Cell Migration and Proliferation During the In Vitro Wound Repair of the Respiratory

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Cell Migration and Proliferation During the In Vitro Wound Repair of the Respiratory Epithelium Jean-Marie Zahm,* Herve Kaplan, Anne-Laure Herard, Fabrice Doriot, Denis Pierrot, Pascal Somelette, and Edith Puchelle INSERM U314, IFR53, CHU Maison Blanche, Reims, France The respiratory epithelium is frequently injured by inhaled toxic agents or by micro-organisms. The epithelial wound repair represents a crucial process by which surface respiratory cells maintain the epithelial barrier integrity. The repair process involves both cell migration and proliferation, but as yet, the kinetic of these two mechanisms has not been extensively studied. Using an in vitro model of human respiratory epithelium wound repair, proliferative cell immunofluorescent staining and a computer-assisted technique allowing the tracking of living cells, we studied the cell proliferation and migration during the wound repair process. Respiratory epithelial cells were dissociated from human nasal polyps and cultured on a collagen I matrix. At confluency, a chemical wound was made on the culture. We observed that the cell mitotic activity peaked at 48 h after wounding (23% of the cells) and mainly concerned the cells located 160 to 400 µm from the wound edge. The migration speed was highest (35 to 45 µm/h) for the spreading cells at the wound edge and progressively decreased for the cells more and more distant from the wound edge. The temporal analysis of the cell migration speed during the wound repair showed that it was almost constant during the first 3 days of the repair mechanism and thereafter dropped down until the wound closure was completed (after 4 days)

  • respiratory epithelium

  • immediately after

  • epithelial cells

  • methods based

  • epithelial cell

  • wound repair

  • culture medium


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CellMotilityandtheCytoskeleton37:33±43(1997)

CellVitMriogrWatoiounndanRdePpraoirlifoefrtahtieonReDsupriirnagtotrhyeIn
Epithelium

Jean-MarieZahm,*HerveÂKaplan,Anne-LaureHÂerard,FabriceDoriot,
DenisPierrot,PascalSomelette,andEdithPuchelle
INSERMU314,IFR53,CHUMaisonBlanche,Reims,France
Therespiratoryepitheliumisfrequentlyinjuredbyinhaledtoxicagentsorby
micro-organisms.Theepithelialwoundrepairrepresentsacrucialprocessby
whichsurfacerespiratorycellsmaintaintheepithelialbarrierintegrity.Therepair
processinvolvesbothcellmigrationandproliferation,butasyet,thekineticof
thesetwomechanismshasnotbeenextensivelystudied.Usinganinvitromodelof
humanrespiratoryepitheliumwoundrepair,proliferativecellimmuno¯uorescent
stainingandacomputer-assistedtechniqueallowingthetrackingoflivingcells,we
studiedthecellproliferationandmigrationduringthewoundrepairprocess.
Respiratoryepithelialcellsweredissociatedfromhumannasalpolypsandcultured
onacollagenImatrix.Atcon¯uency,achemicalwoundwasmadeontheculture.
Weobservedthatthecellmitoticactivitypeakedat48hafterwounding(23%of
thecells)andmainlyconcernedthecellslocated160to400mfromthewound
edge.Themigrationspeedwashighest(35to45m/h)forthespreadingcellsatthe
woundedgeandprogressivelydecreasedforthecellsmoreandmoredistantfrom
thewoundedge.Thetemporalanalysisofthecellmigrationspeedduringthe
woundrepairshowedthatitwasalmostconstantduringthe®rst3daysoftherepair
mechanismandthereafterdroppeddownuntilthewoundclosurewascompleted
(after4days).Wealsoobservedthatovera1-hourperiod,theintra-individualand
interindividualvariationofthecellmigrationspeedwas43%and37%,respec-
tively.Theseresultsdemonstratethatcellproliferationandcellmigrationduring
respiratoryepithelialwoundrepairaredifferentlyexpressedwithregardtothe
celllocationwithintherepairingarea.CellMotil.Cytoskeleton37:33±43,
1997.
r
1997Wiley-Liss,Inc.

Keywords:airwaywound;migratingcells;proliferativecells;spreadingcells;imaginganalysis;cell
tracking

INTRODUCTION
capacityoftherespiratoryepitheliumtorepairfollowing
injury.Mostofthesemodelshavebeendevelopedinvivo
Therespiratoryepitheliumisfrequentlyinjuredbyindifferentanimalspeciesandhaveallowedthededuc-
inhaledtoxicagentsormicro-organisms,leadingtothetionthatdenudationoftheairwayepithelialsurfaceleads
alterationoftheepitheliumbarrierintegrity.Whateverthe
sourceofinjury,theresponseoftherespiratorysurface
Contractgrantsponsors:AssociationFrancËaisedeLutteContrela
epitheliumtoanacuteinjurycanbecharacterizedbya
Mucoviscidose(AFLM),PoÃleTechnologiqueRÂegionalGBM,IN-
successionofcellulareventsvaryingfromthelossof
SERM(ContratNormaliseÂd'EtudePilote),MinistÁeredel'Enseignement
surfaceepithelialimpermeabilitytothedesquamationof
SupeÂrieuretdelaRecherche.
cellsfromtheepithelium.Theepithelialwoundrepair
representsacrucialprocessbywhichtheremaining
*Correspondenceto:Jean-MarieZahm,INSERMU314,CHUMaison
surfacerespiratorycellsrestoretheepithelialbarrier
Blanche,45,rueCognacq-Jay,51092ReimsCedex,France.
integrity.Numerousmodelshavebeenusedtoexplorethe
Received19July1996;accepted18November1996.
r
1997Wiley-Liss,Inc.

