Retinopathy of prematurity: A review of pathophysiology and signaling pathways

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Review article

Retinopathy of prematurity: A review of pathophysiology and signaling pathways

Mariza Fevereiro-Martins, MD

^a^,^b^,^c^,^∗

, Carlos Marques-Neves, MD, PhD

^d^,^e

, Hercília Guimarães, MD, PhD

^f

, Manuel Bicho, MD, PhD

^a^,^b

aLaboratóriodeGenéticaandGrupoEcogenéticaeSaúdeHumana,InstitutodeSaúdeAmbiental,Faculdadede Medicina,UniversidadedeLisboa,Portugal

bInstitutodeInvestigaçãoCientíficaBentodaRochaCabral,Lisboa,Portugal

cDepartamentodeOftalmologia,HospitalCufDescobertas,Lisboa,Portugal

dCentrodeEstudosdasCiênciasdaVisão,FaculdadedeMedicina,UniversidadedeLisboa,Lisboa,Portugal

eGrupoEcogenéticaeSaúdeHumana,InstitutodeSaúdeAmbiental,FaculdadedeMedicina,Universidadede Lisboa,Lisboa,Portugal

fDepartamentodeGinecologia-ObstetríciaePediatria,FaculdadedeMedicina,UniversidadedoPorto,Porto, Portugal

a r t i c l e i n f o

Articlehistory:

Received23December2021 Revised15November2022 Accepted18November2022 Availableonlinexxx

a b s t r a c t

Retinopathyofprematurity(ROP)isavasoproliferativedisorderoftheretinaandaleading causeofvisualimpairmentandchildhoodblindnessworldwide.Thediseaseischaracter- izedbyanearlystageofretinalmicrovasculardegeneration,followedbyneovascularization thatcanleadtosubsequentretinaldetachmentandpermanentvisualloss.Severalfactors playakeyroleduringthedifferentpathologicalstagesofthedisease.Oxidativeandni-

Abbreviations: AA,arachidonic acid;ADAM, “a” disintegrin and metalloproteinase; Akt, protein kinase B; Ang, angiopoietin;

APE1/Ref-1,apurinic/apyrimidinicendonuclease1/reduction-oxidationfactor1;BDNF,brain-derivedneurotrophicfactor;bFGF,basicfibroblastgrowthfactor;DHA,docosahexaenoicacid;eNOS,endothelialnitricoxidesynthase;EPO,erythropoietin;GPCR,Gprotein-coupled receptor;GPR91,Gprotein-coupledreceptor91;HIF,hypoxia-induciblefactor;IGF-1,insulin-likegrowthfactor-1;IGFBP3,insulin-like growthfactor-bindingprotein3;IL-1,interleukin1;JAK,Januskinase;MAPK,mitogen-activatedproteinkinase;MMP,matrixmetallopro- teinase;NADPH,nicotinamide-adenine-dinucleotidephosphate;NO,nitricoxide;NOS,nitricoxidesynthase;NOX,nicotinamide-adenine- dinucleotidephosphateoxidase;Nrf2,nuclearfactorerythroid2-relatedfactor2;OIR,oxygen-inducedretinopathy;PDGF,platelet-derived growthfactor;PEDF,pigmentepithelial-derivedfactor;PHD,prolylhydroxylasedomain;PLGF,placentalgrowthfactor;PI3K,phosphatidyli- nositol3-kinase;PUFAs,polyunsaturatedfattyacids;ROP,retinopathyofprematurity;ROS,reactiveoxygenspecies;RPE,retinalpigment epithelium;Sema,semaphorinsclass;shRNAs,short-hairpinRNAs;siRNAs,smallinterferingRNAs;STAT3,signalingtransducerand activatoroftranscription3;TIMPs,tissueinhibitorsofmetalloproteinases;VEGF,vascularendothelialgrowthfactor;VEGFR,vascular endothelialgrowthfactorreceptor.

∗ Correspondingauthor:MarizaFevereiro-Martins,MD,LaboratóriodeGenéticaandGrupoEcogenéticaeSaúdeHumana,Universi- dadedeLisboa,InstitutodeSaúdeAmbiental,AvenidaProfessorEgasMoniz,Piso1C,1649-028Lisboa,Portugal.

E-mail addresses: [email protected] (M. Fevereiro-Martins), [email protected] (C. Marques-Neves), [email protected](H.Guimarães),[email protected](M.Bicho).

https://doi.org/10.1016/j.survophthal.2022.11.007

Pleasecitethisarticleas:MarizaFevereiro-Martins,CarlosMarques-Neves,HercíliaGuimarãesetal.,Retinopathyofprema- turity:areviewofpathophysiologyandsignalingpathways,SurveyofOphthalmology,https://doi.org/10.1016/j.survophthal.

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Keywords:

Retinopathyofprematurity oxidativestress

preterminfant neovascularization pathophysiology

trosativestressandinflammatoryprocessesareimportantcontributorstotheearlystage ofROP.Nitricoxidesynthaseandarginaseplayimportantrolesinischemia/reperfusion- inducedneurovasculardegeneration.Destructiveneovascularizationisdrivenbymediators ofthehypoxia-induciblefactorpathway,suchasvascularendothelialgrowthfactorand metabolicfactors(succinate).Theextracellularmatrixisinvolvedinhypoxia-inducedreti- nalneovascularization.Vasorepulsivemolecules(semaphorin3A)intervenepreventingthe revascularizationoftheavascularzone.Thisreviewfocusesoncurrentconceptsaboutsig- nalingpathwaysandtheirmediators,involvedinthepathogenesisofROP,highlightingnew potentiallypreventiveandtherapeuticmodalities.Abetterunderstandingoftheintricate molecularmechanismsunderlyingthepathogenesisofROPshouldallowthedevelopment ofmoreeffectiveandtargetedtherapeuticagentstoreduceaberrantvasoproliferationand facilitatephysiologicalretinalvasculardevelopment.

ThisisanopenaccessarticleundertheCCBY-NC-NDlicense (http://creativecommons.org/licenses/by-nc-nd/4.0/)

1. Introduction

Firstdescribedin1942asretrolentalfibroplasia,retinopathy ofprematurity(ROP)isaretinalneovasculardisorderimpact- ing30–50%ofverylowbirthweightpreterminfants,making it oneofthemostprevalentcausesofblindnessand child- hoodvisualimpairmentworldwide.¹⁸⁷^,²⁰²^,³⁰⁶Ofthose affected byROP,25–30%developseriouseyecomplications,including severeametropia,strabismus,abnormalitiesofretinalfunc- tion,and,inthe mostseverecases,blindness.¹⁸⁷ Advances in neonatal care have helped improve the survival rate of preterminfants,resultinginanincreasednumberofpreterm infantsat-riskforROP.²⁰²

ROP is a multifactorial disease, with short gestational period, low birth weight, and hyperoxia being the most frequently associated factors.³⁴⁹ Itspathogenesis has been widely studiedin humansand animalmodels.¹⁴² Develop- ment ofhuman retinalvasculature begins during the 16^th weekofgestationand concludesatthe40^th week.⁹⁴There- fore,preterminfantsexhibitincompletedevelopmentofthe retinalvasculatureandaperipheralavascularzone.⁹⁴Inad- dition,anincreaseinoxygenbioavailabilityatbirthexposes preterm infantstoarelativelyhyperoxicenvironmentthat, coupledwiththeinfant’simmatureantioxidantsystem,leads tooxidativestress.²⁴Therelativehyperoxiaalsoinhibitsthe expressionofhypoxia-induciblefactor(HIF)andvascularen- dothelialgrowthfactor(VEGF),disruptingthegrowthofreti- nalbloodvessels.⁶²Duringphase1ofROP,retinalmicrovascu- lardegenerationoccurs,associatedwithanarrestinthepro- gressivevascularizationoftheperipheralretina.⁹³Inphase2, thesevascularchangesresultinretinalischemiaandtrigger thereleaseofgrowthfactorsleadingtoabnormalintravitreal neovascularization.⁹³

