The antiviral drug tenofovir, an inhibitor of Pannexin-1-mediated ATP release, prevents liver and

22 Pages·2017·12.18 MB·English

RESEARCHARTICLE
The antiviral drug tenofovir, an inhibitor of
Pannexin-1-mediated ATP release, prevents
liver and skin fibrosis by downregulating
adenosine levels in the liver and skin
JessicaL.Feig1☯,AranzazuMediero1,2☯,CarmenCorciulo1,HailingLiu1,JinZhang1,3,
MiguelPerez-Aso1,LauraPicard1,TuereWilder1,BruceCronstein1*
a1111111111 1 DivisionofTranslationalMedicine,DepartmentofMedicine,NYU-LangoneMedicalCenter,NewYork,New
York,UnitedStatesofAmerica,2 BoneandJointResearchUnit,IIS-Fundacio´nJime´nezD´ıazUAM,Madrid,
a1111111111
Spain,3 DepartmentofImmunologyandRheumatology,LiHuiliHospital,MedicalSchoolofNingbo
a1111111111
University,Ningbo,China
a1111111111
a1111111111 ☯Theseauthorscontributedequallytothiswork.
*[email protected]
Abstract
OPENACCESS
Citation:FeigJL,MedieroA,CorciuloC,LiuH,
Background
ZhangJ,Perez-AsoM,etal.(2017)Theantiviral
drugtenofovir,aninhibitorofPannexin-1-mediated
Fibrosingdiseasesarealeadingcauseofmorbidityandmortalityworldwideand,therefore,
ATPrelease,preventsliverandskinfibrosisby
thereisaneedforsafeandeffectiveantifibrotictherapies.Adenosine,generatedextracellu-
downregulatingadenosinelevelsintheliverand
skin.PLoSONE12(11):e0188135.https://doi.org/ larlybythedephosphorylationofadeninenucleotides,ligatesspecificreceptorswhichplay
10.1371/journal.pone.0188135 acriticalroleindevelopmentofhepaticanddermalfibrosis.Resultsofrecentclinicaltrials
Editor:MatiasAAvila,UniversityofNavarraSchool indicatethattenofovir,awidelyusedantiviralagent,reverseshepaticfibrosis/cirrhosisin
ofMedicineandCenterforAppliedMedical patientswithchronichepatitisBinfection.Belongingtotheclassofacyclicnucleosidephos-
Research(CIMA),SPAIN
phonates,tenofovirisananalogueofAMP.Wetestedthehypothesisthattenofovirhas
Received:July26,2017 directantifibroticeffectsinvivobyinterferingwithadenosinepathwaysoffibrosisusingtwo
Accepted:November1,2017 distinctmodelsofadenosineandA2AR-mediatedfibrosis.
Published:November16,2017
Methods
Copyright:©2017Feigetal.Thisisanopen
accessarticledistributedunderthetermsofthe Thioacetamide(100mg/kgIP)-treatedmiceweretreatedwithvehicle,ortenofovir(75mg/kg,
CreativeCommonsAttributionLicense,which SubQ)(n=5–10).Bleomycin(0.25U,SubQ)-treatedmiceweretreatedwithvehicleorteno-
permitsunrestricteduse,distribution,and
fovir(75mg/kg,IP)(n=5–10).AdenosinelevelsweredeterminedbyHPLC,andATP
reproductioninanymedium,providedtheoriginal
releasewasquantitatedasluciferase-dependentbioluminescence.Skinbreakingstrength
authorandsourcearecredited.
wasanalysedandH&Eandpicrosirusred-stainedslideswereimaged.Pannexin-1expres-
DataAvailabilityStatement:Allrelevantdataare
sionwasknockeddownfollowingretroviral-mediatedexpressionofofPannexin-1-specific
withinthepaperanditsSupportingInformation
files. orscrambledsiRNA.
Funding:Thisworkwassupportedbygrantsfrom
Results
theNationalInstituteofArthritis,Musculoskeletal
andSkinDiseases(RO1-AR054897andRO1-
Treatmentofmicewithtenofovirdiminishedadenosinereleasefromtheskinofbleomycin-
AR056672),theNYU-HHCClinicaland
treatedmiceandtheliverofthioacetamide-treatedmice,modelsofdiffuseskinfibrosisand
TranslationalScienceInstitute(UL1-TR000038
fromtheNationalCenterforAdvancing hepaticcirrhosis,respectively.Moreimportantly,tenofovirtreatmentdiminishedskinand
PLOSONE|https://doi.org/10.1371/journal.pone.0188135 November16,2017 1/22
Tenofovir,fibrosis,PANX-1andadenosinelevels
TranslationalSciences)andGilead liverfibrosisinthesemodels.Tenofovirdiminishedextracellularadenosineconcentrations
Pharmaceuticals.DrMedieroisfundedbyInstituto byinhibiting,inadose-dependentfashion,cellularATPreleasebutnotincellslackingPan-
deSaludCarlosIIIandFondoEuropeode
nexin-1.