34Zahmetal.

tothesurroundingepithelialcellmigrationandprolifera-inRPMI1640culturemedium(Gibco,GrandIsland,
tion[GordonandLane,1976;Keenanetal.,1982;NY).Thenasaltissuewasthendissociatedbyenzymatic
McDowelletal.,1979;Nikulaetal.,1988;Shimizuetal.,digestion(pronase0.1%)for12h.Thedissociated
1994].However,thekineticofthesetwomajorprocessessurfaceepitheliumwasremovedfromthetissuebygentle
duringthewoundrepairhasnotbeenextensivelystudied,agitationandenzymaticdigestionwasstoppedbyadding
duetothedifficultyintroducedbyinvivomodels.In10%calfserum(Seromed,Biochrom,Berlin,Germany).
addition,inalltheseanimalmodelsofinjuryandrepair,Thecellularpelletcollectedaftercentrifugationat150
3
thein¯ammatorystimulusengagescellularfactorsandgfor10minwasconstitutedbyisolatedmucouscells,
serum-derivedmoleculesintotheprocessofrepair.ciliatedorbasalcells,smallclumpsofreaggregatedcells
Toovercomethecomplexassociationoffactorsinandsmallepithelialsheets.Thesecellswereresuspended
theinvivomodels,culturemodelsofrespiratoryepithe-inaserum-freemediumandwereplatedonatypeI
lialcellsallowmoreclearlytheidenti®cationofthecollagengelmatrixpreparedfromrattailtendonsaccord-
cellularandmolecularalterationsassociatedwiththeingtothetechniquedevelopedinourlaboratoryand
woundrepairprocesses[Zahmetal.,1991,1993].AttheearlierdescribedbyChevillardetal.[1993].Thecells
molecularlevel,alterationsincytoskeletalproteinpat-wereculturedinanhumidi®edatmospherecontaining
terns,bindingofgrowthfactorsandinteractionswith
extracellularmatrixproteinsandmetalloproteinases95%airand5%CO
2
inRPMI1640mediumsupple-
throughspeci®ccellsreceptors(integrins),cangovernmentedwith1g/mlinsulin(Sigma,L'IsleD'Abeau,
thecellmigrationintherepairingarea.The¯attenedcellsFrance),1g/mltransferrin(Sigma),10ng/mlepidermal
whichmigrateintothewoundedsitechangetheirpheno-growthfactor(Serva,Heidelberg,Germany),0.5g/ml
type,becomingpoorlydifferentiatedcellsexpressinghydrocortisone(Sigma),10ng/mlretinoicacid(Sigma),
keratin14andvimentinintermediate®laments[Shimizu100U/mlpenicillin,and100g/mlstreptomycin.Theuse
etal.,1994;Buissonetal.,1996a].ThecellspreadingandoftypeIcollagengelandgrowthfactor-supplemented
migrationwhichoccurduringwoundrepairdependonculturemediumisthemostsuitableconditiontoachieve
cell-matrixinteractionsmediatedby®bronectinandonedifferentiatedculturesofhumannasalepithelialcells
ofitscellularreceptors,the
a
5
b
1integrin[HeÂrardetal.,[Chevillardetal.,1993].
1996].Recentresultshavealsodocumentedtheincrease
andlocalizationofgelatinolyticmetalloproteinasesasso-
InVitroEpithelialWounding
ciatedwiththemigratoryprocessofepithelialcellsAfter3daysinculture,whenthecellshadreached
duringtherespiratoryepitheliumrepair[Buissonetal.,con¯uency,theculturemediumwasremovedfromthe
1996b].However,uptonow,thespeci®croleofcellculturedish.A2-ldropofNaOH0.1Mwasdepositedin
migrationandcellproliferationinthewoundrepair
processoftherespiratoryepitheliumhasnotbeenexten-thecenteroftheculturedishandimmediatelydilutedin1
sivelydocumented.mlofculturemedium.ThecellsincontactwiththeNaOH
Inthepresentstudy,weusedaninvitrowoundingdropdesquamatedfromthecollagenImatrix,creatinga
modelofdissociatedrespiratoryepithelialcellsculturedcircularwoundofabout30mm
2
inarea.Thewounded
fromhumannasalpolypstoevaluatethedynamicsoftheculturewasthenrinsedwith1mlofculturemediumand
cellmigrationandproliferationduringwoundrepair.Weincubatedinairwith5%CO
2
at37°C.
alsoreportedinthepresentwork,anovelmethod,using
imageanalysis,toprovidedataonthemigrationkinetics
WoundAreaDetermination
ofeitherindividualcellsoracellpopulationduringtheEvery12hduringthewoundrepairprocess,the
woundrepairprocess.Weobservedthatthecellmitoticwoundedculturedishwasplacedonthestageofan
activitypeakedat48hafterwounding.Thetemporalinvertedmicroscope(NikonTMS-F)equippedwitha
3
1
analysisofthecellmigrationspeedshowedthat,despiteobjectiveandavideoCCDcamera(Cohu4700).The
largeintra-individualandinterindividualvariations,thevideoimageofthewoundwasdisplayedonavideo
overallmigrationspeedofthecellpopulationatthe
woundedgeremainedfairlyconstantduringthe®rst3monitorandthewoundedgewasdrawnmanually.From
daysofrepairanddecreasedprogressivelyuptothethisdrawing,thewoundareawasdeterminedandex-
woundclosure.pressedasafunctionoftime.
CellProliferation
MATERIALSANDMETHODS
Forcellproliferationquantitation,woundedcul-
RespiratoryEpithelialCellCulture
tureswerecryo®xedevery24hthroughoutthewound
Nasalpolypswereobtainedfrompatientsundergo-repairprocess.Thecentralareaoftheculture(about200
ingnasalpolypectomyandwereimmediatelyimmersedmm
2
inarea)inwhichthewoundwasperformed,wascut

