Role of extracellular vesicles in hypoxia-induced hepatic ...

University of Groningen

Role of extracellular vesicles in hypoxia-induced hepatic injury in non-alcoholic fatty liverdiseaseHernandez Villanueva, Alejandra

DOI:10.33612/diss.180853744

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Roleofextracellularvesiclesinhypoxia-

inducedhepaticinjuryinnon-alcoholic

fattyliverdisease

PhDThesis

toobtainthedegreeofPhDatthe

UniversityofGroningen

ontheauthorityofthe

RectorMagnificusProf.C.Wijmenga

andinaccordancewith

thedecisionbytheCollegeofDeans.

Thisthesiswillbedefendedinpublicon

Wednesday6October2021at14.30hours

by

AlejandraAndreaHernándezVillanueva

bornon22March1990

inSantiago,Chile

2

ROLEOFEXTRACELLULARVESICLESIN

HYPOXIA-INDUCEDHEPATICINJURYINNON-

ALCOHOLICFATTYLIVERDISEASE

TESISPARAOPTARALGRADODEDOCTOR

ENCIENCIASMÉDICASENCOTUTELACONLA

UNIVERSIDADDEGRONINGEN

ALEJANDRAHERNÁNDEZVILLANUEVA

SUPERVISORES:

MARCOARRESEJIMENEZ

HANMOSHAGE

SANTIAGODECHILE

2020

3

Supervisors

Prof.A.J.Moshage

Prof.M.Arrese

Assessmentcommittee

Prof.J.W.Jonker

Prof.H.vanGoor

Prof.A.Feldstein

4

TomyparentsVictorandGloriaforpushingmetospreadmywingsandforalwaystrustingme,

Tomylovelyfamily,brothers,nephewsandauntsfortheirunconditionalsupport,

TomysoulfriendswhoaremyotherfamilythatIchose,

Tomytutor,speciallyHanandMarco,thatIoweeveryeducationandprofessionaltraining,

TomyboyfriendPedroforbeingthehappinessofmylife,

Toeachoneofyou...Ialwayscarryinmyheart.

5

Amispadresporimpulsarmeaabrirmisalasyporconfiarsiempreenmí,

Atodamifamiliaporsuamorincondicional,

Amisamigasdelalmaquesonmiotrafamiliaqueelegí,

Amisprofesoresquelesdebocadaenseñanzayformaciónprofesional,

Amipololoporserlaalegríademivida,

Acadaunodeustedes...losquieroyllevosiempreenmicorazón.

6

TableofContentsPag

Preface&Scopeofthisthesis 7

Chapter1:GeneralIntroductionandaimthethesis 9

Chapter2:ExtracellularvesiclesinNAFLD/ALD:frompathobiologytotherapy.

Cells9,817.2020 15

Chapter3:Chemicalhypoxiainducespro-inflammatorysignalsinfat-laden

hepatocytesandcontributestocellularcrosstalkwithKupffercellsthrough

extracellularvesicles.(BBA)MolecularBasisofDisease1866,165753.2020

65 30

Chapter4:Extracellularvesiclesderivedfromfat-ladenhepatocytesundergoing

chemicalhypoxiapromoteapro-fibroticphenotypeinhepaticstellatecells.

(BBA)MolecularBasisofDisease1866,165857.202061

Chapter5:HypoxiaincreasedCaspase-1inextracellularvesiclesderived

fromexperimentalnon-alcoholicsteatohepatitismodelsandpromotes

inflammasomeactivationinKupffercells.82

Chapter6:Discussion,ConclusionandFuturePerspectives99

Appendices:Summaries(EnglishandSpanish)andNederlandse 104

samenvatting,Acknowledgements;Abbreviations;

Supplemmentarymaterial,andListofpublications.

7

Preface&Scopeofthisdoctoralthesis

PREFACE

Non-alcoholicfattyliverdisease(NAFLD)isahighlyprevalentchronicliverdiseasethataffects30%of

thegeneralpopulation.TheincidenceofNAFLDisstillincreasing.NAFLDencompassesapathological

spectrumof liver injury, ranging from isolatedsteatosis toan inflammatorycondition termednon-

alcoholic steatohepatitis (NASH), that can progress to liver fibrosis and subsequent cirrhosis and

hepatocellular carcinoma (HCC). Clinical observations have indicated that obstructive sleep apnea

syndrome (OSAS) is a significant risk factor that predisposes patients to the progression of liver

steatosis,inflammationandfibrosis.OSASisasleepbreathingdisordercharacterizedbyintermittent

hypoxia(IH)duringsleep.IthasbeenshownthatIHinpatientsandinexperimentalrodentmodelsis

linkedtooxidativestress,steatosis,inflammationandliverfibrosis.

In recent years, extracellular vesicles (EV) have been implicated in intercellular communication in

variouspathophysiologicalconditions,includingNASH.However,themechanismsunderlyingtherole

ofEVinNASHinthecontextof(intermittent)hypoxiaremainsunexplored.

This doctoral thesis focuses on the role of extracellular vesicles in the pathogenesis of hypoxia-

induced hepatic injury in different models of NASH. To validate our hypothesis we used in vitro

modelstoinvestigatecellularcrosstalkbetweenfat-ladenhepatocytesexposedtochemicalhypoxia

andnon-parenchymalcells,suchashepaticstellatecellsandKupffercells.Also,weusedan invivo

model of NASH to evaluate whether IH promotes liver injury and increases circulating EV. Our

findings reveal new insights on the pathophysiological effects of hypoxia on lipotoxicity,

inflammation and fibrosis. Moreover, we provide novel insights with regard to the presence of

caspase-1inEV,suggestingcaspase-1asapotentialnovelbiomarkertomonitorNAFLDandOSA.

SCOPEOFTHETHESIS

Theresearchdescribedinthisthesisfocusesonthecombinationoflipotoxicityandhypoxiaininvitro

and in vivo models of NASH. We investigate whether hypoxia promotes the activation of

inflammatory and fibrotic pathways, with a special emphasis on the role of EV in inflammasome

activation.

InChapter1wepresentageneralintroductionofNAFLD,intermittenthypoxia-relatedOSA,therole

ofEVinNAFLD/NASHandthegeneralaimsofthisthesis.

In Chapter 2, the role of EV in liver pathophysiology is reviewed and discussed, including their

potentialapplicationsasbiomarkersandtherapeutics.

8

InChapter3we introduceourexperimental invitromodelofNASHusingprimary rathepatocytes

exposed to free fatty acids (FFA) and subjected to chemically-induced hypoxia (CH) using the

hypoxia-induciblefactor1alpha(HIF-1α)stabilizercobaltchloride(CoCl2).Weobservedthathypoxia

aggravateshepatocellular injuryviaamechanismthat involves inflammasome/caspase-1activation

infat-ladenhepatocytesandcellularcrosstalkwithKupffercellsthroughEV.Futhermore,we

InChapter4,westudythecellularcrosstalkbetweenhypoxichumanHepG2cellsexposedtoFFAand

humanhepaticLX-2stellatecells.Weobservedthathypoxiaexacerbateshepatocellulardamageand

pro-fibroticsignalingandthatthisiscorrelatedwithincreasedEVreleasefromfat-ladenHepG2cells.

Interestingly,EVfromhypoxicfat-ladenHepG2evokedapro-fibroticresponseinLX-2cells.

InChapter5weshowanimportantfindingaboutcaspase-1asoneofthecargocomponentsofEV

from both hypoxic fat-laden hepatocytes as well as from serum of mice fed the CDAA diet and

exposed to IH. Moreover, we observed that silencing of HIF-1α in hepatocytes abolished the

induction of inflammasome-related genes in Kupffer cells. These data suggest that IH could be an

aggravatingfactorintheprogressionofNASHviaHIF-1αinduction

Finally,inChapter6wesummarizeanddiscussourresultsandprovideanoutlookforfuturestudies.

9

Chapter1

1.-GENERALINTRODUCTIONANDAIMSOFTHEDOCTORALTHESIS

Non-alcoholicfattyliverdisease(NAFLD)isthemostcommonliverdiseaseworldwide,affectingupto

30% of the current population (1). NAFLD is used as an umbrella term that describes a clinico-

pathologicalentitydefinedbythepresenceofaspectrumofhepatichistologicalchanges.Observed

phenotypes vary in severity from non-inflammatory isolated steatosis (also termed non-alcoholic

fattyliver[NAFL])toamoreaggressiveformnamedsteatohepatitis(ornon-alcoholicsteatohepatitis,

NASH), which is characterized by inflammatory changes and hepatocellular ballooning associated

withvaryingdegreesofliverfibrosis(2,3).NASHisamultisystemdiseaseassociatedwithasignificant

risk for thedevelopmentof cirrhosisandhepatocellular carcinoma,aswell as liver transplantation

andliver-relateddeath(4,5).

The pathogenesis of NAFLD involves a complex interaction between nutritional factors, obesity,

insulin resistance, changes in the microbiota, genetic and epigenetic factors. These factors are

involved inthedevelopmentandprogressionofsteatosisresulting inanexcessiveaccumulationof

lipids, especially triglycerides, in the hepatocytes (6-8).Moreover, these factors contribute, either

simultaneouslyorsequentially,totheinflammatoryandfibroticresponseofthelivertosteatosis(9).

At the cellular level, the increased influx of free fatty acids (FFA) into the liver exceeds the

physiological capacity, leading to reactive oxygen species (ROS) overproduction, mitochondrial

dysfunctionandcellulardeathinaprocesscalledlipotoxicity,ultimatelyleadingtoinflammationand

fibrosis(10,11).

Several studies have shown that lipotoxicity appears to be the central driver of hepatocellular

damagethatcontributestotheinflammatoryresponseandthedevelopmentofliverfibrosis(12,13).

The crosstalk between hepatocytes and non-parenchymal cells plays a crucial role in this

inflammatory and fibrotic response (14-17). It is believed that fat-laden (steatotic) hepatocytes

release damage signals to the extracellular environment, resulting in paracrine effects on

neighboring cells such as resident macrophages of the liver (Kupffer cells) and stellate cells that

promote the activationof inflammatory and fibrogenicpathways, respectively (18,19). In addition,

fat-laden hepatocytes trigger various pathways leading to hepatocellular dysfunction by autocrine

effectsthatinitiateandperpetuatelipotoxicity(20-22).

Inrecentyears,obstructivesleepapneasyndrome(OSAS),acommonsleepdisordercharacterizedby

recurrentclosureoftheupperairwaysduringsleep,hasbeenassociatedwiththedevelopmentand

progressionofNAFLD(23)inbothobeseandnon-obesesubjects(24-25).Themostnotedhallmarkof

OSAS is intermittent hypoxia (IH), which leads to tissue hypoxia and can result in ROS

10

overproduction,mitochondrialdysfunctionandinflammation(26).Theseeventsarealsoinvolvedin

theinitiationofNASH(27).SeveralstudieshaveindicatedalinkbetweenOSASorIHwithincreased

liver TG accumulation in NAFLD through modulation of β-oxidation of fatty acids and de novo

lipogenesis inhepatocytes (28-31). Inaddition, inexperimentalmodelsofNASH ithasbeenshown

that mimicking OSAS with IH has pro-inflammatory effects as indicated by increased levels of

inflammatorycytokinessuchastumornecrosisfactoralpha(TNF-α)andinterleukin-6(IL-6)(30,31).

Likewise,invitrostudiesusinghepatocytesdemonstratedthathypoxia-induciblefactor1alpha(HIF-

1α)increaseshepatocyteapoptosisandgenerationofpro-inflammatorysignalsininflammatorycells

(32-34). Moreover, a link between HIF-1α and hepatic inflammation and fibrosis has also been

described inanimalstudiesof IH (35-37).Takentogether, theexperimentalevidencesuggests that

hypoxia,inaHIF-1αdependentmanner,contributestothetransitionfromisolatedsteatosistomore

advancedstagesofNAFLDandcanaggravatelipotoxicity,inflammationand/orfibrosise.

ThepathophysiologyofNASHisknowntoinvolvehepatocellulardamageassociatedwithlipotoxicity,

triggeringlocalinflammatoryresponsesandcontributingtoliverfibrosis.However,thereisalackof

knowledgeregardingthemechanismsofthedetrimentaleffectofhypoxiaonfat-ladenhepatocytes

andtheroleof intercellularcommunicationbetweenhepatocytesandnon-parenchymalcellstypes

suchasKupfferandstellatecells.

Recent studies have indicated that extracellular vesicles (EV) play a key role in intercellular

communicationinliverpathobiology(38-40).Inparticular,EVhavebeenimplicatedinhepatocellular

injury,inflammationandhepaticfibrosisinNAFLD,bothinhumansaswellasinexperimentalmodels

ofNASH(41-45).

EVarenanoparticlesdefinedbyalipidbilayer.ThecargoofEVcanhavemanyeffectsontargetcells,

bothphysiologicalaswellaspathophysiological.ClassificationofEVdependontheirsizeandsiteof

biogenesis:Exosomes(40-150nm)arereleasedfrommultivesicularbodies,microvesicles(MVs)(50-

1000nm)arereleasedfromthebuddingplasmamembraneandapoptoticbodies(50-5000 nm)are

releasedfromblebbingcells(47).ExosomesandMVsmaycontainavarietyofbioactivemolecules,

including cytoplasmic proteins, lipids, specific lipid raft-interacting proteins, messenger RNAs,

noncodingRNAsandmetabolites(39-46).Ingeneral,apoptoticbodiesareexcludedfromstudieson

EV (46). EVhavebeen implicated inmanypathophysiologicalprocessesand recently, the fieldhas

expandedintoEV-baseddiagnostics,prognosisandtherapeutics(48).

SeveralresearchgroupshavedemonstratedtheinvolvementofEVinintercellularcommunicationin

invivoaninvitromodelsofliverdiseases(42-44,48,49).Recentstudieshaveshownthathepatocyte-

derivedEV activatepro-inflammatory signals andpro-fibrotic signals in non-parenchymal cells (48,

11

49). Additionally, increased levels of circulating EV in in vivo models of NAFLD correlate with

histologicfeaturesofNASHthatindicateliverdamage(42-44,50).Therefore,itisimportanttostudy

thecontentofEVthatexertparacrineeffectsontargetcells.Interestingly,recentdatasuggestthat

EV released from fat-laden hepatocytes activate the inflammasome via caspase-1 activation in

hepatocytesandmacrophagesleadingtoaninflammatoryresponse(51).

TheNOD-likereceptorPyrinDomainContaining3 (NLRP3) inflammasome isspecificandcritical for

the activation of caspase-1 and the processing of pro-inflammatory cytokines. The NLRP3

inflammasomehasrecentlybeendemonstratedtocontributetothetransitionfromNAFLDtoNASH

(52,53). Studies have also shown that expression of inflammasome components is increased in

mousemodelsofNASHand inhumanswithNASH(54,55).Moreover,downregulationofNLRP3or

caspase-1inflammasomecomponentsalleviatehepaticsteatosis, inflammationandfibrosis(55-58),

suggestingthattheinflammasomeisapotentialtherapeutictargetinNASH.

Theresearchpresentedinthisdoctoralthesisinvestigateswhetherhypoxiaexacerbateslipotoxicity

in fat-laden hepatocytes and liver injury in a mice model of non-alcoholic steatohepatitis via the

release of extracellular vesicles and cellular crosstalk between hepatocytes and non-parenchymal

cells.Additionally,weexaminedinflammasomeactivationinconditionsofhypoxia,bothinvitroand

invivo.Takentogether, theresultsof this thesisestablisha linkbetweenextracellularvesiclesand

hepatocellular damage in hypoxia models that mimics OSA. This link involves EV-mediated

intercellular communication between hepatocytes and non-parenchymal cells in non-alcoholic

steatohepatitis.

12

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steatohepatitis.LabInvest.2012;92:713-23.

57. Yang G, Lee HE, Lee JY. A pharmacological inhibitor of NLRP3 inflammasome prevents non-alcoholic fatty liver disease in a

mousemodelinducedbyhighfatdiet.SciRep.2016;6:24399

58. CabreraD,WreeA,PoveroD,SolísN,HernandezA,PizarroM,MoshageH,TorresJ,FeldsteinAE,Cabello-VerrugioC,BrandanE,

Barrera F, Arab JP, Arrese M. Andrographolide Ameliorates Inflammation and Fibrogenesis and Attenuates Inflammasome

ActivationinExperimentalNon-AlcoholicSteatohepatitis.SciRep201714;7:3491.

15

Chapter2

EXTRACELLULARVESICLESINNAFLD/ALD:FROMPATHOBIOLOGYTOTHERAPY

ABSTRACT

In recent years, knowledge on the biology and pathobiology of extracellular vesicles (EV) has

exploded. EV are submicron membrane-bound structures secreted from different cell types

containingawidevarietyofbioactivemolecules[i.e.,proteins, lipids,andnucleicacids(codingand

non-codingRNA,andmitochondrialDNA].EVhaveimportantfunctionsincell-to-cellcommunication

and are found in awide variety of tissues and body fluids. Better delineation of EV structure and

advancesintheisolationandcharacterizationoftheircargohaveallowedtoexplorediagnosticand

therapeutic implications of these particles. In the field of liver diseases, EV are emerging as key

playersinpathogenesisofbothalcoholicliverdisease(ALD)andnonalcoholicliverdisease(NAFLD),

the most prevalent liver diseases worldwide and their complications, including development of

hepatocellularcarcinoma.Inthesediseases,stressed/damagedhepatocytesreleaselargequantities

ofEVthatcontributetotheoccurrenceofinflammation,fibrogenesisandangiogenesisthatarekey

pathobiologicalprocessesinliverdiseaseprogression.Moreover,thespecificmolecularsignaturesof

released EV in biofluids have allowed to consider EV as promising candidates to serve as disease

biomarkers. Also, different experimental studies have shown that EV may have potential for

therapeutic use as a liver-specific delivery method of different agents taking advantage of their

hepatocellularuptake through interactionswith specific receptors. In this review,wewill focuson

the most recent findings concerning the role of EV as new structures mediating autocrine and

paracrine intercellular communication in both ALD and NAFLD as well as their potential use as

biomarkers of disease severity and progression. Emerging therapeutic applications of EV in these

liverdiseasesarealsoexaminedandthepotentialforsuccessfultransitionfrombenchtoclinic.

1.-INTRODUCTION

Knowledgeofthepathobiologyofextracellularvesicles(EV)hasexpandedsignificantlyinthelastdecade

(1,2).Indeed,significantadvanceshavebeenmadeindelineatingthemechanismsofassemblyandrelease

ofEVaswellastheirsubsequentmembranefusionwithtargetcells(3,4).Moreover,powerfulanalytical

techniqueshavemadepossibletheextensivecharacterizationofthecargoofEVthatincludesamyriadof

molecules including growth factors, metabolic enzymes, microRNAs and transcription factors, certain

proteins,lipidsandmetabolites,amongothers,thatmodulateintercellularandinter-organcommunication

(3,5,6).Ofnote,high-throughputdatasetsofvesicularcomponentsarenowavailableinpublicdatabases,

whichstronglysupportsEVresearch(7,8).

16

NovelinsightsintothebiologyofEVshowthattheseparticlesregulatecriticalbiologicalfunctionsandmay

act as contributors to disease pathogenesis andmay also serve as disease biomarkers in virtue of the

relative simplicityofEV isolation fromdifferentbiofluids (9). Inaddition,EVaregaining interest froma

therapeuticpointofviewduetotheirpotentialasauniquedrugdeliverysystem(10).