DesarrolloRegional(FEDER)throughthe“Miguel
Servet”program.
Conclusions
Competinginterests:A.M.andB.C.havefileda
patentonuseofadenosineA2ARagoniststo Thesestudiessuggestthattenofovir,awidelyusedantiviralagent,couldbeusefulinthe
preventprosthesisloosening(pending)anda
treatmentoffibrosingdiseases.
patentontheuseofAntibodiesagainstNetrin-1for
thetreatmentofbonediseases.C.C.andB.C.have
filledapatentontheuseofA2ARagonistto
preventosteoarthritis.J.F.,M.P-A,T.W.donot
haveanydisclosures.B.C.holdspatentsnumbers
5,932,558;6,020,321;6,555,545;7,795,427; Introduction
adenosineA1RandA2BRantagoniststotreatfatty
liver(pending);adenosineA2ARagoniststo Fibrosingdiseasesarealeadingcauseofmorbidityandmortalityworldwide[1].Fibrosis,the
preventprosthesisloosening(pending).B.C.isa excessaccumulationofextracellularmatrix(ECM),affectsavarietyoforgansincluding,
consultantforBristol-MyersSquibb,AstraZeneca, amongothers,theliver,lung,andskin.ThestructuralcomponentsoftheECM,growthfac-
Novartis,CanFiteBiopharmaceuticals,Cypress
tors,cytokines,chemokines,andproteases,aswellascentralsignalingcascadesimplicatedin
Laboratories,Regeneron(Westat,DSMB),
fibrogenesisandfibrolysis,arenearlyidenticalinthesedifferenttissues[2].Thespecificetiol-
Endocyte,Protalex,Allos,Inc.,Savient,Gismo
Therapeutics,AntaresPharmaceutical,Medivector, ogyoffibrosisinmostorgansisonlyincompletelyunderstoodnonetheless,anantifibrotic
KingPharmaceutical,Celizome,Tap agent,pirfenidonewasrecentlyapprovedforthetherapyofinterstitialpulmonaryfibrosis.No
Pharmaceuticals,PrometheusLaboratories, suchagentshaveenteredtheclinicforthetreatmentofhepaticcirrhosisorskinfibrosis.
Sepracor,Amgen,Combinatorx,KyowaHakka,
Agrowingbodyofworkfromdifferentlaboratorieshasimplicatedadenosineanditsrecep-
Hoffman-LaRocheandAvidimerTherapeutics.BNC
torsinthepathogenesisoffibrosisinthelung,skin,liverandheart[3–5].Adenosine,anextra-
hasstockinCanFiteBiopharmaceuticals.Onbehalf
cellularnucleosidewhichisgeneratedbythedephosphorylationofATP,hasbeenshownto
ofmyroleasaconsultantforseveralcompanies
doesnotalterouradherencetoPLOSONEpolicies bothstimulatewoundhealingandpromotefibrosis[6–8].CellstransportATPintotheextracel-
onsharingdataandmaterials. lularspaceviathespecifictransporterPannexin-1andothertransportersandmanycelltypes
expressnucleosidetriphosphatephosphohydrolase(NTPP,CD39)andecto-5’nucleotidase
Abbreviations:3TC,Lamivudine;AOPCP,α,β-
Methylene-ADP;CD73,ecto-5’nucleotidase;ECM, (CD73)ontheirsurfacewhich,sequentiallyconvertATPtoAMPandadenosine.Extracellular
extracellularmatrix;TAA,Thioacetamide. adenosineactsateitherA (A2AR)orA (A2BR)receptors,membersofthelargefamilyofG
2A 2B
proteincoupledreceptors,todirectlystimulatefibroblastproductionofextracellularmatrixand
growthfactors,leadingtofibrosis[3].IthasbeenobservedthatPannexin-1andextracellular
ATPareupstreamregulatorsofAngiotensinIIandTGF-βandtriggerfibrosisinmechanical
stretch-inducedcardiacfibrosis[9].Inducedischemiarapidlyincreasestheglycosylationof
Pannexin-1andincreasesitstraffickingtotheplasmamembrane.ATPreleasethroughPan-
nexin-1channelsisinvolvedincardiacfibrosisfollowingmyocardialinfarction[10].Baoetal.
demonstratedthatPanx-1hemichannelsprovidestheligandfortheadenosineA2Areceptors
thatplaysaroleintheinhibitorysignalatthebackoftheneutrophil,andinhibitionofPanx-1
hemichannelsblockedA2AreceptorstimulationpreventingcAMPaccumulationandimpair-
ingmigrationandpolarizationofneutrophils[11].Deletionoftheenzymesinvolvedinadeno-
sineproduction(eitherCD73orCD39orboth)preventshepaticandskinfibrosisinmurine
modelsanddeletionorblockadeofeitherA2AorA2Breceptorspreventshepatic,dermalor
peritonealfibrosisaswell[12].Moreover,micelackingadenosinedeaminasehaveamarked
increaseinextracellularadenosineandsufferfromexcessfibrosisinlung,skinandotherorgans
andblockadeordeletionofA2ARandA2BRpreventsthisfibrosis[13,14].Evidencefromepi-
demiologicandcasecontrolstudiesalsosupportstheroleofadenosinereceptorsinfibrosis.