CellProliferationandMigrationinWoundRepair35

outfromtheculturedish,embeddedinOCTcompound,recordedfor2severy5min,the¯uorescentnucleusof
immersedinliquidnitrogenfor5minandthenkeptatcellsinthesamemicroscopic®eldatthewoundedge.
2
80°C.Thicksections(5m)perpendiculartotheFromthesevideorecordings,weusedanimageanalysis
woundedculturewereobtainedusingaReichertCryocut,techniquethatwedevelopedtoanalyzethemigrationof
slicedonaglassslideanddehydratedinair.Forindividualcellsformingcontinuoussheets.Inoneexperi-
immunostaining,thesectionswereimmersedinmethanolment,usingphase-contrastillumination,wecontinuously
at
2
20°Cfor5minandrinsedin0.1Mphosphate-recordedthecellsatthewoundedgefora10-minperiod
bufferedsaline(PBS).Eachsectionwasthenincubatedinordertovisualizethecellspreading.
for1hwithamousemonoclonalantibody(MIB-1,
Immunotech,France)whichrecognizestheKi67nuclear
CellTrackingandCellMigrationMeasurement
antigenandwhichwasdiluted1/50in0.1MPBSand
thereafterincubatedwithabiotin-conjugategoatanti-Theimagesweredigitizedasa766
3
574pixels
mouseIgM,diluted1:25in0.1MPBSfor1h.Thecellsand24-bitarrayfromthevideorecordings,usinga
werethenstainedwithstreptavidin-FITCfor30min.TheSparc-ClassicworkstationequippedwithaXVideocard
sectionsweremountedinglycerol-PBS-1,4diazabicyclo(ParallaxGraphics,SantaClara,CA).Beforetreatment,
2-2octaneandobservedunderaZeissAxiophotmicro-theimageswerereducedto512
3
512pixelsand8-bit
scopeequippedwithepi¯uorescenceillumination.Succes-greylevels.WedevelopedasoftwareinClanguageusing
sivemicroscopic®eldsof40minlengthradiatingouttheXlibrary(version1.1oftheXWindowsystem)anda
fromthewoundedgewereobserved.Ineach®eld,thetoolkitavailableinXView(SunMicrosystems,Moun-
numberofcellsbeingpositivelystainedfortheKi67wastainView,CA).Thesoftwareisuser-interfacedandhas
quanti®edandexpressedasapercentageofproliferativethreemainfunctions:(1)thedetectionofthecellnucleus,
cellswithregardtothetotalnumberofcells.(2)thecomputationofthetrajectoriesofthenucleus,and
(3)theanalysisofthenucleustrajectories.
VideoRecordingoftheCellMigration
Thedetectionofthecellnucleuscanbedone
Immediatelyafterwounding,cellculturesweremanuallybyusingthecomputermouseorautomatically
incubatedfor15minwithHoechst33258(Sigma)at0.1byusingimagesegmentationmethodsbasedonthreshold-
mg/mlinculturemedium,allowingthe¯uorescentstain-ing.Thetrackingofseveralnucleitogetherisdoneby
ingofthenucleusinlivingcells.Theculturewasthenusingtrajectory-basedmethods[AggarwalandNandhaku-
washedtwicewithculturemediumtoremovetheexcessmar,1988].Thegeneralschemeofthesealgorithmsisto
of¯uorescentdye.Followingthat,thewoundedcultureminimizethemeancostofallthecomputedtrajectories,
dishwasplacedonthestageofaZeissIM35invertedusingoneorseveralparameterssuchastheregularityof
microscopeandwasenclosedinasmalltransparentcellmotion,thelowdeviationofthecelltrajectoryorthe
culturechamber(Climabox)with5%CO
2
inairat37°C.nearnessofthesuccessivepositionsofthecells.Forthe
Themicroscopewasequippedwithanepi¯uorescencemainpart,weusednearnessandlowdeviationofthe
illumination(Hglamp)throughanexcitation®lterat360trajectoriesasparametersofminimizationforseveral
nm,andanemission®lterat510nm,andwithalowlevelsuccessiveimagesandnearnessparametersforonlytwo
SITcamera(Lhesa4036).Aspecialelectronicallyoper-successiveimages.Aftertrajectorycomputation,the
atedshutterintheexcitationlightpathwasdevelopedinsoftwaredrawsallthetrajectoriesoftheselectednucleus.
ordertoautomaticallyilluminatethecultureatshortFromthisdrawing,eachnucleustrajectorycanbese-
periodsoftime,andtosimultaneouslyrecordthe¯uores-lectedindividuallyandthemigrationspeediscalculated
centimages.Thisdevice,basedontimingsemiconductorfromonenucleuspositiontotheother.Themean
circuits,allowedthede®nitionoftherecordingtimeofmigrationspeedforallthenucleustrajectoriesisalso
eachvideosequence(from1to99s)andthetimeintervalcalculated.Thesoftwareisabletotrackacellpopulation
betweentwosuccessivesequences(from1to99min).ofabout100cellstogether.Thecomputationtimefor100
ExperimentalProcedure
cellsineightimagesisabout2susinganUnix
Workstation(SunMicrosystems).
Videorecordingsofthe¯uorescent-stainedcell
nucleuswereperformedfollowingthreesetsofexperi-
mentsduringthewoundrepairprocess.Inthe®rstsetof
StatisticalAnalysis
experiments,werecordedfor2severy12hthecellAllthedataareexpressedasmean
6
S.D.(standard
nucleusatthewoundedge,throughoutthewoundrepairdeviationofthemean).TheunpairedStudent'sttestwas
process.Inthesecondsetofexperiments,atday2oftheusedtodeterminesigni®cance(de®nedas
p
,
0.05).
woundrepair,werecordedsuccessivemicroscopic®eldsCurve®ttingwasperformedonaMacintoshQuadra650
whichwereradiatingoutfromthewoundedge.IntheusingCA-CricketGraphIII(ComputerAssociates,New
thirdsetofexperiments,atday2ofthewoundrepair,weYork,NY).

