Studies On The Association Of Endothelial Nitric Oxide Synthase With ?-catenin And Their Roles In Oxidized LDL Induced Endothelial Dysfunction

Background:Endothelial dysfunction(ED)is closely related to the development of many cardiovascular diseases,mainly due to enhanced oxidative stress and reactive oxygen species(ROS).ED is associated with reduced endothelial nitric oxide(NO)synthase(eNOS)activity and increased expression of inducible NO synthase(iNOS).Oxidized low density lipoprotein(ox-LDL)is atherogenic and is the initial factor in the process of vascular endothelial cells injury.NO and its derivatives can modify protein cysteines by S-nitrosylation.?-catenin is a dual functional protein.The activation of classical Wnt signaling can promote the entry of ?-catenin into the nucleus.Then ?-catenin increases the transcriptional activity of T cell factor/lymphocyte enhancing factor(TCF/LEF)which would induce the expression of proinflammatory cytokines,and finally,aggravates the damage of atherosclerotic endothelial cells.There is bidirectional cross-regulation between eNOS and ?-catenin in endothelial cells;however,the interaction and mechanism of eNOS and ?-catenin in vascular endothelial dysfunction are still unclear.Objectives:The objectives of this study are to investigate the interaction and mechanism of eNOS and ?-catenin in ox-LDL-induced endothelial dysfunction,and to clarify the key regulatory role of S-nitrosylated protein modification in the endothelial cells.Methods and results:In vitro,Human umbilical vein endothelial cells(HUVECs)or EA.hy926 cells were exposed to ox-LDL.Ox-LDL induced decrease in eNOS activity,but increase in superoxide level,endothelial cell migration,iNOS activity and NO production.The association of eNOS and ?-catenin was increased in ox-LDL treated endothelial cells.eNOS and ?-catenin nuclear translocation,eNOS S-nitrosylation and ?-catenin target genes were all induced higher in ox-LDL treated endothelial cells.But the increases were inhibited by pretreating with NO synthase inhibitor L-NAME or iNOS-specific inhibitor 1400W.Wild type eNOS or mutants S1177A(which is inactive and cannot undergo phosphorylation at position 1177)or S1177D(which is constitutively active and mimics the increased catalytic activity caused by phosphorylation)were transfected into human embryonic kidney(HEK)293 cells.Immunoprecipitation was performed,and we found that the binding of?-catenin and eNOS was significantly reduced in S1177D transfection but increased in S1177A transfection compared with wild type transfection.Furthermore,endothelial cells were transfected with wild type eNOS or its mutant plasmids Cys94s or Cys99s.Transfection with Cys94s or Cys99s significantly reversed ox-LDL induced eNOS and ?-catenin association,eNOS S-nitrosylation and improved endothelial cell function.In vivo,8 weeks old male ApoE-/-mice were given a high-fat diet(HFD)for 4 weeks to induce atherosclerotic vascular endothelial dysfunction.The wild type C57BL/6J mice were fed a normal diet as control.Aortic root frozen slices were determined by oil red O staining.The expression levels of eNOS,?-catenin and the target protein of p-catenin c-myc were detected by immunofluorescence in the aorta endothelial cells.The results showed that,the expression levels of eNOS and?-catenin in the atherosclerotic aorta endothelial cells were significantly higher than those in the controls.The expression of c-myc was increased in the nucleus.Conclusions:Our data indicate that ox-LDL-induced iNOS-derived NO contributes to S-nitrosylation of Cys94 and Cys99 sites in eNOS.The S-nitrosylated inactive eNOS binds with ?-catenin and they transfer to nucleus together.Nuclear translocation of ?-catenin is transcriptionally activated;its co-activator TCF/LEF and target genes(such as c-myc)are activated.The interaction of eNOS and ?-catenin participates in ED.This finding suggests a new mechanism for the regulation of ox-LDL-induced endothelial dysfunction.