Theprincipalpharmacologiceffectofthemostwidelyconsumeddrugintheworld,caffeine,is
non-selectiveblockadeofadenosinereceptors.Caffeineisthemajorpharmacologicagentin
coffeeandconsumptionofcoffeeandcaffeine(insoftdrinks)isassociated,inadose-dependent
fashion,withreducedriskofdeathandfibrosisfromliverdisease(Reviewedin[15]).
PLOSONE|https://doi.org/10.1371/journal.pone.0188135 November16,2017 2/22
Tenofovir,fibrosis,PANX-1andadenosinelevels
Arecentreportonthelong-termfollow-upofpatientsinaclinicaltrialoftenofovirforthe
treatmentofhepatitisBsuggeststhattenofovirtherapy,incontrasttootherantiviralagents,
reducesorreverseshepaticfibrosis[16].Moreover,histologicdatafromaprospectivestudyin
HBV/HIVco-infectedpatientstreatedwithtenofovirforjustover2yearsdemonstratedfalling
fibrosisscores,atrendnotobservedwithotherantivirals[17].Oncetenofovirisinternalized
intocellsitissubsequentlyphosphorylatedtotheactivemetabolite,tenofovirdiphosphate
[18].InamechanismsimilartothatofNRTIs(Nucleosidereversetranscriptaseinhibitors),
tenofovirdiphosphatecompeteswithdeoxyadenosine50-triphosphate,forincorporationinto
newlyformingHIVDNA.Onceincorporated,terminationoftheelongatingDNAchain
ensues,andDNAsynthesisisinterrupted[19].Theseobservationssuggestthattenofovir,,
mighthavedirectanti-fibroticeffects.
Therefore,wetestedtheeffectoftenofovirtreatmentonhepaticanddermalfibrosisintwo
differentmurinemodelsandexploredthemechanismbywhichtenofovirtreatmentblocks
fibrosisinthesemodels.WereportherethattenofovirblocksPannexin-1-mediatedATP
releaseandpreventshepaticanddermalfibrosisinmurinemodelsinassociationwitha
markedreductionofadenosinereleasebythoseorgans.
Materialsandmethods
Reagents
Bleomycinsulfate(Hospira)waspurchasedas15Ulyophilizedpowder,andreconstituted
accordingtothemanufacturer’sguidelineswithPBSat2.5U/mL.Thioacetamide(TAA)was
purchasedfromSigma.TenofovirwaspurchasedfromSequoiaResearchProducts(UK).
Tenofovirwasdissolvedbyadditionof0.1NNaOHtoafinalpHof7.0andfiltersterilized
(poresize,0.2μm)aspreviouslydescribed[20].Tenofovir(10mMstocksolution)wasalso
kindlyprovidedbyGilead.MalachitegreenkitwaspurchasedfromAnaspec.Alkalinephos-
phatasecolorimetrickitwaspurchasedfromAbcam.ATPdeterminationkit(A22066)was
purchasedfromMolecularProbes.
Mice
InvivoexperimentswereconductedusingC57BL/6malemicethatwereupto14weeksoldby
thetimeofsacrifice.Animalcareandexperimentalprocedureswereperformedinaccordance
withtheInstitutionalAnimalCareandUseCommitteeofNYUSchoolofMedicineand
NationalInstitutesofHealthguidelinesandwereapprovedbytheInstitutionalAnimalCareand
UseCommitteeofNYUSchoolofMedicine.Experimentswereperformedinmalemice,since
wehavepreviouslyobservedthatbreakingtensionandhydroxyprolinecontentweremorecon-
sistentintheskinofmalemicethantheirfemalecounterparts,inagreementwithpriorstudies
[21].Micehadfreeaccesstofoodanddrinkandwerehousedinafacilitywitha12hlight/dark
cyclewithcontroledhumidityandtemperature.Animalsweremonitoreddailytoassesstheir
healthandbehavior.Micethatweremoribund,appearedhunchedoverorshowedothersignsof
severestresswereeuthanized.Wecarriedouttheseexperimentswith10miceineachgroupfor
eachexperimentaswiththisnumberofmicewehave80%powertodetectaslittleasa25%dif-
ferencewithastandarddeviationof15%usinga2sidedanalysisanda95%confidenceinterval.
