高级病生6次课动脉硬化讲座硕士班课件

高级病生第6次课动脉硬化讲座硕士班高级病生第6次课动脉硬化讲座硕士班1“Chronicinflammatorydisorderofintimaoflargebloodvesselscharacterisedbyformationoffibrofattyplaquescalledatheroma”.Hardeningofarteries-ArteriosclerosisArteriosclerosis“Chronicinflammatorydisorder2MacrophagesT-lymphocytesAtheromatousplaqueLumenFibrouscapEndothelialcellsMacrophagesAtheromatousLumenF3AtherosclerosisLipidmetabolismimbalanceInflammationImmuno-responseAtherosclerosisLipidmetabolis4LipidmetabolismimbalanceLipidmetabolismimbalance5ClassificationoflipidsandlipoproteinsClassificationoflipidsandl6CharacteristicsoflipoproteinsCharacteristicsoflipoprotein7DigestionandmetabolismofdietaryfatDigestionandmetabolismofdi8HDLmetabolismandreversecholesteroltransportHDLmetabolismandreversecho9Cholesteroleffluxandreversecholesterol

transportismodulatedbytworeceptorsCholesteroleffluxandreverse10PPARαactivatorsinducecholesterolefflux

andreversecholesteroltransportPPARαactivatorsinducecholes11HDLmetabolism:

5keygenes

HDLmetabolism:

5keygenes12PPARα:apoA-l,apoA-ll,LPL,

ABCA-1andSR-BIexpressionPPARα:apoA-l,apoA-ll,LPL,13PPARaactivatorsinducecholesterolefflux

fromhumanmacrophagesPPARaactivatorsinducecholes14CLA-1/SR-BIproteinmaypromotecholesterol

removalfromperipheralcellsCLA-1/SR-BIproteinmaypromot15CLA-1expressionisregulatedbyPPARαactivators

indifferentiatedhumanmacrophagesCLA-1expressionisregulated16PPARαactivatorsinducecholesterolefflux

andreversecholesteroltransportPPARαactivatorsinducecholes17LDLLDL18Theearlylesion:modifiedlipoproteinsandfoamcellformationTheearlylesion:modifiedlip19ClassesofScavengerReceptorsPluddemannetal.Methods43(2007)207-217ClassesofScavengerReceptors20TheScavengerReceptorFamiliesformodifiedLDLCollagen-likeSRCRSR-AMARCOClassACD36SR-B1immunodominantClassBEGF-likeSR-ECLOX-1Lectin-likeSR-PSOXChemokine-domainMucin-stalkTheScavengerReceptorFamilie21高级病生6次课动脉硬化讲座硕士班课件22ThemacrophageasaninflammatorymediatorThemacrophageasaninflammat23StatinsinducePPARαactivityStatinsinducePPARαactivity24MacrophagesT-lymphocytesAtheromatousplaqueLumenFibrouscapEndothelialcellsMacrophagesAtheromatousLumenF25InflammationInflammation26RoleofmacrophagesMCSFinducesmonocytesenteringlesionto differentiateintomacrophagesMacrophagedifferentiationassociatedwithTLRs andscavengerreceptorsMCSF/apoEKOshowsreducedatherosclerosisScavengerreceptorsbindbacterialendotoxins, apoptoticcellsandoxLDLSR/apoEKOsshowreducedatherosclerosisRoleofmacrophagesMCSFindu27Toll-likeGENESIL-1RTNF-RIFN-gRIRFNFkBMAP

