The endothelial cell tube formation assay on basement membrane turns 20_ state of the science

Angiogenesis(2009)12:267–274DOI10.1007/s10456-009-9146-4

REVIEWPAPER

Theendothelialcelltubeformationassayonbasementmembraneturns20:stateofthescienceandtheart

IrinaArnaoutovaÆJayGeorgeÆ

HyndaK.KleinmanÆGabrielBenton

Received:3January2009/Accepted:11April2009/Publishedonline:28April2009ÓSpringerScience+BusinessMediaB.V.2009

AbstractIthasbeenmorethan20yearssinceitwas rstdemonstratedthatendothelialcellswillrapidlyformcap-illary-likestructuresinvitrowhenplatedontopofareconstitutedbasementmembraneextracellularmatrix(BME,Matrigel,EHSmatrix,etc.).Subsequently,thismorphologicaldifferentiationhasbeendemonstratedwithavarietyofendothelialcells;withendothelialprogenitorcells;andwithtransformed/immortalizedendothelialcells.Thedifferentiationprocessinvolvesseveralstepsinbloodvesselformation,includingcelladhesion,migration,alignment,proteasesecretion,andtubuleformation.Becausetheformationofvesselstructuresisrapidandquanti able,endothelialcelldifferentiationonbasementmembranehasfoundnumerousapplicationsinassays.Suchdifferentiationhasbeenused(1)tostudyangiogenicandantiangiogenicfactors,(2)tode nemechanismsandpathwaysinvolvedinangiogenesis,and(3)tode neendothelialcellpopulations.Further,theendothelialcelldifferentiationassayhasbeensuccessfullyusedtostudyprocessesrangingfromwoundrepairandreproductiontodevelopmentandtumorgrowth.Theassayiseasytoper-formandisthemostwidelyusedinvitroangiogenesisassay.

KeywordsAngiogenesisÁBasementmembraneÁEndothelialcelltubesÁMorphologicaldifferentiationÁQuantitativeassayÁHighthroughputscreen

EndothelialcelltubeformationonbasementmembraneInvivo,endothelialcellsareincontactontheirbasal(nonluminal)surfacewithathin,highlyspecializedextracel-lularmatrix:thebasementmembrane.Thismatrixformsacontinuoussleevearoundtheendothelialcells,andmain-tainsthetube-likestructuresofthebloodvessels[1,2].Thisbasementmembranematrixwasdemonstratedtobebiologicallyfunctionalmorethan20yearsago,afterKubotaetal.[3]platedendothelialcellsonareconstitutedbasementmembranematrixandfoundthattheyrapidlyattached,aligned,andformedcapillary-liketubules.Thecellsdonotproliferate.Theprocessisrapid(within2–3hforimmortalizedendothelialcellsand6–20hforprimaryendothelialcells).Thevesselsthatareformedcontainalumenandtightcell–cellcontacts.Thecellsarepolarizedwiththenucleibasallylocatedtowardsthebasementmembranematrix.Furthermore,thecapillary-likestruc-turestakeupacetylatedLDL,whichisamarkerofdif-ferentiationforthesecells.SuchLDLuptakeisnotobservedwhenthesecellsareculturedinmonolayeroneitherplasticorcollagenIsubstrates.

Theformationofthecapillary-liketubesisspeci ctoendothelialcells;othercelltypesformotherstructures[4].Forexample,mammaryepithelialcellsandsalivaryglandcellsformsphereswithacentrallumen,whilemetastatictumorcellsmigrateandinvadethematrix.Varioustypesofendothelialcellsthathavebeenderivedfromdifferenttissues,fromtransformedendothelialcells,andfromendothelialprogenitorcells,formtheinterconnectingcapillary-likestructureswhenplatedonbasementmem-brane[5–9].Inthisway,endothelialcelldifferentiationonbasementmembranerecapitulatesmanystepsinangio-genesisandisahighlyspeci cprocess.Thebasementmembranematrix,whichcontainsmanycriticalfunctional

I.ArnaoutovaÁJ.GeorgeÁH.K.KleinmanÁG.BentonTrevigenInc.,Gaithersburg,MD,USA

H.K.Kleinman(&)

NIH,NIDCR,30ConventDrMSC4370,Building30,Room400,Bethesda,MD20892,USAe-mail:hkleinman@dir.nidcr.nih.gov

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andstructuralcomponents,isthekeytothisdifferentiationprocess.