Dosedeterminationofantiviraltherapy
Dosedeterminationwasperformedviaconversionofanimaldosestohumanequivalentdoses
basedonbodysurfacearea[22].ConsultationwithGileadSciencessupportedtheuseofsuch
dosesinmurinemodels.
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Tenofovir,fibrosis,PANX-1andadenosinelevels
Experimentaldesign:Antiviraltherapy/administrationoftenofovirto
micesubjectedtofibrosingagents
Inanestablishedmodelofadenosine-mediatedskininjury[7],skinfibrosiswasinducedwith
bleomycinchallenge(2.5U/mL,0.1ml).Usinga27-gaugeneedle,100μLoffilter-sterilized
bleomycin(dosedeterminedfrompilotexperiments)orPBSwasinjectedsubcutaneouslyinto
theupperbackofthemice.Injectionsinthesamesitewerecarriedoutonalternatedaysfor18
days.C57BL/6miceweretreatedwithtenofovir(75mg/kg)administeredintraperitoneally
daily,duringthecourseofbleomycintreatment(n=10micerandomizedforeachtreatment).
Todeterminethespecificityoftenofovir,othergroupsofanimalsweretreatedwithlamivu-
dine(3TC)(100mg/kg),anotherreversetranscriptaseinhibitorwhichisananalogueofcyti-
dine,administeredsubcutaneously(n=10).
Inanestablishedmodelofadenosine-mediatedliverinjury,liverfibrosiswasinducedby
thioacetamide(TAA[23]).Briefly,C57BL/6miceweretreatedwiththeknownhepaticfibro-
sis-inducingagentTAA(100mg/kginPBS,intraperitoneally,threetimesweeklyfor8weeks).
Dailyantiviraltreatmentswithtenofovir(75mg/kg)wereadministeredsubcutaneously,dur-
ingthecourseofinjury.Controlmicereceivedintraperitonealinjectionsofvehicleorappro-
priatedrugtreatment(n=10eachtreatmentinarandomizedprocedure).
Dermalmorphometricmeasurements
Dermalmorphometricmeasurementswereperformedaswehavepreviouslydescribed[7,24].
Followingsacrificethedorsalskinoftheanimalswasshaved.Skinthicknesswasmeasuredon
12mmpunchbiopsiesobtainedfromtheback.Fourdifferentskinmeasurementswere
recordedpermouse,andanaverageofthoserecordingsweremade.Breakingstrengthofthe
skinwasmeasuredonthepunchbiopsiesusingatensiometer(Mark-10SeriesEGDigital
ForceGauge,Mark-10CorporationCopiague,NY).Forcepswereclampedatthefurthermost
extremesofthebiopsysampleandthepointofmaximalstressbeforetearingofthebiopsywas
recorded[7,24].
Histology,picrosiriusredstaining,andscarindexdetermination
Paraffinsectionswerestainedwithhematoxylinandeosin(H&E)andpicrosiriusredasper
thePuchtlermethodaspreviouslydescribed[24]orimmunohistochemistrywithantibodies
α-SMA(Abcam,Cambridge,MA).Endogenousperoxidaseactivitywasblockedwithhydro-
genperoxide.Tissuesectionsweredigestedwithalkalineendopeptidasefor12minutesat
42˚Candprimaryantibodieswereincubatedovernightatroomtemperature.Primaryanti-
bodywasdetectedwithsecondaryanti-rabbit(SantaCruzBiotechnologyInc,SantaCruz)fol-
lowedbyFast3’3’-Diaminobenzidine(Sigma-Aldrich,MOUSA)development,with
hematoxylinnuclearcounterstaining.Appropriatenegativecontrolswereincludedwiththe
studysections.SlideswereacquiredusingaLeicaSCN400DigitalSlideScanner.Cellswere
countedusingtheSlidepathDigitalImageHub(DIH).Siriusredstainingwasperformedto
evaluatethedegreeoffibrosisinthespecimens,asestablished[25].Birefringencepatternand
huewereevaluatedusingpolarizedlightmicroscopytodeterminecollagendensity,patternof
deposition,andmaturity.Normaltissuesdisplayfinecollagenbundleswithayellow-green
birefringenceinarandompatternwhilesofttissuefibrosisresultsinformationofthicker,par-
allelcollagenbundleswithanorange-redbirefringence[26].Thescarindexisaquantitative
analysisoffibrosiscalculatedbycomparingtheratiooforange-redtoyellow-greenstaining
(highervaluesrepresentincreasedscarring)usingSigmaScanPro5softwarewithaminimum
of3–6sectionsperanimal(n=5animals/group)[27,28].Thesameredorgreencolor
PLOSONE|https://doi.org/10.1371/journal.pone.0188135 November16,2017 4/22
Tenofovir,fibrosis,PANX-1andadenosinelevels
thresholdwasappliedtosectionsofthesamesizeforalltheimages.Resultswereexpressedas
redorgreenpixels/highpowerfield(hpf)aswellasacomprehensivescarindexindicativeof
thedegreeoffibrosis.