KINASEJAK/STATInflammation,Anti-MicrobialResponse,Apoptosis,…Toll-likeGENESIL-1RTNF-RIFN-gR28TLRreceptorsandatherosclerosis10familymembersRecognizepathogenassociatedmolecularpatterns (e.g.LPS,dsRNA)aswellasoxLDL,HSP60etc.Initiatesignalingcascadesleadingtoproductionof inflammatorycytokines,proteases,reactiveoxygen speciesInadditiontomacrophages,expressedbydendritic cells,mastcells,endothelialcellsTLRreceptorsandatherosclero292004Science3031481-1482InflammatorycytokinesToll-likeReceptors(TLRs)2004Science3031481-1482Infl30nLDLmmLDLoxLDL15-LO,Fe2+Cu2+Contd.ox.?CD14?TLR4CD36InflammationFoamcellsAirox.nLDLmmLDLoxLDL15-LO,Fe2+Cu2+C31PAFRGPCRsCD36SR-ATLRsCRPNaturalIgMsBindinganduptakeCytokinesCo-stimulatorymoleculesMDALPCPAF-likelipidsoxLDLOxidizedphospholipidsExposedPCheadgroupsLipidmediatorsComplementproteinsChemokinesInflammatorycellrecruitmentComplementactivationAdaptiveimmuneresponseFoamcellformationPAFRGPCRsCD36SR-ATLRsCRPNatura32TLRsCytokinesCo-stimulatorymoleculesAdaptiveimmuneresponseMyD88AdjuvantsAutoimmuneligandsLipidligandsTregoxLDLCD1LipidantigensAtheroprotectiveimmunity?TLRsCytokinesCo-stimulatorymo33mmLDLPCMCMyD88TLR4LPSoxPLTLR4[61]mmLDL[35,36]AdhesionmoleculesCD14/TLR4[63,64]MyD88Inflammatorycytokines[60]CD36TLR2/TLR6oxLDLApoptoticcellsBacterialdiacylatedlipopeptidesandlipoteichoicacidTLR2[66][65]TRIFIRF-3Cholesterolefflux[74]TLR1/TLR2EndogenousligandBacterialtriacylatedlipopeptide[26][26]”Infectiousburden”ExogenousligandsTLR4TLR4[23,24]LPSoxPLImmunologicalpriming[86]TLR4Co-stimulatorymolecules[51][20]MacrophagefoamcellECMonocyterecuitmentInflammatorycytokinesLipiduptakeBjörkbacka(2006)CurrOpinLipidol17:527-53mmLDLPCMCMyD88TLR4LPSoxPLTLR4[34InnateimmunityN-fMetpeptidesChemokinesLipidmediatorsMyD88BacterialandviralPAMPsGPCRsTLRsAntibodyopsonizationoxLDLIncreasedintegrinavidityCytoskeletalchangesRespiratoryburstPhagocytosisAdhesiontoendotheliumMigrationintotissuesKillingofmicrobeROIsCellularresponseFunctionaloutcomeNaturalIgMsCRP,SAPBacterialphosphoryl-cholineComplementactivationComplementopsonizationScRsMannosereceptorFcRCRsMicrobialglyco-proteins/lipids(mannose)AntigenpresentationCytokinesandchemokinesreleasedC5a,C3a,C4aSignalingInnateimmunityN-fMetpeptides35Immuno-responseImmuno-response36ReducedatherosclerosisinMyD88nullmiceBjörkbackaetal.(2004)Nat.Med.10:416-421ReducedatherosclerosisinMyD37TLRsandatherosclerosisCD14TLR4MD2MyD88TRAMTRIFMal/TirapTLR1TLR2TLR6TLR2TLR4MyD88MyD88Björkbackaetal.Nat.Med.(2004)10:416-421Michelsenetal.PNAS(2004)101:10679-10684Mullicketal.JCI(2005)115:3149-3156CD36Hoebeetal(2005)Nature433:523-527TLRsandatherosclerosisCD14TL38高级病生6次课动脉硬化讲座硕士班课件39高级病生6次课动脉硬化讲座硕士班课件40高级病生6次课动脉硬化讲座硕士班课件41WhydoesoxidizedLDLbecomeimmunogenic?FragmentationofapoB-100OxidationofphospholipidsFormationofcovalentaldehyde(MDA)andaminoacidadductsWhydoesoxidizedLDLbecomei42高级病生6次课动脉硬化讲座硕士班课件43HowDoesImmunizationWithOxidized

LDLAffectAtherosclerosis?Reduceddevelopmentofatherosclerosisinrabbits(PalinskietalPNAS1995,AmelietalATVB1996)ReduceddevelopmentofatherosclerosisinapoEdeficientmice(GeorgeetalAtherosclerosis1998,FreigangetalATVB1998,ZhouetalATVB2001)HowDoesImmunizationWithOxi44Isitpossibletoproduceavaccineagainstatherosclerosis?WhatstructuresinoxidizedLDLarerecognizedbytheimmunesystem?Aretheseimmuneresponsesassociatedwithcardiovasculardiseaseinhumans?Canprotectiveimmuneresponsesbeselectivelyactivatedbyavaccine?Canantibodiesbeuseddirectlytotreatatherosclerosis?Isitpossibletoproduceava45CharacterizationofepitopesinoxidizedLDLbyalibraryofpeptideELISAs

