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PMC3260085

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Publishedonline2012Jan11.

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Matrixmetalloproteinase2and9activities
inthehumanlensepithelialcellsandserum
ofsteroidinducedposteriorsubcapsular
cataracts
BhagwatV.Alapure, MamidipudiR.Praveen,DevarshiU.Gajjar,Abhay
R.Vasavada,TrilokJ.Parmar,andAnshulI.Arora
AuthorinformationArticlenotesCopyrightandLicenseinformation

ThisarticlehasbeencitedbyotherarticlesinPMC.

Abstract

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Purpose
Toevaluatethelevelofmatrixmetalloproteinase(MMP)2and
MMP9activitiesinpatientswithsteroidinducedposterior
subcapsularcataract(PSC).
Methods
Thisprospective,observationalstudycomprisedof156patients
havingeithersteroidinducedPSC(n=50)ornonsteroidalPSC
(n=106)wereperformedtoevaluatethelevelofMMP2andMMP
9activitiesinthelensepithelialcells(LECs)andtheserum.
AnteriorlenscapsulesharboringLECswereobtainedduring
phacoemulsificationandperipheralbloodwascollectedfrom
patientsbeforeadministrationofanesthesia.Serumwasseparated
bycentrifugationat10,000gfor15minat4C.TheLECsand
serumsampleswereprocessedtoanalyzeMMP2andMMP9
activitiesusingsuccinylatedgelatinassay.Quantitativerealtime
PCR(qRTPCR)wasperformedtodeterminethemRNAlevelsof
MMP2andMMP9inLECs.ThemRNAlevelswereexpressedas
aratio,usingthedeltadeltamethodforcomparingtherelative
expressionresultsbetweencaseswithsteroidinducedPSCand
caseswithnonsteroidalPSC.MMP2andMMP9levelswerealso
comparedinthetwogroupsusingimmunolocalization.
Results
ThelevelofMMP2andMMP9activitywasfoundtobehighin
LECsandserumofcaseswithsteroidinducedPSC.Furtherinall
steroidinducedcases,a1.4foldincreasewasobservedinMMP2

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activityinLECsanda1.4foldincreaseinMMP9activityinthe
serum.BothqRTPCRandimmunolocalizationshowedincreased
expressionofMMP2andMMP9activity.
Conclusions
MMP2andMMP9activityinbothLECsandserumwas
significantlyhigherincaseswithsteroidinducedPSC.Thepossible
useofMMP9asanoninvasivebiomarkerinascertainingthe
presenceofsteroidinducedPSCshouldbeevaluatedusingalarger
samplesize.

Introduction

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Posteriorsubcapsularcataract(PSC)isanopacitythatoccursatthe
rearendofthelenscapsule.Ultrastructuralstudieshavesuggested
thattheevolutionofPSCisassociatedwithfiberswellingand
breakdown,whichinturnisaccompaniedbytheformationofa
complexandpeculiarmembranestructure[1].
In1960,Blackandcolleaguesfirstnotedtherelationshipbetween
steroidusageandPSC[2].ItwasalsoobservedthatPSC
developedonlyafterapatienthadbeenonahighdosesteroid
treatmentforlongerthanoneyear[3].Moreover,thedevelopment
ofPSChasbeenobservedinpatientswithdiseasessuchas
rheumatoidarthritis,asthma,aswellaskidneytransplantrecipients
whohavecontinuouslytakensystemicsteroidand
immunosuppressivedrugtreatment.Topicallyappliedsteroidsand
inhaledsteroids,takenforasthma,havealsoresultedinPSC[4].
ThisformofPSCaswellascorticalchangesduetothe
administrationofsteroidaldrugsoccurmoreoftenasaresultof
diffusionofsuchsubstancesfromtheposteriorchamberbycrossing
thebloodaqueousbarrier,uvealinflammation,anddueto
disturbanceofthevitreoushumor[5].Thecharacteristicfeatureof
steroidinducedPSCisthatitshowsaberrantmigrationoflens
epithelialcells,proliferation,andsuppressionofdifferencesinthe
lens[6].
JoblingandAugusteyn[4]suggestedthatadministrationofsteroids
causesanalterationinthegrowthfactorsthatarereachingthelens.
Thesealterationsintheexpressionofgrowthfactorsmightbeone
ofthecausesforsteroidinducedcataract.Further,fromother
studies,itwasunderstoodthatthesegrowthfactorsareresponsible
fortheinductionofmatrixmetalloproteinases(MMPs)inthecells
[7,8].Reportsfromseveralotherinvitroandinvivostudiesusing
animals,humanlensepithelialcells,andcelllineculturehave
shownthattransforminggrowthfactorbeta(TGF)andepidermal
growthfactor(EGF)upregulatetheexpressionofMMP2and
MMP9[913].
MMPsrepresentafamilyofendopeptidasesthatarecapableof
degradingtheextracellularmatrix(ECM)moleculesandthereby
maintainingnormalphysiologicprocessessuchasmorphogenesis
andinfluencingcellbiologicactivities[14].Wongandcolleagues
[15]haveshownthatinhibitionofMMPspreventsmigrationoflens
epithelialcells(LECs)andcontractionofthelenscapsule.Itechoes
asimilarfunctionasthatofsteroids,whichinducetheaberrant
migrationoflensepithelialcells.SimilarlyMMPsarealsofoundto