Hepaticsections(5cross-sectionsperliver)wereharvestedandstainedwithH&Eorpicro-
siriusred,asdescribedpreviously[29].Digitizedphotomicrographs(entirecross-sectionsat
×20;5sectionsperliver)werequantitatedfortotalareaofpicrosiriusredstainingusingSig-
maScanProsoftwarev.5.0.0(SPSS,Chicago,IL,USA).Colorthresholdswereappliedfor
fibroticandtotalhepaticarea.Fibrosiswascalculatedasapercentageoftotalhepaticareaand
expressedastheaverageofrandomlyselectedtissuesectionsfromeachliver(n=9-10/experi-
mentalgroup);(n=5/controlgroup).Resultswereexpressedas%fibrosisoftotalliverarea.
Toxin-inducedadenosinereleaseinmice
C57BL/6micewerepretreatedwithvehicleortenofovir(75mg/kg)intraperitoneallyorsubcu-
taneouslyfortheskinandliverexperiments,respectively.Sincethehalf-lifeoftenofoviris
between16–18hours[18,30,31],itwasimperativeforlevelstoachievesteadystateafter5
half-lives.Aftertwodaysofpretreatmentwithantivirals,aninjectionofbleomycin(0.25U,
SubQ)orTAA(100mg/kg,intraperitoneally)wasadministeredtoinduceanacutetoxin-
inducedadenosinereleaseresponse.Experimentswereperformedwithatleast5animals/
group.
Twenty-fourhoursaftertheinsult,12mmskinbiopsiesweretakenandweighedsothattis-
suespecimensof~40mgwereanalyzed.Briefly,skinbiopsieswerewashedinPBScontaining
antibiotics(penicillin200U/L,streptomycin200ug/L,andfungizone50ug/L),cutintosmall
pieces,andincubatedinDMEMat37˚C,5%CO .IntheanimalstreatedwithTAAthelivers
2
wereharvestedandspecimensof~40mgwereanalyzed.HepaticsectionswerewashedinPBS
containingantibiotics,asabove,cutintosmallpiecesandincubatedinDMEMat37˚C,5%
CO .After4hoursofincubation,supernatantswerecollectedforadenosinedetermination.
2
Quantificationofadenosinelevelsbyhigh-pressureliquid
chromatography(HPLC)
AdenosinewasextractedfromsupernatantsandquantitatedbyHPLC,aswehavepreviously
described.Proteinsinthesupernatantsweredenaturedbyadditionoftrichloroaceticacid
(10%vol/vol).Thetrichloroaceticacidwasextractedwithfreon-octylamine,andthesuperna-
tantswerecollectedandstoredat-80˚Cbeforeanalysis.Theadenosineconcentrationofthe
supernatantswasdeterminedbyreverse-phaseHPLC[32].Briefly,sampleswereappliedtoa
CI8μBondapackcolumn(WatersChromatographyDiv.,Milford,MA)andelutedwitha
0–40%-lineargradient(formedover60min)of0.01Mammoniumphosphate(pH5.5)and
methanol,witha1.5ml/minflowrate.Adenosinewasidentifiedbyretentiontimeandthe
characteristicUVabsorbancespectrum,andtheconcentrationwascalculatedbycomparison
tostandards.Insomeexperiments,theadenosinepeakwasdigestedbytreatmentwithADA
(0.15IU/ml,30minat37˚C)toconfirmthatthepeakidentifiedcontainedonlyadenosine.All
sampleswereruninduplicate,andresultswereexpressedasadenosineconcentration(nM)
per12mmbiopsy,tonormalizeadenosinetothenetweightofthesample.
Quantificationofdermalhydroxyprolinecontent
Hydroxyprolinecontentintissuespecimenswasmeasuredcolorimetricallyasdescribedprevi-
ously[33,34].Tissuespecimenswerehydrolyzedin12NHClat120˚C.Hydrolysateswerefil-
teredandneutralizedtopH7withNaOH(phenolphthaleinaddedtoensureadequate
neutralization).Samplesweremixedwithchloramine-Tsolution(1.4%chloramine-T,10%N-
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Tenofovir,fibrosis,PANX-1andadenosinelevels
propanol,and80%citrate-acetatebuffer).Themixturewasincubatedfor20minutesatroom
temperature.Ehrlich’ssolution(DimethylaminoBenzaldehydeP-Dmba,Isopropanol,Per-
chloricAcid)wasaddedandthesampleswereincubatedat60˚Cfor20minutes.Absorbance
wasmeasuredat560nm.Standardcurves(0to50μg)weregeneratedforeachexperiment
usingreagenthydroxyprolineasastandard.Resultswereexpressedasμgofhydroxyproline
permgoftissue.