InthefieldofhepatologyEVhaverecentlyemergedasnovelplayersinthepathogenesisandprogression

ofseveralconditions(11-13)includingthetwomostcommonliverdiseasesworld-wide:non-alcoholicliver

disease(NAFLD)andalcoholicliverdisease(ALD)(14).Specifically,recentstudiespointtoasignificantrole

ofEVinmodulating injury,amplifying inflammationandpromoting liverfibrosis inbothNAFLDandALD

(15-17).Sinceinformationonthistopicisdynamicandrapidlyevolving,weaimtoprovideanup-to-date

overviewofthecurrentknowledgeontheroleofEVinthecontextofbothNAFLDandALDwithemphasis

ontheirpotentialdiagnosticandtherapeuticimpactinthesediseases.Weexcludefromthisreviewdata

regardingEVinlivercancersincethishasbeenrecentlyreviewedelsewhere(18,19).

2.-GENERALCONCEPTSOFEVINTHELIVER:EVBIOGENESIS,SECRETIONANDCARGO

DetailsontheformationandsecretionpathwaysofEVhavebeenrecentlyreviewedelsewhere(15,20,21)

andcanalsobefoundinothercontributionsinthisspecialissueofCells(22).Onlybasicconceptswillbe

providedhereaswellasinformationonaspectsthatareofparticularimportanceforliverphysiologyand

pathophysiology.

Ingeneral,EVareclassifiedaccordingtosizeandbiogeneticpathway,suchasexosomes,microvesiclesand

apoptoticbodies(23).Exosomesarebilayerlipidvesicleswithadiameterof30-150 nmthatarederived

fromendosomalmultivesicularbodies(MVBs)(15,23).Itsformationresultsfromtheinvaginationofthe

plasmamembrane (early endosome) and the subsequent fusion of endocytic vesiclesmediated by the

endosomalsortingcomplexresponsiblefortransport(ESCRTs)andothercomponents(suchasceramides

andtetraspanins)(24).TheMVBscanreleasetheintraluminalvesiclesknownasexosomesbythefusionof

MVBstotheplasmamembrane,aprocessmediatedinpartbyRabGTPases(25).Microvesicles(MVs)have

adiameterof50-1000nmandoriginatefromtheplasmamembranebybuddingandfissionfollowedby

releaseintotheextracellularspace(15,23).MVscontainasubsetofcellsurfaceproteinsdependingonthe

compositionoftheparentalplasmamembrane(26,27).Apoptoticbodieshaveadiameterof100-5000 nm

and originate from the budding of cellmembranes andmay contain nuclearmaterial,which is quickly

phagocytosedduringprogrammedcelldeath (15,28).UnlikeexosomesandMVs, the roleof apoptotic

bodiesisnotrelatedtointercellularcommunication.Therefore,whenstudyingEVundercellulardamage

conditions,apoptoticbodiesareexcluded(29).

All EV transport a variety of bioactive molecules, including cytoplasmic proteins, lipids, specific lipid

raft-interactingproteins,messengerRNA(mRNA),microRNA(miRNA),ribosomalRNA(rRNA),transferRNA

17

(tRNA),noncodingRNAs(ncRNAs),DNA,mitochondrialDNA(mtDNA)andmetabolites(24,30).Lipidomic

analysis has shown that EV, independently of their biogenesis, contain a myriad of lipids such as

cholesterol, sphingomyelin, ceramide, saturated fatty acids, phosphatidylcholine,

phosphatidylethanolamine and phosphatidylserine (4, 23, 29, 30). In addition, proteomic analysis has

shown that EV contains different types of proteins, such as heat shock proteins (Hsp70 and Hsp90),

tetraspanins (CD9,CD63,CD81,CD82), endosomal sorting complexproteins required for transport (Alix

andTsg101),receptorsincludingepidermalgrowthfactorreceptor(EGFR),membranetraffickingproteins

(GTPases,FlotillinandAnnexins),cytoskeletalproteins(tubulinandactin)andcytosolicproteins(5,26).Of

note, thecargoofEVvariesdependingnotonlyontheircellularoriginbutalsoontheconditionunder

whichtheyarereleased(i.e.physiologicalvs.pathological).

In the liver, both parenchymal (hepatocytes) and non-parenchymal cells (i.e. hepatic stellate cells,

endothelial,cholangiocytes,Kupfercellsandliverendothelialcells)havebeenfoundtoreleaseEVinboth

physiologicalandpathologicalstates(20).However, informationontargetcell repertoire, receptorsor

other specific actions is still limited and incomplete. It has been shown that healthy hepatocytes

producelimitedamountsofexosomescontainingproteinspotentiallyrelevantforcellsurvival,growthand

proliferation(31),whereasstressedhepatocytesboostexosomerelease(32)andenrichtheircontent in

specific proteins, lipids and microRNAs that modulate the transcriptional program of neighboring

hepatocytesandnon-parenchymalcells,thusmodulatinginflammationandfibrosiswhicharecriticalfor

theprogressionofliverdiseases(Figure1).InterestinglyrecentevidencesuggeststhatEVfromfat-laden

hepatocytecanalsosignaltootherorganssuchasadiposetissueinfluencingadipogenesisandtissue

remodeling(33).

2.2EVandliverinflammation

Hepatocellular damage determines the release of a number of signals into the extracellular

environment that can contribute to tissue inflammation (34). Some of these signals (collectively

termed damage-associated molecular patterns [DAMPs]) are packaged in EV and signal between

hepatocytesandnon-parenchymalcells suchas liver-residentmacrophages (Kupffercells,KC) (35).

Indeed, EVmay evoke synthesis and release of proinflammatory cytokines such as pro-interleukin

(IL)-1b and IL-6 (36) by KCs, thus contributing to local inflammation (37). Also, EV released from

hepatocytes can promote therecruitmentof additional immune cells (i.e.

proinflammatorymonocyte-derived macrophages) into theliver maintaining and amplifying

inflammation. EV can also signal to endothelialcells and can contribute to vascular inflammation

(38). Furthermore, to add complexity, EV canalsobe secreted fromother cells and influence liver

inflammation. In this regard, some evidence suggests that platelet-derived EV may have

18

proinflammatoryeffects inthe liverbutthisneedsfurtherconfirmation(39,40).Finally, ithasalso

been shown that EV are released frommonocytic cells and induce polarization towards the anti-

inflammatory M2 phenotype of neighboring naive monocytes by delivering cargo miR-27a, thus

contributingtoresolutionofinflammation(41).Collectively,thesefindingssuggestthatEVreleased

from injuredhepatocyteshavean important role inmodulating the inflammatory responseduring

liver damage through intercellular communication between different cell types with potential

contributions fromother cell-derivedEV (35). Specificmechanisms involved inNAFLDandALDare

reviewedbelow.

2.3EVandliverfibrosis

Persistent liver fibrogenesis and the development of cirrhosis are responsible for the liver-related

morbidityandmortalityassociatedwithchronic liverdiseases (42).Activationor trans-differentiationof

HSCsresultingininsolublecollagendepositionanddistortionofthenormalmacro-andmicro-anatomical

structureoftheliveristhemajordriverofliverfibrogenesis(43).Theroleofparacrinesignalsoriginating

from injuredepithelial cells (hepatocytes) that candirectlyor indirectly induceHSCactivationhasbeen

recognized in recent years (44). In this regard, EV seem to play a role as shown by previous reported

studies.Ofnote,lipid-inducedhepatocyte-derivedEVseemtoregulateHSCactivationbyshuttlingspecific

microRNAs (i.e. miR-128-3p) that suppress PPAR-γ expression in HSC leading to a marked increase of

profibrogenicgeneexpression(45).Otherauthorshaveshownthatinternalizationofendothelial-derived

exosomesbyHSCsenhancescellmigrationinaprocessmediatedbysphingosine1-phosphate(S1P)(46).

Additionally, intercellular communicationbetweenquiescent andactivatedHSCs via exosomes canalso

modulatefibrosis.Inthisregard,aroleforshuttledmicroRNA214(miR-214)inregulatingtheexpressionof

alpha-smoothmuscleactinandcollageninactivatedHSCshasbeendemonstrated(47,48).Moreover,EV

derived from non-resident cells such as platelets or granulocytes have been shown to increase

angiogenesis and tohaveprocoagulantproperties, thuspromoting fibrogenesis (49, 50). These findings

suggest that EV appear to be key modulators in fibrosis as signals from both parenchymal and non-

parenchymallivercellscaneitherdriveorslowdownHSCactivation.Inaddition,circulatingEVmaybe

usedasabiomarkerofhepaticfibrosisandhavepotentialimplicationsforthedevelopmentofnovelanti-

fibrotictargets(51)asreviewedinthefollowingsections.

3.-EVASBIOMARKERSINLIVERDISEASES

DynamicchangesofEVgenerationinpathologicalconditionsandtheaccessibilitytomeasureandanalyze

theminbiologicalsamples(i.e.blood,urine,bileandotherbiofluids)renderEVgoodcandidatesasdisease

biomarkers.Therelativeaccessibilityfortestingandperformingrepeatedmeasurementsovertimecould

facilitate early diagnosis, disease monitoring and development of personalized medicine.

19

Furthermore,refinementoftechniquesallowingisolationandindepthcharacterizationofEVcargoes

can lead to identification of disease-specific molecular signatures or profiles and provide ample

opportunitiesforEVtobeusedassuitable,non-invasivebiomarkers(52).

EV have been studied as potential biomarkers of liver injury in the setting of ALD, NAFLD, drug-

inducedliverdiseaseandcholangiopathies(13,27,53,54)aswellasdiagnostictoolsforlivercancer

(i.e.hepatocellularcarcinomaandcholangiocarcinoma)(19,54).Inthisregard,determinationofthe

numberofcirculatingEVandtheuseofnucleicacidbased,lipid-basedandprotein-baseddiagnostics

have been performed tomeasure EV enriched in liver-derivedDNA,microRNAs, lipids or proteins

(13,27).However,beforeimplementingEV-basedbiomarkersintheclinic,standardizationofsample

processing(i.e.collection,transportation,storageandhandling)andassaysystemsisneededaswell

aslargereplicativestudiestoallowEVmolecularsignaturestobeconclusivelylinkedtospecificliver

diseases.RecentadvancesrelatedtoNAFLDandALDarereviewedbelow.

4.-THERAPEUTICPOTENCIALOFEV

From a therapeutic perspective, EV either unmodified or engineered, can be utilized for therapeutic

purposes(55).Withregardtoliverdiseases,effortshavebeenfocusedontwomajorareas:a)theuseof

EVasdeliveryvehiclesofdrugstotheliver(56)andb)theuseofEVthemselvesastherapeuticagentsto

stimulateliverregeneration,modulateinflammation,reduceliverfibrosisorhalthepatocarcinogenesis(57,

58). The formerapproach involves theuseofdifferent techniques to loadEVwithadesired cargo (i.e.

miRNA, siRNA, chemotherapeutic agents) to act as a “Trojan horse” on target cells. Theoretically,

membranepropertiesofEValloworganandcell-specificdelivery,immune-evasionandtargetingofdistinct

intracellulartraffickingpathways.However,anumberofchallengesrelatedtothemanufacturingofEV(i.e.

production,coating, loading,etc.)stillneedtobesolvedbeforecontrolledclinicalstudiescanbecarried

out(59,60).WithregardtotheuseofEVastherapeuticagents,mostoftheavailableevidencehasbeen

generatedusingmesenchymalstemcell(MSC)-derivedEVobtainedfromhumanumbilicalcordorhuman

embryos that have been tested in numerous preclinical liver diseasemodels (i.e. carbon tetrachloride,

thioacetamide,D-galactosamine/TNF-α-inducedlethalhepaticfailureandbileductligation)withpromising

results.DataforNAFLDandALDaremorelimitedandarereviewedinthecorrespondingsectionsbelow.

Athird,andstillnascent,approachrelatedtoEV-basedtherapyisbasedontheconceptthat interfering

withEVsecretionoruptakemayattenuateharmfuleffectsontargetcells(11,61).Inthisregard,several

pharmacological agents are being explored that havebeen shown to inhibit EV trafficking,modify lipid

metabolism or decrease EV secretion. However, the complexity of EV biogenesis poses significant

challengestothedevelopmentofspecificagentsabletoblockEVproductionselectively[seeref.(61)for

anin-depthreviewofthistopic].

20

5.-EVINNAFLD

AnumberofstudieshavedemonstratedaroleofEVinbothpathogenesisandprogressionofNAFLD

(14,15).Triggeringof inflammationandfibrosisdevelopmentarekeyforprogressionfromisolated

steatosis (also referred as NAFL or non-NASH fatty liver) to nonalcoholic steatohepatitis (NASH),

which ishallmarkedbythepresenceofhepatocyteballooningasreflectofongoing liver injuryand

death (36). Data from different diet-induced animal models of NASH have shown that EV

concentration increases with disease progression in a time-dependent manner.(17, 62, 63). This

seemstobearesponsetotheaccumulationof toxic lipidsandtheirdownstreammediators in the

liver,which increasethecapacityofhepatocytestoformandreleasedifferenttypesofEV(37,62-

64). In vitro treatment of hepatocytes with non-esterified fatty acids evokes the release of EV

containingnumerousmolecules includingC-X-Cmotif ligand10 (CXCL10), sphingosine-1-phosphate

(S1P), mitochondrial DNA (mtDNA), micro-RNAs, ceramides and tumor necrosis factor-related

apoptosis-inducingligand(TRAIL)(15).Thesemoleculesmayamplifyinflammationthroughmultiple

mechanisms such as macrophage activation and monocyte chemotaxis as well as inflammasome

activationandmodulationoftheNF-κBpathwayintargetcells(64,65).Asmentionedearlier,EVmay

bereleasedbydifferentmechanismsincludingacaspase-3-dependentmechanism(63)oractivation

ofdeath receptor5 (DR5) inhepatocytes.Hepatocyte-derivedEVare able to induceexpressionof

pro-inflammatorycytokinesandpromoteM1polarizationofhepaticmacrophages(37,66).Ofnote,

CXCL10-bearing EV can also serve as chemotactic stimuli formacrophages as shown recently (64).

Moreover,EVreleasedfromhepatocytescancontributetohepaticrecruitmentofmonocyte-derived

macrophages by promotingmonocyte adhesion via integrin β1(ITGβ1)-dependentmechanisms as

showninmurineNASH(67).AdditionalstimulicanalsostimulateEVreleasefromhepatocytes.Inthis

regard,ithasbeenobservedthathypoxiadeterminesthatfat-ladenhepatocytesreleaseEVableto

signal KC evoking proinflammatory phenotypes in this cells, a phenomena that may explain may

underlietheaggravatingeffectofobstructivesleepapneasyndromeonNAFLD(68).Thus, itseems

clear that lipotoxic injury of hepatocytes determines EV release, promoting inflammation through

activation and recruitment of macrophages (14) with clear implications for the triggering of

inflammationinNAFLD/NASH(36). Inaddition,hepatocyte-derivedEVmaypromoteHSCactivation

(45,69)inexperimentalmodelsofNAFLD/NASH.Interestingly,bothmouseandhumanHSCsrelease

EVthattargethepatocytesandHSCsthemselves(47,48,70).Collectively,thesedataimplicateEVas

partofthecellulareventstriggeringhepaticfibrogenesis,akeyprocessinNAFLDprogression(71).In

additiontosignalingtomacrophagesandHSCs,EVmayactonendothelialcells (38,63)promoting

vascularinflammationwithpotentialimplicationsforNAFLD-relatedatherosclerosis.Inthisregard,it

has been shown that EV carryingmiR-1 as cargomediates proinflammatory effects in endothelial

21

cells in mice via downregulation of KLF4 and activation of the NF-κB pathway (38). Finally, other

organ-derived EV (i.e. visceral adipose tissue-derived exosomes) can contribute to NAFLD

pathogenesisandprogressionandinfluencefibrogenicpathwaysinbothhepatocytesandHSCs(72)

underscoringtheroleofotherinsulin-sensitiveorgansinNAFLD.

Studiescarriedout inmousemodelsofNASHhaveshownthat totalcirculatingEVandparticularly

hepatocyte-derived EV are elevated early in the disease process while other cell-derived EV (i.e.

macrophage-andneutrophil-derivedEV)appearinthecirculationlater,likelyreflectingtheongoing

inflammatoryprocess(62,73).ProteomicprofilingofcirculatingEVinexperimentalNAFLDhasbeen

demonstrated to allow differentiation between NAFLD vs. control animals (17). These findings

underscorethepotentialofEVasminimallyinvasivebiomarkersforNAFLD(74),whichareurgently

forclinicaltrialsandintheclinic.SincecirculatingEV(mainlyexosomes)arealsoincreasedinhuman

NAFLDandhavebeenfoundbysomeauthorstocorrelatewithdiseasehistologicalfeatures(75),EV

analysis in serum involving quantitative and qualitative determinations (including cell surface

markersassessmentandmeasurementofdifferentcargoes [i.e.proteins, lipidsandmicroRNAs]) is

nowafocusofintenseresearch.Severalstudieshavebeenpublishedinthisregardshowingthatthe

number of CD14+ and CD16+ EV is inversely associated with the severity of NAFLD-related liver

fibrosis,while also increasing the diagnostic capability of the enhanced liver fibrosis score (LFS) in

patientswithNAFLD(AUC:0.948and0.967forCD14+andCD16+EV,respectively,vs0.915forLFS

alone) (76). Other efforts include detection of circulating EV containing C16:0 ceramide- and S1P-

enriched lipid species that progressively increase in the plasma of obese patients with simple

steatosis and inNASHpatientswithearly fibrosis (62).Unfortunately,diagnostic accuracyof these

determinationsremainsincompletelyexploredinthefieldofNAFLD/NASHandrigorousvalidationof

this approach is needed (13, 74). Moreover, significant challenges remain regarding isolation,

reproducibilityanddefinitionofnormalcontrols(13,77).

Therapeutic efforts involving EV in the field of NAFLD/NASH are nascent. Attempts to halt

inflammation and fibrosis in rodent models of NAFLD/NASH using EV as therapeutic agents have

been published recently. EV obtained from amnion-derivedMSC (AMSC) to treat rats with either

NASHor liver fibrosis inducedby the hepatic toxicant CCL4. AMSC-EVwere given intravenously in

one or two doses and amelioration of inflammation and fibrogenesis was observed (78). More

recently,humanliverstemcells(HLSCs)-derivedEVhavebeenusedtotreatmicewithdiet-induced

steatohepatitis(79).TheauthorsfoundthatEV-HLSCtreatmentsignificantlydownregulatedhepatic

pro-fibroticandpro-inflammatorygeneexpressionandamelioratedthehistologicalabnormalitiesin

mice with NASH. Proteomic analysis of EV-HLSCs showed that their cargo included various anti-

22

inflammatoryproteinssuchasInterleukin-10,thatmaycontributetotheobservedbeneficialeffects.

TheseresultsunderscoretheconceptthatEVcanbeexploitedfortherapyinNAFLD/NASH.

6.-EVANDALD

Recentstudieshave focusedon the roleofEV inALD (11,14,80,81).Bothhepatocyteandmonocyte-

derivedEVhavebeenpostulatedtoregulatemacrophagedifferentiation,therebypromotinginflammation

inalcoholichepatitis(AH)(24,41,53,69,82).Severalmoleculeshavebeenproposedtoberesponsiblefor

EV-mediatedcell-to-cellsignaling, includingmiRNAs(inparticular,miR-122andmiR-155)(41,81,83-88).

TheCD40ligandwasproposedasanEVcargothatcouldpromotemacrophageactivationinvitroandin

vivoinexperimentalmodelsofAH(81).Also,inamousemodelofALDinvolvinggastricinfusionofethanol,

amicroRNAbarcode (let7f,miR-29a, andmiR-340) that can be detected in blood,which is specific for

alcohol-relatedliverinjury(16).

