NOX4基因缺失對內皮功能紊亂和動脈粥樣硬化的影響

NADPHoxidase4protectsagainstdevelopmentofendothelialdysfunctionandatherosclerosisinLDLreceptordeficientmiceEuropeanHeartJournalNovember17,2015

IntroductionEndothelialdysfunctionisanearlyeventinatherosclerosisthatprecedesclinicalsymptoms.TheroleofdifferentNoxisoformsincardiovascularphysiologyandpathophysiologyiscontroversial.IncreasedformationofsuperoxideanionsbyNox1,2,and5reducesNOavailabilityandcanpromoteendothelialdysfunction.recentevidencesupportsavasoprotectiveroleofH2O2producedbyNox4.Therefore,weanalysedtheimpactofgeneticdeletionofNox4onendothelialdysfunctionandatherosclerosisintheLdlr-/-model.Results1.Nox4-/-Ldlr-/-micedevelopendothelialdysfunction.

2.Nox4-/-Ldlr-/-miceonhigh-fatdietdevelopincreasedatherosclerosis.3.LossofNox4-derivedH2O2couldbepartiallycompensatedforbynNOSupregulation4.SaphenousarteryofNox4-/-miceshowimpairedflow-mediateddilationinvivo.1.Nox4-/-Ldlr-/-micedevelopendothelialdysfunction(A)Concentration–responsecurveforAchinaorticsegmentsof10-week-oldwild-type,Ldlr-/-,Nox4-/-,andNox4-/-/Ldlr-/-mice(n≥10)precontractedwithphenylephrine.(B)Maximaleffectof30mmol/LAChonaorticsegmentsofwild-type,Ldlr-/-,Nox4-/-,andNox4-/-/Ldlr-/-mice(n≥10).(C)Concentration–responsecurvesforsodiumnitroprussideinaorticsegmentsbetweenthestrainsofmice.1.Nox4-/-Ldlr-/-micedevelopendothelialdysfunctionConcentration–responsecurveforacetylcholineinaorticsegmentsof10-week-oldwild-typeandLdlr-/-micewithcatalase.1.Nox4-/-Ldlr-/-micedevelopendothelialdysfunctionConcentration–responsecurveforacetylcholineinaorticsegmentsof10-week-oldwild-typeandLdlr-/-micewithpaxilline.1.Nox4-/-Ldlr-/-micedevelopendothelialdysfunction(H)AmplexredassayforH2O2generationofAortathoracalissegmentsofwild-type,

Ldlr-/-,Nox4-/-,andNox4-/-/Ldlr-/-

.H2O2formationwasmeasuredasthecatalase-sensitivepartofthesignal(a.u.:arbitraryunits).(IandJ)Real-timepolymerasechainreactionofmurineaortafromwild-type,Ldlr-/-,Nox4-/-,andNox4-/-/Ldlr-/-

mice.1.Nox4-/-Ldlr-/-micedevelopendothelialdysfunction(K)WesternblotanalysisofpAKT1inhumanumbilicalveinendothelialcellstransducedwithshControlorshNOX4for48h.Cellswereeitherkeptunderstaticconditionsorexposedtolaminarshearstress(30dyn/cm2)for5min.(L)Real-timepolymerasechainreactionofNOX4inhumanumbilicalveinendothelialcells.Cellsweretransducedfor72hwithshControlorshNOX4.2.Nox4-/-/Ldlr-/-miceonhigh-fatdietdevelopincreasedatherosclerosis

(A)Atheroscleroticplaqueburden:PlaqueareaintheaorticarchofNox4-/-/Ldlr-/-micewassignificantlyhigher.(B)Galectin-3stainingofaorticarchsectionsofLdlr--andNox4--/Ldlr-/-mice.(C)CollagencontentbySiriusRedstainingwassignificantlyincreasedinNox4-/-/Ldlr-/-mice3.CompensationofH2O2releaseinolderNox4-/-micedecreasedaorticH2O2(Aortathoracalis)levelsinyoungNox-/-micewererestoredtolevelsofwild-typemiceat26weeksofage.

aroleofnNOSaspotentialsourceofH2O2.3.CompensationofH2O2releaseinolderNox4-/-mice

nNOSmRNAexpressionwassignificantlyincreasedinaortasof26-week-oldbutnot10-week-oldNox4-/-mice3.CompensationofH2O2releaseinolderNox4-/-miceE：Aorticendothelialfunctionof26-week-oldNox4-/-wasnotaltered.F：However,NOavailabilityseemedtobedecreasedinaorticsegmentsofNox42/2mice.ThesemiceshowedatrendtodecreasedL-NAME-inducedendothelium-dependentconstriction.AlthoughexvivoanalysisofvascularfunctionintheaortaofNox4-/-miceshowednodifferenceinendothelium-dependentrelaxationcomparedwithwild-typemice,FMDinsaphenousarteryintheNox4-/-micewassignificantlyreduced.Thissupportsamuchhighersensitivityoftheinvivomethodtoobjectifyalterationsinvesselfunction.4.SaphenousarteryofNox4-/-miceshowimpairedFMDinvivoNox42/2miceshowalteredFMDinvivo.(A)OCTofArteriasaphenaandevaluationofimagesof26-week-old(A)wild-type,(B)Ldlr-/-,and(C)Nox4-/-miceunderrestingcondition,FMDandafterapplicationofSNP.significantincreaseinvesseldiameterduetoFMDinWTmice,whilenosignificantincreaseinvesseldiameterafterclampreleasecouldbedetectedineitherNox4-/-orLdlr-/-mice.SNPcausedsimilarincreasesofvesseldiameter.4.SaphenousarteryofNox4-/-miceshow

impairedflow-mediateddilationinvivoOCTscanoftheArteriasaphenaof26-week-oldWT,Ldlr-/-,andNox4-/-miceshowingtheprocessofFMDduring10minafterocclusion.Discussion1.Nox4→H2O2→pAKT→eNOS→NO↑→vasodilation2.H2O2mightactasanEDHF(內皮超極化因子)inmiceandman.H2O2（EDHF）→BKCa↑→hyperpolarizedmembrane→vasodilation3.InflammationmarkerGalectin-3andcollagencontentwasalsoincreasedinNox4-/-/Ldlr-/-comparedwithLdlr2/2mice.4.olderNox4-/-mice：代償性nNOS↑→H2O2↑5.amuchhighersensitivityoftheinvivomethodtoobjectifyalterationsinvesselfunction.ConclusionsNox4playsanimportantroleinmaintainingendothe