高级病理生理学课件 高级病生-血管新生与疾病 -孟丹

1ANGIOGENESISDanMeng,MDDepartmentofPhysiologyandPathophysiology,ShanghaiMedicalCollegeFudanUniversity

2Contacttome上海市东安路130号西7号楼212室

复旦大学上海医学院生理与病理生理学系

Tel/p>

Email:dmeng@

DanMeng,M.D.,AssociateProfessorDepartment

of

Physiology

&

Pathophysiology

Fudan

University

Room212,West7thBuilding,

130

Dongan

Road

Shanghai

200032,

China.

Tel:

86-21-54237392

Email:dmeng@

Contacttome3Outline1-Angiogenesisandvasculogenesis2-Importantfactors,receptorsandsignalingpathways3-Tumorangiogenesis

4-Angiogenesis-relateddiseases5-Anti-angiogenesistherapiesandtherapeuticAngiogenesis6-Angiogenesisassaymodel4AngiogenesisandvasculogenesisAngiogenesisSproutingorNon-sproutingofcellsfrommatureendothelialcellsofthevesselwallVasculogenesisFormationofbloodvesselsbydifferentiationfrom(hem)angioblasts5Angiogenesisandvasculogenesis61Ferrara.NatRevCancer2002

2Folkman.NEJM19711990sAngiogenesisfirstdescribedbyDrJohnHunter1787SeveralGermanpathologistsobservethatsomehumantumoursarehighlyvascularised,leadingtothesuggestionthatthenewvesselshaveanimportantpathogenicroleinthecancerprocess11800Landmarkpublication:JudahFolkmanproposestumourgrowthisangiogenesis-dependent21971DiscoverybyNapoleoneFerraraandcolleaguesofpossiblythemostimportantangiogenicfactor,VEGF1989Keylandmarksinunderstandingangiogenesis7StructureofvesselsandcapillariesSmallartery:Capillary:

endothelialcell,basallamina,pericytes8Angiogenesis:Sproutingangiogenesis9Angiogenesis:SproutingangiogenesisMousecornea:woundinginducesangiogenesis,chemotacticresponsetoangiogenicfactors10SmoothMuscleCellsEndotheliumBasementMembraneProteasesAngiogenicStimulus(VEGF)Angiogenesis-BasementMembraneBreakdown11EndotheliumNascentVascularSproutsVEGFSmoothMuscleCellsBasementMembraneAngiogenesis-EndothelialCellMigration12EndotheliumVEGFSproutElongationSmoothMuscleCellsBasementMembraneAngiogenesis-EndothelialCellProliferation13EndotheliumVEGFNewLumenFormationSmoothMuscleCellsBasementMembraneAngiogenesis-CapillaryMorphogenesis14EndotheliumVEGFSMC,pericyterecruitmentSmoothMuscleCellsBasementMembraneAngiogenesis-VascularMaturation15EndotheliumEndothelialcell-celljunctionsSmoothMuscleCellsBasementMembraneAngiogenesis-VascularMaturation16Non-SproutingAngiogenesis(Intussuseptiveangiogenesis)17NormalAngiogenesisinChildren18NormalAngiogenesisinAdultsAngiogenesisintissueduringwoundhealingAngiogenesisinuterinelining19Capillariessproutingin

theretinaofanembryonicmouse20Capillarylumenopeningupbehind

thetipcell(reddyeinjected)2122NormalmacularmaculardegenerationAngiogenesisinretina23Outline1-Angiogenesisandvasculogenesis2-Importantfactors,receptorsandsignalingpathways3-Tumorangiogenesis

4-Angiogenesis-relateddiseases5-Anti-angiogenesistherapiesandtherapeuticAngiogenesis6-Angiogenesisassaymodel24AngiogenesisisatightlyregulatedbalanceHanahan&Folkman.Cell1996=Pro-angiogenicfactors(e.g.VEGF,bFGF,angiopoietin)=Anti-angiogenicfactors(e.g.angiostatin,endostatin,thrombospondin-1)OnOffTheangiogenicswitchVEGF=vascularendothelialgrowthfactorbFGF=basicfibroblastgrowthfactor