Fromaclinicalpointofview,thereiscurrentlynobiomarkerabletoassessearlystagesofALD.EVhave

beenshowntocorrelatewiththediagnosisandprognosisofalcoholichepatitis(89).Additionally,EVhave

beenproposedaspotentialbiomarkerstodifferentiatemildandsevereformsofALDandtheircargo,such

assphingolipids,todiscriminatebetweendifferentliverdiseaseetiologies(89).Earlyidentificationofthese

subjectsmightleadtotimelyinterventioninthediseaseprocess.Atpresent,thediagnosisofAHreliesona

historyofalcoholconsumptioninacompatibleclinicalscenario.Therearenobiomarkerswhichpermitthe

earlydiagnosisofAHin“at-risk”populationsnorscreeningteststhatcananticipatethoseatriskformore

severe manifestations of AH. This at-risk group is particularly important to identify since there is no

effectivetherapyforsevereAHandeffortstoavoid itcouldbe implemented.Severalclinicalprognostic

markershavebeenproposedbutthevariablesusedreflecttheseverityofliverdisease.Theseindices(i.e.

theMaddreydiscriminantfunction,modelforend-stageliverdisease(MELD)andtheLilleScore)arebased

on the non-specific biochemical assessment of liver and renal function and rely heavily on serum total

bilirubin,prothrombintime,andcreatinine(90).Therelianceonbilirubinlimitstheirdiagnosticutility,as

specificity is confounded by hyperbilirubinemia in co-existent cholestatic liver diseases such as drug-

induced liver disease or cholestatic hepatitis such as primary sclerosing cholangitis and primary biliary

cholangitis. None of these prognostic scores utilizes a pathophysiologically validated biomarker that

reflectstheunderlyingmolecularandsignalingmechanismsofthedisease.Furthermore,theinflammatory

responseispredictiveofmortalitybutisnottakenintoaccountinmathematicalmodels.Theycanidentify

subjectsathighestmortalityriskwithanareaunderthereceiveroperatingcurve(AUROC)ofunder0.8;

idealsurvivalmodelsshouldhaveanAUROC>0.8(91).Thereasonthesescoresareimperfectmaybethat

they are not based on pathophysiologic mechanisms that mediate liver injury. Liver biopsy, the gold

standard for diagnosis ofAH, remainsunderutilizeddue to the concurrent coagulopathy,which greatly

23

increasestheriskofbiopsy-relatedcomplications(92).Liverbiopsy-basedhepatichistologyscoreperforms

asanAUROCof0.73inpredicting90-daymortality(91).InthisregardtheuseofEVasbiomarkerinAHis

promising.Thus,ithasbeenrecentlyreportedthatthetotalnumberofEVwassignificantlyincreasedin

patientswithAHandthattwomicroRNAs(i.e.miRNA-192andmiRNA-30a)weresignificantlyincreasedin

plasmaofsubjectswithAH(82).Mostrecently,EVhavebeenusedasasurrogatemarkerofimprovement

inclinicaltrialsofpatientswithAH(93).FurtherstudieswillberequiredtovalidateEVasabiomarkerfor

AHdiagnosisand/orprognosis.

7.-CONCLUDINGREMARKSANDFUTUREPERSPECTIVES

ThefieldofEVinfattyliverdiseasesisrapidlyevolvingastheimportantfunctionsoftheseparticlesincell-

to-cellcommunicationandinthepathogenesisofbothALDandNAFLD,themostprevalentliverdiseases

worldwide, are unveiled. The release of large quantities of EV by stressed/damaged hepatocytes

contributestoinflammation,fibrogenesisandangiogenesis,fuelingliverdiseaseprogressionandprovides

opportunities for intervention. The identification of specific molecular signatures of released EV is

promising in the search fordisease-specificbiomarkersalthoughmoredata isneeded tovalidate these

markersinlargercohortsandinarigorousmanner.EVmayhavepotentialfortherapeuticusebutthisfield

isstillnascent.MoreresearchisneededforasuccessfultransitionofcurrentEVknowledgefromthebench

totheclinic.

24

Figure1.Extracellularvesicles(EV)canbereleasedbyhepatocytesuponlipotoxicoralcohol-inducedinjury.EVcargoes

includeamyriadofmoleculesthatcanactontargetcellsevokinginflammatoryandfibrogeniceventsandpromoting

neoplastictransformationthuscontributingtotheprogressionofbothalcoholicliverdisease(ALD)andnonalcoholic

liverdisease (NAFLD) to their inflammatoryandmoreaggressive formsnonalcoholic steatohepatitis (NASH) and

alcoholicsteatohepatitis(ASH).

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25

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Chapter3

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CHEMICAL HYPOXIA INDUCES PRO-INFLAMMATORY SIGNALS IN FAT-LADEN HEPATOCYTES AND

CONTRIBUTESTOCELLULARCROSSTALKWITHKUPFFERCELLSTHROUGHEXTRACELLULARVESICLES

ABSTRACT

Background: Obstructive sleep apnea syndrome (OSAS), which is characterized by occurrence of

intermittent hypoxia (IH), is an aggravating factor of non-alcoholic fatty liver disease (NAFLD).We

investigatedtheeffectsofhypoxia inboth invitroandinvivomodelsofNAFLD.Methods:Primary

rathepatocytestreatedwithfreefattyacids(FFA)weresubjectedtochemicallyinducedhypoxia(CH)

usingthehypoxia-induciblefactor-1alpha(HIF-1α)stabilizercobaltchloride(CoCl2).Triglyceride(TG)

content, mitochondrial superoxide production, cell death rates, pro-inflammatory cytokines and

inflammasomecomponentsgeneexpressionandproteinlevelsofcleavedcaspase-1wereassessed.

Also,Kupffer cells (KC)were treatedwithconditionedmedium (CM)andextracellularvesicles (EV)

fromhypoxicfat-ladenhepatocytesandthecholinedeficientL-aminoaciddefined(CDAA)-fedmice

modelusedtoassesstheeffectsofIHonexperimentalNAFLDinvivo.Results:CHinduceda2-fold

increase in HIF-1α protein levels. Hepatocytes exposed to FFA and CoCl2 exhibited increased TG

content and higher cell death rates aswell as increasedmitochondrial superoxide production and

mRNAlevelsofpro-inflammatorycytokinesandofinflammasome-componentsinterleukin-1β,NLRP3

andASC.Proteinlevelsofcleavedcaspase-1increasedinCH-exposedhepatocytes.CMandEVfrom

hypoxic fat-laden hepatic cells evoked a pro-inflammatory phenotype in KC. Livers fromCDAA-fed

miceexposedtoIHexhibitedincreasedmRNAlevelsofpro-inflammatoryandinflammasomegenes

aswellasincreasedlevelsofcleavedcaspase-1.Conclusion:Hypoxiapromotesinflammatorysignals

including inflammasome/caspase-1 activation in fat-laden hepatocytes and contributes to cellular

crosstalkwithKupffercellsbyreleaseofEV.Thesemechanismsmayunderlietheaggravatingeffect

ofOSASonNAFLD.

1.-INTRODUCTION

Inrecentyears,obstructivesleepapneasyndrome(OSAS),acommonsleepdisordercharacterizedby

recurrentclosureoftheupperairwaysduringsleep,hasbeensuggestedtomodulatetheseverityof

differentmetabolicdisorders(1,2).ThehallmarkofOSASistheoccurrenceofintermittenthypoxia

(IH) leading to tissuehypoxiaandpromotingoxidative stress, inflammationandamyriadofmulti-

organ pathophysiological effects (3). Among conditions in which OSAS acts as both a potential

inducerandasanaggravatingfactorisnon-alcoholicfattyliverdisease(NAFLD),thecommonestliver

diseaseworldwide (4-6).NAFLDdescribesa clinicopathologicalentitydefinedby thepresenceofa

spectrumofhepatichistologicalchangesranginginseverityfromisolatedsteatosis(alsotermednon-

alcoholic fatty liver [NAFL]) tosteatohepatitis (namednon-alcoholicsteatohepatitis,NASH)through

31

to advanced fibrosis and cirrhosis (7, 8). Development of steatosis is closely linked to both

overweightandobesityaswellastoinsulinresistanceandtransitionfromisolatedsteatosistomore

advancedstagesofthediseaseoccursonlyinaminorityofNAFLDpatients.Multiplefactorsinfluence

NAFLD development and progression including individual’s genetic background, environmental

factorsandthepresenceofamyriadofcomorbiditiesincludingOSAS(6,9).

The main mechanisms of hepatocellular damage in NAFLD include those related to lipotoxicity,

mitochondrialdysfunction, formationof reactiveoxygenspecies,endoplasmic reticulumstressand

disturbed autophagy ultimately leading to hepatocyte injury and death that triggers hepatic

inflammation, hepatic stellate cell activation, and progressive fibrogenesis, thus driving disease

progression(8,10-12). Inaddition,recentstudieshaveshownthat inflammasomeactivationisalso

oneofthekeyeventsinNAFLDprogression(13-15).Specifically,theNOD-likereceptorPyrinDomain

Containing3 (NLRP3) inflammasomehasbeenrecognizedtobe involved intheprogressionof liver

damage in experimental in vitro (hepatocytes and immune liver cells) and in vivomodels ofNASH

(16-18)andalso inhuman studies (19).NLRP3 inflammasomemediates thematurationof inactive

pro-caspase-1 into active cleaved caspase-1, which cleaves gasdermin D (GSDMD) that in turn

determine the activation of the pro-inflammatory cytokines interleukin [IL]-1β and IL-18, which

amplifythepathologicalphenomenainNAFLDbypromotinginflammationandpyroptoticcelldeath

(20). Of note, the progression of damage in NAFLD also involves the participation of other liver-

residentcellssuchasmacrophagesorKupffercells(KC)aswellastherecruitmentof inflammatory

cellsfromtheperiphery(21-23).

WithregardtothepathophysiologicalconnectionsbetweenOSASandNAFLD,existingdataindicate

that OSASmay relate to both NAFLD occurrence as well as disease progression. Of note, several

studies have shown that IH is able to inducemetabolic alterations such as insulin resistance and

increased liver triglyceride (TG) accumulation as well as increased oxidative stress and increased

inflammation,whichare related tosteatosisdevelopmentandhepatocellulardamage, respectively

(24-26).HepaticTGaccumulationinthesettingofOSASmayresultfromhypoxia-relatedchangesin

lipid metabolic pathways such as a decrease in fatty acid β-oxidation and increased de novo

lipogenesis(5,27).Also,IHhasbeenshowntopromotepro-inflammatoryeffectsinanimalmodelsof

NAFLDmodulating inflammatory cytokineproduction (i.e. tumornecrosis factor-alpha [TNF-α] and

IL-6) (28,29).Althoughsomestudiessuggestarelevantroleofhypoxia, likelythrough inductionof

the hypoxia inducible factor 1 alpha (HIF-1α), in promoting hepatocellular cell death and the

generation of pro-inflammatory signals in several experimental settings (30), assessment of these

phenomenainthecontextofNAFLDhasbeenlessexplored(5,31).

32

In the present study, we aimed to assess the effects of hypoxia on cellular lipid accumulation,

hepatocellular death and pro-inflammatory signals including those related to the NLRP3

inflammasomeleadingtocaspase-1activationininvitroandinvivomodelsofNAFLD.Moreover,we

explored whether hypoxia modulates cellular crosstalk between hepatocytes and resident

macrophages (i.e. KCs) in the setting of NAFLD and if this crosstalk involves the release of

extracellularvesicles(EV)fromhepatocytes.Ofnote,EVhavebeenrecentlyrecognizedasplayingan

importantrole inamplifyingthe inflammatoryresponse inNASH(32,33)butfewdataexistonthe

potentialroleofhypoxiainmodulatingtheirrelease.Wefoundthathypoxiainduceshepatocellular

damageinfat-ladenhepatocytesthatinvolvesNLRP3inflammasome-associatedcaspase-1activation

and increasedmitochondrialsuperoxideproduction leadingto increasecelldeathratesbymultiple

mechanisms including apoptosis and pyroptosis. Also, hypoxia contributes to evoking a pro-

inflammatory response in Kupffer cells by a mechanism that involves EV release from fat-laden

hepatocytes.Theseobservationspartiallyclarifythemechanismsunderlyingtheaggravatingeffectof

OSASonNAFLD.

2.-MATERIALSANDMETHODS

2.1Animals

Specified pathogen-freemaleWistar rats (220–250 g; Charles River Laboratories Inc.,Wilmington,

MA,USA)andmaleC57bl6mice[purchasedfromJacksonLaboratories(BarHarbor,ME,USA)]were

used in the present study. Animals were housed under standard laboratory conditions with free

access to standard laboratory chowdiet andwater.All experimentswere carriedoutaccording to

the Dutch and Chilean laws on the welfare of laboratory animals and guidelines of the local

institutional animal care and use committees of the Pontificia Universidad Católica de Chile and

ethicscommitteeofUniversityofGroningenforcareanduseoflaboratoryanimals.Alleffortswere

madetominimizeanimalssufferingandtoreducethenumberofanimalsused.

2.2 Cell isolation techniques, culture conditions, cell surfacemarkers detection and use of liver-

derivedcelllines

ToconducttheexperimentsdescribedbelowweusedbothprimaryrathepatocytesandKC.Isolation

techniquesusedaredescribedelsewhere(34,35)andintheSupplementarymaterialfile.Detailsof

cultureconditionsandKCcellsurfacemarkersdetectionarealsoprovidedinthatsection.Thehuman

hepatocellular carcinoma cell line HepG2 (American Type Culture Collection [ATCC] HB-8065,

Manassas,VA,USA)wasused tocarryoutexperiments involvingEV isolationafter treatmentwith

FFA.Cultureconditionsaredescribedinthesupplementarymaterialfile.

33

2.3Treatmentwithfreefattyacidsandchemicalhypoxiainduction

Inordertoassess theeffectsofhypoxia in fat-laden livercells,hepatocyteswere incubatedwitha

mixtureoffreefattyacids(FFA)consistingofoleicacid(500μmol/L)andpalmiticacid(250μmol/L)

inanaqueoussolutionofBSAasdescribed(36).IncubationswerecarriedoutwithorwithoutCobalt

(II) chloride (CoCl2; Sigma Aldrich, Saint Louis, MO, USA) (200 μmol/L) for 24 hours to induce

chemicalhypoxia (37).CoCl2 isawell-knownhypoxia-mimeticagent, thatmimicshypoxia/ischemic

conditionsby stabilizationofhypoxia-inducible factorHIF-1α (38). Control cellswere treatedwith

BSA alone. To obtain the corresponding conditioned medium (CM), after treatment, hepatocyte

culturemediumwas replaced by FBS-freemedium for an additional 24 hours. CM from different

treatmentswereaddedtoKCfor24hoursinordertoexplorethepossibilitythathypoxiamodulates

hepatocytes-KCcellularcrosstalk.

2.4OilredOcellstainingandTGmeasurement

Primary rat hepatocytes cultured on glass cover slideswere treatedwith FFA and CoCl2 for 24 h.

Hepatocyteswerethenwashedwithphosphate-bufferedsaline(PBS)twice,andthenfixedwith4%

formalin for10minutes.Cellswerewashedwith60% isopropanol twiceandstainedwithoil-red-O

solution for 10minutes at room temperature. Then, cellswerewashedwith 60% isopropanol and

washedindistilledwaterfor5minutes.Thencellswereincubatedwithhematoxylin/eosinsolution

for 1minute andwashedwithdistilledwater for 5minutes. Slicesweremountedusing 1 dropof

glycerin-gelatin solution. Stained lipid droplets in cells were examined using a slide scanner,

NanozoomerTM (Hamamatsu Photonics K.K., Shizuoka, Japan). Intracellular TG content was

determined using TG Quantification Assay kit (ab65336, Abcam, Cambridge, UK) according to the

manufacturer’sinstructionsandnormalizedtoproteinconcentration.

2.5Apoptosismeasurement

Caspase-3 fluorometric assay was used to determine apoptosis induced by FFA and/or CoCl2 in

freshlyisolatedmousehepatocytes.Aftertreatment,hepatocyteswerescrapedandcelllysateswere

obtained by three cycles of freezing (−196°C) in liquid nitrogen and thawing (37°C) followed by

centrifugation for 5 minutes at 13,000g. Caspase-3 enzyme activity was assayed as described

previously(39).Arbitraryunitsoffluorescence(AUF)werequantifiedinaspectrofluorometeratan

excitationwavelengthof380nmandanemissionwavelengthof430nm.

2.6Assessmentofcelldeathassociatedtodisruptedcellularmembraneintegrity

SYTOXTMGreennucleicacidstain(Invitrogen,S7020,Carlsbad,CA,USA)wasusedtodeterminecell

deathinducedbyFFAand/orCoCl2inhepatocytes(40).Cellswereculturedin12-wellplates.After

34

treatment, diluted SYTOXGreen solution (1:40.000/HBSS)was added to the plates for at least 15

minutesat37°C,5%CO2.SYTOXTMGreenentersthecelluponlossofmembraneintegrityandbinds

toDNAactingasacounterstainthatcanbeanalyzedwhenexcitedat488nm.ALeicafluorescence

microscopewasusedat thatwavelengthof fordetectionof necrotic cells,whichwerequantified

using Image J software. Lactate dehydrogenase (LDH) release was assessed as described in the

Supplementary materials file. Additionally, we assessed the caspase-cleaved gasdermin-N domain

(GSDMD-N) to confirm pyroptotic cell death by Western blot to evaluate the occurrence of

pyroptoticcelldeath(19).

2.7Mitochondrialsuperoxidedetection

At the end of 24h-long incubations, hepatocyteswerewashed oncewithwarmHBSS followed by

incubation with 200 nmol/LMitoSOX™ RedMolecular Probes (InvitrogenTM, Carlsbad, CA,USA) in

William's E medium for 15 min at 37°C protected from light in order to detect mitochondrial

superoxideproduction(41).Then,cellswerewashedwithwarmHBSSandmountedontoglassslides

using DAPI staining solution (InvitrogenTM, Carlsbad, CA,USA). The fluorescence analyses were

immediatelyrecordedusingafluorescentmicroscopeatawavelengthof510/580nm(Ex/Em)bya

Leicamicroscope (LeicaMicrosystemsGmbH, �Wetzlar�,Germany).Quantificationof fluorescence in

microscopicimagesofMitoSOXwasperformedusingImageJsoftware.

2.8WesternBlotanalysesandantibodies

Protein levels expressionwere detected by total cell lysate subjected towestern blot as previous

described (42).The followingantibodieswereused:Monoclonalmouseanti-HIF1α1:1000 (Abcam,

USA); monoclonal mouse anti-GSDMDC1 1:500 (Santa Cruz Biotechnology, Dallas, TX, USA);

polyclonal rabbit anti-CASP-3 (Cell Signaling Technology, Leiden, The Netherlands); monoclonal

mouseanti-CASP11:500 (SantaCruzBiotechnology,Dallas,TX,USA);monoclonalmouseanti-CD81

1:1000(InvitrogenTM,Carlsbad,CA,USA)andmonoclonalrabbitanti-Bcl-21:1000(Abcam,UK)were

used in combination with appropriate peroxidase-conjugated secondary antibodies. Monoclonal

mouse anti-tubulin or actin were used as loading controls (Sigma, Life Sciences, Merck KGaA,

Darmstadt,German).Blotswereanalyzed inaChemiDocXRS system (Bio-Rad,Hercules,CA,USA).

ProteinbandintensitieswerequantifiedbyImageLabsoftware(BioRad,Hercules,CA,USA).

2.9RNAisolationandquantitativereal-timereversetranscriptionpolymerasechainreaction(qRT-

PCR)

Total RNA was isolated with TRI-reagent (Sigma-Aldrich, Saint Louis, MO, USA) according to the

manufacturer’s instructions. Quantification of RNA was measured with the Nanodrop

35

spectrophotometer (Thermo Scientific, Wilmington, DE, USA). Reverse transcription (RT) was

performed using 2.5 µg of total RNA. Quantitative Real Time PCR (qRT-PCR) was carried out in a

StepOnePlus™(96-well)PCRSystem(AppliedBiosystems,Thermofisher, Waltham,MA,USA)using

TaqManmethodorSYBRGreenmethod.Thesequencesoftheprobesandprimersetsaredescribed

intheSupplementarymaterialsfile.mRNAlevelswerenormalizedtothehousekeepinggene18Sand

furthernormalizedtothemeanexpressionlevelofthecontrolgroup.Relativegeneexpressionwas

calculatedviathe2ddCT.