Basementmembranestructureandfunctionalcomponents

Althoughthebasementmembraneinvarioustissuesappearssimilarwhenexaminedthroughtheelectronmicroscope,itsmolecularcompositionisdifferentineachtissue[1].Theuniquenessofeachtissue-speci cbasementmembraneislikelyimportantinprovidingdistinctfunc-tionalitytoeachtissue.Thematrixcontainsmanyproteinsthatvaryinbothamountandtype,dependingonthetissueoforiginandfunctionofthevessel(Table1).Themajorcomponentsofthevascularbasementmembranearelam-inins8and10,collagenIV,andnidogens/entactins1and2.Manyisoformsoftheseandothermoleculesarealsofoundindifferentbasementmembranematricesandfurthercontributetotheircomplexity.Forexample,therearesixisoformsofcollagenIVchainsresultingin56possiblecombinationsoftrimers.CollagensVIII,XV,andXVIII,heparansulfateproteoglycans,growthfactors(FGF,TGF,PDGF,andVEGF),matrixmetalloproteinaseproenzymes(MMPs),BM40/SPARC/osteonectin, bulins1and2,thrombospondins1and2,and bronectin,arealsoreportedinbasementmembranematrices[10–12].Whenthebase-mentmembraneisdegradedbytraumaortissuedamage,manyoftheaforementionedcomponentsaredegradedexposingcrypticsitesorgeneratingactivefragmentsthatpromoteorinhibitangiogenesis.Inthisway,speci cpathologiesmakeadditionalregulatoryfactorsavailable.DegradationofthebasementmembranecollagensIVandXVIII,forexample,leadstothegenerationofarrestin,endostatin,canstatin,andtumstatin,whichinhibitangio-genesis.Thereleaseofcertainstoredgrowthfactors,onthe

otherhand,wouldbeexpectedtoincreaseangiogenesis[1,10–12].Thebasementmembraneisthereforestructur-allydynamic,uniqueforeachtissue,andfunctionallyspeci c.

Thecomplexityanddynamicnatureoftheendothelialbasementmembranecontributetoitsvariousfunctions(Table2).CollagenIVcontributestothebasementmem-brane’sstructuralintegrityandpromotescelladhesionandmigration[1].FragmentsofcollagenIVareantiangio-genic.Heparansulfateproteoglycanslinkcollagenandlamininnetworks,bindsolublecomponents,suchasgrowthfactors,andregulatethe ltrationactivityofthebasementmembranematrix[13].Micewithdeletionofperlecanhavebleedingproblems,suggestingthatperlecanplaysapartinmatrixstability.Thelamininsareconsideredthemajorbiologicallyactivecomponentsofthebasementmembrane[14,http://wendang.chazidian.commininsorganizeandestablishthebasementmembranematrixandpromoteendothelialcelladhesion,migration,anddifferentiation.Someoftheactivesitesinlamininshavebeenidenti edwithfragmentsandwithsyntheticpeptides[16].Fibulinsbindtomanymatrixmoleculestopromotebasementmembranestability,adhesion,andmigration[17].Micede cientin bulin-1developbleedinginvarioustissues,whichsuggeststhatthisproteinalsoregulatesbasementmembranestability.

Table2BasementmembranefunctionsintheendotheliumAnchorendothelialcells:tissueintegrityMaintainendothelialcelldifferentiationMaintaintightcell–cellcontactsFiltrationofwastesandnutrientsBarriertocellinvasion

Separatedifferenttissuetypes(endotheliumfromstroma)Storagedepotforgrowthfactorsandproenzymes

Transductionofmechanosensingsignalsfromlumentovesselwall

Table1ComponentsofendothelialcellbasementmembraneComponent

CollagensIV,VIII,XV,XVIIIEntactins/nidogens1and2HeparansulfateproteoglycansLaminins8and10