Severalmulticenterclinicaltrialsconductedoverthepast 70yearshavefailedtofindanidealoxygensaturationrange that preventsROPinPhase 1without increasingmorbidity andmortality,andhigh-oxygentreatmentinPhase2didnot showanybenefit.⁷⁸^,²⁷¹Theinsightsresultingfromthesestud- ies,however,havesignificantlyimprovedtheclinicalmanage- mentofROP.⁷⁸Althoughtheidealoxygensaturationrangere- mainsunknown,datafromrandomizedclinicaltrialssuggest

thatmaintaininganoxygensaturationrangeof90–95%ap- pearstobesaferthan85–89%.⁷⁵Anotherinsightfromthese trialsis thatrigorous managementtoavoidfluctuations in oxygensaturationisimportantinreducingtheriskofROP.⁷⁸ Moreclinicaltrialsareneededtooptimizeoxygentherapyand helppreventROP.⁷⁸

Currently,laserphotocoagulationandablativecryotherapy aretheprimarytreatmentsforROP.Theyworkbydestroying theavascularretinathatproducesthegrowthfactorsrespon- sibleforneovascularization;however,neitherofthesethera- piestargetthemainmechanismsofpathologicalneovascu- larization.²³^,⁷⁸Both treatmentscanreducethe incidenceof blindness,but oftendonotimprove visualacuityandhave potentialadversesideeffects,namelyinflammation,myopia, peripheralvisionloss,andscarinduction.⁶¹^,¹⁵⁴Ofthe2treat- ments,laserphotocoagulationismoreconvenienttoadmin- isterandresultsinlesspainandinflammation,andrelatively fewsystemiccomplications.Itisthestandardtherapy,espe- ciallyforROPstage3withplusdiseaseinzone2.²³ Recent studieshaveshownpromisingresultsforantiangiogenicther- apywithanti-VEGFagents.Oneofthem,ranibizumab,wasap- provedforthetreatmentofROPintheEuropeanUnionin2019, buthasyettobeapprovedforthisspecificusebytheUSFood andDrugAdministration.⁷⁸Whilestudieshaveshownanti- VEGFtobethemosteffectivetreatmentforsevereROP(zone I),²⁵⁷reportsofrecurrentintravitrealneovascularizationpre- sentingaslateas60weekspost-menstrualage,¹⁶⁷long-term defectsinvisualacuityorsizeofthevisualfield,disordersin photoreceptorfunctions,andthepossibilityofadverse out- comesinotherorgansduringtheneonatalperiod,remaina concern.¹¹^,¹¹⁸^,¹⁴⁶

Inalmost8decadesofclinicalandlaboratoryresearch,ad- vanceshavebeenmadeinclarifyingthepathogenesisofROP;

however,abettercomprehensionofROPpathogenesisandthe mechanismsthatregulateangiogenesismayleadtomoreef- fectiveandtargetedtherapeuticagentsforthepreventionand treatmentofsevereROPwhilepreservingphysiologicalreti- nalangiogenesis.Wefocus onexisting informationon me- diatorsinvolvedinthepathogenesisofROPanditssignaling pathways.Basedoncurrentknowledgeofmolecularmecha- nisms,wedescribenewpotentiallypreventiveandtherapeu- ticmodalitiesthatcanconstituteimportantlinesofinvestiga-

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tion.Webeginwithadescriptionoftheanimalmodelsusedto studythepathogenesisofROP,followedbynormalandpatho- logicalretinalvascularizationand,lastly,themolecularmech- anismsinvolved inROPthat constituteour mainobjective, highlightingpotentialtherapeutictargets.

2. Animal models of oxygen-induced retinopathy

Toavoidtheethicalandsafetyconcernsinherentinexperi- mentationwitheyesofhumanpreterminfants,animalmodels of oxygen-induced retinopathy(OIR) are typically used tostudythe pathophysiologyofROPandevaluate potential treatments.¹⁴¹Animalssuchasmice,rats,andcatsvascular- izetheirretinasafterbirth,resultinginaretinalvascularde- velopmentsimilartopreterminfantsineyesobtainedatterm intheseanimals³⁴⁹;however,alimitationtoallanimalmod- elsisthattheycannotfullysimulatecomplicationsthatmay ariseafterbirthinpreterminfants.¹⁴¹

Inoneofthemostcommonmodels,¹¹⁹^,¹⁴⁰themouseOIR modeldesignedbySmith,³⁴⁸newbornmiceareexposedtoa highoxygen(75%)environmentfrompostnatalday7–12,¹³⁰ causingvaso-obliterationofcapillariesalreadydevelopedin thecentralretina.Theythenreturntoambientairtodevelop OIRthat leadstovasoproliferation into the vitreousatthe junctionsofthevascularizedandavascularcentralretina.¹⁴⁰ Oneadvantageofthemicemodelistheeaseofgeneticma- nipulationwhichfacilitatesthestudyofthemolecularmech- anismsofROP.²³¹

Penn’sratmodeloffluctuatingoxygen(50/10OIRmodel) is another commonly used OIR model.¹¹⁹^,¹⁴⁰ The oxygen- controlledenvironmentischangedfrom50%to10%oxygen, every24hours,frombirthtopostnatalday14.Thisleadsto adelayindevelopmentoftheperipheralavascularretinafol- lowedbyvasoproliferationatthejunctionofvascularizedand avascularretina;similartowhatisseeninhumanpreterm infants.¹⁴⁰ Thismodelisthemostrepresentative ofhuman ROP.¹¹⁹^,¹⁴⁰Unlikemice,geneticmanipulationofratsiscompli- cated,makinganalysisofmolecularmechanismsmorechal- lenging.Newertechniques emergedto address this limita- tion.¹⁴¹Onesuchtechniqueinvolvestheintroductionofshort- hairpinRNAsorgeneticmutationsthroughgenetherapyto silenceRNAthroughsmallinterferingRNAsorknockoutspe- cificgenes.¹⁴¹Forexample,lentivirushasbeenusedtolinkcell specificpromotorswithshort-hairpinRNAstotargetspecific typesofcellsintheretina,resultingintheknockdownofgene productsinthetargetedcellsonly.¹⁸¹Thisnewtechniquehas beenusefultodeterminetheeffectsofangiogenicsignaling onpathologicalandphysiologicalretinalangiogenesis.³⁰^,¹⁸¹

InanotherOIRanimalmodelnewbornbeaglesareexposed to100% oxygen up to postnatal day 4,causing a delay in physiologicalretinalvasculardevelopmentandvasoprolifer- ation.²³⁷ Theeyesofthe beagle puppyhaveasizecloseto thatofhumanpreterminfants,andthistranslationalaspect isusefulwhentestingpharmaceuticalagentstotreathuman ROP.¹⁴²

ThephasesofROPinanimalmodelsofOIRaresimilarto thoseofhumanROP.HumanROPisdividedintotwophases, whicharesubdividedintofivestages.Phase1ofhumanROP

Figure1– Timelineofnormalvasculardevelopmentversus pathologicalvasculardevelopment(ROPphases)byweeks ofgestationalage.Thedevelopmentofthechoriocapillaris startsbetween5.5and8weeksandiscompletedat20–22 weeks.Retinalvascularizationstartsataround16weeks.

Retinalbloodvesselsgrowradiallyfromtheopticdisc towardstheoraserrata.Vascularizationofthenasalretina iscompletedataround36weeksandthatofthetemporal retinaat40weeks.Thetransitionbetweenphase1and phase2ofROPgenerallyoccursaround32weeks.

(stages1and2)isdefinedbydelayedphysiologicretinalvas- culardevelopmentandcorrespondstomouseandratmodel phase1.Thestage3ofphase2ofhumanROPisdefinedby neovascularizationandcorrespondstophase2ofthemouse and rat models.²⁴⁰^,³⁵⁸ While part ofphase 2,the final two stagesofROPinhumans(stages4and5)aresometimescon- sideredapseudo-phase3.Theyarecharacterizedbyfibrovas- cularchangeswithretinaldetachment.¹⁴¹^,²³⁷^,²⁴⁹This“phase- 3isabsentfromthemouseandratmodels,asneithermodel reproducestheretinaldetachmentseeninhumanROP;however,thebeaglemodelshowssomecharacteristicsofthead- vancedstagesofhumanROP,specificallytheretinalfolding seeninstage4.¹⁴²^,²⁴⁹

3. Normal and pathological retinal vascularization

3.1. Retinalvascularization

Nutrientsandoxygenaresuppliedtotheretinaviatwo-vessel systems: the choroidal circulation that receives the largest bloodflow(65–85%)andnourishestheouterpartoftheretina (photoreceptors)and the retinalcirculation,responsiblefor a smallerpart ofthe flow (20–30%)and supplies the inner layers.¹³Thechoroidalcirculationiscompleteataround20 weeksofgestation(Fig.1).³⁶Thedevelopmentofretinalblood vesselsinhumansbeginsataround16weeksofgestationand spreadsfromthemiddleoftheopticaldiscoutward.¹⁷³ The nasalretinaisvascularizedat36weeksofgestationandthe temporalretinaataround40weeks.⁹⁴Forthisreason,preterm infantspresent incompletelyvascularizedretinas, withthe areaoftheperipheralavascularzonedependentontheges- tationalage.¹⁷³^,³⁴⁹

Vasculardevelopmentoccursintwophases:vasculogenic andangiogenic.¹⁷²Thevasculogenicphaseconsistsofthefor- mationofnewbloodvesselsfrombonemarrow-derivedan- gioblastsandisusuallyseenduringembryogenesis.⁵⁵^,¹⁰⁵^,¹⁷²

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Figure2– Schematicrepresentationofthemaineventsimplicatedinthepathogenesisofretinopathyofprematurity.