2.10Enzyme-linkedimmunosorbentassay(ELISA)ofInterleukin-1beta

Levels of pro-inflammatory cytokine of IL-1β in primary rat KC were assessed by the ELISA kit

(ab100768,Abcam,Cambridge,UK),accordingtothemanufacturer’sinstructions.

2.11EVisolationandcharacterization

EVwerecollectedfromculturemediaofHepG2cellsasdescribedpreviously(43)andsummarizedin

the Supplementary materials file. Nanoparticle tracking analysis (NTA) was performed using

NanoSightNS300instrumentation(Marvel,Egham,UK)thatusesbothlightscatteringandBrownian

motionanalysis fornanoparticlecharacterization.We furthercaharacterizedEVusingTransmission

electron microscopy. Sample preparation and details of these analysis are described in the

Supplementarymaterialsfile.

2.12TreatmentofKCcellswithandextracellularvesicles

RatprimaryKCwere incubatedwithFBS-freeWilliam'sEmediumandexposedto15μgofEVthat

wereisolatedfromHepG2cellstreatedwithCoCl2+FFAfor24h.After24hofEVtreatment,KCwere

harvestedtocontinuewithanalysesbyquantitativePCR.

2.13EffectsofintermittenthypoxiainexperimentalNASH

Animalexperimentswereapprovedbythe institutionalanimalcareandusecommittee(Comitéde

ética y bienestar Animal, Escuela de Medicina, Pontificia Universidad Católica de Chile, CEBA

100623003).MaleC57BL/6miceaged10weeksatthebeginningofthestudyanddividedintofour

experimental groups (n = 4–8) receiving either choline-deficient amino acid-defined (CDAA) diet

(Catalog#518753,DyetsInc.Bethlehem,PA)toinduceNASHorthecholine-supplementedL-amino

acid defined (CSAA, Catalog # 518754, Dyets Inc. Bethlehem, PA) diet as control for 22 weeks as

previouslydescribed(16,44).AnimalswereexposedtoIHornormoxia(chambers41x22x35cm,COY

labproducts™,GrassLake,MI,USA)duringthelast12weeksoftheexperimentalorcontrolfeeding

period.IHregimenconsistedin30events/hourofhypoxicexposuresfor8hour/dayduringtherest

36

cycle, between9 amand5pm. This cyclewas repeated7days aweek for 12 consecutiveweeks.

After ending the study, mice were anesthetized (ketamine 60 mg/kg plus xylazine 10 mg/kg

intraperitoneally) and then euthanized by exsanguination. Serum and liver tissue samples were

collected and processed or stored at −80 °C until analyzed. Gene expression and protein analyses

were carried out as described above. Positive control of hypoxia induction was evaluated by

measurementofhepaticgeneexpressionofHIF-1α.

2.14HistologicalStudies

Liversteatosiswasanalyzedonparaformaldehyde-fixedliversectionsstainedwithOilRed-Ostaining

that show lipiddeposits in red coloron frozen7 μm liver cryosections.Ablindedpathologist (J.T.)

assigneda score for steatosis, inflammationand fibrosis asdescribed (44). Scoresweregivenas it

follows: Steatosis: grade 0, none present; grade 1, steatosis of ≤25% of parenchyma; grade 2,

steatosisof26–50%ofparenchyma;grade3,steatosisof51–75%ofparenchyma;grade4,steatosis

of≥76%ofparenchymaandinflammation:grade0,noinflammatoryfoci;grade1,1–5inflammatory

foci per high power field; grade 2, >5 inflammatory foci/high power field. Fibrosis was estimated

qualitativelyinSiriusred-stainedsamples.

2.15Hepatictriglyceridedetermination

Hepatic triglyceride content (HTC)was using 40-50mg of homogenized liver tissue in 1.5ml of a

CHCl3-CH3OHmixture(2:1,v/v),followedbyaFolchextractiondescribedpreviously(16,31).

2.16StatisticalAnalyses

AnalyseswereperformedusingGraphPadsoftware(version5.03,GraphPadSoftwareInc.,CA,USA).

Statistical analyses were performed using one-way analysis of variance (ANOVA) with a post-hoc

Bonferronicorrectionwithmultiple-comparisontestorbyparametrict-testswhenneeded. Allthe

results are presented as a mean of at least 3 independent experiments ± SEM. Values were

representedas absolutenumber, normalizeddata respect to control or percentage for categorical

variables.Regardinginvitroassays, independentexperimentsmeansdifferentcellplatesharvested

fromatleastthreeanimals.Regardinginvivoassays,independentexperimentsmeansatleastthree

differentanimalsamples.AlltheresultswereanalyzedandplottedusingGraphPadsoftware(version

5.03,GraphPadSoftwareInc.,CA,USA).Statisticswithavalueofp<0.05wereconsideredsignificant.

3.-RESULTS

3.1HypoxiainducesHIF-1αandpromotestheincreaseoflipiddropletsinfat-ladenhepatocytes:To

investigatewhetherhypoxiaexacerbateslipotoxicityinan invitromodelofexperimentalNASH,we

37

treated fat-ladenprimary rat hepatocyteswith CoCl2, a hypoxiamimetic agent that promotes the

accumulationofHIF-1α(45,46).Asexpected,chemicalhypoxiaincreasedproteinlevelsofHIF-1αin

both control and fat-laden hepatocytes as determined by Western Blot technique (Figure 1).

Interestingly,wealsoobservedanincreaseinthenumberofintracellularlipiddroplets,determined

byOilRedO staining (Figure2a) andofhepatocyteTG content (Figure2b) after treatmentwitha

mixture of FFA, which was higher in cells concomitantly treated with CoCl2 indicating that the

hypoxicconditionpromotesanincreaseintracellularlipiddepositioninthismodel.

Figure1.ChemicalhypoxiastabilizesHIF-1α.Primaryhepatocyteswereincubatedfor24hwitholeicacidand

palmitic acid (2:1 ratio) (FFA) in thepresenceor absenceof CoCl2 200μmol/L. Protein levels ofHIF-1αwas

determinedbyWesternBlotasdescribedinMaterialandMethods.α-Tubulinwasusedasloadingcontrol.Data

wereshownasmean±SEM(n≥3)*indicatesP<0.05and**indicatesP<0.01.

38

Figure 2. Steatosis in vitro: Free fatty acids and chemical hypoxia treatment increase the content of

triglycerides.Primaryhepatocyteswereincubatedfor24hwitholeicacidandpalmiticacid(2:1ratio)(FFA)in

thepresenceorabsenceofCoCl2200μmol/L.a)OilRedOstaining(scalebar:40μm)andb)Triglycerides(TG)

contentweremeasuredasdescribedinMaterialsandMethods.Datawereshownasmean±SEM(n≥3)***

indicatesP<0.005and****indicatesP<0.001.

3.2 Hypoxia increases apoptotic and pyroptotic cell death in fat-laden hepatocytes: To evaluate

whether the induction of chemical hypoxia exacerbates lipotoxic cell damage and death, we

determinedproteinlevelsandactivityofcaspase-3toassessapoptoticcelldeathandusedSYTOXTM

Green nucleic acid stain and LDH leakage to evaluate cell death associated to disrupted cellular

membraneintegrity.Additionally,weassessedthecaspase-cleavedgasdermin-Ndomain(GSDMD-N)

toevaluatepyroptoticcelldeath(19).WhileCoCl2didnotinfluencecleavedcaspase-3andcaspase3

activityinnormalhepatocytes,steatotichepatocytesundergoingchemicalhypoxiadisplayedatwo-

fold increase incleavedcaspase-3 (Figure3a)andsix-fold increase incaspase-3activity (Figure3b)

comparedtocontrolcells.Ofnote,treatmentwithamixtureofFFAaloneledtoatwo-foldincrease

(n.s.) in caspase 3 activity suggesting that chemical hypoxia exacerbates lipotoxicity and apoptotic

cell death in fat-laden hepatocytes. Also, using SYTOXTM Green nucleic acid stain (Figure 4a) and

measurementofcellularLDHleakage(Figure4b),wefoundthatchemicalhypoxiaalsoincreasedcell

death rate associated to losing plasmamembrane integrity of fat-laden hepatocytes compared to

control cells or cells treated only with FFA. Furthermore, protein levels of GSDMD-N, which is

considered a pyroptosis executor (19), increased three-fold in hypoxic fat-laden hepatocytes

comparedtocontrolcells(Figure4c).

39

Figure3.Chemicalhypoxiapromotesapoptoticcelldeathevaluatebya)cleavedCaspase-3andb)Caspase-3

activityinfat-ladenhepatocytes.Primaryhepatocyteswereincubatedfor24hwitholeicacidandpalmiticacid

(2:1ratio)(FFA) inthepresenceorabsenceofCoCl2200μmol/L. a)CleavedCaspase-3protein levelsandb)

Caspase-3activityweremeasuredasdescribedinMaterialsandMethods.Datawereshownasmean±SEM(n

≥3)*indicatesP<0.05;***indicatesP<0.005and****indicatesP<0.001.

40

Figure 4. Hypoxia promotes pyroptotic cell death

evaluate by a) SYTOX green, b) Lactate

dehydrogenase leakage and c) Gasdermin-N in fat-

laden hepatocytes. Primary hepatocytes were

incubated for 24h with oleic acid and palmitic acid

(2:1 ratio) (FFA) in thepresenceorabsenceofCoCl2

200 μmol/L. a) Sytox green (Representative images

andquantification),b)LDHleakagepercentageandc)

GSDMD-N protein levels determinated by Western

Blot were measured as described in Materials and

Methods.Datawereshownasmean±SEM(n≥3)*

indicatesP<0.05and**indicatesP<0.01.

41

3.3Hypoxia increases superoxideproductionbymitochondria in steatosis invitro:Recent studies

on the pathogenesis of NASH have described an important role formitochondrial oxidative stress

(47-50).Toevaluatethisissue,weexaminedthelevelsofmitochondrialsuperoxidegenerationinfat-

ladenhepatocytestreatedwithCoCl2.Fat-ladenhepatocytesundergoinghypoxiaexhibitedten-fold

higher levels of mitochondrial superoxide fluorescence determined by MitoSOXTM compared to

control cells. CoCl2 or FFA treatment alone did not determine significant changes in MitoSOXTM

fluorescenceintensity(Figure5).Thesedataindicatethatchemicalhypoxiainfat-ladenhepatocytes

promotesoxidativestressbyanincreaseofmitochondrialsuperoxideproduction.

Figure5. Hypoxia increasesmitochondrial superoxide levels in steatotichepatocytes. Primaryhepatocytes

wereincubatedfor24hwitholeicacidandpalmiticacid(2:1ratio)(FFA)inthepresenceorabsenceofCoCl2

200 μmol/L. Superoxide generation bymitochondria was determined usingMitoSOXTM fluorogenic probe as

described inMaterialsandMethods.Datawereshownasmean±SEM(n≥3)** indicatesP<0.01and***

indicatesP<0.005.

3.4EffectofCoCl2-inducedchemicalhypoxiaontheexpressionofpro-inflammatorycytokinesand

inflammasome components in fat-laden hepatocytes: To evaluate whether chemical hypoxia

promotes an inflammatory phenotype in fat-laden hepatocytes,wemeasuredmRNA levels of the

pro-inflammatorycytokinesTNF-a, IL-6aswellasofNLRP3 inflammasomecomponents incultured

cells treated or notwith FFA and CoCl2. As shown in figure 6, steatotic hepatocytes treatedwith

CoCl2displayedamarkedincrease inmRNAlevelsof IL-1β(2.5-fold),TNF-α(2-fold),andof IL-6(9-

fold) compared to non-treated cells. Also,mRNA levels ofNLRP3 andApoptosis-Associated Speck-

Like Protein Containing CARD (ASC) were also significantly increased in fat-laden hepatocytes

undergoinghypoxiacomparedwithcellstreatedsolelywithFFA(Figure6).

42

Figure6.Hypoxia increasestheexpressionofpro-inflammatorycytokines insteatotichepatocytes.Primary

hepatocytes were incubated for 24h with oleic acid and palmitic acid (2:1 ratio) (FFA) in the presence or

absenceofCoCl2200μmol/L.mRNAlevelsof IL-1β,TNF-αandIL-6weremeasuredasdescribedinMaterials

andMethods.Datawereshownasmean±SEM(n≥3)*indicatesP<0.05;**indicatesP<0.01;***indicates

P<0.005and****indicatesP<0.001.

3.5 CoCl2-treatment of fat-laden hepatocytes increases protein expression of caspase-1: The

inflammasome is a multiprotein complex needed for caspase-1 processing and the subsequent

activation of the inflammatory cytokines IL-1β and IL-18, which has been involved in the

pathogenesisofNAFLD/NASH (20).Therefore,weexamined theproteinexpressionofcaspase-1 in

both freshly isolated control hepatocytes and hepatocytes treated with FFA as described above

treated or not with CoCl2 in order to explore the effects of chemical hypoxia on inflammasome

activation.WhileFFAtreatmentdidnotinfluenceproteinexpressionofcaspase-1,CoCl2treatment

increased the protein levels of pro-caspase-1 (inactive form) and cleaved caspase-1 (p20 and p10

activeforms)inhepatocytesirrespectiveiftheywerefat-ladenornot(Figure7)Thesedatasuggest

that chemical hypoxia is able to activate the inflammasome complex in hepatocytes resulting in

caspase-1activation.

43

Figure7. Hypoxia increasescleavedcaspase-1protein levels insteatotichepatocytes.Primaryhepatocytes

wereincubatedfor24hwitholeicacidandpalmiticacid(2:1ratio)(FFA)inthepresenceorabsenceofCoCl2

200 μmol/L. Protein levels of pro-caspase-1 and cleaved caspase-1 (p10 and p20) were determinated by

WesternBlotasdescribed inMaterialsandMethods.α-Tubulinwasusedas loadingcontrol.Thesumofp20

and p10 caspase-1 was calculated to determine the generation of cleaved Caspase-1. Data were shown as

mean±SEM(n≥3)*indicatesP<0.05;**indicatesP<0.01and***indicatesP<0.005.

3.6Conditionedmediumofsteatotichepatocytessubjectedtohypoxiaincreasespro-inflammatory

cytokines in Kupffer cells: To explore whether hypoxic, fat-laden hepatocytes can trigger pro-

inflammatory responses inKC,weperformedexperimentsexploring theeffectsofexposingKCs to

CMobtainedfromfat-ladenhepatocytesundergoingchemicalhypoxia.WefirstprofiledisolatedKC

assessingtheexpressionoftwotypicalmacrophagemarkers[CD-68asmacrophagemarkerandCD-

163 as KC marker (51)] by immunofluorescence (Figure 8) confirming the quality of KC isolation.

Then,weobserved thatCM fromsteatotichepatocytes subjected tohypoxia increasesKC’smRNA

levelsofpro-inflammatorygenesanddecreasedmRNA levelsof theanti-inflammatorycytokine IL-

10,without affecting gene expressionof the classic KCM2marker gene arginase-1 (Arg-1) (Figure

9a). Furthermore, protein levels of IL-1βwere significantly higher in KC treatedwithCM from fat-

laden hepatocytes undergoing chemical hypoxia compared with all other groups (Figure 9b).

Treatment of cultured rat KC with CM obtained from hepatocytes treated with FFA alone also

resulted inhigherprotein levelsof IL-1β inKCcompared tocontrolcells (Figure9b)but thiseffect

44

was less intense than that observed with CM from steatotic hepatocytes undergoing chemical

hypoxia, suggesting that hypoxia exacerbates the expression of this pro-inflammatory cytokine.

These data support the hypothesis that hypoxic and steatotic hepatocytes release signals that

promoteapro-inflammatoryphenotypeinKC.

Figure8.CharacterizationofKupffercellsbyimmunofluorescence.ProteinexpressionofspecificKupffercell

green fluorescentmarker CD163 (A) andmacrophage red fluorescentmarker CD68 (B)were determined by

immunofluorescence and nuclei were stained blue with DAPI as described in Materials and Methods

(Supplementarymaterials).Scalebar:10μm

45

Figure9.Conditionedmediumofsteatotichepatocytessubjectedtohypoxiaincreasesa)pro-inflammatory

genesand)IL-1βproteinlevelsinKupffercells.Culturemediumofprimaryhepatocyteswasreplaced24hours

aftertreatmentbyFFAandCoCl2-freemediumforanadditional24hours(conditionedmedium;CM).Kupffer

cellsweretreatedfor24hourswiththeCMfromhepatocytes.a)mRNAlevelsofIL-1β,TNF-α,iNOS,IL-6,IL-10

andArg-1 andb) IL-1βprotein levels by ELISAweremeasuredasdescribed inMaterials andMethods.Data

wereshownasmean±SEM(n≥3)* indicatesP<0.05;** indicatesP<0.01;*** indicatesP<0.005;****

indicatesP<0.001.

3.7Extracellularvesicles increased inCMfromfat-ladenhepatocellularcell lineexposedtoCoCl2

andpromotedpro-inflammatorygenesexpression inKC:Toexplore if EV released from fat-laden

hepatocytesundergoinghypoxiaplayaroleinpromotingapro-inflammatoryeffectsinKC,EVwere

collected from culture media obtained from cultured HepG2 cells. We first explored if CM from

HepG2 cells exposed to the different treatments evokedproinflammatory signals in KC. Results of

these experiments are shown in Supplementary figure 2. As shown in this figure, similar to the

effects observed in experiments involving hepatocytes, CMdid determine increased expression of

proinflammatorygenesinKC,adecreaseintheexpressionofIL-10andhadnoeffectonArginase-1

expression.We thenexplored theeffectsofHepG2-derivedEVonKCandcompared theobserved

effects with the effect of treatment with the EV-free fraction. We first characterized EV by

transmission electronmicroscopy aswell as byWestern blot detection of the presence of the EV

markerCD81andtheabsenceofthemitochondrialproteinBcl-2 inallEVfractions(Figure10aand

SupplementaryFigure1).Ofnote,thesizeandconcentrationofHepG2-derivedEVweredetermined

byNTA. Interestingly,we foundan increaseofEV inculturemedia fromfat-ladencellsexposed to

CoCl2(6.0x1011particles/mL)comparedtoculturemediafromcontrolcells(9.5x1010particles/mL)

(Figure10b).Asshowninthisfigure,theaveragesizeofEVobtainedfromculturemediaofcontrol

46

cellswas150nmwhilethoseEVobtainedfromfat-ladenHEPG2cellsundergoingCoCl2was130nm

(n.s.). Next, we examined the response of KCs to treatment with HepG2-derived EV. EV from

steatoticHepG2cellstreatedwithCoCl2determinedamarkedincreaseinmRNAlevelsinKCofthe

followingproinflammatorygenes: IL-1β (2.0-fold),TNF-α (2.5-fold), iNOS (1.6-fold)andof IL-6 (2.2-

fold)incomparisontotreatmentwithEVfromnon-treatedcells,withoutaffectingsignificantlygene

expressionofIL-10andArg-1(Figure10c).However,EV-freeCM,asnegativecontroltoevaluatethe

specificeffectofEV,promotedanincreasedoniNOSgeneexpressioninKC(1.4-fold),demonstrating

that CM EV-freemay contains soluble substances that induce the expression of this specific pro-

inflammatorycytokine.Ofnote,treatmentofKCwithEVobtainedfromHepG2cellstreatedwithFFA

or CoCl2 alone did not evoke significant changes in KC expression of pro-inflammatory genes

(SupplementaryFigure3).