enhancethemigrationoflensepithelialcellsbyanundefined
mechanism.ItwasalsoshownthatmultipleMMPsareexpressedin
thelensandtheirexpressionisalteredinseveralcataract
phenotypes[16].
Intheeye,MMPshavebeenexaminedinseveraloculardiseases
includingretinaldiseases,glaucoma,cornealdisorders,scleritis,
uveitis,pterygium,andcataract[14,1719].Themostwidely
studiedMMPsintheoculartissuesareMMP2andMMP9.Inour
previousstudy,weobservedthatMMP9activitywashigherin
corticalcataractfollowedbyPSCandnuclearcataracts[20].
SincetheroleofMMPsinthegenesisofPSCwasnotunderstood,
inthepresentstudy,weattemptedtoassessthelevelofMMPs
especiallyMMP2andMMP9inlensepithelialcellsaswellasthe
serumofpatientswithPSC.

Methods

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Patientselection
Thisprospectiveobservationalstudycomprised156patientswith
uncomplicatedagerelatedcataractwhounderwent
phacoemulsificationatIladeviCataract&IOLResearchCentre
Ahmedabad,IndiafromMarch,2004toFebruary,2011.Informed
consentwasobtainedfromallpatients.Onlypatientsintheage
groupof1275yearswithpupilsdilatingmorethan7.0mmand
withotherwisenormaleyeswereincludedinthestudy.Inclusion
criteriawereeyeswithPSCandsteroidinducedPSC.Exclusion
criteriawereeyeswithdiabetesmellitus,hypertension,
glaucoma,shallowanteriorchambers,uveitis,highmyopia(axial
length<27.0mm),pseudoexfoliation,traumaticcataract,subluxated
cataract,previousocularsurgery,oculardisease,andallergyto
dilatingdrops.Thetypeofcataractinallthepatientswasrecorded
accordingtothezoneofopacification.Underaslit
lampobservation,thegradeofthecataractwasrecordedbasedon
thedegreeofhardnessusingtheEmeryandLittleclassification
[21].Anexperiencedsurgeon(A.R.V.)performedallthesurgeries
undertopicalanesthesiausingastandardizedtechniquethathas
beendescribed[22]byanexpert.Thesamplesaredistributed
accordingtoexperimentalrequirementasshowninTable1.
Table1
Sampledistributionofsteroid
inducedPSCandnonsteroid
inducedPSCbytechnique.
Patientsmedicalhistory
Adetailedmedicalhistorywascollectedfromthe50steroid
inducedPSCpatients.Themeanageofthepatientswas
40.5716.67(1268years).Toqualifyforthisstudy,thepatients
hadbeonsteroidtherapyforatleast2years.Ofthesethefollowing
patientswereonoralsteroidtherapy:21patientswithrenal
transplantation,8withasthma,2withlungdisease,3witharthritis,
and16withallergy.Thefollowingsteroidswereadministered:
Wysolone(PrednisoloneLupinLtd,Mumbai,MH,Indiaand