Abbreviations:Ang-2=angiopoietin2;BDNF=brain-derivedneurotrophicfactor;bFGF=basicfibroblastgrowthfactor;

EPO=erythropoietin;HIF=hypoxia-induciblefactor;IGF-1=insulin-likegrowthfactor-1;MMPs=matrix

metalloproteinases;ω3-PUFA=omega-3polyunsaturatedfattyacids;ROP=retinopathyofprematurity;ROS=reactive oxygenspecies;Sema=semaphorin;VEGF=vascularendothelialgrowthfactor.ImagesadaptedfromServierMedicalArt byServierarelicensedunderaCreativeCommonsAttribution3.0UnportedLicense.¹⁸

Theangiogenicphaseischaracterizedbythedevelopmentof newbloodvesselsthatbudfromexistingbloodvessels.¹⁰⁵^,¹⁷² Angiogenesisisdrivenbyphysiologicalhypoxia.¹⁷²

Duringfetaldevelopment,relativetissuehypoxiaactsas astimulusforHIFtotriggerthetranscriptionofangiogenic genestoproducegrowthfactors,suchasVEGF,itsanalogpla- cental growthfactor(PLGF),and theproangiogenicerythro- poietin (EPO).²²³ Tissue hypoxiathat stimulatesangiogene- sisisnot thoughttobenecessaryforthe vasculogenicpe- riod.⁵⁵^,¹²⁰Maternal-fetalinteractioninuteroprovidesunique factorsandidealoxygenlevelstostimulatethegrowthofthe retinalvasculature.³⁸¹

3.2. ROPphases

ROPprogressesin2phases:thevascularattenuationphase (phase 1) and the fibrovascular proliferative phase (phase 2).⁹³^,¹⁷³Thefirst phase is vaso-obliterativeandcharacterized byaninterruptionanddelayinretinalvasculargrowthasso- ciatedwithmicrovasculardegeneration.⁹³ Itoccursbecause preterm infantsare exposedtohigheroxygentensionafter birthcomparedtothatinutero,²⁴eliminatingthephysiological hypoxia.⁸⁹ThehyperoxialeadstoadownregulationofVEGF, aswellasanincreaseinvaso-obliterationofimmatureretinal capillariesthroughtheactionsofoxidativestressandinter- twinedinflammation.⁸²^,⁸⁸^,⁹⁴Duringthisphase,levelsofHIF-1, VEGF,insulin-likegrowthfactor-1(IGF-1),andEPOareallde- creased(Fig.2).¹⁵⁴

Thelossofbloodvesselsinanincreasinglymetabolically activeretinacausesittobecomegraduallyhypoxic.¹⁵⁴Toen- sureadequateperfusion,anoverproductionofgrowthfactors, particularlyVEGF,inducethegrowth,differentiation,andmi- grationofendothelialcells.³⁰⁰Thisleadstoabnormalgrowth ofnewbloodvesselsatthejunctionbetweenthevascularand avascularretina,correspondingtothevasoproliferativephase ofROP(phase2).³⁵⁰Thisphaseoccursataround32–34weeks

ofpostmenstrualage.²⁴^,³⁴⁹Thetransitionbetweenphases1 and 2corresponds morecloselytopostmenstrualage than topostnatalage,²⁸⁴however,thisassociationmaynotbeob- servedinextremeprematurity.¹⁶Thesenewbloodvesselsfail toreperfusetheavascularretina.Insteadofgrowingintoareas ofneed,theygrowchaoticallyintothevitreousandcanlead tothedevelopmentofafibrousscarthatcancauseretinalde- tachmentandleadtovisionloss.⁶¹ThiscriticalphaseofROP (stages 4–5)occurs mostfrequently around34–36weeksof postmenstrualage¹⁶¹;however,thetimingoftheROPphases canbemodifiedbyexposuretoveryhighconcentrationsof oxygen.³³¹ Prenatalfactors,suchasinflammatoryprocesses andchorioamnionitis,canalsoaffectintrauterineretinalneu- rovasculardevelopmentandpredisposethefetalretinatose- vereROP.⁴¹⁰UnderstandingROPphasesandtheircausescan allowtheidentificationoftheidealpostnatalenvironmentfor theimmaturepreterminfant.¹⁵⁴

TheInternationalClassificationofROPwasoriginallypub- lishedin1984,¹⁰expandedin1987,andrevisitedin2005³⁰⁸and 2021,⁶⁵anddescribes5stagesofROP.ROPinitiallyappearsas afinedemarcationlinebetweenthevascularandavascular retina– stage1.Itthenprogresses,elevatingthisjunctioninto aridge– stage2.Thesefirsttwostagesmayregresssponta- neouslyandareconsideredasinitialormildROP;however, thepathologicalvasculaturecancontinuetogrowoutsidethe retinalplane,leadingtothevascularstage– stage3.These neovesselsarefragileandcanbleedintothevitreous,causing fibrotizationandtraction,ultimatelyresultingintheretinal detachmentthatdefinesfibrovascularstage– stages4and5.

Retinaldetachmentmayhavepermanentblindnessasapos- sibleconsequence.¹⁴⁰

3.3. Theroleofretinalandchoroidalbloodflow

As a result of the incomplete retinal vascularization in preterm infants, retinal oxygenation depends mainly on

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choroidalcirculation,whichseemstoplayafundamentalrole inthepathogenesisofROP.⁷³Preterminfantsaresubjectto risesinbloodoxygentensionthat,intheabsenceofafullyde- velopedautoregulatorycontrolofocularbloodflow,mayresult inanincreaseofretinaloxygenation.¹³⁶Anelaborategenetic andepigeneticinterplaybetweenprostanoidsandnitricoxide (NO)invasomotorregulationleadstotheabsenceofvascular autoregulationandexcessiveoxygendeliverytotheeyes.¹³⁴

Choroidalbloodflowandretinalbloodflowareautoregu- latedoveranarrowrangeofperfusionpressureinthenew- born,partlyduetoinsufficientconstrictionbutmainlyfrom highperinatallevelsofprostaglandins,suchasprostaglandin D2andprostaglandinE2producedbycyclooxygenase(COX), and NO produced byNO synthase (NOS).²^,¹³⁵ NO is a po- tent signaling molecule in blood vessels and its increased formationfromendothelialcellsactivatesguanylatecyclase in the underlying smooth muscle cells. This leads to the generationofcyclicguanosinemonophosphatethatinduces vasodilation and masks the effect of constrictors involved inautoregulatory responsesin newborns.¹³⁶^,¹³⁷ The effects ofvarious prostaglandins are NO-dependent,¹ and specific prostaglandins regulate the expression and activity of en- dothelialNOS(eNOS)inocularbloodvessels.⁸⁷NOSinhibition improveschoroidalbloodflowresponsetohyperoxia,stabi- lizesoxygensupply,andpreventsthehyperoxia-inducedin- creaseinretinalperoxidation.¹³⁶

Inpreterminfants,theincreaseincarbondioxidetension (hypercapnia)isanotherfactorthatcontributestotheinter- ruptionofregulationofretinalbloodflowandchoroidalblood flow.⁵⁷ BecauseoftheBohreffect,asthepressureofcarbon dioxideincreases,theoxygendissociationcurveisshiftedto theright,allowingmoreoxygentobedelivered,increasingits negativeeffectonthedevelopingretina.³⁷⁹Duringsustained hypercapnia,theincreaseinprostaglandinE2inducestheex- pressionofeNOSthatreleasesNOandfurtherreducesocular bloodflowautoregulation.⁵⁷Hypercapniahasbeenassociated withROPinhumansandOIRanimalmodels.³⁷⁹