47

Figure 10. EV obtained from culture media of fat-laden HepG2 cells exposed to CoCl2 increases pro-

inflammatory genes inKupfferCells. EVobtained fromculturemediaof fat-ladenHepG2 cells exposed to

CoCl2 increases pro-inflammatory genes in Kupffer Cells (KC). Presence of EV was confirmed by electron

microscopy (a) transmissionandquantifiedusingNanotrackinganalysis (b).Free fattyacids (FFA)andCoCl2

treatmentofHepG2cellsdeterminedan increase in thenumberof EV in culturedmediawithno significant

changesintheirsize.(c)KCweretreatedwithhypoxicandfat-ladenHepG2cells-derivedEV(15μg)andEV-

free culture media as negative control. mRNA levels of interleukin (IL)-1β, tumor necrosis factor TNF-α,

inducible nitric oxide synthase (iNOS), IL-6, IL-10 and Arginase−1 (Arg-1) were measured as described in

MaterialsandMethods.Dataisshownasmean±SEM(n≥3)*indicatesP<0.05;**indicatesP<0.01;***

indicatesP<0.005;****indicatesP<0.001.

3.8 Intermittent hypoxia in an experimentalmodel of diet-induced non-alcoholic steatohepatitis

increasestheinflammatoryphenotype:Tovalidateourinvitrofindings,weinvestigatedtheeffectof

IH for12weeks inananimalmodel, theCDAAdiet-inducedNASH (16).Toevaluatesteatosis, liver

sections were stained with Oil red O staining (Figure 11a) (Abcam, Cambridge, UK). Steatosis,

inflammation and fibrosiswas gradedblindly by an experiencedpathologist (J.T.) according to the

NASscore.Specifically,theamountofsteatosis(percentageofhepatocytescontainingfatdroplets)

was scored as 0 (<5%), 1 (5–33%), 2 (>33–66%) and 3 (>66%). Although pathological scoring

suggestedahigherdegreeofsteatosisinanimalsonCDAAdietandexposedtohypoxiacomparedto

controlanimalsandcomparedtoanimalsonCDAAdietandnormoxicconditions(Figure11b),wedid

not observed significant differences in the hepatic TG levels between the CDAA-fed mice groups

48

(Figure 11c). Also, no differences were found in NAS score when comparing CDAA-fed groups.

However, mRNA levels of pro-inflammatory genes and NLRP3 inflammasome components

significantly increased in animalswith hypoxia onCDAAdiet compared to all other groups (Figure

12). Finally, we tested the caspase-1 protein levels, which significantly increases in animals with

hypoxia on CDAA diet (Figure 13). With these results, we verified that hypoxia promotes an

aggravated the inflammatory phenotype in NASH and specifically influences inflammasome

activation.

Figure11.IntermittenthypoxiaincreasedsteatosisinmicewithCDAA-inducedliverinjurydeterminedbyOil

Red-Ostaining,withoutchangesinhepatictriglycerides.Micewereplacedoncholine-supplementedL-amino

aciddefined(CSAA)dietascontrol,ordefineddietwithcholinedeficiencyaminoacids(CDAA)for22weeksto

induce liver injuryandintermittenthypoxia(IH)ornormoxiawasappliedforthe last12weeksofthedietas

described in Materials and Methods. a) Liver sections were stained with Oil Red O and analyzed by a

pathologist in a blinded fashion to determine b) steatosis score as described inMaterials andMethods. c)

HepaticTriglycerideswasmeasuredasdescribedinMaterialsandMethods.Datawereshownasmean±SEM

(n≥3)**indicatesP<0.01;***indicatesP<0.005and****indicatesP<0.001.

49

Figure 12. Intermittent hypoxia in mice with CDAA-induced liver injury increases the expression of pro-

inflammatorycytokinesandinflammasomecomponents.Themicewereplacedoncholine-supplementedL-

aminoaciddefined (CSAA)dietascontrol,ordefineddietwithcholinedeficiencyaminoacids (CDAA) for22

weekstoinduceliverinjuryandintermittenthypoxia(IH)ornormoxiawasappliedforthelast12weeksofthe

dietasdescribedinMaterialsandMethods.mRNAlevelsofIL-1β,IL-18,NLRP3,caspase-1,IL-6andIFN-γwere

measuredasdescribedinMaterialsandMethods.Datawereshownasmean±SEM(n≥3)*indicatesP<0.05;

**indicatesP<0.01;***indicatesP<0.005;****indicatesP<0.001.

50

Figura13.IntermittenthypoxiainmicewithCDAA-inducedliverinjuryincreasescleavedcaspase-1.Themice

wereplacedoncholine-supplementedL-aminoaciddefined(CSAA)dietascontrol,ordefineddietwithcholine

deficiencyaminoacids (CDAA) for22weeks to induce liver injuryand intermittenthypoxia (IH)ornormoxia

was applied for the last 12weeks of the diet as described inMaterials andMethods. Protein levels of pro-

caspase-1and cleaved caspase-1 (p10andp20)weredeterminedbyWesternBlot asdescribed inMaterials

andMethods.α-Tubulinwasusedasa loadingcontrol.Thesumofp20andp10caspase-1wascalculatedto

determinethegenerationofcleavedcaspase-1.Datawereshownasmean±SEM(n≥3)**indicatesP<0.01

and***indicatesP<0.005.

4.-DISCUSSION

NAFLD representsahighly frequent causeof liverdisease,which is closelyassociatedwithobesity

and insulin resistance (7, 52).Clinicalobservations suggest thatpatientswithOSAShavea greater

predispositiontodevelopNAFLDandNASH,theaggressiveformofNAFLD,whichischaracterizedby

the presence of necro-inflammatory and fibrotic phenomena in the liver (5). Several studies have

shown that OSAS, mainly through the occurrence of IH, can modulate hepatocellular damage by

triggeringproinflammatoryandprofibroticsignals(25,26),butpathwaysunderlyingthiseffectsare

ill-defined.Thepresent studyusedcellularmodelsofNAFLD/NASH toexplorepotential synergistic

interactionsbetweenhypoxiaandFFAexposure.Tothatend,thehypoxiamimeticagentCoCl2was

usedtotreatfat-ladenhepatocytes.Ourfindingssuggestthat indeedhypoxiadeterminesafurther

increaseincellularTGcontent inFFAtreatedhepatocytesaswellas increasedmRNAlevelsofpro-

inflammatory cytokines as well as those of the NLRP3 inflammasome components. These

phenomenawerealsoassociatedwithincreasedmitochondrialsuperoxidelevelsandincreasedrates

ofcelldeathduetoapoptosisanddisruptionofcellmembrane,likelypyroptosisassuggestedbythe

increase in GSDMD levels. Moreover, conditioned medium obtained from hypoxic fat-laden

hepatocytes promoted an inflammatory phenotype in KC, which is known to be decisive for the

amplificationofthepathologicalphenomenonofNASH(12,23).Additionally,inordertodetermineif

theobservedeffectsonKCcouldbemediatedbyEVreleasefromhepatocytes,weconductedfurther

experimentsinthehumanhepatomacelllineHepG2.Wechoosethisapproachforpracticalreasons

since using this cell line the EV isolation efficiency is high. Our results showed that treatment of

HepG2withFFAandCoCl2determinesanincreaseinthereleaseofEVtothemediumandthatthese

EValsoevokeapro-inflammatoryresponseinratKCinlinewithpreviousobservationsshowingthat

released EV from fat-laden hepatocytes can act in in macrophages and contribute to pro-

inflammatoryresponseinaparacrinefashion(15).

Finally,wefoundcorrelatesofourinvitroNASHmodelfindingsinananimalmodelofNASHinduced

by feeding a CDAA diet. In the latter experiments, we also confirmed that IH promotes NLRP3

51

inflammasome activation in the setting of NASH. Collectively taken, these findings indicate that

hypoxiamay influenceNAFLD development and progression by acting at different levels including

promotion of further lipid accumulation as well as enhancement of liver inflammation and

hepatocellulardeath.Ofnote,recentrodentstudiesindicatethathypoxiamaybealsolinkedtoliver

fibrosis, a key prognostic feature of NASH (8). In fact, Mersarwi et al. demonstrated that HIF-1α

deletioninhepatocytesprotectsagainstthedevelopmentofliverfibrosisinamousemodelofNAFLD

(53).ThisobservationaddstoourdataandsupporttheconceptthathypoxiacontributestoNAFLD

progressioninfluencingcriticalstepsofthedisease.

ExperimentaldataontheeffectsofhypoxiainNAFLDmodelsisscarceandmainlyrestrictedtowhole

animal studies (53, 54). No data is available on the effects of hypoxia in cellular models of

NAFLD/NASH (36). To assess this, we used the hypoxia mimetic compound CoCl2, which induces

chemical hypoxia by stabilizing HIF-1α and 2α (38). This model is well accepted and has several

advantagesoveralternativessuchasthehypoxiachamberoraCO2incubatorwithregulatedoxygen

levels,whichare lessavailable,mostcostlyandprovidea less stableexperimental conditions (38).

CoCl2 has been previously used in normal isolated hepatocytes (38, 45) but not in fat-laden cells

underlying the novelty of our approach. Of note, previous studies as well as experiments of our

laboratory on hepatocyte cell lines have demonstrated the non-toxic effect of CoCl2 at

concentrations not exceeding 400 μmol/L for 24 hours as previously described (55). On the other

hand, FFA treatment using combination of OA and PA effectively induced TG accumulation in the

absence of lipoapoptosis (56). In spite of these advantages and, although CoCl2-induced chemical

hypoxiaisasuitablemodeltoassesstheeffectsofhypoxiaincellularmodels,weacknowledgethat

thismodel reproduces only some of the effects generated by a decrease in oxygen supply,which

limitthegeneralizabilityofourresultsuntiltheyareconfirmedinothermodels.

The observed hypoxia-related increases in intracellular lipid droplets and TG content in fat-laden

hepatocytesisconsistentwithpreviousstudiesthatindicatethathypoxia,throughHIF-1α,promotes

an increase in lipid biosynthesis causing TG accumulation (57, 58).We did not observe amarked

increase in lipidcontent incellstreatedsolelywithCoCl2,whichmayrelateto lengthoftreatment

and experimental conditions.We also did not find an increase in the in vivo CDAA-feeding NASH

model.We think that thismaybe related to the fact that thisparticularmodeldetermines severe

steatosisandinflammation(59)andthatthereforeisdifficulttoobservesubtlechanges.

WithregardtotheeffectsofchemicalhypoxiaoncelldeathofFFAtreatedhepatocytes,qualitative

determination of apoptosis, necrosis and pyroptosis using determination of caspase-3 cleavage,

caspase-3activity,SYTOXgreenstaining,LDHreleaseandGSDMD-Nwerecarriedoutasperformed

52

inprevious studies (56,60).While, treatmentwithCoCl2or FFAalonedidnot inducea significant

increaseinapoptosiscomparedtocontrolconditions,CoCl2treatmentoffat-ladenhepatocytesdid

increase cleaved caspase-3 and caspase-3 activity reflecting ongoing apoptosis. The same

observationwasmaderegardingcelldeathassociatedtoadisruptedplasmamembrane,bySYTOX

GreenandLDH leakagemeasure, asan increasednumberofnecroticdeathcellswasobserved in

steatotichepatocytessubjectedtohypoxia.Giventhefactthathypoxiawasassociatedtoincreaseof

GSDMD cleavage and up-regulation of inflammasome components, it is likely that pyroptosis also

contributedtocelldeathoffat-ladenhepatocytesundergoingchemicalhypoxia.Ofnote,ithasbeen

suggested that pyroptosis may be an inflammatory link related to the progression from bland

steatosistoNASH,asNLRP3activation isnotpresent inanimalmodelof isolatedsteatosiswithout

inflammation(61).Additionally,weobservedasignificantincreaseinmitochondrialsuperoxidelevels

in the fat-laden hepatocytes exposed to chemical hypoxia. Interestingly, increased mitochondrial

superoxidegenerationhasbeencorrelatedtomitochondrialdysfunction,oxidativedamage,cellular

deathandpro-inflammatoryeffectsinNASH(49,62).Thus,thissynergisticeffectofhypoxiaandFFA

in hepatocytes with regard to mitochondrial superoxide generation, likely contributes to the

observedincreaseincelldeathwhenhepatocytesareexposedtobothstimuli.

Inadditiontotheobservedeffectsoncelldeathandoxidativestress,chemicalhypoxiaalsoinduced

theexpressionof several inflammatorycytokines, includingTNF-a, IL-6and IL-1β,aswell asof the

componentsoftheNLRP3inflammasome(caspase-1,NLRP3andASC),insteatotichepatocytes.The

NLRP3inflammasomeactivatespro-caspase-1intoactivecaspase-1(p10andp20cleavedcaspase-1)

that in turn cleaves pro-IL-1β into the mature form of IL-1β promoting amplification of liver

inflammation and cell death (12). In recent years, caspase-1has been studied extensively andhas

been shown to contribute to liver damage during the development of NASH (63, 64) and several

studieshaveshownprotectiveeffectsagainst liverdamagewhencaspase-1 is inhibited(63). Inour

study,weevaluatedtheproteinlevelsofcaspase-1(pro-caspase-1andactivecleaved-caspase-1)in

hepatocytes.Anincreaseinthetotallevelofpro-caspase-1wasobservedinhypoxichepatocytesand

hypoxichepatocytestreatedwithFFAcomparedtothecontrolconditions. Interestingly,cleaved-p-

20 andp-10, the active isoformsof caspase-1,were only detected in hypoxic hepatocytes treated

with FFA. These results are consistent with the observed up-regulation of inflammasome

components and similar to thoseobtainedbyotherauthors inprostateepithelial cellsundergoing

hypoxia, showing increased NLRP3 inflammasome activation as determined by increased cellular

levelsofcleavedcaspase-1(65).Takentogether,ourresultssuggestthattheinductionofhypoxiain

hepatocytes promotes susceptibility to damage by FFA, resulting in increased caspase-3mediated

53

apoptosis, (secondary) necrosis, pyroptosis and an inflammatory phenotype, characterized by

increasedexpressionofpro-inflammatorygenesandproteinlevelsofcaspase-1.

Kupffercells(KC)areinvolvedinthepathogenesisofvariousliverdiseases,includingsteatohepatitis

(66). However, their role in the context of hepatocellular damage due to hypoxia remains poorly

explored.KCarederivedfromcirculatingmonocytesand,onceestablishedintheliver,fulfillmultiple

functions related to the immune system. KC maintain constant paracrine communication with

neighboring hepatocytes, stellate cells and endothelial cells through the signals that they receive

from the extracellular environment (66). To verify the paracrine involvement of KCwith steatotic

hepatocytes in the context of hypoxia, KC were exposed to conditioned medium of hepatocytes

subjected to different treatments. We documented that conditioned medium from steatotic and

hypoxichepatocytesinducedapro-inflammatoryprofileinKC.Thesenovelresultscorrelatewellwith

recent studies showing micro-RNA-mediated cellular crosstalk between hepatocytes treated with

ethanolandmonocytes/macrophages(67).Asmentionedbefore,hepatocytesareabletomodulate

signaling pathways in macrophages, stellate cells and endothelial cells in a paracrine manner by

cytokines, micro-RNAs and EV (32). Our experiments involving isolation of EV from HEPG2 cells

strongly suggest that EV participate in the cellular communication between steatotic and hypoxic

hepatocytesandKC.Thisobservationsmayhavediagnosticandtherapeuticimplications(33).

In the present study, we also explored whether IH influences hepatic steatosis and the pro-

inflammatoryphenotypeinaninvivomodelofNASH.Tothisend,weassessedtheeffectsofhypoxia

for 22 weeks in CDAA-fed mice by evaluating liver histology and hepatic mRNA levels of pro-

inflammatorycytokines.Ourresultsshowedaworseningofhistologicalsteatosis inCDAA-fedmice

subjected to IHcompared to thoseanimals thatdidnotundergohypoxic conditions.However,we

didnotobserveacorrespondingincreaseinthepaticTGcontent.Wethinkthatthismayberelated

to the fact that this particularmodel determines severe steatosis and inflammation (59) and that

thereforeisdifficulttoobservesubtlechanges.

With regard to inflammatory markers, CDAA-fed mice exposed to IH exhibited an increase of all

inflammasome components (IL-1β, IL-18, NLRP3 and caspase-1) and other pro-inflammatory

cytokines (IL-6, IFN-γ). Also, active caspase-1 level protein increased in livers from CDAA-fedmice

exposedto IH,whichcorrelateswiththefindings inthe invitromodel.Theseobservationssupport

theconcept thathypoxiamaycontribute to liverdamage in thesettingofNAFLDbyactivating the

NLRP3inflammasome.ArecentstudyshowingthatIHactivatestheNLRP3inflammasomeinkidneys,

whichresultinprogressiverenalinjury(68)providessupporttothisnotion.Althoughnotexploredin

54

this study, thismight have implicationsnotonly for hepatocellular injurybut also for liver fibrosis

developmentandhepatocarcinogenesis(69,70).

In summary, our results in in vitro and in vivo models of NAFLD/NASH support the notion that

hypoxiaplaysanaggravatingroleinthesettingofexcessivelipidloadinlivercellsbyinfluencinglipid

deposition, hepatocellular death and pro-inflammatory signals, involving NLRP3 inflammasome

activation. Moreover, our novel findings demonstrate that hypoxia modulates cellular crosstalk

between steatotic hepatocytes and inflammatory cells (KCs). Future studies should focus on the

characterizationoftheextracellularenvironmentofhypoxicandsteatotichepatocytestoidentifythe

hypoxia-specificfactorsandthemechanismsofhepatocellulardamageatplayinNASH.

5.-SUPPLEMENTARYFIGURES

FigureS1.CharacterizationofHepG2-EV.EVwerecharacterizedinculturemediumfromcontrolHepG2cells

by detection of EV-positive marker CD81 and EV negative mitochondrial marker Bcl-2 by Western blot as

describedinMaterialandMethods.

55

Figure S2. Conditionedmediumof steatoticHepG2 cells subjected to hypoxia increases pro-inflammatory

genes in Kupffer cells. Culture medium of HepG2 cells was replaced 24 hours after treatment by FFA and

CoCl2-freemedium for an additional 24 hours (conditionedmedium; CM). Kupffer cellswere treated for 24

hourswiththeCMfromHepG2cells.mRNAlevelsofIL-1β,TNF-α,iNOS,IL-6,IL-10andArg-1weremeasured

as described in Materials and Methods. Data were shown as mean ± SEM (n ≥ 3) *indicates P < 0.05; **

indicatesP<0.01;***indicatesP<0.005.

56

Figure S3. EV obtained from culture media of fat-laden HepG2 cells exposed to CoCl2 increases

proinflammatoyrygenesinKupffercells.KCcellswereexposedto15μgofEVthatwereisolatedfromHepG2

cellsthathadbeentreatedwithCoCl2,FFAandCoCl2+FFAfor24h.mRNAlevelsofIL-1β,TNF-α,iNOS,IL-6,IL-

10andArg-1weremeasuredasdescribedinMaterialsandMethods.Datawereshownasmean±SEM(n≥3)

*indicatesP<0.05;***indicatesP<0.005;****indicatesP<0.001.

Figure S4. Intermittent hypoxia inmice increases the gene expression ofHIF-1α. Themicewere placed on

choline-supplemented L-amino acid defined (CSAA) diet as control, or defined diet with choline deficiency

aminoacids(CDAA)for22weekstoinduceliverinjuryandintermittenthypoxia(IH)ornormoxiawasapplied

forthelast12weeksofthedietasdescribedinMaterialsandMethods.mRNAlevelsofHIF-1αwasmeasured

as described inMaterials andMethods. Datawere shown asmean ± SEM (n ≥ 3) * indicates P < 0.05; ***

indicatesP<0.005;****indicatesP<0.001.