Permanentknockdownofcellulartransportersandenzymes
RAW264.7andHepG2cellswereused.TotransfectshRNA,RAW264.7cellsandHepG2cells
(15,000cells/ml)wereplatedand24hourslatercellswereincubatedinthepresenceofHexadi-
methrineBromide(4μg/ml)and108lentiviraltransductionparticlescorrespondingtomouse
Pannexin-1(SHCLNV-NM_019482),mouseConnexin-43(SHCLNV-NM_010288.3),human
Pannexin-1(TRCN0000155349),humanAK4(TRCN000037554),humanNME2
(TRCN000010110)andhumanscrambled(SHC002)withpuromycinselectionmarker,for
another24hourstoallowtransfection.MediawasthenreplacedwithαMEMcontainingpuro-
mycin(1μg/ml),changingthemediaeverythreedaysuntilselectedclonesformed.These
cloneswereisolatedandexpandeduntilconfluence.ScrambledshRNA(SHC002V)wasused
ascontrol.Permanentlysilencedclonesarekeptincultureunderpuromycinselection.
DeterminationofATPReleaseintothesupernate
ExtracellularATPwasdeterminedinRAW264.7,murinemacrophages,andHepG2,human
hepatocytes,celllinesusingabioluminescenceassayinwhichATP-dependentgenerationof
lightbyrecombinantfireflyluciferaseanditssubstrateD-luciferinwasmeasuredusingan
ATPdeterminationkit(A22066)purchasedfromMolecularProbesandassayswereper-
formedaccordingtomanufacturer’sprotocol.Allassayswereperformedintriplicate.
Enzymeactivityassays
Extracellularphosphataseassayswereperformedusingthemalachitegreenassay,accordingto
themanufacturer’sprotocol.Briefly,AMPorATPsubstratewasaddedat100μMtocellsor
recombinantenzyme.AOPCP,aknownCD73inhibitor,wasusedasacontrol.Aphosphate
freebuffercontaining2mMmagnesiumchloride,120mMsodiumchloride,5mMpotassium
chloride,10mMglucose,and20mMHEPESwasused.Resultswereobtainedcolorimetrically
(λ=620nm)andcomparedtoastandardcurveofknownphosphateconcentrations.Alkaline
phosphataseassayswereperformedusingacolorimetrickitsuppliedbyAbcam,accordingto
themanufacturer’sprotocol.Briefly,recombinantenzymewaspretreatedwithadefoviror
tenofovirfor15minutes,andtheconversionofp-nitrophenylphosphatetop-nitrophenolwas
determinedcolorimetrically(λ=405nm).Opticaldensitywasrecordedandcomparedtoa
standardcurveofknownp-nitrophenolconcentrations.
Collagen-1determination
NHDFcellsgrewto70–80%confluenceandmediumwaschangedtoediumwith0.2%BSA
foratleat16hours.CellsweretreatedwithTenofovir10μMaloneorincombinationwith
CGS2168010μMfor24hours.PwasharvestedandquantifiedbyBCA(Bio-Rad).15ugpro-
teinswereloadedforSCS-page.Afterbeingblockedwith3%BSAinTBST,blotswereincu-
batedwithanti-collagenIantibody(SouthernBiotech)for1hour.
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Tenofovir,fibrosis,PANX-1andadenosinelevels
Statisticalanalysis
Datawereexpressedasmeans±SDor±SEwhereverappropriate.ResultswereanalyzedbyStu-
dent’sttestoranalysisofvariance(ANOVA),asappropriate,withGraphPadsoftwarev.4.02
(GraphPad,SanDiego,CA,USA).ValuesofP<0.05wereconsideredsignificant.Foreachsta-
tisticalanalysis,theexperimentalunitwasasingleanimal,andstatisticalanalysiswasper-
formedtocomparegroupsofanimals.
Results
Tenofovirprotectsagainstbleomycin-induceddermalfibrosis
Toconfirmthattenofovirhaddirectantifibroticeffectsindependentofitseffectsonviral
infectionwetestedtheeffectofthedruginmicewithbleomycin-induceddermalfibrosis,an
establishedmurinemodelofscleroderma.[35].Tenofovirtreatmentmarkedlyreducedskin
thicknessinbleomycin-treatedmicecomparedwiththeskinofmicetreatedwithbleomycin
alone(4.3±0.mmversus6.2±0.3mm,respectively,n=5/group,p<0.01)(Fig1A).Moreover,
tenofovirtreatmentreducedbreakingtensioninthebleomycin-treatedmice(0.30±0.02kg
versus0.46±0.04kg,respectively[n=5/group];P<0.01)(Fig1A).Whenanimalsweretreated
withlamivudine,nosignificantchangeswereobservedineitherskinthickness(3.12±0.3mm
versus3.2±0.4mmrespectively,n=5/group,p=NS)orbreakingtension(0.65±0.6Kgversus
0.56.5±0.3Kg,respectivelyn=5/group;p=ns)(Fig1B).
