inhibitor of Wnt/?-catenin signaling pathway,attenuated AGE-induced proliferation,migration and tube formation in HUVECs.Taken together,our results suggested that AGEs induce ?-catenin Y142 phosphorylation,favor its nuclear translocation and hence activate ?-catenin-dependent transcription,which contributes to angiogenesis.Conclusions:1.AGEs mediate ?-catenin Y654 phosphorylation through Src,which contributes to the dissociation of VE-cadherin/?-catenin,leading to the impairment of cell-cell adhesive force and hence endothelial hyperpermeability.2.AGEs mediate ?-catenin Y142 phosphorylation through FAK,which favors to the disruption of ?-catenin/?-catenin.The cytoplasmic-free ?-catenin contributes to actin filament remolding,leading to the formation of stress fibers and hence endothelial hyperpermeability.3.The phosphorylation of ?-catenin favors its dissociation from adherence junctions,and ?-catenin Y142 phosphorylation contributes to its nuclear translocation and is involved in AGE-induced HUVEC angiogenesis. |
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