Growthfactors(FGF,TGFbeta,VEGF)Fibronectin

Fibulins1and2Thrombospondins1and2BM40/SPARC/osteonectin

Matrixmetalloproteinaseproenzymes

Function

Stability,adhesion,migrationLinker,adhesion

Filtration,growthfactorbinding,matrixstabilityAdhesion,migration,proteasesGrowthandmigration

Adhesion,migration

Matrixstability,adhesion,migration

Inhibitsangiogenesis,proliferation,andmigration,andinhibitsTGFbetaCellrounding,migration,inhibitsangiogenesisDegradation

Thesecomponentsaredifferentiallyexpressedandtheamountsvarydependingontheendothelialcelltype,developmentalstage,orpathologicalsituation

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Angiogenesis(2009)12:267–274BoththrombospondinandBM40/SPARC/osteonectinareantiangiogenicbydifferentandcomplexmechanisms[18,19].Thrombospondinsinhibitendothelialcellproliferationandmigration.Thrombospondinsbindtomanymoleculesinthebasementmembraneandactiveantiangiogenicfragmentshavebeenidenti ed.BM40/SPARC/osteonectinpromotesendothelialcellroundingandmigration,andactiveproangiogenicfragmentshavebeenidenti ed.Thus,thecomponentsofthebasementmembranehavemultiplefunctionsthatarebothstructuralandbiological.

Cellsaretightlyanchoredtothismatrixviacellsurfacereceptors,includingintegrins,syndecans,anddystrogly-can.Multipleinteractionswithvariousmatrixcomponentsresultinatightadhesiontothematrix,whichmaintainstissueintegrityandpreventscelllossfromblood owpressure.Thebasementmembranematrixservesasabarriertocellsandmolecules.Itformsastructuralbarrierbetweendifferenttissuesandit lterswastesandnutrients.Thematrixisalsoimportantinmechanosensingsignalsfromthelumentothevesselwall.Theendothelialbase-mentmembranebindsnumerousgrowthfactors,whicharestoredinthematrixprimarilyviatheirinteractionswiththeproteoglycancarbohydrateregions[4].Themultifunctionalbasementmembranealsoplaysanimportantpartinangiogenesis.

Sourcesofbasementmembranematrix

Basementmembranematrixiscommerciallyavailableorcanbepreparedfromtissuesortumors[20].Themostwidelyusedform,asolubleextractfromtheEHStumor,ishighlyenrichedinlaminin-1,collagenIV,heparansulfateproteoglycan,entactin/nidogen,andvariousgrowthfactors.Althoughitdoesnotcontainallofthesignaturecompo-nentsofanendothelialbasementmembrane,thismatrixisactivewithallendothelialcellstestedtodateforthepro-motionoftubeformationinvitro.Basementmembranematrixcanalsobepreparedwithreducedlevelsofgrowthfactorsbyselectiveprecipitation[21,22].Basementmembraneextractcanbeobtainedfromseveralcommer-cialsources,includingBDBiosciences(USA),TrevigenInc.(USA),AMSBiotechnologyLtd(Europe),StratechScienti cLtd(UK),Gentaur(Europe),AmlogDiagnostic(Israel),KormedCorp.(Korea),Sanbio(Europe),Chin-aGenInc.(China),andFunakoshiCo.,Ltd(Japan).

Endothelialcellcapillary-likeformationonbasementmembranehaswideuseasanassay

Becauseendothelialcelltubeformationonbasementmembranereplicatesmanyofthestepsinangiogenesis,it