Tenofovirdiminishesskinalterationsofbleomycin-treatedmice
Tobetterevaluatethedegreeoffibrosisintheskinspecimens,wefurtheranalyzedskinsec-
tionsfollowingstainingwithpicrosiriusred.Birefringencepatternandhuewereevaluated
usingpolarizedlightmicroscopytodeterminecollagendensity,patternofdepositionand
maturity.AsshowninFig2A,biopsiesfromvehicletreatedmiceshowapredominantlygreen
birefringencewhereastherewasanincreaseinredbirefringencewithadecreaseingreenbire-
fringenceinbleomycin-treatedmice(46±14%increasedredbirefringenceand36±6%
decreasedingreenbirefringencevsnon-treatedanimals,p<0.05respectively)[n=5/group],a
patterntypicaloffibrosis(Fig2B).Skinfromtenofovir-treatedmicedemonstratedmarkedly
diminishedfibroticchanges;indeed,theskinresembledthatofthevehicle-treatedmicenot
treatedwithbleomycin(Fig2A).Furtherquantitationofthered/greenbirefringencedemon-
stratestheeffectofbleomycintreatmentondevelopmentoffibrosisandthereductioninfibro-
sisinthetenofovir-treatedmice(Fig2B).Bleomycin-treatedmicehadoveratwo-foldhigher
“scarindex”thanvehicle-treatedmice,whereastenofovir-treatedmicedidnotsufferan
increaseintheirscarindexafterbleomycintreatment(0.94±0.18scarindexversus2.03±009,
p<0.001,n=5)(Fig2B).
Tofurtherconfirmtheeffectoftenofovirtreatmentonbleomycin-inducedfibrosiswemea-
sureddermalhydroxyprolinecontentasareflectionoftotalcollagencontentanddegreeof
fibrosis(Fig2B).Aswiththeothermeasuresofskinfibrosis,tenofovirtreatmentpreventedthe
bleomycin-inducedincreaseindermalcollagen.Hydroxyprolinecontentwasincreasedin
bleomycintreatedmice(173±37μghydroxyproline/mgtissuecomparedto66±4μghydroxy-
proline/mgtissueforuntreatedmice,respectively,p<0.05)andlevelswererestoredtonearly
thoseofuntreatedmiceinthepresenceoftenofovir(94±9μghydroxyproline/mgtissue,
p=nsvs.untreatedmice)[n=5/group].Noreductionofbleomycin-inducedfibrosiswas
observedwhenanimalsweretreatedwith3TC(Fig2C).
Consistentwiththechangesinskinmatrix,bleomycintreatmentstimulatedamarked
increaseofthemyofibroblastpopulation(22±1α-SMA+cellsversus15±1α-SMA+cellsfor
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Tenofovir,fibrosis,PANX-1andadenosinelevels
Fig1.Tenofovirpreventsliverandskinfibrosisintwomodelsofadenosine-mediatedinjury.A)C57BL/6
micetreatedwithtenofovirtherapy(intraperitonealinjections)areprotectedagainstbleomycin-induceddermal
fibrosis.Bleomycin(0.25U;SubQinjections,dailyfor18days)wasusedtoinducefibrosisinC57BL/6mice.
Intraperitonealtenofovirtherapy(75mg/kg)wasadministeredduringbleomycinchallenge.B)C57BL/6mice
treatedwith3TCtherapyarenotprotectedagainstbleomycin-induceddermalfibrosis.Imagesfrom
representativehematoxylinandeosinstainedslidesarepresented.Skinthicknessmeasurementswere
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Tenofovir,fibrosis,PANX-1andadenosinelevels
performedasdescribedinmaterialsandmethods.Breakingtensionresultswereperformedwithatensiometer
andtherecordingatthepointofmaximalstressbeforetearingofthebiopsywasrecorded.C)Picrosiriusred
stainingoffibroticliver.TAA(100mg/kg;intraperitonealinjections,alternatedaysfor8weeks)wasadministered
toinducefibrosisinC57BL/6mice.Subcutaneoustenofovirtherapy(75mg/kg)wasadministeredduringTAA
challenge.Paraffin-embeddedlivertissuesectionswerestainedin1%Siriusredandcollagen-positivesiteswere
indicatedinred.Digitizedimages(10xand40x)arepresentedandshowrepresentativeliversections(n=5–10).
D)QuantificationofpicrosiriusredstainingwasperformeddigitallyusingSigmaScanProv.5.0.0;datarepresent
thepercentageoftotalliverareastainedbypicrosiriusred.Resultsareexpressedasmean±SEM.