302peptides,correspondingtothecompleteaminoacidsequenceofApoB-100Eachpeptideconsistsof20aminoacidswith5aminoacidsoverlapModificationbymalondialdehyde(MDA)Characterizationofepitopesi46ActiveimmunizationofapoEnullmicewithapoB-100peptidesequencesImmunizationwith100mgpeptidesat6,9and11weeksofageusingAlumasadjuvantHighcholesteroldietat10weeksofageAtherosclerosisassessedbyOilRedOstainingofthedescendingaortaat25weeksofageActiveimmunizationofapoEn47EffectofoxidizedLDLpeptidevaccinesonatherosclerosisinmice%atherosclerosisP<0.01P<0.00500,050,10,150,20,250,3ControlPeptide45Peptide210Peptide240FredriksonGNetalEffectofoxidizedLDLpeptide48EffectofimmunizationwithMDA-apoB-100peptide45onspecificIgM,IgG,IgG1(Th2)andIgG2a(Th1)inapoEnullmice*p<0.05,***p<0.005012CarrierIgM

IgGIgM

IgGFragment

I***FredriksonGNetal,Autoimmunity2005.EffectofimmunizationwithMD49ImmunizationusingMDA-apoB-100peptidesinmiceReductionofatherosclerosisbyupto70%RemainingplaquescontainmorecollagenandlessmacrophagesIncreaseinIgGbutnotinIgMSwitchfromTh1toTh2-specificIgGNosignificanteffectsonplasmalipidsImmunizationusingMDA-apoB-150PassiveimmunizationstrategyHumanIgG1againstpeptides45and210wereproducedthroughscreeningofasinglechainantibodyfragmentlibraryandsubsequentcloningintoapcDNA3vector

ApoEknockoutmiceweregivenhighcholesteroldietfrom6weeksofageTreatedwith0.5mgantibodyat21,22and23weeksofageandsacrificedtwoweekslaterEffectsassessedbyOROstainingofdescendingaorta,TrichromeMassonstainingandmacrophageimmunostainingofsubvalvularplaquesPassiveimmunizationstrategyH51EffectofpassiveimmunizationwithrecombinanthumanIgGagainstpeptide45onearlyatherosclerosisinmiceSchiopuetal,Circulation2004Effectofpassiveimmunization52RegressionofaorticplaqueareainantibodytreatedApobec-1-/-/LDLr-/-miceSchiopuAetal.,JACC2007Regressionofaorticplaquear53RecombinanthumanantibodiestoMDA-apoB-100peptidesupregulateABCA-1expressioninvivoandinvitroSchiopuAetal.,JACC2007Recombinanthumanantibodiest54RecombinanthumanantibodiestoMDA-apoB-100peptidesblockoxidizedLDL-inducedmonocyteMCP-1productioninvitroSchiopuAetal.,JACC2007Recombinanthumanantibodiest55ConclusionsTheidentificationoftheimmunogenicstructuresinoxidizedLDLhasmadeitpossibletodevelopnoveltherapeuticapproachesfortreatmentofatherosclerosis.Studiesperformedinatherosclerosis-pronemicedemonstratethatbothpeptide-basedvaccinesandrecombinantIgGtargetingepitopesinoxidizedLDLsignificantlyreduceatherosclerosis.ConclusionsTheidentification56AtherosclerosisLipidmetabolismimbalanceInflammationImmuno-responseAtherosclerosisLipidmetabolis57Thankyou!!!Thankyou!!!58高级病生第6次课动脉硬化讲座硕士班高级病生第6次课动脉硬化讲座硕士班59“Chronicinflammatorydisorderofintimaoflargebloodvesselscharacterisedbyformationoffibrofattyplaquescalledatheroma”.Hardeningofarteries-ArteriosclerosisArteriosclerosis“Chronicinflammatorydisorder60MacrophagesT-lymphocytesAtheromatousplaqueLumenFibrouscapEndothelialcellsMacrophagesAtheromatousLumenF61AtherosclerosisLipidmetabolismimbalanceInflammationImmuno-responseAtherosclerosisLipidmetabolis62LipidmetabolismimbalanceLipidmetabolismimbalance63ClassificationoflipidsandlipoproteinsClassificationoflipidsandl64CharacteristicsoflipoproteinsCharacteristicsoflipoprotein65DigestionandmetabolismofdietaryfatDigestionandmetabolismofdi66HDLmetabolismandreversecholesteroltransportHDLmetabolismandreversecho67Cholesteroleffluxandreversecholesterol

transportismodulatedbytworeceptorsCholesteroleffluxandreverse68PPARαactivatorsinducecholesterolefflux

andreversecholesteroltransportPPARαactivatorsinducecholes69HDLmetabolism:

5keygenes

HDLmetabolism:

5keygenes70PPARα:apoA-l,apoA-ll,LPL,

ABCA-1andSR-BIexpressionPPARα:apoA-l,apoA-ll,LPL,71PPARaactivatorsinducecholesterolefflux

fromhumanmacrophagesPPARaactivatorsinducecholes72CLA-1/SR-BIproteinmaypromotecholesterol

removalfromperipheralcellsCLA-1/SR-BIproteinmaypromot73CLA-1expressionisregulatedbyPPARαactivators

indifferentiatedhumanmacrophagesCLA-1expressionisregulated74PPARαactivatorsinducecholesterolefflux

andreversecholesteroltransportPPARαactivatorsinducecholes75LDLLDL76Theearlylesion:modifiedlipoproteinsandfoamcellformationTheearlylesion:modifiedlip77ClassesofScavengerReceptorsPluddemannetal.Methods43(2007)207-217ClassesofScavengerReceptors78TheScavengerReceptorFamiliesformodifiedLDLCollagen-likeSRCRSR-AMARCOClassACD36SR-B1immunodominantClassBEGF-likeSR-ECLOX-1Lectin-likeSR-PSOXChemokine-domainMucin-stalkTheScavengerReceptorFamilie79高级病生6次课动脉硬化讲座硕士班课件80ThemacrophageasaninflammatorymediatorThemacrophageasaninflammat81StatinsinducePPARαactivityStatinsinducePPARαactivity82MacrophagesT-lymphocytesAtheromatousplaqueLumenFibrouscapEndothelialcellsMacrophagesAtheromatousLumenF83InflammationInflammation84RoleofmacrophagesMCSFinducesmonocytesenteringlesionto differentiateintomacrophagesMacrophagedifferentiationassociatedwithTLRs andscavengerreceptorsMCSF/apoEKOshowsreducedatherosclerosisScavengerreceptorsbindbacterialendotoxins, apoptoticcellsandoxLDLSR/apoEKOsshowreducedatherosclerosisRoleofmacrophagesMCSFindu85Toll-likeGENESIL-1RTNF-RIFN-gRIRFNFkBMAP