4. Oxidative and nitrosative stress, reduced anti-oxidative reserve

Oxidativestressisaconsequenceofanimbalanceinthegen- erationandquenchingofreactiveoxygenspecies(ROS)and hasbeenimplicatedinthepathophysiologyofROP.¹⁴⁰ With highoxygenconsumption,²⁵²constantexposuretolightand rich content of easilyoxidable long-chain polyunsaturated fattyacids(PUFAs),retinaltissueispronetolipidperoxidation andhighlysusceptibletooxidativedamagebyROS.⁶³^,²²⁵Hy- peroxia,inflammatoryresponseduetohypoxia-reperfusion, infection,long-termparenteralnutrition,bloodtransfusions andincreasedlevelsofnon-protein-boundironproducehigh levelsofROS.²⁹⁸Fluctuationsinoxygensaturationappearto bemoredamagingthansustainedhyperoxia.³¹

Qanungoandcoworkersdemonstratedanotableincrease intheproductionofenzymatic and nonenzymatic antioxi- dantssuchassuperoxidedismutase,catalase,andglutathione peroxidaseinthelatestageofgestation.³⁰⁴Therefore,preterm infantspresent arelativedeficiency inantioxidant systems

andlowlevelsofvitaminEandascorbicacidtocounterbal- anceROSincrease.²⁹⁸

ROS,namely superoxide anion(O2 ̅), hydrogen peroxide (H2O2), and hydroxyl radical (HO^●)⁹² are by-products from normal aerobic metabolism that activate signaling pathways.¹²³^,³²⁰ The mitochondrion is the main intracellular source responsible for the production of superoxide radical, though someenzymes also havea role in ROS gener- ation,namelynicotinamide-adenine-dinucleotidephosphate (NADPH)oxidase(NADPHoxidase/NOX)andNOS.⁸^,³¹⁷Inex- perimental OIRmodels,several isoforms ofNOX,including NOX1,⁴⁰⁹ NOX2,⁴²⁴ and NOX4,³⁹⁶ are implicated inthe pro- ductionofROSthatinterferewithperipheralretinalvascular- izationandareinvolvedinlaterintravitrealneovasculariza- tion.³²¹Retinalvascularobliteration,seeninthefirstphase ofROP,isthoughttobepartlyduetoendothelialcellsapop- tosisinduced byoxidativestress³²⁰^,³⁸² which isalso associ- atedwithdelayedretinalvasculardevelopmentinmodelsof ROP.¹²³^,³²⁰

Inadditiontohyperoxia,hypoxiathatdevelopsafterhyper- oxiadamagesnewlydevelopedcapillariescanalsoleadtothe activationofNOXandNOS.³⁹⁷TheenhancedNOSisdysfunc- tional(uncoupled)andcontributestooxidativestress(Fig.3).⁸⁹ SaitoandcoworkersfoundthatactivationofNOXandJanus kinase(JAK)/signalingtransducerandactivatoroftranscrip- tion3(STAT3),involvedinpathwaysofapoptosis,istriggered inhypoxia-exposedretinalmicrovascularendothelialcells.³²¹ Inthe50/10OIRmodel,theincreasedNOXactivityinducedby supplementaloxygencausedintravitrealneovascularization mediatedbyJAK/STAT3activity.³²¹InhibitionofNOXactivity withapocyninreducedthepercentageoftheareaofintravit- realneovascularizationtothetotalretinalarea,buttherewas nodecreaseinVEGF,suggestingthatNOXcanalsoactinde- pendentlyfromVEGF.³²¹

Saitoandcoworkersalsodemonstratedthatinhibitionof NOXledtoareductioninapoptosisandavascularretinainan animalmodelofROP,butreducedvasoproliferationwasonly observedifthehypoxicstimulusforangiogenesiswaslimited whenpupswereplacedintosupplementaloxygen.¹⁴²^,³²⁰This showedthatROSmayactivatesignalingofangiogenesis,indi- rectlythroughNOXactivation³²¹ordirectlythroughVEGF.⁹²

Burstsofsuperoxidegeneratedbyleukocytesareimpor- tant to combatinvading microorganisms, and this may be particularlyimportantintheimmune-suppressedpretermin- fant.¹⁴²Therefore,NOXinhibitioncanhaveundesirablecon- sequencessuchassepsisandnecrotizingenterocolitis.³⁹^,¹⁸⁵ Inaddition,studiesdonotsupporttheuseofcertainantiox- idantssuchasn-acetylcysteineinpreterminfants.³⁵²While antioxidantsmaybeeffectiveinquenchingexternalROS,they may not be able toaccess intracellular oxidative signaling mechanisms.³⁹⁷Tofindsaferpotentialtherapies,itisuseful tostudythesignalingcascadesactivatedbyROSthatmediate thepathologicalcharacteristicsofROP.³⁹⁷

Nuclearfactorerythroid 2-relatedfactor2(Nrf2),acyto- protectivetranscriptionfactor,isupregulatedinresponseto oxidativestress.¹³⁸ Hemeoxygenase-1,agene regulatedby Nrf2,catalyzeshemedegradationand,inresponsetooxida- tivestress,isupregulatedtoprotectcellsfromROS.¹³⁸Previ- ousstudieshavesuggestedthemodulationofNrf2andHeme oxygenase-1expressionbythephosphatidylinositol3-kinase

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Figure3– Diagramshowingtherelationshipbetweenoxygenstressandretinopathyofprematurity.Intheearlylifeof preterminfants,oxygenstressleadstoactivationofNADPHoxidase,eNOSandarginase.Theretinalhypoxiathatoccurs duetofluctuatingoxygentensionsorwhenhyperoxiainjuresnewlydevelopedcapillaries,triggerstheoverproductionof ROSandHIFstabilization.ThesignalingpathwaysactivatedbyROS/nitro-oxidativestress,andHIF-1αcontributeto pathogenesisofretinopathyofprematurity.Abbreviations:eNOS=endothelialnitricoxidesynthase;EPO=erythropoietin;

JAK=Januskinase;HIF=hypoxia-induciblefactor;PI3K=phosphatidylinositol3-kinase;ROS=reactiveoxygenspecies;

STAT3=signalingtransducerandactivatoroftranscription3;VEGF=vascularendothelialgrowthfactor.

(PI3K)/proteinkinaseB(Akt)andextracellularsignal-regu- latedkinase(ERK)pathways.⁸⁶^,²⁰¹Recently,Dongandcowork- ers demonstrated inexperimentalstudies that polypyrimi- dinetract-bindingprotein-associatedsplicingfactorinduced the expressionofNrf2andHemeoxygenase-1viaPI3K/Akt andERKsignaling,resultingintheeliminationofintracellular ROSandsuppressionofthedevelopmentofpathologicalvas- cularization.⁸⁶Protein-associatedsplicingfactorappearstobe apotentialantioxidantcapableofregulatingpathologicalreti- nalangiogenesis.⁸⁶

4.1. Nitricoxideandendothelialnitricoxidesynthase NOisagas-signalingmolecule,⁸⁹synthesizedbyNOSenzyme catalysisinwhichL-arginineisconvertedtoL-citrullineus- ingmolecularoxygenandreducedNADPHasco-substrates.⁵⁰ NObindstotheonlyknownNOreceptor,theenzymeguany- latecyclase.⁷¹TherearethreemainisoformsofNOSinver- tebrates:neuronalNOS(nNOS/NOSI),inducibleNOS(iNOS/