36Zahmetal.

Fig.1.Setoflowmagni®cation(
3
1objective)micrographsshowing
theinvitrowoundrepairprocessoftherespiratoryepithelium.The®rst
micrograph(t0)wastakenimmediatelyafterwounding.Thefollowing
micrographsweretakenevery30hduringthewoundrepair.The
progressiveclosureofthewoundisobserved.Bar
5
1mm.Fig.2.Temporalevolutionofthewoundsurfaceduringthewound
repairprocessoftherespiratoryepithelialcells,whosemicrographsare
RESULTS
showninFigure1.Eachpointrepresentsthemean
6
S.D.ofthree
experiments.Thecurvecorrespondstoathird-degreepolynomial®t
WoundRepairProcess
throughtheexperimentaldata.Thedecreaseinwoundarea(0.37mm
2
Humansurfacerespiratoryepithelialcellscultured
perhour)appearslinearupto60handthenrapidly¯attensoutupto
onatypeIcollagenreachedcon¯uencywithin2to3
woundclosure.
days.ThecontactofadropofNaOHwithacon¯uent
respiratoryepithelialcellcultureinducedalocalized
injurycharacterizedbythecelllysisanddesquamationwhereWaisthewoundareaandtthetimeofrepair.As
andthedenudationofthecollagenmatrixonwhichtheshownbytheequationofthe®tcurve,the®rst-order
cellsweregrowing.Thechemicallyinducedwoundsincoefficientcorrespondedtothelinearpartofthe®tcurve.
therespiratoryepithelialcellcultureconsistedofcircularThedecreaseinwoundareaappearedthereforelinearup
to50hofrepairduetothefactthatthesecond-and
holes.Microphotographstakenusinganinvertedmicro-third-ordercoefficientsofthe®tequationinterferedonly
scopeatlowmagni®cation(
3
1objective),immediately
afterwoundingandthereafterevery30hduringwoundabout10%intherateofdecrease.However,onandafter
repair,showtheprogressiveclosureofthewoundedthe60thhourofrepair,thethird-ordercoefficientactsasa
surface(Fig.1).Theearlyreepithelializationpatternfactor¯atteningoutthecurveandalterstherateof
displayedbytherespiratorycellsimmediatelyafterdecreaseby16%,39%and77%at60,80and100hof
injury,appearedtoinvolvethespreadingofthecellsatrepair,respectively.Thisobservationindicatesthatthe
thewoundedgeandthemovementofcellsheetstowardrepairprocessbecomesslowerwhenmostoftheinjured
thewound[Zahmetal.,1991].Themigrationprocessissurfacewasrecoveredbytherepairingcells.
uniformlydistributedaroundthewoundsurfaceassug-
CellProliferation
gestedbytheconcentricityofthesuccessivewound
surfaces.AregularfeaturewhichprogressivelyappearedInordertoassesswhethertheinvitromodelof
duringthewoundrepairisthepuckeringofcellsheetsinrespiratoryepitheliumwoundrepairinvolvedcellprolif-
therepairedarea(Fig.1,t90).Theobservationoferation,woundedculturesatdifferentdaysoftherepair
semithinsectionsofwoundedculturesrevealedthattheprocesswereimmuno¯uorescentlystainedforthenuclear
puckersradiatingaroundthewoundaremadeofmultilay-antigenKi67whichisamarkerofcyclingcells.The
eredcells.micrographinFigure3ashowsanumberofKi67-
Themeanwoundsurfaceplottedversustimede-positivecellsintheculture2hafterwounding(day0).As
creasedsharplyuptothe50thhourofrepair(Fig.2).AshowninFigure3b,thenumberofproliferativecellsin
thirddegreepolynomial®tthroughtheexperimentaldatatherepairingareaincreaseddramaticallyat48hafter
wounding(day2).Whenthewoundclosurewascom-
gavethefollowingcurveequation:pleted(day4),thenumberofproliferativecellsfelldown
Wa
(t)
5
27.99
2
0.37t
2
1.28
3
10
2
3
t
2
2
2.14
3
10
2
5
t
3
toavaluesimilartothatobtainedatday0(datanot