Scale=100μm.**p<0.005,***p<0.001comparedtovehicle(ANOVA).
https://doi.org/10.1371/journal.pone.0188135.g001
control,p<0.001,n=5)thatwasreversedbytreatmentwithtenofovir(17±1α-SMA+cells,
p<0.001,n=5)(Fig3).Similartocollagencontentandorganizationandskinthickening,
myofibroblastaccumulationwaspreventedbytenofovir75mg/Kg(Fig3).
Tenofovirpreventsfibrosisinamurinemodelofhepaticfibrosis
Tenofovirantiviraltherapyisassociatedwithcontinuedimprovementinliverhistologyinsev-
eralreports[16,17,36].Tofurtherconfirmwhethertherewasadirectanti-fibroticeffectof
tenofovirwecharacterizedtheeffectsofthisagentonhepaticfibrosisinanestablishedmurine
modelofhepaticfibrosisinducedbythioacetamide(TAA,[37]).Consistentwithourfindings
intheskin,intheTAA-treatedmicetherewasclearformationoffibrousseptaintheharvested
livers(Fig1C).TenofovirtreatmentmarkedlyreducedhepaticfibrosisintheTAA-treated
mice(Fig1C).Histomorphometricmeasurements(Fig1D)wereconsistentwiththehistologi-
calappearanceinthetreatmentgroups;theTAA/tenofovirgroupofmicewasprotectedfrom
fibrosisascomparedtotheTAA/vehiclegroup(1.01±0.05%fibroticareaversus4.44±0.37%
forTAA,p<0.001,n=10)(Fig1Cand1D).
TenofovirdoesnotaffecteitherbasalorA2Areceptor-stimulated
collagen1expression
TenofovirhaspreviouslybeenreportedtobealigandforadenosineA1,A2BandA3,butnot
A2Areceptorsalthoughtheconcentrationsrequiredarehigherthanaregenerallyachievedin
vivo[38,39].Todeterminewhetheradenosinereceptoractivation/inhibitionbytenofovir
couldplayaroleinthediminishedfibrosiswestudiedtheeffectoftenofovironcol1a1expres-
sionbyprimaryhumandermalfibroblasts.Tenofovirneitherinhibitedbasalcollagenexpres-
sionnorA2Areceptor-stimulated(CGS21680,1μM)byfibroblasts(S2Fig).
Tenofovirtreatmentofmicediminishesadenosinereleasefromskinex
vivo
Aswehavepreviouslyreportedthatadenosine,generatedbytheactionofCD39andCD73,
playsacriticalroleindevelopmentofbothhepaticanddermalfibrosisinmurinemodelsof
cirrhosisandscleroderma,respectively,[7,23]wetestedthehypothesisthattenofovir’santifi-
broticeffectsaremediatedbyinhibitionofadenosineproduction.Miceweretreatedwith
tenofovirfortwodaysandthenbleomycinwasadministered,skinwasharvestedandcultured
overnightexvivo,adenosinelevelsinsupernatesweremeasuredbyHPLC,asdescribed.Bleo-
mycintreatmentinducedatwofoldincreaseinadenosinerelease(4.13±0.53nM/mgtissue
comparedto2.26±0.37nM/mgtissueforvehicle,p<0.005),aneffectwhichwasmarkedly
reducedinskinfrommicetreatedwithtenofovir(2.47±0.18nM/mgtissue,p=nsvs.vehicle)
(Fig4A)[n=5/group].Similarly,aswehavepreviouslyreported,TAAtreatmentincreases
releaseofadenosineintotheextracellularspacebyaCD73/CD39-mediatedmechanism(872
±120nM/mgtissueforTAAcomparedto345±55nM/mgtissueforcontrol,p<0.05)and
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Tenofovir,fibrosis,PANX-1andadenosinelevels
Fig2.Tenofovirdiminishesdensecollagenfibrils.A)RepresentativeSiriusredbirefringenceimagesareshownat10x,20x,
and40xoriginalmagnification.Orange-redisindicativeoffibrosis;yellow-greenisconsistentwithnormaltissue.Biopsysiteis
nearthesiteofinduration18daysaftertreatment.B)Quantitativeanalysisofredpixels/HPFandgreenpixels/HPFwas
performedwithSigmaScanProsoftware.Scarindexmeasurements,quantitativeanalysisoffibrosiscalculatedbycomparingthe
ratiooforange-redtoyellow-greenstainingusingSigmaScanProsoftware.Hydroxyprolinecontentisshownnormalizedtothe
mass(inmg)ofthesample.C)RepresentativeSiriusredbirefringenceimagesareshownat10xoriginalmagnification.Orange-
PLOSONE|https://doi.org/10.1371/journal.pone.0188135 November16,2017 10/22