25Angiogenesis:thekeyroleofVEGF26EGFHypoxiaPDGFIL-8bFGFCOX-2NitricoxideOncogenesVEGFreleaseBindingandactivationofVEGFRIGF-1ProliferationSurvivalMigrationANGIOGENESISPermeabilityIncreasedexpression(MMP,tPA,uPA,uPAr,eNOS,etc.)VEGFisakeyregulatorofangiogenesisFerrara,etal.EndocrRev1997Kerbel,etal.NatRevCancer2002COX=cyclooxygenase;EGF=epidermalgrowthfactor;

IGF=insulin-likegrowthfactor;

PDGF=platelet-derivedgrowthfactor;

VEGFR=vascularendothelialgrowthfactorreceptor27Biochem.Soc.Trans.(2003)31,1171-1177VEGF:VEGF-A:initiationofvasculogenesisandsproutingangiogenesis,Immaturevessels,Vascularpermeabilityfactor

PLGF:remodelingofadultvesselsVEGF-B:heartvascularizationVEGF-C:lymphaticvesselsVEGF-D:lymphaticvesselsVEGFR:(TKR)VEGFR-2:growthandpermeabilityVEGFR-1:negativeroleVEGFR-3:lymphaticvesselsVEGF/VEGFRfamilyPLGF:placentagrowthfactorNRP:neurophilin28VEGFsignalinginangiogenicECs29Sproutingtowardschemotacticgradient:VEGF30TheroleofVEGFintumorangiogensisVEGFstimulatesgrowthofendothelialcellsunderinvitroconditions.VEGFenhancesvascularpermeability.Itcontributestotumorcellpenetrationintovascularnetworksandmetastasis.VEGFstimulatestheproductionofplasmiongenactivator(tPA,uPA)andinterstitialcollagenaseinEC,thuspromotesthedegradationofmatrix.31SemenzaGLidentifiesaDNA-bindingactivityinducedbyhypoxiain1992NamethenuclearfactorasHypoxicinduciblefactor-I(HIF-1)MolCellBiol1992;12:5447-54I:inducibleC:constitutiveIdentificationofHIF-132Aheterodimericmemberofbasichelix-loop-helix(bHLH)family,containingPASdomainComposeHIF-1αandHIF-1βPersistentexpressionofHIF-1β,identicaltoarylhydrocarbonreceptor(AHR)nucleartranslocator(ARNT)BindingtoDNAsequence5’-(A/G)CGTG-3’TrendsCardiovascmed1996;6:151-7HIF-133HIF-1bindspecificallytoVEGFsequencesthatmediatedhypoxiainducibletranscriptionTransfectionofHIF-1aplasmiddramaticallyincreaseVEGFtranscriptioninnon-hypoxicandhypoxiccellExpressionofadominantnegativeformofHIF-1ablockreportergenebearingVEGFenhancerinhypoxiccellTrendsCardiovascmed1996;6:151-7HIF-1andVEGF34JCO2005;23:4776-89O2Fe(II)HIF-1αisdestroyedbyubiquitylationbyVHL35CCR2003;9:4641-52RASRafMEKERKHIF-1signalpathway36InHIF(+)xerograft,tumorbleedprofuselyandseverelyHIF(-)xerograft’svasculatureismoreuniform,withfewerlargevesselandmoreavascularzoneNature1998;394:485-90HIF-1andtumorangiogenesis37FGF-2andPDGFinangiogenesis38AngiopoietinsandTieReceptorsAng1:RemodelingandmaturationSupportsinteractionwithothercellsandmatrix,Vesselsize,RepairofdamagedvesselsAng2:NaturalAng1antagonist,OverexpressionsimilarAng-1k.o.Ang3:?Ang4:?Tie2:bindsAng1-4Tie1:?39Angiopoitin-1signalingpathway40InmammalstherearefourNotchreceptors,Notch1,Notch2,Notch3andNotch4.TherearefiveligandsnamedJagged1,Jagged2,Delta-like(Dll)1,Dll3andDll4.TheNotchfamilyiscriticallyimportantfortheproperconstructionofthevascularsystem.GlobalknockoutsofNotch1aloneorNotch1/4togetherareembryoniclethalwithseverevasculardefects.NotchligandshavecometorepresentanalternateorcomplementarytargettoVEGF,basedonthepreclinicalstudiescarriedoutwithDll4inhibitorsandtheNotch1decoy.Notchsignalingregulatesangiogenesis41M.Slevin,TherapeuticAngiogenesisforVascularDiseases,2011,Pg.85RoleofVEGFandDII4/Notchintumorangiogenesis42Wntsaresecretedcysteine-richglycoproteins,theseproteinsbindtofrizzled(Fz)receptors.Atleast19Wnthomologsand10Fzhomologsareexpressedinmammals.IntheabsenceofaWntsignal,cytosolic-cateninisphosphorylatedbyaproteincomplexcontainingglycogensynthasekinase-3(GSK3),Axinandadenomatouspolyposiscoli(APC).Thisphosphorylated-cateninisrapidlyubiquitinatedanddegraded.BindingofcertainWntstoFzproteins,resultsinactivationofdishevelled(Dsh).DshinhibitstheGSK3-containingphosphorylationcomplex,therebypromotingaccumulationofcytosolic-catenin,whichthentranslocatestothenucleus,bindstotranscriptionfactorsoftheT-cellfactor(TCF),andactivatesthetranscriptionofnumerousgenesimplicatedinproliferation,migration,differentiationandapoptosis.Wntsignalingpathway43CanonicalWNTsignalingTargetgenesMYCCCND1