57

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61

Chapter4

EXTRACELLULARVESICLESDERIVEDFROMFATLADENHYPOXICHEPATOCYTESPROMOTEAPRO-

FIBROTICPHENOTYPEINSTELLATECELLS

ABSTRACT

Background:Transitionfromsteatosistonon-alcoholicsteatohepatitis(NASH)isakeyissueinNon-

alcoholicfattyliverdisease(NAFLD).Observationsinpatientswithobstructivesleepapneasyndrome

(OSAS),suggestthathypoxiacontributestoprogressiontoNASHandliverfibrosisandthereleaseof

extracellularvesicles(EV)byinjuredhepatocyteshasbeenimplicatedinNAFLDprogression.Aim:to

evaluatetheeffectsofhypoxiaonpro-fibroticresponseandthereleaseofEVinNAFLDandtoassess

cellularcrosstalkbetweenhepatocytesandhumanhepaticstellatecells(LX-2).Methods:HepG2cells

were treated with free fatty acids and subjected to chemically-induced hypoxia (CH) using the

hypoxia-inducible factor-1alpha (HIF-1α) stabilizer cobalt chloride (CoCl2). Lipid droplets, oxidative

stress, apoptosis and pro-fibrotic-associated genes were assessed. EV were isolated by

ultracentrifugation.LX-2cellsweretreatedwithEVfromhepatocytesandpro-fibrogenic-associated

markers were determined. CDAA-fed mice model was used to assess the effects of intermittent

hypoxia (IH) on experimental NASH.Results: CH-treatment in fat-laden HepG2 cells increased the

steatosis, oxidative stress, apoptosis, pro-fibrotic gene expression and increased the release of EV

compared to non-treated HepG2 cells. Treatment of LX2 cells with EV from fat-laden hypoxic

hepatocytesincreasedpro-fibroticmarkerscomparedtoEVfromnon-treatedhepatocytes.CDAA-fed

animalsexposedtoIHexhibitedincreasedfibrosisthatcorrelatedwithanincreaseofcirculatingEV.

Conclusion:Hypoxiapromoteshepatocellulardamageandpro-fibroticsignalingthatcorrelateswith

increasesofEVreleasefromhepatocytesandfromour invivomodelofNASH.EVfromhypoxicfat-

ladenhepatocytesevokepro-fibroticresponsesinLX-2cells.

1.-INTRODUCTION

Non-alcoholicfatty liverdisease(NAFLD)iscurrentlythemostcommonliverdiseaseandisamajor

globalhealthproblem(1).NAFLDischaracterizedbyfataccumulationintheliverwhichmayprogress

to hepatitis, cirrhosis and liver-relatedmorbidity andmortality (2). Recent evidence suggests that

saturated fatty acids (FFAs) contribute to the phenomenon of lipotoxicity and can trigger

inflammation in the liver and an abnormal wound-healing response or fibrosis leading to the

developmentofnon-alcoholicsteatohepatitis(NASH)(3-5).

ItisstillnotclearwhysomepatientswithfattyliverdevelopadvancedstagesofNASHmorerapidly

andseverelythanothers.ClinicalobservationsinpatientswhosufferfromObstructiveSleepApnea

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syndrome (OSAS) have revealed that these patients have a higher risk of developingmore severe

NAFLD associated with significant liver damage (6,7). The pathophysiology of this condition is

characterizedbyintermittentairwayobstructionthataltersgasexchangeleadingtoperiodichypoxia

(8). Several studies have demonstrated that hypoxia inducesmetabolic alterations such as insulin

resistance, increased oxidative stress, increased liver triglyceride accumulation and increased

inflammation,hepatocellulardamageandfibrogenesis(9-14).

During hypoxia, the transcription factor hypoxia inducible factor 1 alpha (HIF-1α) is stabilized and

translocated to the nucleus to activate its target genes bymeans of binding ofHIF-1α toHypoxia

ResponsiveElements (HREs) located in the target genepromoters (15). These target genesmodify

hepatocytelipidmetabolismandalsoenergymetabolism,cellsurvival,inflammationandfibrosis(16-

18).InrodentmodelsofNASH,intermittenthypoxiawasshowntohaveapro-inflammatoryandpro-

fibroticeffectas indicatedby increased levelsofNF-κB-dependent inflammatorycytokines,suchas

TNF-α,IL-6andIL-1β,andanincreasedexpressionofcollagentypeIintheliver(20,21).Likewise,in

vitro studies using hepatocytes and stellate cells showed that HIF-1α canmodulate pro-fibrogenic

signaling(17,21-23),whichcouldbekeyforthedevelopmentandprogressionofNASH.

Theinvolvementofextracellularvesicles(EV)intheprogressionofNAFLDhasbeenstudiedrecently

in invitroand invivomodels (24-27).EVcorrespondtosmallparticles releasedafter the fusionof

multivesicularbodiesordirectlyfromthecellmembranewithasizeof50to150nm(28).EVplayan

important role in cellular communication innormalphysiological andpathophysiological situations

duetotheircontentofproteins,mRNAsand/orlipids(29).Thereareenoughevidencethatindicate

theinvolvementofEVinNASH(30,31),buttheirroleinthecontextofhypoxiaandeffectsonnon-

parenchymal cells, notably stellate cells, the principal cell type involved in matrix deposition in

fibrogenesis,remainstobeelucidated.

Therefore, the aim of this study was to test the hypothesis that hypoxia leads to hepatocellular

damagethatinvolvesreleaseofEVthathaveapro-fibroticeffectonstellatecells.Weusedboth in

vitroandinvivomodeltotestthishypothesis.

2.-MATERIALSANDMETHODS

2.1Animals

Animalexperimentswereapprovedbythe institutionalanimalcareandusecommittee(Comitéde

ética y bienestar Animal, Escuela de Medicina, Pontificia Universidad Católica de Chile, CEBA

100623003).MaleC57BL/6miceaged10weeksatthebeginningofthestudyanddividedintofour

experimental groups (n = 4-8) receiving either choline-deficient amino acid-defined (CDAA) diet

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(Catalog#518753,DyetsInc.Bethlehem,PA)toinduceNASHorthecholine-supplementedL-amino

acid defined (CSAA, Catalog # 518754, Dyets Inc. Bethlehem, PA) diet as control for 22 weeks as

previouslydescribed(32,33).AnimalswereexposedtoIHornormoxia(chambers41x22x35cm,COY

labproducts™,GrassLake,MI,USA)duringthelast12weeksoftheexperimentalorcontrolfeeding

period.IHregimenconsistedof30events/hourofhypoxicexposuresfor8hour/dayduringtherest

cycle, between9 amand5pm. This cyclewas repeated7days aweek for 12 consecutiveweeks.

After ending the study, mice were anesthetized (ketamine 60 mg/kg plus xylazine 10 mg/kg

intraperitoneally) and then euthanized by exsanguination. Serum and liver tissue samples were

collectedandprocessedorstoredat−80 °Cuntilanalyzed.

2.2Histologicalstudies

Liver fibrosis was analyzed on paraformaldehyde-fixed liver sections stained with Sirius Red on

frozen7 μmlivercryosections. Stainingwasquantitatedbydigital imageanalysis(ImageJ,NIH,US)

aspreviouslydescribed(32).

2.3Cellcultureandtreatmentwithfreefattyacidsandchemicalhypoxiainduction

The human hepatocellular carcinoma cell line HepG2 (ATCC, USA) was cultured in Dulbecco’s

modifiedEaglemedium(1X) + GlutaMAX™-I(DMEM,10569010,Gibco)supplementedwith10%fetal

bovineserum(FBS;Gibco)and1%penicillin-streptomycin(Gibco)at37°Cand5%CO2.Allcellswere

platedinacellcultureplateatleast24h-36hbeforetreatment.Uponreaching80%confluence,the

cellswereincubatedwithamixtureoffreefattyacids(FFA)consistingofoleicacid(500μmol/L)and

palmiticacid(250μmol/L)inanaqueoussolutionofbovineserumalbumin(BSA)asdescribed(34).

IncubationswerecarriedoutwithorwithoutCobalt(II)chloride(CoCl2;SigmaAldrich)(200μmol/L)

for 24 hours to induce chemical hypoxia (35). CoCl2 is a well-known hypoxia-mimetic agent by

stabilizationofhypoxia-induciblefactor(HIF)-1α(36).ControlcellsweretreatedwithBSAalone.

2.4Westernblotanalyses

Cell lysateswere resolved onMini-PROTEAN® TGX Stain-Free™ Precast Gels (BioRad, UK, Oxford).

Semidry-blottingwasperformedusingTrans-BlotTurboMidiNitrocelluloseMembranewithTrans-

Blot Turbo System Transfer (BioRad). Ponceau S 0.1% w/v (Sigma) staining was used to confirm

protein transfer. Anti-HIF1α (610958, Biosciences), anti-CASP1 (SC-56036, Santa Cruz), anti-CD81

(10630D,Invitrogen),anti-Bcl-2(ab32370,Abcam),anti-TypeICollagen(1310-01;SouthernBiotech)

and anti-αSMA (A5228, Sigma)were used in combinationwith appropriate peroxidase-conjugated

secondaryantibodies.Tubulin(T9026,Sigma)oractin(A5228,Sigma)wereusedasloadingcontrols.

64

The blots were analyzed in a ChemiDoc XRS system (Bio-Rad). Protein band intensities were

quantifiedbyImageLabsoftware(BioRad).

2.5Nileredstaining

IntracellularlipiddropletsintheHepG2cellsweredetectedwithNileRedfluorescentprobe(N1142,

ThermoFisher).HepG2cellsweregrownin96-wellplatesandtreatedwithFFAand/orCoCl2.After

treatment,thecellswerewashedtwicewithPBSandstainedwithNileRedsolutionfor10 mininthe

dark. The cells were then washed twice with PBS and stained with Hoechst dye (33342, Thermo

Fisher).Thefluorescenceintensityofeachwellwasanalyzedwithexcitation/emissionwavelengthat

488nm/550nmusingamicroplatereader.Thefluorescenceimageswererecordedusinganinverted

fluorescencemicroscope

2.6Determinationofreactiveoxygenspecies

The intracellular generation of reactive oxygen species (ROS) in HepG2 cells was monitored with

DCFH-DAfluorescentprobe.HepG2cellsweregrownin96-wellplatesandtreatedwithFFAand/or

CoCl2. Cells were washed twice with PBS and incubated with the cell permeable reagent 2’,7’–

dichlorofluorescindiacetate(DCFDA;Abcam,USA)for45min.CellswerethenwashedtwicewithPBS

andthefluorescenceintensityofeachwellwasanalyzedwithexcitation/emissionwavelengthat495

nm/529nmusingamicroplatereader.

2.7Apoptosismeasurement

Caspase-3 fluorometric assay was used to determine apoptosis induced by FFA and/or CoCl2 in

HepG2cells.Aftertreatment,HepG2werescrapedandcelllysateswereobtainedbythreecyclesof

freezing(−80°C)andthawing(37°C)followedbycentrifugationfor5minutesat13,000g.Caspase-3

enzymeactivitywasassayedasdescribedpreviously (37).Thearbitraryunitsof fluorescence(AUF)

was quantified in a spectrofluorometer at an excitation wavelength of 380 nm and an emission

wavelengthof430nm.

2.8Assessmentofcelldeathassociatedtodisruptedcellularmembraneintegrity

SYTOX®Greennucleicacidstain(Invitrogen,S7020)wasusedtodeterminecelldeathinducedbyFFA

and/or CoCl2 in HepG2 (38). Cells were cultured in 12-well plates. After treatment, diluted Sytox

Green solution (1:40.000/PBS) was added to the cells for at least 15 minutes at 37°C, 5% CO2.

Necrotic cells have ruptured plasma membranes, allowing entrance of non-cell permeable Sytox

Greenintothecellsandbindingtonucleicacids.NecrosiswasdeterminedusingaLeicafluorescence

microscopeatawavelengthof488nm.

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2.9RNAisolationandquantitativereal-timereversetranscriptionpolymerasechainreaction(qRT-

PCR)

HepG2cellswereharvestedoniceandwashedtwicewithice-coldPBS.TotalRNAwasisolatedwith

TRI-reagent (Sigma) according to the manufacturer’s instructions. Reverse transcription (RT) was

performedusing2.5 µgoftotalRNA,1XRTbuffer(500 mmol/lTris-HCl[pH8.3];500 mmol/lKCl;30

mmol/l MgCl2; 50 mmol/l DTT), 1 mmol/l deoxynucleotides triphosphate (dNTPs, Sigma), 10 ng/µl

random nanomers (Sigma), 0.6 U/µl RNaseOUT™ (Invitrogen) and 4 U/µl M-MLV reverse

transcriptase(Invitrogen) inafinalvolumeof50 µl.ThecDNAsynthesisprogramwas25°C/10min,

37°C/60minand95°C/5min.ComplementaryDNA(cDNA)wasdiluted20X innuclease-freewater.

Real-Time qPCR was carried out in a StepOnePlus™ (96-well) PCR System (Applied Biosystems,

Thermofisher)usingTaqManprobes.Thesequencesoftheprobesandprimersetsaredescribedin

Supplementary Material. For qPCR, 2X reaction buffer (dNTPs, HotGoldStar DNA polymerase, 5

mmol/LMgCl2) (Eurogentec,Belgium,Seraing),5µmol/Lfluorogenicprobeand50µmol/Lofsense

and antisense primers (Invitrogen)were used.mRNA levelswere normalized to the housekeeping

gene18Sandfurthernormalizedtothemeanexpressionlevelofthecontrolgroup.

2.10Extracellularvesiclesisolation

ForEVcollection,HepG2cellsweregrowninculturedishesof100mmtoobtain70mlofserum-free

conditionedmedium after different treatments for an additional 24 hours. EVwere isolated from

conditionedmediumbydifferential ultracentrifugation (UCFThermo-Sorvall 80wx+) according to a

modifiedpreviousprotocol(39).Atotalvolumeof70mlmediumpertreatmentwasdepletedofcells

andcelldebrisbyconsecutive,low-speedcentrifugations(2,000×gfor30minand12,000×gfor45

min).Theresultingsupernatantswerecarefullycollectedandcentrifugedfor70minat120,000×gat

4°C.PelletsfromthiscentrifugationstepwerewashedinPBS,pooledandcentrifugedagainfor60

minat 100,000× g at 4°C. For EV collection fromanimals, serumsampleswere reconstituted in a

totalvolumeof4.4mLandwerecentrifugatedat2,000×gfor30minand10,000×gfor30min.The

resulting supernatants were carefully collected and centrifuged for 70min at 120,000 × g at 4°C.

PelletsfromthiscentrifugationstepwerewashedinPBS,pooledandcentrifugedagainfor60minat

100,000×gat4°C.TheobtainedpelletsfromHepG2cellsorserumwereresuspendedinlysisbuffer

or PBS solution depending on subsequent experiments and stored in aliquots at -80 °C. Protein

concentrationinEVpelletweremeasuredusingBCAproteinassaykit(Pierce,Rockford,IL).

2.11Nanoparticletrackinganalysis

Concentration and size distribution of isolated EVwere assessed by nanoparticle tracking analysis

(NTA)usingNanoSightNS300 instrumentation (Marvel, Egham,UK). EV samplesweredilutedwith

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PBS to a concentration of 108 to 109 particles/mL in a total volume of 1 ml. Each sample was

continuously run througha flow-cell top-plate setupat 18°Cusinga syringepump.At least three

videos of 20 seconds documenting Brownianmotion of nanoparticles were recorded and at least

1000ofcompletedtrackswereanalyzedbyNanoSightsoftware(NTAv3.2).

2.12Transmissionelectronmicroscopy

Isolated EV were fixed in 2% paraformaldehyde in 0.1M phosphate buffer overnight at 4°C. The

sampleswerethenplacedonFormvar/Carbon300Mesh(FCF300-CU,EMS)gridandairdriedfor10

min.Thegridswerefirstcontrastedwithuranyl-oxalatesolutionandthencontrastedandembedded

in amixtureof 4%uranyl acetate. ThegridswereairdriedandvisualizedwithaPhilips Tecnai 12

(Biotwin,Eindhoven,Netherlands)electronmicroscopeat80kV.Theimageswerecapturedwiththe

softwareItemOlympusSoftImagingSolutions(WindowsNT6.1).

2.13TreatmentofLX-2cellswithextracellularvesicles

ToinvestigatethecrosstalkbetweenEVfromHepG2andLX-2humanhepaticstellatecells,atypical

cell linetostudyhepaticfibrogenesis(40),LX-2cellsweretreatedwith isolatedEV.LX-2cellswere

culturedinDMEMsupplementedwith10%FBSand1%penicillin-streptomycinat37°Cand5%CO2.

All cellswere plated in a cell culture plate at least 24-36 h before treatment.Upon reaching 80%

confluence, thecellswere incubatedwithFBS-freemediumandexposed to15μgofEV thatwere

isolatedfromHepG2cellsthathadbeentreatedwithCoCl2,FFAandCoCl2+FFAfor24h.After24hof

EVtreatment,LX-2cellswereharvestedtocontinuewithanalysesbyquantitativePCR,WesternBlot

andimmunofluorescence.

2.14Immunofluorescencemicroscopy

LX-2 cellsweregrownonglass cover slipsplaced in12-wellplates.After treatment, culturemedia

wereremovedandcoverslipswerecarefullywashedtwicewithPBS.Cellswerethenfixedusinga4%

paraformaldehyde solution in PBS for 10 min at room temperature and washed twice with PBS.

Permeabilizationwasperformedby incubationof thesamples for10minutes in0.1%TritonX-100

(Sigma). The cells were washed twice with PBS and incubated with 2% BSA (Sigma) in PBS/0.1%

Tween 20 (Sigma) solution for 30 minutes to block non-specific binding sites. Goat anti-Type I

Collagen(1310-01;SouthernBiotech)wasusedatadilutionof1:200in2%BSA/PBSinahumidified

chamber for 1 hour at room temperature. Samples were subsequently washed twice with 2%

BSA/PBS.Finally,cellswereincubatedwithrabbitanti-goatAlexaFluor®568atadilutionof1:500in

2%BSA/PBSfor30minatroomtemperatureinthedark.SlidesweremountedwithProLongantifade

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with DAPI (Molecular Probes) and images were evaluated using fluorescence microscopy and

analyzedbyLeicaALSAFSoftware(Leica).

2.15Statisticalanalyses

AnalyseswereperformedusingGraphPadsoftware(version5.03,GraphPadSoftwareInc.,CA,USA).

All results are presented as a mean of at least 3 independent experiments ± SEM or as absolute

number or percentage for categorical variables. The statistical significance of differences between

themeansof theexperimental groupswas evaluatedusingone-wayanalysis of variance (ANOVA)

with a post-hoc Bonferroni multiple-comparison test; P<0.05 was considered as statistically

significant.

3.-RESULTS

3.1 Chemical hypoxia increases lipid droplet content in fat laden hepatocytes: To investigate

whether hypoxia exacerbates lipid accumulation in an in vitro model of experimental NASH, we

treatedHepG2cellswithCoCl2,ahypoxiamimeticagentthatpromotestheaccumulationofHIF-1α.

HepG2 cells treatedwith CoCl2 showed a significant increase ofHIF-1α, independent of treatment

withFFA (Figure1).To inducesteatosis,HepG2cellsweretreatedwithamixtureofoleicacidand

palmitic acid (FFA) as described previously (41). Hepatocytes showed increased formation of lipid

droplets(Figure2a).Interestingly,asignificantincreaseinthecontentoflipiddropletswasobserved

inthesteatotichepatocytestreatedwiththechemicalinducerofhypoxia(Figure2b),indicatingthat

hypoxiaincreasessteatosisinthismodel.

Figure1.ChemicalhypoxiastabilizesHIF-1α.HepG2cellswereincubatedfor24hwitholeicacidandpalmitic

acid2:1 (FFA) in thepresenceorabsenceofCoCl2200μmol/L.Protein levelsofHIF-1αweredeterminedby

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WesternBlotasdescribedinMaterialandMethods.α-Tubulinwasusedasloadingcontrol.Datawereshown

asmean±SEM(n≥3)****indicatesP<0.001.