NOSII),andeNOS(NOSIII).³⁶¹^,³⁷⁴TheisoformiNOSisusually expressedfollowingtheexposuretoproinflammatorystim- uli¹²¹ and is ahallmark molecule ofthe pro-inflammatory M1 macrophages.⁴¹⁸ NO generatedby eNOS has the func- tionofvasodilation,amongotherimportantvasculareffects, whilenNOSisexpressedinneurons,modulatesneurogenesis

andsomeneurophysiologicalfunctions,andregulatesvascu- lartone.¹⁰⁷^,¹²¹

Amongallisoforms,eNOSisthemostabundantenzyme in vascular endothelial cells.³⁷⁴ NO produced by eNOS is crucialforthe inductionofangiogenesis.¹²⁷ VEGFactivates eNOSthroughtheAktsignalingpathway,andAkt-dependent eNOS phosphorylationappears toplayakey role inangio- genesis.¹⁰⁷^,¹²⁷ThecontributionofeNOS-derivedNOtoVEGF- inducedvascularpermeability²⁷³andtopromotingendothe- lialcellsurvivalandmigrationisalsowelldocumented.²⁶³^,³⁵¹ Recently, Ninchoji and coworkers demonstrated that NO causes vascular hyperpermeability by destabilizingthe ad- herensjunctionsofendothelialcells.²⁷³InhibitionofeNOSin micestabilizedtheadherensjunctionsofendothelialcellsin vasculartufts,decreasingvascularleakageand,consequently, reducing pathological neovascularization without affecting thegeneralblood perfusionofthe retina.²⁷³ These findings showthatitispossibletopreventvascularleakagethrough pharmacologicalinhibitionofNOproduction.²⁷³ In another recentstudy,Smithand coworkers showedthateNOS con- trolsthepolarizationofendothelialcells,influencingVEGF- induced migrationand the sprouting of angiogenesis in a mouse model ofOIR.³⁵¹ The results ofthis study indicate thateNOS inhibitionincreasesendothelialcell polarization and redirectsrevascularizationoftheavascularretina,pre- venting misdirectedvesselsfromgrowing into thevitreous

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body.³⁵¹Furtherstudiesareneededtoassessthepotentialof eNOStopreventretinalneovascularizationandasastrategy forvascularregenerationoftheischemicretina.³⁵¹Combina- tiontherapyofNOinhibitorsandanti-VEGF,givenatlowbut stilleffectivedoses,mayprovetobebeneficial forvascular retinopathies.²⁷³LocaladministrationofNOSinhibitorsmay benecessarytoavoidadverseeffectsofsystemicadministra- tion,suchashypertensionorothercomplicationsassociated withvasoconstriction.²⁷³

NOcanhaveprotectiveand proangiogenic propertiesin theeyewithdifferenteffectsonOIRdependingonthereti- nalredoxstate.²⁸OxidativestressmayconverteNOSfroma NO-producingenzymetoadysfunctionalenzymethatgen- eratesO2̅.⁸⁹ThisprocessisreferredtoasNOSuncoupling.⁸⁹ BeauchampetalshowedinanOIRmodelthateNOSexpres- sionandactivityincreaseswhentheredoxstateisshiftedto- wardsanoxidativeenvironment.²⁸Inthiscircumstancethe oxygenreductionbyeNOSisuncoupledfromtheformationof NOandeNOSproduceslessNOthatcanreactwithROS,result- inginthegenerationofhighlyreactivenitrogenspecies.⁸⁹^,¹²³ Reactivenitrogenspeciescanelicitseveralmodificationsof macromoleculesand leadtonitrosative stress.⁶⁷ NOreacts withsuperoxidetoformperoxynitrite(ONOO_̅),animportant mediatorofhyperoxia-inducedvaso-obliteration,resultingin microvasculardegeneration.⁸⁹ Excessiveproductionofreac- tivenitrogenspeciescanresultinharmfuleffectssuchaslipid peroxidation,DNAdamage,andsuperoxidedismutaseinac- tivation.²²⁵^,²⁷⁹ An increaseinoxidativestress resultsinin- creasedNOdegradationduetonitrosativestress,ultimately leadingtoendothelialdysfunction.¹⁰⁷Theimportantroleof nitrosativestressinROPwasdemonstratedbyreducingthe severityofoxygen-inducedretinalmicrovasculardegenera- tioninmiceaftergeneticablation⁴⁰and pharmacological in- hibitionofeNOS.¹⁹⁴

Insummary,inretinalhypoxia,agreaterretinaleNOSex- pressionincreasesNOproductionand,consequently,vasodi- lationandangiogenesis.³¹⁷IntheearlystagesofOIR,vasodila- tioninducedbyNOrepresentsacompensatorymechanismto reducevascularobliteration.³¹⁷^,³⁷⁶Thesubsequentimprove- mentinocularbloodflowinducestheproductionofROS.³⁷⁶ Furthermore,althoughNOisessential forrapidly initiating angiogenesis,itplaysacriticalroleinretinalendothelialcells, increasingvascularpermeabilityandpathologicalneovascu- larization.³⁵¹Theevidencedescribedaboveallowustocon- cludethat furtherresearch is neededto assess the poten- tialofregulating NOproductionbyeNOSintheprevention ofvascular retinopathies,including ROP. Inaddition, in an OIRmousemodel,iNOSmodulatedtheactivityofHIF-1via PI3K/AktsignalingandVEGFexpression,presentinganother potentialformofintervention.¹⁴⁴

4.2. Interactionbetweenfree-radicalsandprostanoids Isoprostanesareprimarilyproducedbyfreeradical-mediated oxidationofarachidonicacid(AA).³⁰¹Underoxidizingcondi- tions,the productionofisoprostanes exceeds thatofCOX- derivedprostaglandins and may contribute tomicrovascu- larinjury inROP, astheyinduce the productionofthrom- boxaneA2 which hascytotoxic effects.¹⁶⁵ Beauchamp and coworkers demonstrated in an OIR model that inhibition

ofCOXandthromboxaneA2synthaseselectivelyrestricted retinal oxygen-induced vaso-obliteration.²⁷ Cis- to trans- isomerizationofAAbynitrativestressresultingintrans-AA (TAA) formation was associatedwithvaso-obliteration and retinalendothelialdegenerationinthemodel.²¹ Theforma- tionoftheantiangiogenicandproapoptoticthrombospondin- 1¹⁹⁴viaactivationoflong-chainfattyacidreceptorGPR40is responsiblefortheendothelialcellstoxicityinducedbytrans- AA.¹⁶²

PhospholipaseA2enzymescanbeactivatedinresponse tophysiologicalstimuliortooxidativestressandhypoxia.²⁵ Theseenzymeshydrolyzefattyacidsofmembranephospho- lipidsandcanleadtothereleaseofAA,plateletactivationfac- tor,andlysophospholipids.³⁹⁷Theplateletactivationfactoris apro-inflammatorymediatorthatcontributestomicrovascu- lardamagetotheretina,withitscytotoxiceffectsmediated mainlybythromboxaneA2.²⁶Barnettandcoworkerssuggest thatphospholipaseA2anditsdownstreamsignalingisasso- ciatedwithbothphasesofROPinOIRmodels,eitherindepen- dentlyorinassociationwiththeactivationofVEGFsignaling invascularendothelialcells.²⁵

5. Arginase

Arginasebelongstotheureohydrolaseenzymefamily,with twoisoformsencodedbytwodifferentgenes.²⁶⁹Arginase1, thecytosolicisoform,ismainlyexpressedintheliver,where ithasacentralroleintheureacycle.²⁶¹^,²⁶⁹Arginase2,themi- tochondrialisoform,isexpressedinextrahepatictissues,es- peciallythekidney.³⁴¹Bothisoformsarealsopresentinthe brain,retina,andothertissues.³⁴¹

Arginaseconverts L-arginineto ureaand ornithine.The productionofhepaticureaiscrucialforthedetoxificationof ammonia.³⁶⁷L-ornithineismetabolizedbyornithineamino- transferasetoformprolineneededforcollagensynthesisand byornithinedecarboxylasetoformpolyaminesthatenhance celldifferentiationandproliferation.³¹¹^,³⁶⁷Themetabolismof L-argininebyarginasealsoresultsintheformationofgluta- mate.²⁶⁹Arginasehasanimportantroleinwoundrepairand hasbeenimplicatedinneuroprotectionandneuralregenera- tionviatheproductionofpolyamines.⁹⁵^,¹⁹¹^,²¹³

Preterminfantstendtohavelowlevelsofarginineandglu- tamineduetotheinabilitytomaintainendogenoussynthesis ofthesesemi-essentialaminoacids.³⁷⁸Experimentalstudies showasignificantcontributionoftheseaminoacidstoreti- nalvascularfunction.¹⁰⁸^,¹⁹⁹^,²⁷⁰^,³⁴¹InanOIRmousemodel,in- travitrealneovascularizationandvascularhyperpermeability werereducedbysupplementarytreatmentwitharginineand glutamine.²⁷⁰