JUNTCFLEFPPARDuparMMP7AXIN2NrCAMTF2GatrinCD44METephrinBBMP4ClaudinVEGFID2Twiststromelysin

44Wnt-2-nullmicehaveabnormalplacentalvasculaturewithareducednumberoffetal-derivedplacentalcapillaries.MicedeficientforFz-5displayvascularabnormalitiesandareembryoniclethal.-cateninaccumulationhasbeencorrelatedwithangiogenesisincancer.-cateninisfoundinthecytoplasmandnucleusofendotheliuminneovesselsofgliomas.Thevesselsofthenormaladultbrain,incontrast,donotaccumulate-catenininthecytoplasmornucleus.Wntsignalingpathwayandangiogenesis45

WT-/-Absenceofintra-retinalcapillariesinFz4(-/-)mice46Outline1-Angiogenesisandvasculogenesis2-Importantfactors,receptorsandsignalingpathways

3-Tumorangiogenesis

4-Angiogenesis-relateddiseases5-Anti-angiogenesistherapiesandtherapeuticAngiogenesis6-Angiogenesisassaymodel47Angiogenesis:essentialforthegrowthandmalignantprogressionoftumoursStagesatwhichangiogenesisplaysarolein

tumourprogressionPremalignanttumourMalignanttumourTumour

growthVascular

invasionMicro-metastasesMetastaticgrowthAngiogenic

switch481-Sprouting3-incorporationofBM-derivedprecursors4-LymphangiogenesisGrowthoftumorvessels2-Intussusceptivegrowth49Figure13.32aTheBiologyofCancer(©GarlandScience2007)Recruitmentofcapillariesbyanimplantedtumor50Figure13.34aTheBiologyofCancer(©GarlandScience2007)Chaoticorganizationoftumor-associatedvasculature51Mosaicvessels52Abnormalendothelium53Figure13.33TheBiologyofCancer(©GarlandScience2007)TumorvesselisonlypartiallyoverlaidbypericytesandSMC54Figure13.37TheBiologyofCancer(©GarlandScience2007)Themodelofislettumorcellprogression55Figure13.38bTheBiologyofCancer(©GarlandScience2007)Theangiogenicswitchandrecruitmentofinflammatorycells56InjectedcancercellsIsolatedorgan

(e.g.,thyroidgland)InfusenutrientsolutionWithoutAngiogenesis,TumorGrowthStopsstopgrowingasmassreaches