Ranbaxy,Gurgaon,Haryana,India),Dexamethasone(Taj
PharmaceuticalsLtd.Mumbai,MH,India),Omnipred
(PrednisoloneAcetateALCONchemicalLtd,Bangalore,KA,
India),Doxophyllin(AtmanPharmaceuticals,Mumbai,MH,India),
andCyclosporin(TajPharma,Mumbai,MH,India).Thesteroid
inducedPSCpatientsdetaileddemographicsshownintheTable2.
Therewere106patientswithnonsteroidPSC.Themeanageof
thesepatientswas53.439.92(2575years).
Table2
detailedsteroidinducedPSC
patientsdemographics.
Sampleprocessingandproteinestimation
BloodwascollectedfrompatientswithsteroidinducedPSC(n=33)
andnonsteroidinducedPSC(n=89)beforetheadministrationof
anesthesia.Fromthesecollectedbloodsamples,serumwas
separatedbycentrifugationat10,000gfor15minat4C.The
serumwasstoredimmediatelyat4C,untilused.
Thecurvilinearcapsulorhexis,approximately5mmindiameter,
wasremovedfromtheanteriorregionofthelenscapsuleduring
phacoemulsificationandstoredimmediatelyat20Cuntilused.
Thesampleswerehomogenizedindividuallyat4Cbyadding
350lofcold0.1Mpotassiumphosphatebuffer(pH7.5).Allthe
samplesweregivenanequalnumberofstrokesduring
homogenizationtoobtainuniformity.Thehomogenatewas
centrifuged(HereausCentrifuge,Buckinghamshire,England,UK)
at10,000gfor15minat4C,andthesupernatantwasstored
immediatelyat4C.Thequantityoftotalproteininallthesamples
wasestimatedbytheLowrymethod[23]usingBSAasastandard.
Proteinconcentrationwasexpressedintermsofmicrogramper
microliter(g/l)ofhomogenate.
Succinylationofgelatin
GelatinwaspurchasedfromSigmaAldrich(St.Louis,MO)and
succinylatedusingtheproceduredescribedpreviously[24].Gelatin
wasdissolvedin50mMSodiumBoratebufferwith10mMCaCl2,
pH7.0forMMP2and50mMboratebuffer,pH8.5forMMP9,at
aconcentrationof20mg/ml.Anequalamountofsuccinic
anhydridewasthengraduallyaddedtothesolutionandthepHof
thereactionwasmaintainedat7.0forMMP2and8.5forMMP9.
Thesuccinylatedgelatinwasthendialyzedextensivelyagainst
50mMsodiumboratebuffer,pH7.0and8.5,respectively.Since
dialysisresultedindilutionofthegelatin,thefinalconcentration
wasdeterminedusingtheLowrysmethod[23].
Succinylatedgelatinassay
Succinylatedgelatinassay[25]wasusedfortheanalysisofMMP2
andMMP9activities.Briefly,about50lofenzymesourceand
200gofSuccinylatedgelatinwasaddedandthefinalvolumewas
madeupto1.5mlwith50mMSodiumBoratebufferandwith
10mMCaCl2,pH7.0forMMP2and50mMboratebuffer,pH
8.5forMMP9.Ablankwithoutsubstratebutwithboratebuffer

