1、高级病生第6次课动脉硬化讲座硕士班“Chronic inflammatory disorder of intima of large blood vessels characterised by formation of fibrofatty plaques called atheroma”.Hardening of arteries-ArteriosclerosisArteriosclerosisMacrophagesT-lymphocytesAtheromatous plaqueLumenFibrous capEndothelial cellsAtherosclerosisLipid me
2、tabolism imbalanceInflammationImmuno-response Lipid metabolism imbalanceClassification of lipids and lipoproteinsCharacteristics of lipoproteinsDigestion and metabolism of dietary fatHDL metabolism and reverse cholesterol transportCholesterol efflux and reverse cholesteroltransport is modulated by t
3、wo receptorsPPAR activators induce cholesterol effluxand reverse cholesterol transportHDL metabolism:5 key genes PPAR:apo A-l,apo A-ll,LPL,ABCA-1 and SR-BI expressionPPARa activators induce cholesterol effluxfrom human macrophagesCLA-1/SR-BI protein may promote cholesterolremoval from peripheral cel
4、lsCLA-1 expression is regulated by PPAR activatorsin differentiated human macrophagesPPAR activators induce cholesterol effluxand reverse cholesterol transportLDLThe early lesion:modified lipoproteins and foam cell formationClasses of Scavenger ReceptorsPluddemann et al.Methods 43(2007)207-217 The S
5、cavenger Receptor Familiesfor modified LDLCollagen-like SRCRSR-AMARCOClass ACD36SR-B1immunodominantClass BEGF-likeSR-ECLOX-1Lectin-likeSR-PSOXChemokine-domainMucin-stalkThe macrophage as an inflammatory mediatorStatins induce PPAR activityMacrophagesT-lymphocytesAtheromatous plaqueLumenFibrous capEn
6、dothelial cells InflammationRole of macrophages MCSF induces monocytes entering lesion to differentiate into macrophages Macrophage differentiation associated with TLRs and scavenger receptors MCSF/apoE KO shows reduced atherosclerosis Scavenger receptors bind bacterial endotoxins,apoptotic cells an
7、d oxLDL SR/apoE KOs show reduced atherosclerosisToll-likeGENESIL-1RTNF-RIFN-g gRIRFNFk kBMAPKINASEJAK/STATInflammation,Anti-Microbial Response,Apoptosis,TLR receptors and atherosclerosis 10 family members Recognize pathogen associated molecular patterns(e.g.LPS,dsRNA)as well as oxLDL,HSP60 etc.Initi
8、ate signaling cascades leading to production of inflammatory cytokines,proteases,reactive oxygen species In addition to macrophages,expressed by dendritic cells,mast cells,endothelial cells2004 Science 303 1481-1482Inflammatory cytokinesToll-like Receptors(TLRs)nLDLmmLDLoxLDL15-LO,Fe2+Cu2+Contd.ox.?
9、CD14?TLR4CD36InflammationFoam cellsAir ox.PAFRGPCRsCD36SR-ATLRsCRPNatural IgMsBinding and uptakeCytokinesCo-stimulatory moleculesMDALPCPAF-like lipidsoxLDLOxidized phospholipidsExposed PC head groupsLipid mediatorsComplement proteinsChemokinesInflammatory cell recruitmentComplement activationAdaptiv
10、e immune responseFoam cell formationTLRsCytokinesCo-stimulatory moleculesAdaptive immune responseMyD88AdjuvantsAutoimmune ligandsLipid ligandsTregoxLDLCD1Lipid antigensAtheroprotective immunity?mmLDLPCMCMyD88TLR4LPSoxPLTLR461mmLDL35,36Adhesion moleculesCD14/TLR463,64MyD88Inflammatory cytokines60CD36
11、TLR2/TLR6oxLDLApoptotic cellsBacterial diacylated lipopeptides and lipoteichoic acidTLR26665TRIFIRF-3Cholesterol efflux74TLR1/TLR2Endogenous ligandBacterial triacylated lipopeptide2626”Infectious burden”Exogenous ligandsTLR4TLR423,24LPSoxPLImmunological priming86TLR4Co-stimulatory molecules5120Macro
12、phage foam cellECMonocyte recuitmentInflammatory cytokinesLipid uptakeBjrkbacka(2006)Curr Opin Lipidol 17:527-53Innate immunityN-fMet peptidesChemokinesLipid mediatorsMyD88Bacterial and viral PAMPsGPCRsTLRsAntibody opsonizationoxLDLIncreased integrin avidityCytoskeletal changesRespiratory burstPhago
13、cytosisAdhesion to endotheliumMigration into tissuesKilling of microbeROIsCellular responseFunctional outcomeNatural IgMsCRP,SAPBacterial phosphoryl-cholineComplement activationComplement opsonizationScRsMannose receptorFcRCRsMicrobial glyco-proteins/lipids(mannose)Antigen presentationCytokines and
