Cytokine induced angiogenesis in chick embryos.

Severalgrowthfactorsareinvolvedinthedevelopmentofthebloodsysteminbothdevelopingchickembryosandtheiryolksacs(Gilbert,2003).Theprocessofbloodformation,knownashematopoiesis,isdividedintotwocategories:theembryonicstageandadultstage.Thisexperimentinvestigatestheembryonicstageofhematopoiesis,duringwhichtimebloodvesselsarecreatedandcirculationbegins(Gilbert,2003).Theconstructionofbloodcellsoccursthroughtwoprocesses,thefirstofwhichisvasculogenesis.Duringvasculogenes,"bloodislands"appearintheyolksacoftheembryoandformcapillarynetworks(Gilbert,2003).Priortotheactualformationofthese"bloodislands,"specifiedmesodermcellsmustfirstformhemangioblasts,theprecursorsofbloodcellsandbloodvessels(Gilbert,2001and2003).Hemangioblastsgiverisetobothangioblastsorhematopoieticstemcells,whichformendothelialcellsandbloodcells,respectively(Gilbert,2003).Thesecellsthencondensetoformbloodislands,theinnermostcellsofwhicharecomposedofhematopoieticstemcells,andtheoutermostcellsofangioblasts(whichwilleventiallylinetheinsideofthebloodvessels)(Gilbert,2000and2003).Theendothelialcellswillformtubesandconnecttocreateanetworkofcapillariesknownastheprimarycapillaryplexus(Gilbert,2000).Thesecondstageofbloodformationinchickembryos,knownasangiogensis,ismarkedbythetheformationofamorecomplexvesselsystem.Thisisaccomplishedbya"remodeling"ofestablishedvesselstocompletethecirculatorysystem(Gilbert,2000and2003).Threeparacrinegrowthfactors(alsoknownascytokines)areinvolvedintheprocessofvasculogeneis:basicfibroblastgrowthfactor(bFGF),vascularendothelialgrowthfactor(VEGF),andangiopoietin-1(Ang1)(Gilbert,2000and2003).Overall,thesefactorsareconcentratedbytheextracellularmatrixofthemesenchymalcellsatthesitesofhematopoiesis,andtheycontributetobloodcellandlymphocyteformation.Onasmallerscale,thefirstgrowthfactor,bFGF,isresponisbleforboththespecificationofmesodermcellstoformhemangioblasts,andthevascularizationofthechorioallantoicmembrane(CAM)tissue(Gilbert,2000and2003).TheCAMmembraneislocatedbeneaththeshellmembrane,andisformedbythefusingofthechickallantoicmembranewiththemesodermallayerofthechorion(Gilbert,2000and2003).Itisthismembranethatabsorbscalciumfromtheeggshell,neededfortheformationoftheskeletonofthechickembryo(Gilbert,2003).Thesecondgrowthfactor,VEGF,issecretedbythemesenchymalcellsandinstigatestheformationofbloodislandsintobloodvessels.Lastly,Ang1mediatestheinteractionbetweentheendothelialcellsandthesmoothmusclecellsthatcoverthevessels(Gilbert,2000)Inthefirstoftwoexperiments,theVEGFandbFGFgrowthfactorswereusedtoinduceangiogenesis;bothareappliedtotheCAMmembraneofthechickembryo.Inthesecondexperiment,onlytheeffectofbFGFonbloodvesselformation(orangiogenesis)withintheCAMmembranewasevaluated.

Procedure:1.CutsmallpiecesofWhatmann3MMfilterpaper(3mmX3mm)andsoakthemwith3mlHydrocortison-acetate(3mg/mlinethanol)(Sigma).Air-drythefilterdiscsfor1.5hrs.

2.Obtain4mLeach:bFGF(1.5mg/ml),VEGF(1.5mg/ml),orDMEM(atissueculturemediuminwhichthecontrolfilterdiscsshouldbesoaked).Pourthesolutionsintoseparatepetridishesthatarelabeledwiththenamesofthecompounds.Soakthefilterpaperdisksinthesolutionsfor30minutes.

3.Whilethefilterpaperissoaking,obtainten-day-oldchickeggsandwashthemin70%ethanol.LabeltheeggsaseitherDMEM,bFGF,orVEGFusingapencil.Openthebluntendoftheeggwithforceps.Peelbacktheshellmembrane,beingcarefulnottodamagetheCAMmembrane.