CellProliferationandMigrationinWoundRepair37

Fig.3.Proliferativeactivityofrespiratoryepithelialcellsduringthein(day4),thepercentageofproliferativecellswasnotsigni®cantly
vitrowoundrepair.Theproliferativecellsweredetectedbyimmuno¯u-differentascomparedwithday0.Eachbarrepresentsthemean
orescenceoftheKi67antigen.
a:
AfewKi67-positivecellsarepercentage
6
S.D.ofproliferativecellsquantitatedonatleast12
observedatday0ofwoundrepair.
b:
Forty-eighthoursafterdifferentrepairingareas.
d:
Spatialevolution,atday2,ofthe
wounding,numerousKi67-positivecellsarepresentintherepairingpercentageofproliferativecellsintherepairingarea.Asigni®cant
area.
c:
Percentageofproliferativecellsatday0,day2andday4oftheincrease(
p
,
0.05)inthepercentageofproliferativecellswas
woundrepairprocess.Asigni®cantincreaseinthepercentageofobservedatadistanceof80to320mfromthewoundedge.Each
proliferativecellswasobservedatday2(
p
,
0.05).Atwoundclosurevaluerepresentsthemean
6
S.D.ofthreemeasurements.

shown).Figure3cillustratesthevariationsintheprolifera-increaseinproliferationat320mwassigni®cantly
tiveactivityofthecellsintherepairingarea.Thehigher(
p
,
0.05)ascomparedtotheproliferationat250
percentageofproliferativecellswas7.1
6
7.0%atday0,m.Onthecontrary,theproliferationofcellslocatedata
increasedsigni®cantly(
p
,
0.05)upto23.0
6
25.6%atdistancehigherthan400mfromthewoundedge
day2anddecreasedto2.8
6
3.0%atday4whenthedramaticallydecreasedtoonly1±2%.
woundclosureoccurred.Theseresultsdemonstratethattheproliferative
Inordertoassesswheretheproliferativecellswereactivityismaximalatday2oftherepairprocessand
located,wequanti®edtheirnumberinareasmoreandmainlyconcernsthecellslocatedwithinanareabehind
moredistantfromthewoundedge.Thevariationatday2the®rstcellrowsofthewoundborder.
inthepercentageofproliferativecellsinrelationtothe
distancefromthewoundedgeisrepresentedinFigure3d.
CellSpreading
AslightincreaseinthenumberofproliferativecellswasToanalyzethedynamicsofcellmotilityatthe
observedatashortdistancefromthewoundedge.Thewoundedgeofwoundedrespiratoryepithelialcellcul-
cellslocatedintherepairedareaatadistanceof80to320tures,videorecordingsofthewoundedcultureswere
mfromthewoundedgewerecharacterizedbyamadeunderphase-contrastillumination.From60images
signi®cantincrease(
p
,
0.05)intheirproliferativeactiv-recordedeveryminfor1h,amoviewasproduced,
ity.Thedecreaseinproliferationbetween160mand240allowingthevisualizationoflamellipodiadynamicsof
mwasnotsigni®cantcomparedto160m,butthethecellslocatedatthewoundedge.Fourpictures

83

Zahmetal.