14、chemokines releasedC5a,C3a,C4aSignaling Immuno-responseReduced atherosclerosis in MyD88 null miceBjrkbacka et al.(2004)Nat.Med.10:416-421TLRs and atherosclerosisCD14TLR4MD2MyD88TRAMTRIFMal/TirapTLR1TLR2TLR6TLR2TLR4MyD88MyD88Bjrkbacka et al.Nat.Med.(2004)10:416-421Michelsen et al.PNAS(2004)101:10679-
15、10684Mullick et al.JCI(2005)115:3149-3156CD36Hoebe et al(2005)Nature 433:523-527Why does oxidized LDL become immunogenic?Fragmentation of apo B-100 Oxidation of phospholipids Formation of covalent aldehyde(MDA)and amino acid adductsHow Does Immunization With Oxidized LDL Affect Atherosclerosis?Reduc
16、ed development of atherosclerosis in rabbits (Palinski et al PNAS 1995,Ameli et al ATVB 1996)Reduced development of atherosclerosis in apo E deficient mice (George et al Atherosclerosis 1998,Freigang et al ATVB 1998,Zhou et al ATVB 2001)Is it possible to produce a vaccine against atherosclerosis?Wha
17、t structures in oxidized LDL are recognized by the immune system?Are these immune responses associated with cardiovascular disease in humans?Can protective immune responses be selectively activated by a vaccine?Can antibodies be used directly to treat atherosclerosis?Characterization of epitopes in
18、oxidized LDL by a library of peptide ELISAs 302 peptides,corresponding to the complete amino acid sequence of Apo B-100 Each peptide consists of 20 amino acids with 5 amino acids overlap Modification by malondialdehyde(MDA)Active immunization of apo E null mice with apo B-100 peptide sequences Immun
19、ization with 100 mg peptides at 6,9 and 11 weeks of age using Alum as adjuvant High cholesterol diet at 10 weeks of age Atherosclerosis assessed by Oil Red O staining of the descending aorta at 25 weeks of ageEffect of oxidized LDL peptide vaccines on atherosclerosis in mice%atherosclerosisP0.01P0.0
20、0500,050,10,150,20,250,3ControlPeptide 45Peptide 210Peptide 240Fredrikson GN et alEffect of immunization with MDA-apoB-100 peptide 45 on specific IgM,IgG,IgG1(Th2)and IgG2a(Th1)in apo E null mice*p0.05,*p0.005012IgG1 IgG2aIgG1 IgG2aCarrierFragment I*012CarrierIgM IgGIgM IgGFragment I*Fredrikson GN e
21、t al,Autoimmunity 2005.Immunization using MDA-apo B-100 peptides in mice Reduction of atherosclerosis by up to 70%Remaining plaques contain more collagen and less macrophages Increase in IgG but not in IgM Switch from Th1 to Th2-specific IgG No significant effects on plasma lipidsPassive immunizatio
22、n strategy Human IgG1 against peptides 45 and 210 were produced through screening of a single chain antibody fragment library and subsequent cloning into a pcDNA3 vector Apo E knockout mice were given high cholesterol diet from 6 weeks of age Treated with 0.5 mg antibody at 21,22 and 23 weeks of age
23、 and sacrificed two weeks later Effects assessed by ORO staining of descending aorta,Trichrome Masson staining and macrophage immunostaining of subvalvular plaques Effect of passive immunization with recombinant human IgG against peptide 45 on early atherosclerosis in miceSchiopu et al,Circulation 2
24、004Regression of aortic plaque area in antibody treated Apobec-1-/-/LDLr-/-miceIEI-E3(P=0.004)2D03 (P=0.001)20 weeksBaseline25 weeksControl 29wFITC-8024681012192021222324252627282930WeeksOil Red O stained area(%)IEI-E3(P=0.004)2D03 (P=0.001)20 weeksBaseline25 weeksControl 29wFITC-8024681012192021222
25、324252627282930WeeksOil Red O stained area(%)Schiopu A et al.,JACC 2007Recombinant human antibodies to MDA-apoB-100 peptides upregulate ABCA-1 expression in vivo and in vitroSchiopu A et al.,JACC 2007Recombinant human antibodies to MDA-apoB-100 peptides block oxidized LDL-induced monocyte MCP-1 prod
26、uction in vitroSchiopu A et al.,JACC 2007Conclusions The identification of the immunogenic structures in oxidized LDL has made it possible to develop novel therapeutic approaches for treatment of atherosclerosis.Studies performed in atherosclerosis-prone mice demonstrate that both peptide-based vaccines and recombinant IgG targeting epitopes in oxidized LDL significantly reduce atherosclerosis.AtherosclerosisLipid metabolism imbalanceInflammationImmuno-responseThank you!