1–2mmindiameter57TumorsuspendedinanteriorchamberWithAngiogenesis,TumorGrowthProceedsIrisLensCorneaTumorgrowingontheiris？TumorsizeDaysTumorgrowingontheirisTumorsuspendedinanteriorchamber246

8

1058AngiogenesisChamberCancercellSignalingmoleculePlacechamberbeneathananimal'sskinWhatPromptsAngiogenesis?59Inject

cancercellsAngiostatininjectionsNotreatmentAllowtimeformetastasestoappearManymetastasesFewmetastasesRemove

initialtumorLetinitialtumorgrowfor

severalweeksAngiogenesisInhibitorsandMetastasis60NormalmouseNocancerCancerInjectbreastcancercellsAngiogenesis-deficientmutantmouseCancerinAngiogenesis-DeficientMice61WithoutAngiogenesis,TumorGrowthStopsSummary62Outline1-Angiogenesisandvasculogenesis2-Importantfactors,receptorsandsignalingpathways

3-Tumorangiogenesis

4-Angiogenesis-relateddiseases5-Anti-angiogenesistherapiesandtherapeuticAngiogenesis6-Angiogenesisassaymodel63Angiogenesis-relateddiseasesExcessCancerAtherosclerosisHemangiomasRheumatoidarthritisAge-relatedmaculardegenerationDeficiency:Myocardialischemia

LimbischemiaCerebralischemia64

ATHEROSCLEROSIS:Pathology,Pathogenesis,Complications,NaturalHistory65FibrousPlaquesComplicatedLesions66HemorrhageintoPlaque67Ulceration/Hemorrhage/CholesterolCrystals68Atherosclerosisandangiogenesis

69Mechanismsofcoordinatedangiogenesisandinflammationintheprogressiveenlargementofthenecroticcore70Myocardialischemiaandangiogenesis71Rosenzweig,A.NEnglJMed2003ThreeStagesintheMovementofEndothelialProgenitorCellsfromBoneMarrowtoVascularEndothelium72737475

Hedmanetal,Circulation2003SafetyandFeasibilityofLocalIntracoronaryVEGFGeneTransfer

inthePreventionofPostangioplastyandintheTreatmentofChronicMyocardialIschemia76Improvementinmyocardialperfusion(bloodsupply)afterFGF-1treatmentImprovementinmyocardialperfusion(bloodsupply)afterFGF-1treatmentasdemonstratedbySPECTimaging(singlephotonemissioncomputedtomography).77Limbischemiaandangiogenesis78Cerebralischemia79Outline1-Angiogenesisandvasculogenesis2-Importantfactors,receptorsandsignalingpathways

3-Tumorangiogenesis

4-Angiogenesis-relateddiseases5-Anti-angiogenesistherapiesandtherapeuticAngiogenesis6-Angiogenesisassaymodel80TherapeuticAngiogenesisIsitsafe?Willithaveanimpactonclinicalpractice?81TherapeuticAngiogenesis:

PotentialRisksGenomicincorporationInflammatoryreactionUnwantedvascularproliferationGrowth/spreadoftumorsParadoxicalworseningofIschemicHeartDisease8283TherapeuticAngiogenesisIsitsafe?Willithaveanimpactonclinicalpractice?84(Wollertetal,Circulation2005)Thepreliminaryclinicalevidencesuggeststhatstemcelltherapymightwork.However,weshouldtakeintoaccountthelessonslearnedfromtherapeuticangiogenesisstudies,wheregreatexpectationsraisedbyopenstudieshavenotbeenconfirmedbysubsequentrandomizedtrialsWeadvocatetonolongerperformsmallopenstudies,buttoconductintermediate-size,double-blind,randomized-controlledclinicaltrialstoestablishtheeffectsofstemcelltherapyonsurrogatemarkers,likeLVEF,myocardialperfusion,orexercisecapacityWillithaveanimpactonclinicalpractice?85Anti-angiogenesistherapies86MatrixCancercellVEGF(orbFGF)AngiogenesisInhibitorsMMPsReceptorproteinEndothelialcellAngiogenesisInhibitorsintheTreatmentofHumanCancer87IntegrinCombretastatinA4ApoptosisEndostatinEMD121974TNP-470SqualamineCancercellVEGF(orbFGF)MMPsReceptorproteinMatrixEndothelialcellIntegrininteractswithdrugstodestroyproliferatingendothelialcellsDrugmoleculeDrugsThatInhibitAngiogenesisDirectly88MatrixCancercellVEGF(orbFGF)MMPsReceptorproteinEndothelialcellInterferon-alphaAnti-VEGF