Figure2.Hypoxia aggravates FFA-induced steatosis in vitro. HepG2 cellswere incubated for 24hwitholeic

acidandpalmitic acid2:1 (FFA) in thepresenceor absenceofCoCl2200μmol/L. Lipiddroplets contentwas

measuredby a) Fluorescence intensity andb) Fluorescence imagesofNileRedasdescribed inMaterial and

Methods.Datawereshownasmean±SEM(n≥3)*indicatesP<0.05;****indicatesP<0.001.

3.2 Hypoxia increases oxidative stress and apoptotic cell death in fat laden hepatocytes: To

evaluatewhether the inductionofhypoxiaexacerbatesdamageby lipotoxicity,wedeterminedthe

generationof reactiveoxygen species (ROS)and theextentof celldeath.The increase inROSwas

evaluatedusingthefluorogenicdyeDCF.TheresultsindicatedasignificantincreaseinROSineachof

thetreatmentscomparedtothecontrol(Figure3).Furthermore,hypoxiaexacerbatestheincreasein

ROSinsteatoticHepG2cellscomparedtothehypoxicandFFAconditionsseparately.

Apoptoticcelldeathwasevaluatedbymeasuringtheactivityofcaspase-3.Theresultsdemonstrate

thathypoxiainsteatoticHepG2cellsinducesapoptoticlipotoxicitycomparedtocontrolcells.(Figure

4a).Hypoxiaorsteatosisalonedidnotinduceapoptoticcelldeath.Lipotoxicitywasalsoevaluatedby

Sytox Green assay to detect cell death associated to disrupted cellular membrane integrity. The

differenttreatmentsinHepG2cellsdidnotinduceanynecroticcelldeath(Figure4b).

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Figure3.Hypoxia increasestheproductionofreactiveoxygenspecies insteatoticHepG2cells.HepG2cells

were incubated for 24hwith oleic acid and palmitic acid 2:1 (FFA) in the presence or absence of CoCl2 200

μmol/L. Reactive oxygen species (ROS) were measured as described in Material and Methods. Data were

shownasmean±SEM(n≥3)*indicatesP<0.05;**indicatesP<0.01;***indicatesP<0.005;****indicates

P<0.001.

Figure4.Hypoxiaandsteatosisinvitropromoteapoptoticcelldeathbutdonotpromotenecroticcelldeath.

HepG2cellswere incubatedfor24hwitholeicacidandpalmiticacid2:1(FFA) inthepresenceorabsenceof

CoCl2200μmol/L.Caspase-3activityandnecrosisweremeasuredasdescribedinMaterialandMethods.Data

wereshownasmean±SEM(n≥3)*indicatesP<0.05

3.3Hypoxiainducestheexpressionofpro-fibroticcytokines:Toevaluatewhetherhypoxiapromotes

a profibrotic phenotype in fat laden HepG2 cells, we measured the mRNA levels of different

profibrotic cytokines.Wedemonstrate that chemical hypoxia in fat ladenhepatocytes significantly

70

increases mRNA levels of profibrotic cytokines. Expression of some genes was also increased in

hepatocytestreatedonlywithCoCl2(Figure5).

Figure5.Hypoxiaincreasestheexpressionofpro-fibroticcytokinesinsteatoticHepG2cells.HepG2cellswere

incubatedfor24hwitholeicacidandpalmiticacid2:1(FFA)inthepresenceorabsenceofCoCl2200μmol/L.

mRNA levels of TGF-β1, CTGF, collagen-I, α-SMA and TIMP-1 were measured as described in Material and

Methods.Datawereshownasmean±SEM(n≥3)*indicatesP<0.05;**indicatesP<0.01;***indicatesP<

0.005

3.4IncreasednumberofEVinhepatocyteconditionedmediuminhypoxicandsteatoticconditions:

To better characterize the involvement of hepatocytes in the promotion of the pro-fibrotic

phenotype and possible crosstalkwith stellate cells,we assessed EV from conditionedmediumof

HepG2 cells (HepG2-EV) following different treatments. After isolation of EV from conditioned

medium, we characterized EV according to previous guidelines (42) by their typical structure

visualizedbyTEM(Figure6a)anddetectionofEVpositivemarkerCD81andEVnegativemarkerBcl-2

(mitochondrial marker) by Western blotting (Figure 6b). Also, the size (around 100-150 nm) and

concentrationofHepG2-EV(Figure6c)weredeterminedbyNTA.Notably,thefatladen-hepatocytes

inthepresenceofCoCl2showedasignificantincreaseintheconcentrationofHepG2-EVcomparedto

all conditions (Figure7).These results suggest thatsteatoticandhypoxicconditions inHepG2cells

canmodulatethereleaseofEV.

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Figure 6. CharacterizationofHepG2-EV. EVwere characterized in conditionedmedium from controlHepG2

cells by a) transmission electron microscopy; b) detection of EV-positive marker CD81 and EV negative

mitochondrialmarkerBcl-2byWesternblotc)sizedistributionbyNTAasdescribedinMaterialandMethods.

Figure7. EV release is increased inhepatocyte conditionedmedium inhypoxicand steatotic conditions in

vitro. EVquantification in conditionedmedium fromHepG2afterdifferent treatmentsperformedbyNTAas

describedinMaterialandMethods.Datawereshownasmean±SEM(n≥3)*indicatesP<0.05;**indicatesP

<0.01.

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3.5HepG2-EV fromsteatoticandhypoxic conditionspromoteapro-fibroticphenotype in stellate

cells:ToevaluatewhetherHepG2-EVhaveadirecteffectonstellatecellsweusedthehumanstellate

celllineLX-2.LX-2cellswerestimulatedwith15μg/mlofHepG2-EVfromeachtreatmentandmRNA

andprotein levelsof somepro-fibrotic cytokineswereevaluated.As shown in Figure8,HepG2-EV

from steatotic and hypoxic condition significantly increase the gene expression of TGF-β-1, CTGF,

collagen-I and α-SMA in LX-2 cells. The results also suggest an additive effect of FFA and CoCl2 to

promote a pro-fibrotic phenotype in LX-2 cells. Interestingly, a similar result was observed in a

confirmatoryexperimentassessingproteinlevelsofcollagen-I(Figure9a-9c)andα-SMA(Figure9b)

inLX-2cellstreatedwithHepG2-EVfromsteatoticandhypoxiccondition.

Figure 8. HepG2-derived EV increase the expression of profibrotic cytokines in LX-2 cells. LX-2 cellswere

exposedto15μgofEVthatwereisolatedfromHepG2cellsthathadbeentreatedwithCoCl2,FFAandCoCl2+

FFAfor24h.mRNAlevelsofTGF-β1,CTGF,Collagen-Iandα-SMAweremeasuredasdescribedinMaterialand

Methods.Datawereshownasmean±SEM(n≥3)**indicatesP<0.01;****indicatesP<0.001.

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a) b)

c)

Figure9.EVderivedfromsteatoticandhypoxicHepG2cellsincreasestheexpressionofprofibroticproteins

in stellate cells. LX-2 cellswereexposed to15μgof EV thatwere isolated fromHepG2 cells that hadbeen

treatedwithCoCl2,FFAandCoCl2+FFAfor24h.Proteinlevelsofa)Collagen-Iandb)α-SMAweredetermined

byWesternblotasdescribed inMaterialsandMethods.β-Actinwasusedas loadingcontrol. c) Intracellular

Collagen-I expressionwas determinedby immunofluorescence as described inMaterials andMethods.Data

wereshownasmean±SEM(n≥3)*indicatesP<0.05;**indicatesP<0.01;***indicatesP<0.005.

.

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3.6Hypoxiapromotesapro-fibroticphenotypeandcorrelateswithincreasedreleaseofEVinanin

vivomodelofNASH:Tovalidatethepreviousresultsinaninvivomodel,CDAAdietfeedingfor22-

weekswas used to induceNASH and an intermittent hypoxia regimenwas applied for the last 12

weeks.Asexpected,CDAAdiet-fedmiceshowedsignificant liverfibrosisasshownbyconventional

SiriusRedstaining(Figure10a).Interestingly,collagendepositionareaofIH-treatedmicewithNASH

was even further increased compared with CDAA diet-group, indicating a pro-fibrotic action of

hypoxia.Inaddition,asshowninFigure10b,IHsignificantlyincreasedhepaticmRNAlevelsofseveral

markersoffibrogenesisinducedbytheCDAAdietincludingTGF-β1,CTGF,collagen-I,α-SMA,TIMP-1

andMCP-1.Finally,EVwereisolatedfrommiceserumtodeterminetheconcentrationofEVineach

experimentalgroup.Asshown inFigure10c,CDAAdiet-fedmiceexhibitedasignificant increaseof

EVwhenexposedtoIHcomparedtocontrolorCDAAdiet-fedmiceundernormoxicconditions.These

resultssuggestastrongcorrelationbetweentheinductionofhypoxiawiththeincreaseoftheEVand

thepromotionofamorepro-fibroticphenotypeinaninvivomodelofNASH.

a)

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b)

c)

Figure10. Intermittenthypoxiapromotesapro-fibroticphenotype inmicewithNASHandcorrelateswith

increased levelsof EV in vivo. Themicewereplacedon choline-supplemented L-aminoaciddefined (CSAA)

dietascontrol,ordefineddietwithcholinedeficiencyaminoacids(CDAA)for22weekstoinduceliverinjury.

Intermittenthypoxia(IH)ornormoxiawasappliedforthe last12weeksofthedietasdescribed inMaterials

andMethods.a)LiversectionsstainedwithSiriusRedfrommicefedcontroldiet,intermittenthypoxiaand/or

choline-deficientaminoacid-defined(CDAA)diet. Inaddition, fibrosisareawasquantifiedusingdigital image

analysis of the red-stained area in Sirius red-stained samples (ImageJ, NIH, US);mRNA levels of pro-fibrotic

cytokinesweredeterminedbyRT-qPCRasdescribed inMaterials andMethods; c)quantificationofEV from

serumsampleswasdeterminedbyNTAasdescribedinMaterialsandMethods.Datawereshownasmean±

SEM(n≥3)*indicatesP<0.05;**indicatesP<0.01;***indicatesP<0.005;****indicatesP<0.001.

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4.-DISCUSSION

Inrecentyears, themechanismsunderlyingtheprogressionofNAFLD/NASHhavebeenthoroughly

studied. The early stages of NAFLD are characterized by fat accumulation in the liver resulting in

steatosisthatcanprogresstohepatocellulardamageandinflammationinaconditiontermedNASH

(2,5).Moreover,somepatientsdevelopliverfibrosisassociatingwithincreasedmortalityduetorisk

ofHCCdevelopment (43). Interestingly,clinicalstudieshave indicatedthatobstructivesleepapnea

(OSA) isanewpredictivefactorofhepatic fibrosis inpatientswithNAFLD(44-46).Recentresearch

hasprovidedevidenceregardingtheroleofhypoxia,ahallmarkofOSA,inthedevelopmentofliver

injuryandhepaticfibrosisinanimalmodelsofNAFLD(17,19,47).However,therearestillmajorgaps

inourunderstandingoftheprogressionofNASHtofibrosis,e.g.thecellularmechanismunderlying

thecrosstalkbetweenhepatocytesandstellatecells,theprincipalnon-parenchymalcellresponsible

for liver scar formation (48). In this study, we demonstrate that hypoxia, induced by a HIF-1α

chemical stabilizer in fat-laden hepatocytes, promotes hepatocellular damage, increases pro-

fibrogenicgeneexpressionand increasesthereleaseofextracellularvesicles (EV).Furthermore,EV

fromhypoxicandsteatotichepatocytespromotemRNAandproteinexpressionofimportantfibrosis

markersinstellatecells.

Inthepresentstudy,ourinvitromodelofsteatosiswaskeytotheanalysisofhypoxiainducedbythe

hypoxiamimeticCoCl2,aHIF-1stabilizer.Weusedthehumanhepatocytecell lineHepG2, inwhich

theeffectivenessofthehypoxiamimeticwasevaluatedthroughthequantificationofHIF-1αlevels,

which increaseddue to the intracellular stabilization,asdescribed inother invitro studies (49,50).

Weshowedthattreatmentwithpalmiticacid(PA)andoleicacid(OA)(1:2)increasedthecontentof

lipid droplets in hepatocytes in the absence of lipotoxicity. Previous studies have shown that OA

promotes steatosis inhepatocytes, both inprimaryhepatocyte cultureand inhepatomacell lines,

whilePAinducesacytotoxicresponse(51,52).Ourresultsareinlinewithapreviousreportusinga

combinationofboth fattyacids,usingahigherOAconcentration, thatpromotedananti-apoptotic

effect and triglyceride accumulation (51). Interestingly, the hypoxia mimetic CoCl2 increased the

contentoflipiddropletscomparedtoHepG2cellswithouthypoxia,asdeterminedviaquantification

ofNileRedfluorescence.Theseresultsaresupportedbyaninvivostudyindicatingthathypoxia,via

HIF-1α,promotesanincreaseinlipidbiosynthesisinsteatoticconditions(53).Hepatocytecelldeath

by apoptosis was measured using caspase-3 and as expected, treatment with FFA alone did not

inducecelldeath.However,HepG2cellsexposedtothecombinationofCoCl2andFFApresentedan

increaseof caspase-3 activity that correlatedwith an increase inoxidative stress compared to the

controlgroup.Unexpectedly,HepG2cellstreatedwithFFAsalsoincreasedROScomparedtoCoCl2

andcontrolgroups.A recent studydemonstrated thatOApreventsROSproduction inHepG2cells

77

treatedwith PA (54). Likewise, another study showed that FFA treatment of HepG2 cells induced

TNF-α generation, which is an importantmediator in hepatic steatohepatitis and liver injury (55).

Additionalstudiestofurtherdelineatethemechanism,suchasmeasurementofmitochondrialROS

production,remaintobeperformed.Wealsoanalyzedhepatocytecelldeathassociatedtodisrupted

cellular membrane integrity (necrosis) using SytoxGreen®. None of the treatments we applied

induced a significant increase in necrosis. The divergent results of apoptosis and necrosis can be

explainedbythefactthatdifferentstimulimayinduceddifferentmodesofcelldeathindifferentcell

types(56).Takentogether,ourfindingsindicatethathypoxiapromoteslipotoxicityasdeterminedby

theincreaseinhepatocellularapoptosisandoxidativestress.

To evaluate the pro-inflammatory effects of hypoxia in fat-laden hepatocytes, wemeasured gene

expression of pro-fibrotic cytokines. As expected and in accordancewith our previous results, the

conditionofhypoxiainsteatotichepatocytesincreasedtheexpressionofTGF-β1,CTGF,collagen-I,α-

SMAandTIMP-1.Moreover,wecheckedtheamountofEVreleasedintheculturemediumandwe

observed an increase after treatment of HepG2 cells with CoCl2 and FFA compared to all other

groups. EV are small structures surrounded by membrane and released from the cells to the

extracellularenvironment.Theyhaveapathophysiological role in variousdiseases, includingNASH

andotherchronicliverdiseases(30,31,57).WeperformedacharacterizationoftheisolatedEVfrom

HepG2 cells using positive and negativemarkers byWestern blotting and determinate EV size by

NTA.Finally,isolatedEVfromcontrolhepatocytesweredirectlyvisualizedusingTEM.

Previousstudieshavedemonstratedthatduringhepatocellulardamage,hepatocytesreleaseEVthat

modulatepro-inflammatoryandpro-fibroticsignaling innon-parenchymal livercells (25,58-60).To

confirmwhether EV from ourmodel of hypoxic and steatotic hepatocytes promote a pro-fibrotic

phenotypeinnon-parenchymallivercells,weusedthehumanstellatecelllineLX-2.EVderivedfrom

CoCl2-treatedfat-ladenhepatocytesincreasedgeneexpressionofpro-fibroticcytokinessuchasTGF-

β1, CTGF, collagen-I and α-SMA in LX-2 cells compared to all other groups. In addition, protein

expressionofcollagen-Iandα-SMAwasanalyzedinLX-2cellstreatedwithEVfromHepG2cells.Both

pro-fibrotic proteins increased when LX-2 cells were treated with EV derived from CoCl2-treated

steatotichepatocytes.ThisinterestingresultsuggeststhattheEV-cargoinourmodelisanimportant

topicforfutureanalysis.Recentstudiesthatsupportourfindingsshowthatpalmiticacidincreased

EV from hepatocytes and change their miRNA cargo inducing pro-fibrogenic signals to HSCs (60).

Anotherstudy,providingsimilarresults, indicatedthatEVfromlipotoxichepatocytesactivateHSCs

viamiRNA-128-3p(25).

78

The exact relation(s) between intermittent hypoxia, as hallmark of OSA, and NAFLD/NASH

progression,specificallyindevelopmentofliverfibrosis,remainincompletelyunderstood.Wefound

thatintermittenthypoxiapromotesapro-fibroticphenotypeinourCDAAdiet-fedanimalmodeland

increasescirculating levelsofEV.Previousstudieshavedemonstratedthat IHmimickingOSAalters

EVgenerationandreleaseaswellasEV-cargoandfunction,promotingpathophysiologyindifferent

in vivo models (61-63). This important finding correlates with our in vitro results and provide

evidence for a novel pro-fibroticmechanism, involving EV. Next challengewill be identified if the

mostofcirculatingEVarederivedfromliverorfromotherspecificcellsourceandidentifygeneand

proteinscargo,includingmembranesurfacecomposition.

In conclusion hypoxia promoted hepatocellular damage and increased pro-fibrotic genes that

correlates with increases of EV derived from hepatocytes and from our in vivo model of NASH.

Moreover, EV fromhypoxic fat-laden hepatocytes promoted a pro-fibrotic responses in LX-2 cells,

showinganovel cell to cell communication in thismodel. Finally,micemodelofNASHexposed to

intermittenthypoxiapresentedapro-fibroticphenotypethatcorrelatewithincreasedofcirculating

of EV. Futurework should focus on analysis to profile the genomic, transcriptomic, proteomic an

lipidomiccargo intoEV-derived fromcellsandbiological fluids fromrodentsmodelsunderhypoxia

conditionthatcouldbepromotesfibrosisprofileinthesettingofNAFLDandOSAS.

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82

Chapter5

HYPOXIAINCREASEDCASPASE-1INEXTRACELLULARVESICLESDERIVEDFROMEXPERIMENTAL

NON-ALCOHOLICSTEATOHEPATITISMODELSANDPROMOTESINFLAMMASOMEACTIVATIONIN

KUPFFERCELLS

ABSTRACT

Background:Intercellularcrosstalkbetweenhepatocytesandnon-parenchymalcellsisanimportant

factorinthepathogenesisofnon-alcoholicfattyliverdisease(NAFLD)andthemoreaggressiveform

non-alcoholic steatohepatitis (NASH). Extracellular Vesicles (EV) plays an important role in

intercellular crosstalk in the context of liver diseases, including NAFLD. Clinical observations have

indicated that Obstructive sleep apnea (OSA) is an aggravating factor in NASH. Hypoxia is the

hallmarkofOSAS,however,whetherhypoxiamodulatesthecrosstalkbetweenhepatocytesandnon-

parenchymalKupffer cells (KCs)hasnotbeen investigateddeeplyyet.Aim: to investigatewhether

hypoxiamodulates hepatocellular damage and intercellular crosstalk in the context of NAFLD and

whetherEVandinflammasome/caspase-1areinvolvedinthismodulation.Methods:Hepatomacell

line HepG2 were treated with free fatty acids (FFAs) and chemical hypoxia (CH) was induced by

cobalt (II) chloride (CoCl2). Pro-inflammatory and inflammasome-related gene expressions were

assessedinhypoxicfat-ladenHepG2andinlivertissue.IntermittentHypoxia(IH)wasappliedtomice

fed a CDAA diet (NASH model). EV were isolated from conditioned medium (CM) or serum by

ultracentrifugation and characterized by nanoparticle tracking and electron microscopy. Cleaved

caspase-1and caspase-1activitywasmeasuredbyWesternblot andenzymatic assay respectively.

