AGEs/RAGE Mediate Wnt/beta-catenin Signaling To Promote Human Aortic Smooth Muscle Cells Calcification In Diabetes Mellitus

Abstract： Objective：1.To investigate whether the RAGE has the functionto promote arterial smooth muscle cells calcification；2.To find out therelationship between RAGE and wnt/β-catenin signaling pathway；3.To studywhether the RAGE contribute to the course of smooth muscle cell phenotypictransform into osteoblast.Methods：（1）We took the lentiviral transfectiontechnique to establish human VSMC model which can over express RAGEsteadily.And the results were test by green fluorescent protein，flow cytometry，and western blotting.(2)We divided this group into blank controlgroup,non-transfection group， empty vector transfection group andRAGE-lentiviral transfection group.Cells of blank control group werecultivated in a normol condition.Cells of non-transfection group，empty vectortransfection group and RAGE-lentiviral transfection group were cultivated ina condition of20mmg/L AGEs，10mmol/L sodium β-glycerophosphate andpyruvic acid.The calcification of human aortic smooth muscle cells wasobserved under inverted phase contrast microscope.10days later，we dealt thecells with von kossa staining and quantitative analysis of calcium.(3)Wedivided this group into blank control group,empty vector transfection group andRAGE overexpression group.Cells of blank control group were cultivated in anormol condition.Cells of non-transfection group，empty vector transfection group and RAGE-lentiviral transfection group were cultivated in a condition of20mmg/L AGEs，10mmol/L sodium β-glycerophosphate and pyruvicacid.After that we detected the factors associated with VSMC calcificationby western blotting.(4)In order to inhibit the expression of β-catenin, β-cateninsiRNA was used.This part of our experiment was divided into blank controlgroup,RAGE overexpression group,siRNA control group and β-catenin siRNAinhibiting group.Cells of blank control group were cultivated in a normolcondition.Cells dealt with β-catenin siRNA were cultivated in a condition of20mmg/L AGEs，10mmol/L sodium β-glycerophosphate and pyruvic acid.Theexpression of OPG and cabfa1was test by western blotting.Results:The testresults of green fluorescent protein，flow cytometry，and western blotting provethat the human VSMC model which can over express RAGE was establishedsuccessfully.(2)Compared with blank control group,non-transfection group andempty vector transfection control group,the calcification of cells inRAGE-lentiviral transfection group was enhanced when training withAGEs,sodium β-glycerophosphate and pyruvic acid.(3)When training withAGEs,sodium β-glycerophosphate and pyruvic acid,the expression of RAGE、β-catenin、cabfa1、OPG in blank control group,empty vector transfectioncontrol group and RAGE overexpression group was enhanced.In addition,theexpression of those factors in RAGE overexpression group was much strongerthan that in other groups.(4)Compared with RAGE overexpression group andsiRNA control group, the expression of downstream gene cabfa1and OPG inβ-catenin siRNA inhibiting group was obvious suppressed.Conclusion:1.Theoverexpression of RAGE can promote VSMC calcification,when enhance theexpression ofβ-catenin、OPG、cabfa1.2.RAGE may active the downstreamsignal factors through wnt/β-catenin signal path,then accelerate thetransformation of smooth muscle cell phenotypic into osteoblast,and finallyenhance the calcification.3.The course of RAGE enhancing the expression ofOPG and cabfa1,can be restrained by β-catenin siRNA.