4.DropthefilterpaperovertheembryointheareawiththeleastnumberofvisIBLebloodvessels.CovertheholeintheshellswithScotchtape,andplaceinanincubatorsetat37C(beingcarefulnottojostletheembryos)forfourdays.

5.Afterthefour-dayperiod,removethetapecoveringtheholesandextractthefilterpaperusingapairofforceps.Examinethefilterpaperforthepresenceofbloodvessels.Countallbloodvesselsonthefilterpaperandrecordthenumberswithinyourlabnotebook.Comparetotheotherconditions.

Results:Theembryosofthefirstexperimentweretreatedwithoneoftwogrowthfactors,eitherbFGForVEGF.ThosetreatedwithbFGFshowedthegreatestamountofbloodvesselforma-tionwhencomparedwiththeDMEMandVEGFembryos(picturesoftheresults).AproblemmayhaveoccurredwiththeVEGF,asithadbeeninthelabforanextendedperiodoftimeandmayhavebeentoooldtoproduceaccurateresults.

Ofthetwelveembryospreparedforthesecondexperiment(duringwhichonlytheeffectsofbFGFonbloodvesselformationwasevaluated),sevensurvived.Whenopened,thoseembryosthatdidnotsurvivehadsignificantlydarkeramnionicfluidthantheirlivingcounterparts.Inaddition,theamnionicfluidofthedeadembryosappearedcloudyandgaveoffadistinctodor.Ofthesevensurvivingembryos,onlythefilterpaperdisksofthreewereremovedsuccessfuly(withportionsoftheCAMmembraneattached).Inothercases,whenremovingthedisks,bloodvesselswerecutinsuchawayastocontaminatethefilterpapersquares;someremovedsquaresweredyedredbytherupturedbloodvessels,andtheCAMmembranewaseitherdislodgedorlost.

Figure1.PhotographofCAMmembraneattachedtoafilterpapersquare(3mmx3mm)treatedwithDMEM.TheCAMmembranewasremovedfroma15-day-oldchickembry,afterthemembranewasexposedtoatreatedfilterpapersquareforfivedays.

WhenexaminingthenumbersofbloodvesselsontheCAMmembranestreatedwitheitherDMEMorbFGF,theonetreatedwithbFGFhadagreaternumberofbloodvessels(13)ascomparedwiththevesselsobservedonthetwodiskstreatedwithDMEM(7and12,respectively);however,itmustbetakenintoaccountthatonlythreetestresultswerecollected.Inaddition,asignificantdifferencebetweenthenumberofbloodvesselsofeithertheDMEMorbFGFCAMmembranesdidnotexist.

Discussion:

Fromthefirstexperiment,bFGFwasshowntobeinfluentialinbloodvesselformation.Afterembryoswereexposedprematurelytothegrowthfactor,alargenumber(incomparisonwitheithertheDMEMorVEGFbloodvesselcounts)ofbloodvesselsformed.InsufficientdatafromthesecondexperimentpreventsanydefiniteconclusionsabouttheroleofbFGFonbloodvesselformationwithintheCAMmembrane.However,thebFGFCAMmembranedidexhibitmorebloodvesselsthaneitheroftheDMEMsegements.ThebFGFmembranesegmenthadatotalofthirteenbloodvessels,whereastheDMEMsegmentshadsevenandtwelvebloodvessels,respectively.IncomparisontothebFGFbloodvesselcount(13),theaverageoftheDMEMfiltersquarebloodvessels(7.5)wasmoderatelylower.

ThefirstexperimentshowedastrongcorrelationbetweenthegrowthfactorandincreasedbloodvesselformationontheCAMmembraneofchickembryos.bFGF,producedintheCAMmembrane,isrequiredfortheprocessofvasculogenesis,duringwhichtimehemangioblastsarespecifiedandbloodvesselsarecreated(Gilbert2003).AgreaterconcentrationofbFGFmaycausemorebloodvesselstoappearduringvasculogenesis.WithinanareaofhighbFGFconcentration,moremesodermcellsmaybespecifiedtoformhemangioblasts,andmorebloodislandsandvesselswillbeproduced(Gilbert,2003).Inaddition,theimportanceofthesegrowthfactorsisdemonstratedbymutantembryosthatlacktheproperreceptorsforbFGF(andalsoVEGF),whichhaveasignificantlylowerchanceofsurvivalasvasculardevelopmentwillnotoccurproperly.