| Objective:This study was to determine whether advanced glycation end products modified human serum albumin(AGE-HSA) alters the cellular expression and distribution of the ERM(Ezrin/Radixin/moesin) protein in human umbilical vein endothelial cells(HUVECs) and to explore the mechanism in this pathological procedure.This study was also performed to investigate the effect of AGE-HSA on morphological changes and expression of adherens junction protein vascular endothelial(VE)-cadherin in HUVECs and to understand the mechanism of this alteration.Methods:AGE-HSA was prepared by incubation of 1.75 mg/ml human serum albumin with 100 mmol/L of D-glucose for 8 weeks.Human umbilical vein endothelial cells(HUVEC)-derived from human umbilical vein was planted on gelatin-coated glass bottom micro well Petri dishes and six well plate.The confluence cells were subjected to the stimulation of different concentrations and timing AGE-HSA.Or the cells were pre-administrated with anti-RAGE IgG or MAPK inhibitors(PD98059,SB203580,SP600125) or Rho kinase inhibitor (Y27632),respectively,before exposed to AGE-HSA.In some other cases,the cells were pre-infected with recombinant adenovirus of dominant negative mutants of MEK1 or MKK6b and p38 upstream kinase or recombinant adenovirus of dominant negative mutants of p38α,β,respectively,and then subjected to AGE-HSA adminstration.For the control experiment,the cells were transfected with recombinant adenovirus of constitutively active mutants of MEK1 and MKK6b for 24 h.The morphological changes of ERM protein and VE-cadherin was detected with immunofluorescent and observed with cofocal microscope.The expression of VE-cadherin was detected with anti-VE-cadherin antibody by SDS-PAGE and western blot.Results:1.Phosphorylation of ERM detected by immunofluoscent in HUVECs was increased by AGEs stimulation in a concentration- and time- dependent pattern. Pre-treatmented HUVECs with anti-RAGE IgG did not casue obvious ERM phosphorylation after AGEs applications.These changes of phosphorylation ERM induced by AGEs were also abolished by Rho kinase inhibitor Y-27632,ERK upstream inhibitor PD98059 and p38 MAPK inhibitor SB98059 while HUVECs could preserve the normal morphological characteristics and expression of ERM protein after AGE-HSA stimulation in those treated cells.The phosphorylation of ERM in HUVECs was inhibited by transfecting the cells with recombinant adenovirus of dominant negative mutants MEK1,MKK6b,and p38α,β.2.The distribution of VE-cadherin was significantly changed by AGE-HSA treatment in a dose- and time-dependent manner and was coincident with the AGES-induced reorganization of F-actin and the formation of stress fiber.The protein expression of VE-cadherin in HUVECs was not changed after treatment of 50 mg/1 AGE-HSA for 4,8,and 12 h respectively.The localization of VE-cadherin in HUVECs pre-treated with anti-RAGE IgG did not show much alteration after stimulation by AGEs.The dispersed changes of VE-cadherin induced by AGEs were abolished by Rho kinase inhibitor Y-27632,ERK upstream inhibitor PD98059,p38 MAPK inhibitor SB98059,and JNK inhibitor SP600125,repectively.The transfection of HUVECs with recombinant adenovirus of dominant negative mutants MEK1,MKK6b and p38α,andβblocked the AGE-HAS-induced morphological characteristics of VE-cadherin,while the transfection of HUVECs with recombinant adenovirus of constitutively active mutants MEK1,MKK6b could mimic the alteration caused by AGE-HSA treatment.Conclusions:1.AGEs-HSA could induce a significant ERM phosphorylation in time- and dose- dependent manners.AGE-RAGE interaction plays a central role in mediating the change of ERM phosphorylation induced by AGEs.Rho kinase and ERK and p38 signal pathways are involved in the signaling mechanisms in AGE-induced ERM phosphorylation in HUVECs.2.AGEs-HSA could induce VE-cadherin morphological change in time- and dose- dependent manners.AGE-RAGE interaction plays a central role in mediating the morphological change of VE-cadherin induced by AGEs.Rho kinase and MAPK are involved in the signaling mechanisms in AGE-induced in HUVECs. |