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hasbeenwidelyusedasascreenforangiogenicandanti-angiogenicfactors[23–25].Indeed,mostoftheknownangiogenicfactorsareactiveinthisassay.Itshouldbenotedthatfactorscanbetestedbyexogenousadditionorbytransfectiontoupordownregulatethefactorlevel,ifitisaprotein.Onecanalsotestendothelialcellsisolatedfromgeneticallymodi edanimalsandfrompatientswithgeneticdiseases[9].Asa rstscreenassay,ithasmanyadvantages.Itisrapid,quantitative,andcanbedoneinhighthroughputmodetoscreenlargenumbersofchemi-cals.Moreover,itencompassesallstepsintheangiogenicprocess:adhesion,migration,proteaseactivity,alignment,andtubeformation.Sincetheassaymeasuresmultiplesteps,ithasmanyadvantagesoverotherinvitroassays,suchascellattachment,proliferation,migration,andinvasion,whichmeasurefewerstepsintheangiogenicprocess.Endothelialcellmigrationorinvasionassaysareusefulbutmoredif culttoperform;and,fora rstscreen,itispossiblethattheywouldnotmeasuretheactivityofafactorthatmightregulateonlytubeformation[26,27].Otherinvitrotubeformingassays,suchastubeformationoncollagenIgels,arenotinwideusebecausetheyrequirelongertimeperiods,theircellsdonotallformtubes(makingquantitationdif cult),andthetubesmightnotbeproperlypolarized(insideouttubules)[23,28].Similarly,capillary-liketubeswillforminhigh-densityendothelialcellculturesbut,sincetheyformontopofthecellmonolayer,thetubestakeatleastaweektoformandaredif culttoquantitate[29].Theendothelialcelltubefor-mationonbasementmembraneassayisgenerallynotusedalonetode nefactors.Rather,itisusedasa rstscreenthatisfollowedbyadditionalinvitroassaysandexvivoorinvivoassays[23].Manyresearchlaboratoriesusethetubeformingassayasa rstscreen,largelybecauseitsparescostlyanimaltestingandinvestigatortime.

Theformationofcapillary-liketubesinvitroonbase-mentmembraneisquick,simpletoperform,andwidelyused(Figs.1,2)[30,31].Therearesomeimportantcon-ditionsthatwillensureoptimalresults.Thenumberofcellsplatediscritical.Toofewcellsyieldincompletetubes;toomanyyieldlargeareasofmonolayers(Fig.3).Theopti-mumcellnumberis*4,800cells/cm2(15,000cells/wellona96-wellplate).Ifprimarycellsareused,suchashumanumbilicalveinendothelialcells(HUVECs),thecellsshouldbeearlypassage(passage2–6)andallcellstestedshouldhavebeenpassagedatleasttwiceafterbeingremovedfromliquidnitrogentoobtaintheoptimumtubeformation(Fig.4).Thecellsshouldbeat*80%con u-encebeforeharvestingfortheassay.Inaddition,theamountofbasementmembranematrixappliedtothedishisalsoimportant:toolittlematerialresultsinpoortubeformation(Fig.5).However,toomuchbasementmem-branesubstrateisnotaproblemfortheassayresults

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Fig.1Stepsintheassayofendothelialcelltubeformationonbasementmembrane

matrix

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Fig.2Humanumbilicalveinendothelialcell(HUVEC)tubeformationonabasementmembranesubstratewithtime.Cellswereseededat4,800cells/cm2

(Fig.5).Basementmembranecanbepreparedwithreducedgrowthfactors.Thismaterialisadvantageousfortestingfactorsthatpromoteangiogenesisbecausetubeformationintheabsenceofaddedangiogenicfactorsisgreatlyreducedovercompletebasementmembranematrix.Itisfareasiertoobservethelargerincreaseintubefor-mationwithangiogenicfactorsonthegrowthfactor-reducedbasementmembranematrix(Fig.6).Althoughtheconditionsnecessaryforobtainingtheoptimumresultsmayseemcomplex,theassayisinfacteasytoperformand

reliable.Itspopularityoverthepasttwodecadessupportsthisassertion.

Tubeformationinvitrotostudythegenesandsignalingeventsinearlyangiogenesis

Theformationofendothelialtubulesonbasementmem-braneallowsinvestigatorsnotonlytostudyfactorsthatregulatethisprocess,butalsotode negenesand

signaling

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Fig.3Effectofcellnumberonendothelialcelltubeformationonbasementmembranematrix.DataareshownforHUVECsat16

h

Fig.4Effectofendothelialcellpassagenumberafterthawingontubeformation.DataareshownforHUVECsat20h

pathwaysthatcannotbestudiedeasilyinvivo.Inthisway,itservesasamodelforthemoleculardissectionoftubeformation.Studieswithcyclohexamideshowthatproteinsynthesisisrequiredfortubeformationonbasementmembrane[32].Ithasalsobeenshownthatcollagensyn-thesisinhibitors(cis-hydroxyproline,D609,GPA1734,

and

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