Arginaseactivityandexpressionareincreasedbyinflam- matoryprocessesandROSstates.¹⁹^,⁵⁴Whenarginaseactivity isincreased,itcancompetewithNOSforthecommonsub- strateL-arginine,causingNOStobecomeuncoupled,resulting inadecreaseofNOproductionandcontributingtonitrosative stress.³⁶⁷Forthisreason,arginasecanregulatethefunctionof thethreeisoformsofNOS,eNOS,iNOS,andnNOS.¹⁰⁸

Arginase1upregulationinendothelialcellsdecreasesNO, resultingindecreasedendothelialcell-dependentvasorelax- ation,andultimatelyleadingtoreducedbloodflowandsub-

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sequentischemia¹¹⁰^,²⁶⁹;however,theexpressionofarginase 1 in immune cells can decrease NO production by iNOS, reducing oxidative stress and inflammation.¹¹⁰^,³⁴⁰ In fact, arginase 1is considered amarker ofantiinflammatory M2 macrophages.³⁴⁰Foudaandcoworkersshowedthatintravit- real treatmentwith arginase1reducesretinalneurovascu- lardegenerationinwild-typemiceafterischemia-reperfusion injury.¹¹⁰ This study also demonstrated the importance of arginase1inmacrophagepolarizationtowardsareparative phenotypeallowingneurovascularrecovery.¹⁰⁸^,¹⁹⁹^,²⁷⁰^,³⁴¹In a translationalstudy,Foudaandcoworkersalsodemonstrated thatthesystemicadministrationofthepegylatedarginase1 (polyethyleneglycollinkedtorecombinanthumanarginase) dampens the inflammatory response of macrophages and markedlyprotectedagainstneurovascularinjuryafterretinal ischemia-reperfusion injury.¹⁰⁹ Pegylatedarginase was also reportedtocrosstheblood-retinalbarrieranditspenetration wasenhancedbyimpairmentoftheblood-retinalbarrier.¹⁰⁹

While studies have demonstrated a protective role of arginase 1, other studies provide evidence that arginase 2 playsaroleinretinaldamage.Elevatedarginase2levelshave been associatedwithretinalneurovasculardegeneration in modelsofischemicretinopathythroughmechanismsinvolv- ingincreasedoxidativestress,glialactivation,andchangesin polyaminepathways.³⁴⁰Thecatabolicproductsofpolyamine oxidationandglutamatecanleadtooxidativestressandDNA damagethatcancausecelldamage.²⁶⁹Shoshaandcoworkers suggestedthatarginase2isadownstreamtargetofNOX2.³⁴¹ ThesuperoxideproducedfromactivatedNOX2,alongwithin- creasesinotherROSafterischemia/reperfusion,mayplayan importantroleinarginase2upregulation,leadingtocelldeath and ischemia/reperfusion-induced neurovascular degenera- tion.³⁴¹

L-citrullineisaprecursortoL-arginineviatheL-arginine recycling pathway;howeverL-citrullinehasbeen shown to haveaninhibitoryeffectonarginase.³³²^,³³³Thisinhibitoryef- fectmayfurtherincreaseNOproductionbyprovidingmore L-argininetotheNOSpathway.³³²Shatanawiandcoworkers demonstrated that supplementationof L-citrulline reduces arginase activity andincreases nitric oxid plasmalevels in patientswithtype2diabetes.³³²Inhumans,oralL-arginine treatmentisimpairedbymetabolisminthegutandliver.L- citrulline, theprecursorofL-arginine,isnotsubject topre- systemic elimination, and for this reason, oral L-citrulline supplementation increases the plasma concentration of L- argininemoreeffectively.³²⁷L-citrullineisavailableinoraland intravenous formsandhas beenused safelyinclinical tri- alswithinfantsundergoingcardiacsurgeryandinchildren withsicklecelldiseaseandmitochondrialdisease.⁹¹^,³⁴⁶^,⁴⁰⁰A clinicaltrialaimstoevaluatethesafetyprofile,efficacyand adequate dosage ofenteral L-citrullinesupplementation in preterm infants(NCT03649932).Theinvestigators intendto use the informationfrom thisstudy toconduct arandom- izedplacebo-controlledtrialtoassesstheroleofL-citrulline supplementationtotreatpulmonaryhypertensionassociated withbronchopulmonarydysplasiainpreterminfants.

In summary, more studies are needed to evaluate the arginase pathwayasatherapeutictarget totreat oxidative stress-relatedretinopathy.Thedevelopmentofmethodsfor cell-specifictargetingandspecificinhibitorsofarginaseiso-

formscouldfacilitateprogressinthisarea.³⁴⁰Specificdelivery ofpegylatedarginase1tomicroglialcells/macrophagesmay beanoptiontolimitinflammation,avoidingpotentiallyharm- fuleffectsonvascularendothelium.³⁴⁰Pegylatedarginase1 isbeingclinicallytestedinpatientswithmelanomaandad- vancedhepatocellularcarcinomaandappearstobesafeand well tolerated.¹⁰⁸^,¹⁹⁹^,²⁷⁰^,⁸³^,³⁴¹^,⁴²³ While the role of arginase inROPisclearerthanbefore,many aspectsstillneedtobe explored.Forexample,themechanismsofarginase-induced retinaldamage andthecomplimentaryorcontradictoryac- tionsofthe twoarginaseisoformsneedtobebetterunder- standed.³⁴⁰Potentialsystemicadverseeffectsonimmunere- sponses and endothelialcell functionshould alsobestud- ied.¹⁰⁸

6. Hypoxia-inducible factor

AllresponsestohypoxiaincellsshareHIFasacommonde- nominator.⁶⁶HIFisatranscriptionfactorandaheterodimeric complex composed of two subunits: an oxygen-dependent subunitHIF-1α(oritsanalogsHIF-2αandHIF-3α)andacon- stitutivelyexpressednuclearsubunitHIF-1β(1,2)⁹⁷^,³⁹⁴– also denominatedasaryl hydrocarbonreceptornuclear translo- cator.¹²² In normoxic conditions,HIF-1α ishydroxylated by prolyl hydroxylase domain (PHD) in the cytosol. PHDs be- long to a small family of proteins. In humans, there are 3forms(PHD1,PHD2,PHD3)withtheirownspecializedactiv- ity.²⁵³ ThehydroxylationofHIF-1αbyPHDprovidesabind- ingsignalforVonHippel-Lindau(VHL)tumorsuppressorpro- tein,⁴⁵ thus enabling ubiquitination by the cullin-2 E3 lig- ase complex (CRL2^VHL E3)²⁰³ and degradation by the 26S proteasome.⁴⁵

SincePHDusesoxygenandironascofactors,astateofhy- poxiainhibitstheenzymatic activitiesofPHD,allowingthe stabilization of HIF-1α.²⁰³ As a result, the HIF-1α level in- creasesandbindstoHIF1-βinthenucleustoactivatethean- giogenicmechanisms whichhelp cellsadjustto hypoxia.⁹⁷ In addition, the Krebs cycle activity is linked to oxidative phosphorylationwhichisbothimpactedbyhypoxia.¹⁸⁷Inhi- bitionofsuccinatedehydrogenasebyhypoxiaresultsinthe accumulationofsuccinate,whichisexportedtothecytosol, binds toand activatesits receptor,GPR91,and leadstoin- hibitionofPHDandactivationofHIFexpression.²⁶⁴Another Krebscycleintermediate,fumarate,alsoaccumulatesduring hypoxiaandinhibitsPHD activityleadingtoanincreasein HIF-α.¹²⁸

Consequently,inhypoxia,HIF-1αhydroxylation isinhib- itedanditescapesfrom VHLbindingand26S proteasome- dependent degradation.²⁰³ This results in the stabilization ofHIF-1αandsubsequenttranslocationintothenucleus.³⁸⁴ Theproteindimerizeswithconstitutively expressedHIF-1β toformtheHIF-1complexwhichbindstoE-box-likehypoxia responseelements(HREs)inthepromoterregionofhypoxia- induciblegenes.¹⁷⁷TheHIF-1complexenablesthetranscrip- tion ofhundreds ofgenes, including those involvedinan- giogenesis,suchas VEGF,EPO,platelet-derivedgrowth factor(PDGF),angiopoietin(Ang)-1,andangiotensin-converting enzyme1;glucose/ironmetabolism;stemcellmaintenance;

andcellsurvivalandproliferation.Thisprocesshelpsthecells

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adapttolowoxygenconditions.¹⁷¹^,³⁰³^,⁴⁰⁶^,⁴²⁵Synnestvedtand coworkersfoundthatadenosine,anotherangiogenicfactor,is alsocontrolledbyHIF-1α.³⁶⁸HIF-1and HIF-2regulatemany commontranscriptionaltargets,butsomegenesarenotco- regulated.For example, the expression ofEPO and certain geneslinkedtoironmetabolismarecontrolledbyHIF-2while HIF-1controlsanaerobicglycolysis.¹²⁸

Thereisalsoevidencethatnon-hypoxicstimuliinduceHIF.