antibody

SU5416

SU6668

PTK787/ZK22584Noendothelial

cellgrowthDrugsThatBlocktheAngiogenesisSignalingCascade89Marimistat

AG3340

COL-3

Neovastat

BMS-275291MatrixCancercellVEGF(orbFGF)MMPsReceptorproteinEndothelialcellNoendothelial

cellmigrationDrugsThatBlockExtracellularMatrixBreakdown90DrugsWithOtherMechanismsofAction91OntoClinicalTrials92VEGFVEGFR-2Anti-VEGFantibodies

(e.g.Avastin)Antibodiesinhibiting

VEGFreceptorsSolubleVEGFreceptors(e.g.VEGF-TRAP)SmallmoleculeVEGFRinhibitors(TKIs)(e.g.sunitinib,sorafenib,vatalanib,vandetanib)Ribozymes(e.g.Angiozyme)Migration,permeability,DNAsynthesis,survivalLymphangiogenesisAngiogenesisTKI=tyrosinekinaseinhibitorInhibitionofVEGF:activedevelopmentprogrammeforangiogenesisinhibitors93AvastininhibitsangiogenesisthroughanovelmechanismofactionpreventsthebindingofVEGFtoitsreceptorsistheonlyanti-VEGFagentcurrentlyinclinicaluseforthetreatmentofadvancedNSCLCAvastinVEGFXGrowthProliferationMigrationSurvivalXAvastinisthefirstandonlyanti-angiogenicthatdirectlyinhibitsVEGF94EGFRErbB1HER1HER2ErbB2

neuHER3ErbB3HER4ErbB4Ligand-bindingdomainsTransmembraneTyrosine-kinasedomainsEGFTGF-αAmphiregulinBetacellulinHB-EGFEpiregulinHeregulinsHeregulinsBetacellulinSalomon,etal.Hematol1995Woodburn.PharmacolTher1999HER=humanepidermalgrowthfactorreceptor;

TGF=transforminggrowthfactorEGFRpathwayismediatedbyafamilyofligandsandmembranereceptors951Cohen.JBiolChem19622Cohen,etal.JBiolChem19803Ullrich,etal.Nature19841990sIdentificationofEGFinmicebyDrStanleyCohen1(NobelPrizein1986)1962Isolationofhuman

EGFRbyDrStanleyCohen21980HumanEGFR

geneclonedandsequencedbyDrAxelUllrichandcolleagues31984↑evidenceforEGFR

roleincancertumorigenesisLate1980sKeylandmarksinunderstandingtheEGFRpathwayanditsroleintumourdevelopment96Woodburn.PharmacolTher1999;Lynch,etal.NEJM2004

Knowlden,etal.Endocrinol2003;Chakravarti,etal.CancerRes2002Proliferation,invasion,metastasis,angiogenesisand

inhibitionofapoptosis Src PLCg GAP Grb2 Shc Nck Vav Grb7 CrkPKCRasJNKAblPI3K AktExtracellularIntracellularTransactivationMAPKEGFRpathwayiscomplexbutwellunderstood97Inhibitionoftyrosinekinasephosphorylation

hasarangeofanti-tumoureffectsTarceva,Iressa¯Angiogenesis¯Metastasis¯Adhesion¯InvasionSensitivitytoothertherapies(e.g.chemotherapy)¯Proliferation­ApoptosisEtessami&Bourhis.DrugsFuture2000Moyer,etal.CancerRes1997

Harari&Huang.SeminRadiatOncol2002=inhibition98VEGFandEGFRrepresentrational