andenzymesourcewaspreparedforeachenzymeassay.The
contentswereincubatedat37Cfor30min.About500lof
0.03%solutionof2,4,6trinitrobenzenesulfonicacid(TNBSA
SigmaAldrich)wasthenaddedtothereactionmixtureandallowed
toincubateatroomtemperatureforanother20min.Theoptical
density(OD)ofeachreactionwasobtainedat450nmusinga
PerkinElmerUV/VISspectrophotometer(MolecularDevices,
MenloPark,CA).Theinhibitorycapacity(%)wasevaluatedby
comparingtherelativeactivityoftheenzymeinthepresenceand
absenceoftheMMP2inhibitorIandMMP9inhibitorI.Enzyme
activityintheabsenceoftheinhibitorwasconsideredtobe100%.
AstandardgraphwasplottedusingpurifiedMMP2andMMP9
(SigmaAldrich)andwasusedforcalculationofMMP2and
MMP9activitiesinallthesamples.TheMMP2andMMP9
activitieswereexpressedinnanogrampermicrogram(ng/g)of
totalproteins.
RealtimePCRquantification
TotalRNAwasextractedfromfreshlycollectedcapsulorhexis
samplesusingTRIZOL(InvitrogenGibco,GrandIsland,NY)
accordingtothemanufacturersprotocol.Onemicrogramoftotal
RNAwasreversetranscribedat25Cfor5min,42Cfor60min,
and70Cfor5minin20lof0.5gofoligo(dT),20pmol
primer,3mMofMgCl2,1mMofdNTPs,4lofImpromIITM5
reactionbuffer(Promega,Madison,WI),1unitofRecombinant
RNasinRNaseInhibitorand1lofImpromIIReverse
Transcriptase(Promega).ForquantitativePCR,amplificationswere
performedonaRocheLightCycler480(RocheLtd.,Berlin,
Germany)in20lofreactionusing2lofcDNA,10lofSYBR
GreenIMaster,andgenespecificprimersatthemMconcentration.
Theamplificationwasperformedat95Cfor5minbeforethefirst
cycle,95Cfor5s,and72Cfor30srepeated50times.
QuantitativePCRwasperformedforMMP2andMMP9,and
glyceraldehyde3phosphatedehydrogenase(GAPDH)wasusedas
thehousekeepinggene.Alltheprimersobtainedfrom
SABiosciences(SABiosciences,NewDelhi,India)arelistedin
Table3.ThemRNAlevelswereexpressedasratios,usingthe
deltadeltamethodforcomparingtherelativeexpressionresults
betweensteroidinducedPSCandnonsteroidinducedPSC.The
nonparametricMannWhitneyUtestwasappliedforstatistical
evaluationandpvalues<0.05wereconsideredstatistically
significant.
Table3
DetailsofqRTPCRprimers
obtainedfromSABiosciences.
Immunostaining
AntibodiesforMMP2andMMP9wereobtainedfromNovus
Biologicals(Littleton,CO)andAnaSpec(Fremont,CA),
respectively.Fluorescentlytaggedsecondaryantibodieswere
obtainedfromInvitrogen.Allotherchemicalswerepurchasedfrom
SigmaAldrich(St.Louis,MO).Capsulorhexisspecimenswere
placedonsilane(aminopropyltriethoxysilane)coatedglassslides

withLECsfacingtheslideandfixedin2%paraformaldehydein
phosphatebufferedsaline(PBS)for5min.Thesampleswere
rinsedwithPBSandchilledacetonemethanol(1:1)wasusedfor
furtherfixation.Thecapsuleofthespecimenwaspeeledwiththe
helpofforcepsleavingLECsontheslides.Thesecellswereused
forimmunolocalizationofMMP2andMMP9.Antigenretrieval
ofMMP9wasperformedusingProteinasek(20g/ml)for1min
at37C.AntigenretrievalofMMP2wasperformedusing0.25%
TritonX100for10min.Followingblockingandstandardwashing
procedures,thecellswereincubatedwithRabbitantiMMP2
(1:100)andMouseantiMMP9(1:100)antibodiesfor1hat37C.
ThecellswerethenwashedwithPBST(PBScontaining0.05%
Tween20),andincubatedwithappropriatesecondaryantibodies
taggedwithAlexafluor546andAlexafluor488(1:200dilution
Invitrogen,GrandIsland,NY).4',6diamidino2phenylindole
(DAPI)wasusedtocounterstainthenuclei.Anepifluorescence
microscope(AxioskopeIICarlZeiss,NewYork,NY)wasusedto
observethecellsandtheimagesweredocumentedwithaCCD
camera(Cohu,SanDiego,CA).
Statisticalanalysis
Statisticalcomparisonswereperformedusingnonparametrictests
suchastheKruskalWallisandMannWhitneytests.