Fig.4.Seriesofphase-contrastimages,takenat3-minintervals,ofrespiratoryepithelialcellsmigrating
andextendinglamellipodiaatthewoundedge.Theleadingedgeofthecelllamellipodiahasbeen
visualizedbyawhiteline.Thecellslocatedatthewoundedgearecharacterizedbyafunctionalpolarity
(monopolar)withacontinuouslychanginglamellipodiashape.Bar
5
10m.

extractedfromthemovieevery3minarepresentedinandthereforearemoreeasilyanalyzedwiththeimage
Figure4.Inordertobettervisualizethefrontofthecellanalysissoftwarespeci®callydevelopedforthecell
lamellipodia,itsborderwasunderlinedbyawhitevelocitymeasurement(Fig.5).Themicrographsin
drawing.ThelamellipodiaformationwasacharacteristicFigure5ashowcellnucleistainedwiththeHoechstdyeat
featureofthecellsatthefrontrowofthewoundedge.theedgeofawoundatday2ofthewoundrepair.Figure
Thelamellipodiaremainedstrictlylocalizedatthe``head''5brepresentsthesamenucleirecorded60minlater.The
orfrontofmigratingcells.Wecaneasilyobservethatthetrajectoriesdeterminedfromthe®eldanalyzedevery15
shapeofthecelledgeiscontinuouslymodi®edduringtheminoverthe60-minperiodofobservationwerefairly
spreadingprocess,butthecellfunctionalpolarity(mo-straight(Fig.5candd).AsshowninFigure5d,the
nopolar)remainedconstantthroughouttherepairprocess,softwarethatwedevelopedallowedustocalculatethe
leadingtotheunidirectionalityofcellmovement.angulardeviation(
a
1,...,
a
4)fromthehorizontalofthe
successivetrajectoriesofthenuclei.Themeandeviation
CellMigration
angleforalltheselectednucleiwas0.30
6
0.11rd,which
Totrackthemigratorycellsrepairingthewound,con®rmedtheuniformdirectionalityofthecellmigra-
westainedthecellnucleiwithHoechst¯uorescentdye.tion.Thesetrajectoriesrepresentatypicalbehaviorofthe
The¯uorescentimagesobtainedundertheseconditionsradianmigratingcellswhichactivelyparticipatedtothe
arelesscomplexthanphase-contrastimages(seeFig.4)woundrepairprocess.

Fig.5.Quantitativeevaluationofcellmigrationvelocity.
a:
Fluores-
centstainingwithaDNA¯uorescentdye(Hoechst33258)ofthe
nucleusofrespiratoryepithelialcellsattheedgeofawoundedculture
atday2ofthewoundrepairprocess.
b:
Thesamecellsrecorded1h
later.
c:
Trajectoriesofcellnucleirecordedovera60-minperiod.
d:
Detailedtrajectoriesofaparticularnucleusanalyzedevery15minfor1
h.Theangle
a
representsthedeviationwiththehorizontalofthe
nucleustrajectory.
e:
Temporalevolutionofthemigrationvelocityof

CellProliferationandMigrationinWoundRepair

93

cellsatthewoundedgethroughoutthewoundrepairprocess.A
signi®cantdecreaseinthemigrationspeedwasobservedat72h.Each
barrepresentsthemean
6
S.D.of15measurements.
f:
Temporal
evolutionfor1hofthemigrationspeedofcellsatthewoundedge.The
same15cellswererecordedevery5minfor1h.Hugevariationswere
observedfromonecelltotheother.Eachbarrepresentsthemean
6
S.D.ofthemigrationspeedforthe15cells.

Themigrationspeedofthecellsatthewoundedgequently,thedecreaseinthecellmigrationvelocity
duringthewoundrepairprocessisrepresentedinFigureinducedanalterationinthewoundareadecreaserate.
5e.Wemeasuredanalmostconstantspeed(35to40Figure5frepresentsthemeanmigrationspeedofa
m/h)forupto60hofrepair,followedbyasigni®cantcellpopulationmeasuredevery5minfor1hatthewound
decreasefromhour72untilthewoundclosure.Whenedgeatday2.Thehighstandarddeviationofthe
comparedwiththewoundareadecreasereportedinmigrationspeedcalculatedevery5minforthecell
Figure2,thetimeof60hmaybesigni®cant,becauseitpopulationindicatedthathugevariations(interindividual
roughlycorrespondedtothetimeuptowherethevariation
5
37%)incellmigrationspeedwereobserved
decreaseinwoundareawasroughlyconstant.Conse-fordifferentcellsduringthesametimeperiod.In

40Zahmetal.