Undernormoxic conditions,variousgrowth factorsand cy- tokinessuchasIGF-1andtransforminggrowthfactorβmay stabilizeHIF-1αviaspecifickinasepathways[PI3Kormitogen- activatedprotein kinase (MAPK)].²²⁰ ROS can also increase thetranscriptionalactivityofHIF-1,eveninnormoxia.⁵³The sameisreportedforangiotensin-convertingenzymeandan- giotensinIIreceptors.²³⁰

Asdescribedabove,relativehypoxia,suchasthatseendur- ingfetaldevelopment,inhibitsPHDactivity,increasinglevels ofHIF-1α.²⁸² Thisplaysacrucialroleinretinalvascularde- velopment,bothdirectlyincreasingthetranscriptionofma- jorangiogenicfactors⁴⁰²andindirectlyincreasingtheapop- toticeffectscaused bythe HIF-1–responsivegeneRTP801.³⁷ Preterminfantsareexposed toahigh oxygentensionafter birth,⁹⁴oftenincludingsupplementaloxygentherapy.Hyper- oxiasuppressesHIF-1αlevelsandleadstoareductioninVEGF expressionandinductionofretinalcapillaryobliteration.¹³^,³⁴⁹ Therefore,suppressionofHIF-1αbyhyperoxiaplaysapivotal roleinthe onsetand progressionofthe firstphaseofROP, whileitsupregulationbytissuehypoxiaiscrucialforthesec- ondphase.¹³

SeveralstudieshavedemonstratedthatHIFstabilization duringhyperoxia(hypoxiamimesis)maypreventretinalves- sellossandsubsequently,thesecondphaseofROP.Searsetal demonstratedthatPHDinhibitorsstabilizeHIFinmiceand maintainconditions to stimulate physiological retinalvas- culardevelopmentinphase1ROPinOIRmodels.³²⁸ Hoppe and coworkers tested7 small moleculeHIF PHDinhibitors andreported thatatleast 2ofthem administered system- icallyduringthe hyperoxicphase preventvaso-obliteration andsubsequentpathologicangiogenesisinOIRmice.¹⁶³One ofthesemoleculesisroxadustat,acarbonylglycineapproved inmany countries for the treatment ofanemia associated withchronickidney disease.³²⁹ Theother,AR0,isa benzo- lamideconstructedbymodelingtheoxoglutaratebindingsite ofPHD2.BothmoleculestargetedPHD2betterthanPHD1or 3.¹⁶³TheresearchersobservedthatretinalexpressionofPHD2 becamedominantatpostnatalday8,andsuggestedthatthe inhibitionofPHDatthispointshouldbemaximaltoobtain thebestprotection.¹⁶³ Theyalsodemonstratedthatroxadu- statcanpreventOIRintwoways:directly intheretina,by stabilizingHIFandup-regulatingenzymesforaerobicglycoly- sis,orindirectlyintheliver,bystabilizingHIF-1andstimulat- ingthesecretionofangiogenichepatokines.¹⁶⁴Thesefindings leadtheauthorstosuggestaclinicaltrialwithintermittent useoflowdosesofPHDinhibitorinpreterminfants,start- ingoneweekafterbirthandcontinuingto30weekspostmen- strualage,toallowoxygensupplementationwithoutimpair- ingnormalretinovasculargrowth.¹⁶³Anotherinterestingcon- siderationisthatironandoxygen-dependentPHDsareery- thropoiesisregulators.¹²⁸PHDinhibitors,suchasroxadustat, areusedtotreatanemia,ariskfactorassociatedwithROP.We

thinkthatthiseffectofHIF-PHDinhibitorsshouldalsobein- vestigatedinthepreventionofROP.

Inresponsetohypoxia,HIF-1mediatesmetabolicrepro- grammingforcellularadaptation,increasingfluxthroughgly- colysisanddecreasingglycolyticcarbonentryintothetricar- boxylicacidcycle.²⁰⁰^,³⁴⁴HIFalsoinducesserinesynthesisand metabolism,increasingmitochondrialNADPHproduction.³⁴⁴ One-carbonmetabolismisfolicacidandmethylenetetrahy- drofolateredutasedependent.⁴¹⁶Singhandcoworkersfound thatserine/one-carbonmetabolismaredependentonhepatic HIF-1andmediateHIFprotectionagainsthyperoxia-induced retinalvesselinOIR.³⁴⁴Thisstudysuggeststhatpharmaco- logicalstabilizationofHIFcaninduceaerobicglycolysisand controlserinemetabolism,resultinginaprotectedphenotype inmice.³⁴⁴^,³⁷⁸

Other studies suggest that HIF inhibition during phase 2 OIRmay be anidealstrategy, asit seemsto bedirected towards the pathological action of proangiogenic factors (mainlyVEGF-A),whilemaintainingthephysiologicalroleof thesefactors,essentialinprotectingtheretina.²¹⁷

InonemouseOIRmodel,thepeaklevelsofexpressionof HIF-1αandHIF-2αwerereachedaftertwohoursofexposure tohypoxia.²⁶²Jiangand coworkersdemonstrated thatinhi- bitionofHIF-1αsuppressestheproductionofpro-angiogenic factorsthatcausetheneovascularphase(phase2).¹⁸⁰Studies havedemonstratedtheroleofHIFinthepathogenesisofROP (Table1).

Miwa and coworkers studied two HIF inhibitors with differentmechanisms:topotecan,whichsuppressestheac- cumulationofHIF-1αproteinbutnotmRNAexpressionand, doxorubicin,whichinhibitsHIF-αsbypreventingitsbinding to the hypoxia response element²⁵⁸ (Table 1). Shoda and coworkersscreenedmarineproductsandfoundsixspeciesof fishwithHIFinhibitoryeffects.³³⁹Betweenthem,Decapterus tabl components suppressed retinal neovascularization in a mouse model of OIR, however, the mode of action and effectorcompoundshavenotbeenclarified.²¹⁷^,³³⁹Usui-Ouchi et al demonstrated that intravitreal injection of peptides derivedfromtheintrinsicallydisorderedproteinCITED2,an endogenousnegativefeedbackregulatorofHIF-1αprevented ROPinamousemodelofOIR.³⁸³Itwasalsoreportedthatthe combinationofthispeptideinhibitorofHIFwithareduced concentrationoftheanti-VEGFafliberceptcausessuppression ofneovascularizationandstimulatestherevascularizationof theischemicretina.³⁸³

Apurinic/apyrimidinic endonuclease 1/reduction- oxidation factor 1(APE1/Ref-1) isa multifunctional protein that has a role of redox-transcription activator and of endonuclease.¹³⁸^,²²¹ APE1/Ref-1 is expressed during retinal developmentand inretinaland choroidalendothelialcells, retinalpigmentepithelialcells,and pericytes.²⁸⁷APE1/Ref-1 regulates transcription factors that are involved in retinal neovasculardiseases,includingHIF-1α,STAT3,andNF-κB.¹³⁸ Low oxygen levels and APE1/Ref-1 redox activity induce HIF-1αexpression.¹³⁸ TheinhibitionofAPE1/Ref-1asanew option for the treatment of retinal neovascular diseases, includingROP,isaddressedbyanotherreview.¹³⁸

Phase IIand III clinical trialsofPHD inhibitors forane- miaofchronickidneydisease andofHIFinhibitorsforthe treatmentofcanceraresummarizedinthereviewsbyHaase

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JID:SOP[mNS;December21,2022;23:36]

Table1– Resultsofstudiesinvestigatingmoleculesorpathwaysinvolvedinthepathogenesisofretinopathyofprematurityandrelatedpharmacologicalagents.