Results

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Ofthe156patientswithuncomplicatedcataract,50hadsteroid
inducedPSC(meanage40.5716.67[SD])and106hadnon
steroidalPSC(meanage53.439.92[SD]).
Table4showsthemeanvalueofMMP2andMMP9activitiesin
theLECsandserumofsteroidinducedPSCandnonsteroidal
PSC.Thedifferenceinthemeanactivitybetweensteroidinduced
PSCandnonsteroidalPSCwasstatisticallysignificant(p<0.001).
SteroidinducedPSChadsignificantlyhigheractivityofMMP2
andMMP9intheLECsandserumascomparedwithnonsteroidal
PSC.
Table4
DescriptivestatisticsofMMP2
andMMP9activitiesinLECsand
serumofsteroidinducedPSCand
nonsteroidinducedPSC.
Table5showstheresultsoftheposthocstatisticalanalysisfor
multiplepairedcomparisonsofthemeandifferenceinMMP2and
MMP9activitiesinLECsandserumofsteroidinducedandnon
steroidalPSC.Astatisticallysignificantdifferencewasseen
betweensteroidinducedandnonsteroidalPSC(p<0.001),withthe
highestactivityofMMP2andMMP9intheLECsandserumof
steroidinducedPSC.Theposthocanalysisthereforesupportedthe
ANOVA.

Table5
MultiplecomparisonsusingtheBonferronitestfor
posthocanalysisofMMP2andMMP9activities
inLECsandserumofsteroidinducedPSCand
nonsteroidinducedPSC.
Table6showstheratioofMMP2activityintheLECsandserum
ofsteroidinducedandnonsteroidalPSC.MMP2activitywas
increasedby0.4and0.18foldintheLECsandserumofsteroid
inducedPSCrespectively.Further,thistablealsoshowstheratioof
MMP9activityintheLECsandserumofsteroidinducedandnon
steroidalPSC.MMP9activitywasincreasedby0.4and0.07fold
intheLECsandserumofsteroidinducedPSCrespectively.
Table7comparesthelevelsofMMP2andMMP9activityinthe
LECsandserumofsteroidinducedPSC.TheMMP2ratio
betweentheLECsandserumdecreasedinsteroidinducedPSC
(1:25)ascomparedtononsteroidalPSC(1:29).However,the
MMP9ratiobetweentheLECsandserumincreasedinsteroid
inducedPSC(1:124)ascomparedtononsteroidalPSC(1:98).
Table6
RatioofMMP2andMMP9
activitiesinlensepithelialcells
(LECs)andserumofnonsteroid
andsteroidinducedposterior
subcapsularcataract(PSC).
Table7
RatioofMMP2andMMP9
activitiesinLECsversesserumof
nonsteroidandsteroidinduced
PSC.
Tovalidatetheresultsobtainedfromthesuccinylatedgelatinassay,
wehaveusedtwotechniques,namelyqRTPCRand
immunolocation.ThemRNAexpressionofMMP2andMMP9
wasevaluatedusingqRTPCRintheLECsofsteroidinduced
PSCandnonsteroidalPSC.Therelativequantificationcompared
tothehousekeepinggeneGAPDHshowedasignificant
upregulationinmRNAexpressionofMMP2andMMP9in
steroidinducedPSC(Figure1)comparedtononsteroidalPSC
withpvaluesof0.001and0.04,respectively.Immunolocalization
ofMMP2andMMP9intheLECsofsteroidinducedPSCand
nonsteroidalPSCfurthervalidatedtheresultsofthesuccinylated
gelatinassay.MMP2wasobservedinthenucleusandcytoplasm
oftheLECs(Figure2A,D)andMMP9wasobservedina
punctuatedforminthecytoplasmoftheLECs(Figure3A,D).The
MMP2andMMP9levelswerehigherintheLECsofsteroid
inducedPSC(Figure2DandFigure3D).
Figure1

ExpressionofMMP2andMMP9
mRNAinsteroidandnonsteroid
inducedPSC.Thegraphrepresents
thequantitativeanalysisofMMP2
andMMP9mRNAexpressionand
botharehigherintheLECsof
steroidinducedPSCascomparedto
nonsteroidinducedPSC....
Figure2
LocalizationofMMP2inlens
epithelialcells(LECs)ofhuman
senilecataracts.MMP2levelsare
higherinthecytoplasmofLECsof
steroidinducedPSCandMMP2
expressionwasalsoobservedin
someofthecellnuclei.Nonsteroid
inducedPSCSPSCsteroidinduced
...
Figure3
LocalizationofMMP9inlens
epithelialcells(LECs)ofhuman
senilecataracts.TheMMP9levels
arehigherinthecytoplasmofLECs
ofsteroidinducedPSC.PSCnon
steroidinducedPSCSPSCsteroid
inducedPSCLECslensepithelial
cells,scalebar...