activity(24%ofproliferativecellsat48hafterwound-
ing)intheinvivowoundrepairprocessoftherattracheal
epithelium.
Themajoreventinthewoundrepairprocessisthe
cellspreadingandmigrationwhichallowstheepithelium
recoveryofthedenudedcollagenmatrix.Time-lapse
videorecordingsassociatedwithcomputermethodsal-
lowthecontinuousmonitoringoftheprocessofcell
spreadingandmigration.
AsseeninFigure4,underphase-contrastmicros-
copyobservation,itisdifficulttoindividualizethecells
andtomonitortheirmovementinacon¯uentcellculture.
Toovercomethisproblem,westainedthecellnucleus
withaDNAdye(Hoechst33258)whichisexcitableby
UVillumination.Takingintoaccountwhatwehave
previouslydemonstrated,thatthedurationofUVillumi-
nationcaninducedramaticchangesinthephysiological
stateofthecells[Zahmetal.,1994],weusedan
Fig.6.Spatialevolutionofthemigrationspeedofcellsatday2ofthe
electronictimerwhichallowedustoreducetheUV
woundrepairprocess.Thecellmigrationspeedprogressivelyand
illuminationtimetoonlyafewsecondsforeachvideo
signi®cantly(
p
,
0.05)decreasedwiththeincreaseinthedistance
recording.
fromthewoundedge.Eachbarrepresentsthemean
6
S.D.of15
measurements.
Totrackthemovingcells,weusedtrajectory-based
methods[AggarwalandNandhakumar,1988]which
commendseveralassumptions.Firstly,thenuclei,which
addition,themeancoefficientofvariationofthemigra-areusedasmarkersofthecells,areconsideredasarigid
tionspeedforthesamecellmeasuredevery5minforabodyandareindistinguishablefromeachother.Secondly,
1-hperiodwas43%(
5
intra-individualvariation),indicat-eachnucleusistrackedusingonlyonerepresentative
ingthatthemigrationspeedofonegivencellwasnotpoint:thecenterofthenucleus.Whilethecellsare
constantovera1-hperiod.trackedusingthecenterofthenucleusasasinglemarker,
Themeanmigrationspeedofcellsmeasuredinonlytranslationmovementsofthecellsareobservable.
successiveareasprogressivelymoredistantfromtheOthermovements,suchascellrotation,forexample,are
woundedgeisrepresentedinFigure6.Weobservedthatnotdetectable.Thetrajectory-basedmethodraisesthe
thecellmigrationspeedcontinuouslyandsigni®cantlyproblemoftheocclusionswhichconcerntheobjects
(
p
,
0.05)decreasedwiththeincreaseinthedistancevisibleinanimageandnolongervisibleinthefollowing
fromthewoundedge.Theepithelialcellsatthewoundimageofthesequence.Wealsohavetoconsiderthecase
edgemovedfasterthanthecellswhichweredistantfromofobjectswhicharenotpresentinanimageandbecome
thewoundedge.Atadistancehigherthan1.6mmfromvisibleinthefollowingimages.Somealgorithmsdealing
thewoundedge,thecellmigrationwasnolongerwiththeocclusionshavebeendescribed[Fletcheretal.,
detectable.1991;RangarajanandShah,1991;SethiandJain,1987],
butthesealgorithmsgenerallyonlytakeintoaccountthe
DISCUSSION
missingobjectsinsuccessiveimagesequences.Inthe
imagesfromourwoundrepairmodel,thenumberof
Theexperimentsdescribedinthepresentworknucleusinanimageiscontinuouslyincreasingthrough-
showthattherespiratoryepitheliumisabletorepairoutthesetofimages.Toovercomethisproblem,weused
woundsthroughacombinationofcellmigrationandcellanalgorithmwhichconsideredthatthenumberoftracked
proliferation.Cellproliferationwasquanti®edthroughnucleiwasconstantinallofthesuccessiveimages.With
theKi67antigenwhichisamarkerofcyclingcells,i.e.,thatpurpose,thenucleiwereselectedmanuallyintheset
cellsinG1,S,G2andMphases.Inourinvitromodelofofimages.Thisisdoneeasilyandquicklyusingthe
respiratoryepithelialwoundrepair,cellproliferationtookcomputermouseandisfacilitatedbythecellnucleus
placepreferentiallyintherepairingarea,withamitoticstainingwhichmadethenucleiclearlyvisibleinthe
activitywhichpeakedat48h.Whenthewoundclosureimages.
wascompleted,themitoticactivitysubsided.TheseTwosetsofmethodsforestablishingthecorrespon-
resultsareconsistentwiththerecentresultsofShimizuetdencebetweentheselectedpointsareavailable.The®rst
al.[1994]whodemonstratedasimilarpatternofmitoticmethodsarebasedontheregularityofthemigrationof