Molecule/

Pathway

Pharmacologicalagent Intervention Testedin Effect Adverseeffect Reference

HIF-1 Dimethyloxalylglycine(PHD inhibitor)(Phase1)

Intraperitonealinjection MouseOIRmodel Preventslossofvessels,vascular tortuosity,andtuftsformation

328

RoxadustatorAR0(PHD inhibitors)(Phase1)

Intraperitonealinjection MouseOIRmodel Preventvaso-obliterationand subsequentpathologic angiogenesis

163

Topotecanordoxorubicin (HIFinhibitors)(Phase2)

Intraperitonealinjection MouseOIRmodel Bothpreventpathologicalbutnot physiologicalretinal

neovascularization.Inaddition, topetecanprotectsthevisual function

258

2-azahypoxanthine(HIF inhibitor)(Phase2)

Oraladministration MouseOIRmodel SuppressVEGFupregulationand retinalneovascularization

218

HIF-1+VEGF HIF-1αsiRNA(Phase2) Subretinalinjection MouseOIRmodel Neovasculartuftsandneovascular nucleiweredecreased

180

CITED2(HIF-1αinhibitor) Subretinalinjection MouseOIRmodel Inhibitsvaso-obliterationand pathologicalangiogenesis

383

HIF-1αsiRNAandVEGF siRNA(Phase2)

Subretinalinjection MouseOIRmodel Resultinmaximumeffectsin suppressionofVEGFinvitroand invivo

180

CITED2(HIF-1αinhibitor) +Aflibercept(Phase2)

Intravitrealinjection MouseOIRmodel Suppressneovascularizationand stimulateischemicretinal revascularization

383

VEGF VEGF(Phase1) Intraocularinjection RatOIRmodel Preventionofendothelialcell apoptosisandrescueofretinal vasculature

7

Anti-VEGF(Phase2) Intravitrealinjection RatOIRmodel Reductionofintravitreal neovascularizationarea

Reductioninweightgain 247

Bevacizumab(Phase2) Intravitrealinjection ClinicalStudy EffectivefortreatmentofzoneI ROP(comparedtolasertreatment) butnotforzone2

Persistentavascularretinaand recurrentintravitreal

neovascularization;serumlevels suppressedfor2months

167,256,413

Bevacizumab(Lowdose) (Phase2)

Intravitreal Injection

ClinicalStudy Thelowesteffectivedoseof bevacizumabmaybe0,004mg

Ocularresults(strabismus,high myopia,nystagmus,retinal detachment)in1yearidenticalto thoseofstudieswithhigherdoses

74,393

Ranibizumab(Phase2) Intravitrealinjection ClinicalStudy EffectiveinthetreatmentofROP ReductioninserumVEGFlevelsfor lessthan4weeks

157

Intravitrealinjection ClinicalStudy Ranibizumabatadoseof0.12mg and0.20mgwasshowntobesafe andeffectiveat1and2yearsof follow-up

Recurrenceswerefrequent 357

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Pleasecitethisarticleas:MarizaFevereiro-Martins,CarlosMarques-Neves,HercíliaGuimarãesetal.,Retinopathyofprema-turity:areviewofpathophysiologyandsignalingpathways,SurveyofOphthalmology,https://doi.org/10.1016/j.survophthal.2022.11.007

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Table1(continued)

Molecule/

Pathway

Ranibizumabversuslaser therapy(Phase2)

IntravitrealInjection ClinicalStudy (Clinicaltrial:

NCT02375971)

Ranibizumab0.2mgmaybe superiortolasertherapyandhas anacceptablesafetyprofileof24 weeks

Lessmyopiawithranibizumab thanlasertherapy.Noadverse adventsreportedwith ranibizumab.

243,359

Aflibercept(Phase2) Intravitrealinjection ClinicalStudy SerumIGF-1levelsdidnotchange significantly

SerumVEGFlevelswere suppressedforatleast8weeks.

Afliberceptinsystemiccirculation after4weeks

118

Afliberceptversuslaser therapy(Phase2)

ClinicalStudy Afliberceptiseffectiveinthe treatment

ofROP

Afliberceptrequiresmore additionaltreatmentsthanlaser photocoagulation

90

Afliberceptversus Ranibizumab(Phase2)

ClinicalStudy Bothareeffectiveinthetreatment ofROP

LowerrateofROPrecurrencewith afliberceptthanwithranibizumab

364

Afliberceptversus Bevacizumab(Phase2)

Intravitrealinjection ClinicalStudy HigherrateofROPrecurrencewith

bevacizumabthanwith aflibercept.SerumVEGF

significantlyreducedfor3months 170

Intravitrealinjection ClinicalStudy Regressionratesignificantly higherwithafliberceptcompared withbevacizumab.

Recurrenceratesignificantly higherwithafliberceptcompared withbevacizumab.

312

Afliberceptversus Bevacizumabversus Ranibizumab(Phase2)

ClinicalStudy Aflibercept,bevacizumaband ranibizumabareeffectiveforthe treatmentofROP

Recurrenceismorefrequentand earlierwithranibizumab.

Bevacizumabisassociatedwith myopicshift

366

VEGFAshRNA(Phase2) Subretinalinjection RatOIRmodel ReductionofVEGFexpression;

Inhibitedintravitreal neovascularizationwithout affectingphysiologicalretinal vasculardevelopmentorpuppy weightgain

395

VEGF164shRNA(Phase2) Subretinalinjection RatOIRmodel Maintainedlong-terminhibitionof intravitrealneovascularization, limitedcelldeath,andpreserved theouternuclearlayercompared withshRNAtoVEGFA

181

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12 AR TICLE IN PRESS

Table1(continued)

Molecule/

Pathway

β-adrenergic receptors

Propanolol(Phase2) Subcutaneousinjection MouseOIRmodel Markedlyreduced

neovascularization;reduced upregulatedVEGF

313

Oral(0.25or0.5 mg/kg/6hours)

ClinicalStudy (Clinicaltrial:

NCT01079715)

Effectiveinreducingthe progressionofROP

Seriousadverseeffects (hypotension,bradycardia) associatedwithsepsis,anesthetic inductionortrachealstimulation wereobservedin5of26infants

101

Topicaladministration ClinicalStudy (Clinicaltrials NCT02504944; NCT02014454)

Propanolol0.2%dosebutnot0.1%

dosereducesROPprogression

102,103

IGF-1 IGFBP3(Phase1and2) Intraperitonealinjection MouseOIRmodel Protectsagainstthe

oxygen-inducedretinalvesselloss, increasesvesselregrowth,and decreaseretinal

neovascularization

233

IGF-1 Intraperitonealinjection MouseOIRmodel Earlyadministration(priorto exposuretohyperoxia)reducethe riskofOIR

386

Fresh-frozenplasmaasa sourceofIGF-1/IGFBP3 (Phase1)

Transfusion ClinicalStudy IncreasesserumIGF-1andIGFBP3 levels

131

IGF-1/IGFBP3(Phase1and 2)

Intravenous ClinicalStudy Welltolerated,safe,andefficient inincreasingserumIGF-1levels

234

Intravenous ClinicalStudy(Phase IIclinicaltrial, NCT01096784)

Doesnotaffectthedevelopment ofROP

13.1%emergingadverseeffects possiblyrelatedtostudydrug

224

EPOand derivates

EPOforpreventingred bloodcelltransfusion

Intravenous/subcutaneous Asystematicreview of2clinicalstudies

RiskfactorforROP(anygrade)and atrendforROPstage>3with earlyEPOtreatment

4

EPOfortreatmentof anemia

Intravenous/subcutaneous ClinicalStudy Doesnotinfluencemarkedlythe incidenceandseverityofROP

330

EPO(ROPphase1) Intravenous/subcutaneous ClinicalStudy (Clinicaltrials:

NCT02036073, NCT03919500)

Effectivefortype2ROPininfant boysorpreterminfantswith gestationalagegreaterthan28 weeksandbirthweightgreater than1500g

365

Darbepoetinforpreventing redbloodcelltransfusion

Subcutaneous ClinicalStudy(small samplesize)

Doesnotinfluencemarkedlythe incidenceandseverityofROP

278

Abbreviations:EPO=erythropoietin;HIF=Hypoxia-induciblefactor;IGF-1=insulin-likegrowthfactor-1;IGFBP3=insulin-likegrowthfactor-bindingprotein3;PHD=prolylhydroxylasedomain;

ROP=retinopathyofprematurity;shRNA=short-hairpinRNA;siRNA=smallinterferingRNA;VEGF=vascularendothelialgrowthfactor.