Discussion

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ThedevelopmentofsteroidinducedPSCisacomplexmechanism
andseveralattemptshavebeenmadetounderstanditsgenesis,but
ithasnotbeenconvincinglyexplained.However,inrecentyears,
osmoticimbalance,oxidativedamage,imbalanceinoculargrowth
factors,andsubsequentchangesinthecellularenzymaticlevels
havebeenrecognizedaspromisingfactorsinthedevelopmentof
steroidinducedPSC[4,6].ThereisgrowingevidencethatMMPs
alsoplayanimportantroleinmanydiseaseprocesses,butmostof
theseenzymesarelikelytobeactiveatalocaltissuelevel[26].
Whileotherstudieshavereportedandlocalizedthepresenceof
MMPsinnormalaswellasincataractouslenses,theroleofMMPs
inthegenesisofcataracthasnotbeenreported[27,28].Several
studieshavesuggestedthatgrowthfactors[713]andestrogen,a
steroidalhormone,areresponsiblefortheinductionofMMPsin
cells[29].Afewstudieshavereportedthatoxidativestress[27]and
growthfactors(TGF)induceMMP2andMMP9activitiesin
lensepithelialcells[11,12,30].Oxidativestresshasbeenshownto
upregulatetranscriptionandMMPactivityinseveraltypesofcells,
includinghepaticstellatecells[31],humanmonocytes[32],and
humancoronarysmoothmusclecells[33].YeandAzar[34]
suggestedthatthepenetrationandmigrationofseveralcelltypes
throughtheextracellularmatrix(ECM)requirestheactivityof
MMPs.Thus,itcanbesuggestedthattheabnormalinductionof

MMPsduetotheadministrationofsteroidsorduetogrowthfactors
mayresultinthedegradationofextracellularmatrixsubstancesthat
inturnhelpinthemigrationofundifferentiatedanteriorepithelial
cellstotheposteriorpoleformingPSC.
Inourpreviousstudy,wehavequantifiedthelevelofMMP9
activityindifferenttypesofcataractousLECs,andMMP9activity
wasfoundtobehighincorticalcataract[20].However,nospecial
effortwasmadetoassociatethelevelsofMMP9activitywiththe
clinicallyobservedsteroidinducedPSC.Hencethepresentstudy
wasdesignedtomeasurethelevelsofMMP2andMMP9
activitiesintheLECsandserumofsteroidinducedPSC.Wehave
selectedserumasthesecondsystem,sinceweintendedtoanalyze
whethertheincreasedlevelofMMP2andMMP9activitiesinthe
LECswasduetothenormalphysiologicstatusorduetothe
administrationofdrugs.
Inthepresentstudy,individualanteriorcapsularsamples(LECs)
obtainedbyanteriorcapsulorhexisaswellasserumofthesame
patientswereused.AsLECsareknowntohavelowprotein
content,asensitivemethodwasrequired.Itiswellknownthatthe
levelsofexpressionandactivityofMMPscanalsobedetermined
byELISA[35],flowcytometricanalysis[36],andmRNA
(mRNA)expressionanalysis[28].Weusedsuccynylatedgelatin
assay,qRTPCR,andimmunolocalizationtoanalyzethe
expressionofMMP2andMMP9insteroidinducedandnon
steroidalPSC.Baragietal.[25],whileevaluatingthesuccinylated
gelatinassay,foundthatthismethodreliesupontheexposureof
primaryaminesafterthesubstratehasbeendegradedbygelatinases
andalsoquantifiesthespecificactivity.Further,theother
advantagesofthisassaywerethattheprotocolrequiredonlysmall
quantitiesofsampleanditwasfoundtobesensitiveenoughto
detectquantitiesaslowas0.1ngofMMP2andMMP9.Inthe
presentstudyandfromobservationsmadeinourpreviousstudy,we
foundsuccinylatedgelatinassayasuitableandsensitivemethodfor
measuringMMPactivityinLECs.TheRTPCRresultsinthe
presentstudyshowedhigherexpressionofbothMMPsintheLECs
ofsteroidinducedPSC.Similarlywealsoobservedhigher
immunohistochemicallocalizationofMMP2andMMP9inthe
cytoplasmofsteroidinducedLECs.
TheresultsfromthepresentstudyrevealedanincreaseinMMP2
andMMP9activitiesintheLECsandserumofsteroidinduced
PSC.Thepatientsrecruitedinthepresentstudyreceivedsteroids
suchasDexamethasone,Wysolone(Prednisolone),Omnipred
(PrednisoloneAcetate),andDoxophyllin(topicalaswellasoral)as
wellastheimmunosuppressivedrug(Cyclosporin).Although
severalstudiesreporttantalizingresultsclaimingthatsteroids
influencethemodulationofMMPactivity,areportbyChoeand
coworkers[37]suggeststhatmethylprednisolonecauses
emphysemalikechangesinthelungsofratsduetoanincreasein
MMPactivity.Further,cyclosporineA,whichistakenasan
immunosuppressivedrugafterkidneytransplantation,wasalso
foundtoupregulatetheexpressionofMMP2inrathearts[38]and
bothMMP2andMMP9inculturedhumantrophoblastcells[39].
AnotherstudybyPoulalhonandcolleagues[40]reportedthatthe