CellProliferationandMigrationinWoundRepair41

theobjects[SethiandJain,1987]andarecalled``smooth-weobservedanegativerelationshipbetweenthecell
ingmethods.''Todeterminethetrajectoriesofthenuclei,migrationspeedandthedistancefromthewoundedge:
wehavetomaximizethesmoothestsetoftrajectories.thecellslocatedfarfromthewoundedgemovedmore
Smoothingcanbecontrolledbyseveralfactorssuchasslowlythanthecellslocatedatthewoundedge.Asshown
nearness,speedofmotionordeviationofthetrajectories.inFigure3,themaximumincreaseincellproliferation
Thesecondmethodsarepredictingmethods[Fletcheretoccurredintheintermediateareaoftherepairedwound.
al.,1991],bywhichtheestimationofthepositionoftheLeeetal.[1994]haveshownthatanincreaseincell
objectsintheimageiscomputedusinginformationfromdensityinducedbycellproliferationisimmediately
thepartialtrajectoryoftheobjects.Theobjectwhichisaccompaniedbythedecreaseintheaveragespeedof
nearesttothecalculatedpositionischosenandthemigrationofacellpopulation.Inaddition,severalyears
predictingfactorscorrectedaccordingtotheknowledgeago,AbercrombieandHeaysman[1953]showedasigni®-
ofthisnewposition.Theprocessmustbeinitiatedusingcantinverserelationshipbetweenthespeedofmovement
theoptical¯owmethod[Urasetal.,1988]orasmoothingofacellandthenumberofothercellswithwhichitwas
method,onatleastthreeimages.incontactduringtheobservedmovement.Thedecrease
Severaltechniqueshavebeendescribedforanalyz-inthecellmigrationthatweobservedintheintermediate
ingcellmotility,butmostofthesetechniquesareappliedareaoftherepairingculturescouldthereforeberelatedto
toisolatedmigratingcells.Tatsukaetal.[1989]describedanincreaseintheproliferationrateofthesecells.The
amethodforquantitativemeasurementofpostcon¯uentdecreaseincellmigrationattheendofthewoundrepair
cellpopulations,butthelatterauthorsmeasuredamotilityprocesscouldberelatedtoacontactinhibitionof
indexofacellpopulationandnotthelocomotionspeedofmovementoccurringwhenjunctionhasbeenestablished
individualcells.Themainadvantageofthecomputer-betweenopposingsheetsofcells,asearlierdescribedby
associatedtechniquethatwehavedevelopedisthatweAbercrombieandHeaysman[1954].
cantrackapopulationofcon¯uentcellsoveralongLeadingcells,migratingtowardthewound,are
periodoftime,andmeasuretheirindividualmigrationpolarizedinthattheyextendlamellipodiaonlyalongthe
speed,allowingthestudyofthefactorscontrollingandfreeedgeofthewound.Thisphenomenonhasbeen
coordinatingthelocomotorymachinery.describedas``contact-stimulatedmigration''byThomas
TheabilityofcellstomoveiscrucialformanyandYamada[1992]andcouldgivedirectiontothe
biologicalprocesses,suchasembryogenesisandwoundmigratorypath.Themovementofcellsinagiven
healing.Inthiscurrentstudy,weanalyzedthecelldirectionhasbeenrelatedtothefactthattheydonot
migrationduringthewoundrepairprocessoftherespira-usuallymoveinadirectionthatwilltakethemoverthe
toryepithelium.Thecellsatthewoundedgearecharacter-surfaceofanothercell,buttheyarefreetomoveinany
izedbyameanmigrationspeedrangingfrom35to45otherdirection.Inawoundedarea,cellswillconse-
m/hduringthe®rst60-hperiodofthewoundrepairquentlybereleasedfromcontactinhibitionoflocomotion
process.Wehavepreviouslyshown[Zahmetal.,1992]andmovetowardthedenudedarea[Abercrombieand
thatinasmallermechanicallymadewound(0.03mm
2
inHeaysman,1970].Animportantfeaturetoconsideristhe
surface),thecellmigrationspeedreached26m/h.Butinhighlydynamicformationoflamellipodiaattheleading
thislatterwoundmodel,thewoundclosureoccurrededgeofthecellslocatedatthefrontofthewound.
within6to8hours.ThedecreaseinthemigrationspeedProtrusionofthecellmembraneiscoupledtopolymeriza-
measuredattheendofthewoundrepairprocessismosttionofactin®lamentsattheleadingedge.Different
likelyduetocontactinhibitioneffects.Recently,Leeetmechanismsforgeneratingprotrusiveforcehavebeen
al.[1994],byanalyzingthemeasurementsofcellpath,inproposed[MitchisonandCramer,1996].Eithermotor
conjunctionwithlocalcellvelocityandthedistancetotheproteinscoulddriveprotrusion,oractinpolymerization
nearestneighborofacell,havedemonstrated,indetail,itselfcouldproduceforce.Theprotrusionmechanismis
thebehaviorofacellasitmigratesandinteractswithfollowedbycelladhesionandtraction.Theleadingcells
othercells.Theseauthorsshowedthatastwocellsatthewoundedgeseemtoactasatractorforthecells
approacheachother,theirspeeddecreases,whereaswhenlocatedbehindthem.Cell-cellinteractionsandprobably
acellspeedsawayfromitsneighbor,itsspeedincreasescell-matrixinteractionalterationsgoverntheabilityofthe
quickly.Thisbehaviorofneighboringcellscouldalsocellpopulationtomigrate.Thesemechanismsaredepen-
explainthehugevariationsincelllocomotionthatwedentontheextracellularmatrixwhichformsthesupport
observedwhenanalyzingthelocalmovementofacellforthecells[RomanandMcDonald,1991].Duringthe
populationatthewoundedgeforashortperiodoftimewoundrepair,cellsurfaceadhesionmoleculesplaya
(Fig.5).likelykeyrolebymodulatingthecellcontactwiththe
Inanalyzingthemigrationofcellpopulationsextracellularmatrixorwithneighboringcells.Among
locatedatanincreasingdistancefromthewoundedge,thesecelladhesionmolecules,theintegrinsareknownto

42Zahmetal.
beinvolvedintheregulationofcellmigrationand
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