KINASEJAK/STATInflammation,Anti-MicrobialResponse,Apoptosis,…Toll-likeGENESIL-1RTNF-RIFN-gR86TLRreceptorsandatherosclerosis10familymembersRecognizepathogenassociatedmolecularpatterns (e.g.LPS,dsRNA)aswellasoxLDL,HSP60etc.Initiatesignalingcascadesleadingtoproductionof inflammatorycytokines,proteases,reactiveoxygen speciesInadditiontomacrophages,expressedbydendritic cells,mastcells,endothelialcellsTLRreceptorsandatherosclero872004Science3031481-1482InflammatorycytokinesToll-likeReceptors(TLRs)2004Science3031481-1482Infl88nLDLmmLDLoxLDL15-LO,Fe2+Cu2+Contd.ox.?CD14?TLR4CD36InflammationFoamcellsAirox.nLDLmmLDLoxLDL15-LO,Fe2+Cu2+C89PAFRGPCRsCD36SR-ATLRsCRPNaturalIgMsBindinganduptakeCytokinesCo-stimulatorymoleculesMDALPCPAF-likelipidsoxLDLOxidizedphospholipidsExposedPCheadgroupsLipidmediatorsComplementproteinsChemokinesInflammatorycellrecruitmentComplementactivationAdaptiveimmuneresponseFoamcellformationPAFRGPCRsCD36SR-ATLRsCRPNatura90TLRsCytokinesCo-stimulatorymoleculesAdaptiveimmuneresponseMyD88AdjuvantsAutoimmuneligandsLipidligandsTregoxLDLCD1LipidantigensAtheroprotectiveimmunity?TLRsCytokinesCo-stimulatorymo91mmLDLPCMCMyD88TLR4LPSoxPLTLR4[61]mmLDL[35,36]AdhesionmoleculesCD14/TLR4[63,64]MyD88Inflammatorycytokines[60]CD36TLR2/TLR6oxLDLApoptoticcellsBacterialdiacylatedlipopeptidesandlipoteichoicacidTLR2[66][65]TRIFIRF-3Cholesterolefflux[74]TLR1/TLR2EndogenousligandBacterialtriacylatedlipopeptide[26][26]”Infectiousburden”ExogenousligandsTLR4TLR4[23,24]LPSoxPLImmunologicalpriming[86]TLR4Co-stimulatorymolecules[51][20]MacrophagefoamcellECMonocyterecuitmentInflammatorycytokinesLipiduptakeBjörkbacka(2006)CurrOpinLipidol17:527-53mmLDLPCMCMyD88TLR4LPSoxPLTLR4[92InnateimmunityN-fMetpeptidesChemokinesLipidmediatorsMyD88BacterialandviralPAMPsGPCRsTLRsAntibodyopsonizationoxLDLIncreasedintegrinavidityCytoskeletalchangesRespiratoryburstPhagocytosisAdhesiontoendotheliumMigrationintotissuesKillingofmicrobeROIsCellularresponseFunctionaloutcomeNaturalIgMsCRP,SAPBacterialphosphoryl-cholineComplementactivationComplementopsonizationScRsMannosereceptorFcRCRsMicrobialglyco-proteins/lipids(mannose)AntigenpresentationCytokinesandchemokinesreleasedC5a,C3a,C4aSignalingInnateimmunityN-fMetpeptides93Immuno-responseImmuno-response94ReducedatherosclerosisinMyD88nullmiceBjörkbackaetal.(2004)Nat.Med.10:416-421ReducedatherosclerosisinMyD95TLRsandatherosclerosisCD14TLR4MD2MyD88TRAMTRIFMal/TirapTLR1TLR2TLR6TLR2TLR4MyD88MyD88Björkbackaetal.Nat.Med.(2004)10:416-421Michelsenetal.PNAS(2004)101:10679-10684Mullicketal.JCI(2005)115:3149-3156CD36Hoebeetal(2005)Nature433:523-527TLRsandatherosclerosisCD14TL96高级病生6次课动脉硬化讲座硕士班课件97高级病生6次课动脉硬化讲座硕士班课件98高级病生6次课动脉硬化讲座硕士班课件99WhydoesoxidizedLDLbecomeimmunogenic?FragmentationofapoB-100OxidationofphospholipidsFormationofcovalentaldehyde(MDA)andaminoacidadductsWhydoesoxidizedLDLbecomei100高级病生6次课动脉硬化讲座硕士班课件101HowDoesImmunizationWithOxidized

LDLAffectAtherosclerosis?Reduceddevelopmentofatherosclerosisinrabbits(PalinskietalPNAS1995,AmelietalATVB1996)ReduceddevelopmentofatherosclerosisinapoEdeficientmice(GeorgeetalAtherosclerosis1998,FreigangetalATVB1998,ZhouetalATVB2001)HowDoesImmunizationWithOxi102Isitpossibletoproduceavaccineagainstatherosclerosis?WhatstructuresinoxidizedLDLarerecognizedbytheimmunesystem?Aretheseimmuneresponsesassociatedwithcardiovasculardiseaseinhumans?Canprotectiveimmuneresponsesbeselectivelyactivatedbyavaccine?Canantibodiesbeuseddirectlytotreatatherosclerosis?Isitpossibletoproduceava103CharacterizationofepitopesinoxidizedLDLbyalibraryofpeptideELISAs

302peptides,correspondingtothecompleteaminoacidsequenceofApoB-100Eachpeptideconsistsof20aminoacidswith5aminoacidsoverlapModificationbymalondialdehyde(MDA)Characterizationofepitopesi104ActiveimmunizationofapoEnullmicewithapoB-100peptidesequencesImmunizationwith100mgpeptidesat6,9and11weeksofageusingAlumasadjuvantHighcholesteroldietat10weeksofageAtherosclerosisassessedbyOilRedOstainingofthedescendingaortaat25weeksofageActiveimmunizationofapoEn105EffectofoxidizedLDLpeptidevaccinesonatherosclerosisinmice%atherosclerosisP<0.01P<0.00500,050,10,150,20,250,3ControlPeptide45Peptide210Peptide240FredriksonGNetalEffectofoxidizedLDLpeptide106EffectofimmunizationwithMDA-apoB-100peptide45onspecificIgM,IgG,IgG1(Th2)andIgG2a(Th1)inapoEnullmice*p<0.05,***p<0.005012CarrierIgM

IgGIgM

IgGFragment

I***FredriksonGNetal,Autoimmunity2005.EffectofimmunizationwithMD107ImmunizationusingMDA-