immunosuppressivedrug,rapamycin,enhancedtheexpressionof
MMP1.Thus,wepresumethattheincreasedactivityofMMP2
andMMP9inLECsmaybeduetotheadministrationofsuch
drugs.
InthecaseofPSC,thelevelsofMMP2activitybetweentheLECs
andserumwasintheratioof1:29,whichwasshowntobereduced
to1:25inthecaseofsteroidinducedPSC.ThereductioninMMP
2activityintheserumofsteroidinducedPSCmaybeattributedto
theadministrationofsteroids.However,theratioofMMP9activity
betweentheLECsandserumofsteroidinducedPSCwashigher
thannonsteroidalPSC.Sinceseveralstudieshavedocumenteda
riseinplasmalevelsofMMP9incancer,hepaticandlungdiseases
andrheumatoidarthritis[4144],subjectswithahistoryofthese
conditionswereexcludedfromthisstudy.Thereasonforthe
observedincreaseinthelevelofcirculatingMMP9intheserumof
steroidinducedcataractcouldnotbeunderstood.However,itcould
beattributedtothecombinedeffectofsteroidsand
immunosuppressivedrugs.Nevertheless,thecauseofincreased
MMP9activityinsteroidinducedPSCremainsunclear.Itis
possiblethatcirculatingleukocytes,macrophages[45],vascular
endothelialcells[46],fibroblastsandsmoothmusclecells[47,48]
maycontributetotheincreasedlevelsofMMP9activity.The
absenceofanincreaseinMMP2levelsintheserummayreflectits
moreconstitutiveexpression,whereasMMP9levelsaremore
responsivetoexternalfactorssuchasreactiveoxygenspecies[49],
inflammatorycytokines[49],immunosuppressivedrugs[38],and
othersteroids[37].
Insummary,thisisthefirststudythatrevealsanassociation
betweenMMPsandsteroidinducedPSC.MMP2andMMP9
activitiesintheLECsandserumsignificantlydifferedbetween
steroidinducedPSCandnonsteroidalPSC.MMP9activitywas
higherintheserumofsteroidinducedPSCserumcanbeusedasa
secondarysourcetodetectsteroidinducedPSC.Werealizethatitis
difficulttomakewiderangingconclusions/assumptionsbasedon
theseobservationsinviewofthesmallsamplesizeused.However,
thisisanimportantstartingpoint.Largerscalefuturestudieswillbe
requiredtoclarifythesefindingsincludingthelinkbetweengrowth
factorsandsystemicinflammatorymarkers.

Acknowledgments

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Theauthorsthankthepatientandhisfamilymembersfor
participatinginthestudyandacknowledgetheICIRCresearchlab
membersandRaghudeepEyeClinicsDoctorsandstaff.Thestudy
wassupportedbygrant,DepartmentofSciencesandtechnology
(DST),India.Dr.BhagwatAlapureisarecipientoftheSenior
ResearchFellowship(SRF)fromIndianCouncilofMedical
Research(ICMR),India.

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ArticlesfromMolecularVisionareprovidedherecourtesyofEmory
UniversityandtheZhongshanOphthalmicCenter,SunYatsen
University,P.R.China

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