Results:CHinfat-ladenHepG2increasedexpressionofpro-inflammatoryandinflammasome-related

genescomparedtonon-treatedhepatocytes.Theseresultscorrelatedwiththe invivo results. The

silencing of HIF-1α using siRNA in HepG2 prevented the increased expression of

inflammasome-related genes in KCs. Treatment of KCs with EV from fat-laden hypoxic HepG2

increased pro-inflammatory and inflammasome-related gene expression in KCs compared to KC

treatedwithEV fromnon-treatedHepG2.Cleavedcaspase-1contentandactivityofcaspase-1was

increasedinEVfromhypoxicfat-ladenHepG2andfromserumofCDAA-fedanimalsexposedtoIH.

Conclusion:OurfindingsindicatethatCHpromotesinflammatoryphenotypeinfat-ladenHepG2and

theinflammasome-relatedgenesexpressioninKCsviaHIF-1α.CHinducesthereleaseofcaspase-1-

EVfromfat-ladenHepG2promotingtheinflammatoryphenotypeinKCs.ThesefindingssuggestEV

andtheircontent(caspase-1)asapotentialnovelbiomarkerinNAFLDandOSAS.

83

INTRODUCTION

Significant advances have been made in understanding the importance of intercellular

communication between hepatocytes and non-parenchymal cells in the pathogenesis of liver

diseases (1,2). In particular in the pathophysiology of non-alcoholic steatohepatitis (NASH), the

inflammatoryandprogressive formofnon-alcoholic fatty liverdisease (NAFLD),Kupffercells (KCs),

theliver-residentmacrophages,areconsideredtoplayakeyroleintheinflammatoryresponseand

diseaseprogression (2,3). Indeed, ithasbeendemonstrated thatKCactivationpromotessteatosis,

inflammationandfibrosisinNASH(4-7).AnearlyeventinthepathogenesisofNAFLDistheincreased

accumulation of lipids in the liver, in particular in hepatocytes. This lipid accumulation promotes

cytotoxic effects known as lipotoxicity, resulting in hepatocellular damage and triggering of

inflammatorysignaling(8-9).Emergingdatasuggestthat lipotoxichepatocytesreleaseextracellular

vesicles (EV) toneighboringtargetcells inanautocrine/paracrinemannerand in thiswaypromote

liver damage (10-11). EV are 50-150 nanometer-sized vesicles released after the fusion of

multivesicularbodiesordirectlyfromthecellmembrane(12).Theircargoisdiverseandmaycontain

proteins,variousRNAspeciesand/orlipids(13).SeveralstudieshaveindicatedtheimportanceofEV

inthecrosstalkbetweeninjuredhepatocytesandKCs(14-17).Moreover,increasedlevelsofEVhave

beenreportedinserumofmiceandhumanswithNASHandtheselevelscorrelatewithhistological

liverinflammationandmacrophageactivation(18-21).

Recent data have also indicated that injured hepatocytes release inflammasome components into

theextracellularenvironment,providingamechanismtospreadinflammasomesignalingtoadjacent

cells (22,23). The activation of the inflammasome promotes the maturation and release of pro-

inflammatoryinterleukinsviaactivationofcaspase-1.Theactivecaspase-1iscomposedofatetramer

containing two 20 kDa fragments and two 10 kDa fragments (24). The activation of caspase-1

initiatesanovelformofprogrammednecroticcelldeathnamedpyroptosis.Pyroptoticcellsrelease

proinflammatorycytokines like IL-1βand IL-18andthe intracellularenzyme lactatedehydrogenase

(LDH) via gasdermin-dependent pores in the plasma membrane (25,26). Recent studies have

indicated that caspase-1 activation plays an important role in inflammation and fibrosis in NASH

(27,28). A recent study reported the presence of caspase-1 in EV released from monocytes that

induced endothelial cell death in amodel of lung injury (29). However, the presence and role of

caspase-1inEVinthecontextofNASHhasnotbeenstudiedyet.

Emerging evidence from clinical studies indicates that intermittent hypoxia (IH), the principal

pathophysiological factor inobstructivesleepapnea(OSA),aggravatesNAFLD(30). Indeed,there is

substantialevidencefromrodentmodelsandhumansubjectsthatIHassociatedwithOSAincreases

84

hepatictriglyceride(TG)levelsandexacerbatesinflammationandfibrosisinNASH(31-35).Although

theprecisemolecularmechanismsunderlying theaggravatingeffectof IHonNASHhavenotbeen

elucidated yet, the activation of the transcription factor hypoxia inducible factor 1 alpha (HIF-1α)

appearstobeanimportantfactor.HepaticHIF-1αactivationincreasessteatosisandpromotespro-

inflammatoryandpro-fibroticsignalingpathways(35-38).

Taken together, there is strong evidence that hypoxia via HIF-1α and Inflammasome/Caspase-1

activationisanaggravatingfactorinNASH.However,itisstillnotfullyunderstoodwhethercaspase-

1participatesinhypoxia-inducedliverinjury.

InthepresentstudywehaveinvestigatedtheroleofhypoxiaandEVininvivoandinvitromodelsof

NASH.Wedemonstratethathypoxiapromotesinflammatoryphenotypeinfat-ladenHepG2andthe

inflammasome-related genes expression in KCs via HIF-1α. Moreover, CH induces the release of

caspase-1-EV from fat-laden HepG2 and promotes cellular crosstalk between hypoxic fat laden-

hepatocytesandKC.

2.-MATERIALSANDMETHODS

2.1Animals

Specified pathogen-freemaleWistar rats (220–250 g; Charles River Laboratories Inc.,Wilmington,

MA,USA)andmaleC57bl6mice[purchasedfromJacksonLaboratories(BarHarbor,ME)]wereused.

Animalswerehousedunderstandardlaboratoryconditionswithfreeaccesstostandardlaboratory

chowdietandwater.AllexperimentswerecarriedoutaccordingtotheDutchandChileanlawson

the welfare of laboratory animals and guidelines of the local institutional animal care and use

committeesofthePontificiaUniversidadCatólicadeChileandethicscommitteeoftheUniversityof

Groningenforcareanduseoflaboratoryanimals.Alleffortsweremadetominimizeanimalsuffering

andtoreducethenumberofanimalsused.

2.2Kupffercellisolation

Kupffercellswere isolated fromthenon-parenchymalcell fractionobtainedduring thehepatocyte

isolation. The supernatant was subjected to different low-speed centrifugations at 4°C and

resuspending the pellets in Hank’s Balanced Salt Solution (HBSS) (Gibco, California, USA)

supplemented with 0.3% bovine serum albumin (BSA). KC were purified using a 20% of density

gradient medium OptiPrepTM (Sigma-Aldrich). After different low-speed centrifugations, the final

pellet was dissolved in HBSS supplemented with 10% FBS to allow cells to attach. After the

attachment period, KC were cultured in William's E medium supplemented with 50 μg/ml

gentamycin,10%FBSand1%ofp/s/ffor18hoursuntilthestartoftheexperiments.

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2.3HepG2hepatomacellline

The human hepatocellular carcinoma cell line HepG2 (ATCC, USA) was cultured in Dulbecco’s

modifiedEaglemedium(1X) + GlutaMAX™-I(DMEM,10569010,Gibco)supplementedwith10%fetal

bovineserum(FBS;Gibco)and1%penicillin-streptomycin(Gibco)at37°Cand5%CO2.Allcellswere

platedinacellcultureplateatleast24 h-36hbeforetreatmentuponreaching80%confluence.

2.4Treatmentwithfreefattyacidsandchemicalhypoxia

Inorder toassess theeffectsofhypoxiaon fat-laden liver cells,HepG2cellswas incubatedwitha

mixtureoffreefattyacids(FFA)consistingofoleicacid(500μmol/L)andpalmiticacid(250μmol/L)

inanaqueoussolutionofBSAasdescribed(41).IncubationswerecarriedoutwithorwithoutCobalt

(II)chloride(CoCl2;SigmaAldrich)(200μmol/L)for24hourstoinducechemicalhypoxia(40).CoCl2

isawell-knownhypoxia-mimeticagent, thatmimicshypoxia/ischemicconditionsbystabilizationof

hypoxia-induciblefactorHIF-1α(42).ControlcellsweretreatedwithBSAalone.

2.5EffectsofintermittenthypoxiainexperimentalNASH

Animalexperimentswereapprovedbythe institutionalanimalcareandusecommittee(Comitéde

ética y bienestar Animal, Escuela de Medicina, Pontificia Universidad Católica de Chile, CEBA

100623003).MaleC57BL/6miceaged10weeksatthebeginningofthestudyweredividedintofour

experimental groups (n = 4–8) receiving either choline-deficient amino acid-defined (CDAA) diet

(Catalog#518753,DyetsInc.Bethlehem,PA)toinduceNASHorthecholine-supplementedL-amino

acid defined (CSAA, Catalog # 518754, Dyets Inc. Bethlehem, PA) diet as control for 22 weeks as

previouslydescribed(43,44).AnimalswereexposedtoIHornormoxia(chambers41x22x35cm,COY

labproducts™,GrassLake,MI,USA)duringthelast12weeksoftheexperimentalorcontrolfeeding

period.IHregimenconsistedof30events/hourofhypoxicexposuresfor8hour/dayduringtherest

cycle,between9amand5pm.Thiscyclewasrepeated7daysaweekfor12consecutiveweeks.At

the end of the study, mice were anesthetized (ketamine 60 mg/kg plus xylazine 10 mg/kg

intraperitoneally) and then euthanized by exsanguination. Serum and liver tissue samples were

collected and processed or stored at −80 °C until analyzed. Gene expression and protein analyses

werecarriedoutasdescribedabove.

2.6WesternBlotanalyses

Protein lysateswerecollectedbyscrapingcells in lysisbuffer (HEPES25mmol/L,KAc150mmol/L,

EDTApH8.02mmol/L,NP-400.1%,NaF10mmol/L,PMSF50mmol/L,aprotinin1µg/µL,pepstatin1

µg/µL, leupeptin1µg/µL,DTT1mmol/L).Totalamountofprotein in lysateswasmeasuredbyBio-

Radproteinassay(Bio-Rad;Hercules,CA,USA).ForWesternblot,30-50µgproteinwasresolvedon

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Mini-PROTEAN®TGXStain-Free™PrecastGels(BioRad,UK,Oxford).Semidry-blottingwasperformed

using Trans-Blot Turbo Midi Nitrocellulose Membrane with Trans-Blot Turbo System Transfer

(BioRad).PonceauS0.1%w/v(Sigma)stainingwasusedtoconfirmproteintransfer.anti-CASP-1(SC-

56036,SantaCruz),anti-CD81(10630D,Invitrogen),anti-CD63(sc-5275,SantaCruz)andanti-ASGPR1

(Novus, USA) were used in combination with appropriate peroxidase-conjugated secondary

antibodies.Tubulin(T9026,Sigma)oractin(A5228,Sigma)wereusedasloadingcontrols.Theblots

were analyzed in a ChemiDoc XRS system (Bio-Rad). Protein band intensities were quantified by

ImageLabsoftware(BioRad).

2.7RNAisolationandquantitativereal-timereversetranscriptionpolymerasechainreaction(qRT-

PCR)

Aftertreatment,HepG2cellsandrathepatocyteswereharvestedoniceandwashedtwicewithice-

cold PBS. Total RNA was isolated with TRI-reagent (Sigma) according to the manufacturer’s

instructions.Reversetranscription(RT)wasperformedusing2.5µgoftotalRNA,1XRTbuffer(500

mmol/l Tris-HCl [pH 8.3]; 500 mmol/l KCl; 30 mmol/l MgCl2; 50 mmol/l DTT), 1 mmol/l

deoxynucleotides triphosphate (dNTPs, Sigma), 10 ng/µl random nanomers (Sigma), 0.6 U/µl

RNaseOUT™(Invitrogen)and4U/µlM-MLVreversetranscriptase(Invitrogen)inafinalvolumeof50

µl.ThecDNAsynthesisprogramwas25°C/10min,37°C/60minand95°C/5min.ComplementaryDNA

(cDNA)wasdiluted20X innuclease-freewater.Real-TimeqPCRwascarriedout inaStepOnePlus™

(96-well) PCR System (Applied Biosystems, Thermofisher) using TaqMan probes. The sequences of

theprobesandprimer setsaredescribed inSupplementaryMaterial. ForqPCR,2X reactionbuffer

(dNTPs, HotGoldStar DNA polymerase, 5 mmol/l MgCl2) (Eurogentec, Belgium, Seraing), 5 µmol/l

fluorogenicprobeand50µmol/lofsenseandantisenseprimers(Invitrogen)wereused.mRNAlevels

werenormalizedtothehousekeepinggene18Sandfurthernormalizedtothemeanexpressionlevel

ofthecontrolgroup.RelativegeneexpressionwascalculatedusingtheΔCtmethod.Thesequences

oftheprobesandprimersetsaredescribedintheSupplementaryMaterial.

2.8TransfectionofHepG2cell linewithHIF-1α small interferingRNAor control small interfering

RNA

HepG2cellsweregrown toapproximately60%confluency in6-wellplatesbefore transfectionand

treatments. Small interfering RNA (esiRNA) was employed to silence HIF-1α in HepG2 cells

(EHU151981, Sigma-Aldrich) using standard Lipofectamine (Lipo3000, Invitrogen) in OPTI-MEM™ I

mediumatafinalconcentrationof100nmol/Laccordingtothemanufacturer’sprotocol.Cellswere

transfected with EGFP siRNA as control (EHUEGFP, Sigma-Aldrich). Experiments were performed

after 24 hours of transfection for 24 hours. The efficiency of transfection was determined by

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quantitative RT-PCR and Western blot for HIF-1α (Supplementary Material). To obtain the

corresponding conditioned medium (CM), hepatocyte culture medium was replaced by FBS-free

mediumaftertreatmentforanadditional24hours.CMfromdifferenttreatmentswereaddedtoKC

for24hours.

2.9Extracellularvesiclesisolation

ForEVcollection,HepG2cellsweregrowninculturedishesof100mmtoobtain70mlofserum-free

conditioned media after 24 hours of treatment. EV were isolated from conditioned medium by

differential ultracentrifugation (UCF Thermo-Sorvall 80wx+) according to a modified protocol

describedpreviously(46).Atotalvolumeof70mlmediumpertreatmentwasdepletedofcellsand

cell debris by consecutive, low-speed centrifugations (2,000 × g for 30min and 12,000 × g for 45

min).Theresultingsupernatantswerecarefullycollectedandcentrifugedfor70minat120,000×gat

4°C.PelletsfromthiscentrifugationstepwerewashedinPBS,pooledandcentrifugedagainfor60

min at 100,000 × g at 4 °C. For EV collection from serum, samples were reconstituted in a total

volumeof4.4mLandcentrifugatedat2,000×gfor30minand10,000×gfor30min.Theresulting

supernatantswerecarefullycollectedandcentrifugedfor70minat120,000×gat4°C.Pelletsfrom

thiscentrifugationstepwerewashedinPBS,pooledandcentrifugedagainfor60minat100,000×g

at 4 °C. The obtained pellets fromHepG2 cells or serumwere resuspended in lysis buffer or PBS

dependingonsubsequentexperimentsandstoredinaliquotsat-80°C.ProteinfromHepG2-derived

EV or serum-derived EVwere obtained using the Total Exosome Protein Isolation kit (Invitrogen).

ProteinconcentrationinEVpelletsweremeasuredusingBCAproteinassaykit(Pierce,Rockford,IL).

2.10TreatmentofKupffercellswithextracellularvesicles

RatprimaryKupffercells(KCs)wereincubatedwithFBS-freeWilliam'sEmediumandexposedto15

μg of EV isolated fromHepG2 cells treatedwith CoCl2, FFA or CoCl2 + FFA for 24h. After 24h of

treatment,KCswereharvested.

2.11Caspase-1activity

Cell lysate, HepG2-derived EV or serum-derived EV were assayed for their ability to cleave a

fluorescentCASP-1substrate,followingthemanufacturer’sinstructions(Abcam,USA).

2.12Statisticalanalyses

AnalyseswereperformedusingGraphPadsoftware(version5.03,GraphPadSoftwareInc.,CA,USA).

All results are presented as a mean of at least 3 independent experiments ± SEM or as absolute

number or percentage for categorical variables. The statistical significance of differences between

88

themeansof theexperimental groupswas evaluatedusingone-wayanalysis of variance (ANOVA)

with a post-hoc Bonferroni multiple-comparison test; P<0.05 was considered as statistically

significant.

3.-RESULTS

3.1 Effect of chemical hypoxia on pro-inflammatory, inflammasome-related gene expressionand

caspase-1 levels inan invitromodelofNASH: To investigatewhether theHIF-1αstabilizerCoCl2

increases the expression of inflammatory genes and inflammasome components in fat-laden liver

cells. HepG2 cells were treated with CoCl2 and FFAs to mimick hypoxia and steatosis in vitro as

described in Materials and Methods. Figure 1 shows that hypoxia increased expression of pro-

inflammatory genes like TNF-α and IFN-γ in fat-laden HepG2 cells. Furthermore, CoCl2 increased

expressionofinflammasomecomponentslikeIL-1β,IL-18,NLRP3andCaspase-1,infat-ladenHepG2

cells. Finally, inflammasome activation was determined by measuring cleaved caspase-1 levels in

HepG2 cells byWestern blot as well as caspase-1 activity. The increased protein level of cleaved

caspase-1 (Fig. 2a) and increased caspase-1 activity (Fig.2b) confirmed that the HIF-1α stabilizer

CoCl2couldpromoteinflammasomeactivationinfat-ladenHepG2cells.

Figure 1. CH increases the expressionof pro-inflammatory genes and inflammasome components in an in

vitromodelofNASH.HepG2cellswereincubatedfor24hwitholeicacidandpalmiticacid(2:1ratio)(FFA)in

thepresenceorabsenceofCoCl2200μmol/L.mRNAlevelsofTNF-αIFN-γ,IL-1β,IL-18,NLRP3andCaspase-1

weremeasuredasdescribedinMaterialsandMethods.Datawereshownasmean±SEM(n≥3)*indicatesP

<0.05.

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Figure2.CHincreasesinflammasomecaspase-1levelinaninvitromodelofNASHa)proteinlevelsofpro-

caspase-1andcleavedcaspase-1.b)activityofcaspase-1.HepG2cellswereincubatedfor24hwitholeicacid

and palmitic acid (2:1 ratio) (FFA) in the presence or absence of CoCl2 200 μmol/L. Protein levels of pro-

caspase-1 and cleaved caspase-1 (p20) were determined by Western blot as described in Materials and

Methods. α-Tubulin was used as loading control. Total p20 caspase-1 was calculated to determine the

generationofcleavedcaspase-1.Datawereshownasmean±SEM(n≥3)*indicatesP<0.05;****indicatesP

<0.001.

3.2SilencingofHIF-1αinhypoxicandfat-ladenHepG2preventsinflammasomeactivationinKCs:

To explore whether hypoxic and fat-laden HepG2 cells can increase expression of

inflammasome-relatedgenesinhepaticresidentmacrophages,weisolatedprimaryratKupffercells

(KC) and investigated whether inflammasome activation in KCs depends on HIF-1α in fat-laden

hepatocytes. Conditionedmedium from steatoticHepG2 cells treatedwith CoCl2 increasedmRNA

levelsofNLRP3,Apoptosis-AssociatedSpeck-LikeProteinContainingCARD(ASC),Caspase-1andIL-1β

in KC. The increase of these genes was abolished by HIF-1α knockdown in steatotic HepG2 cells

treated with CoCl2 (Figure 3). Taken together, our results suggest a cellular crosstalk between

hepatocytesandmacrophages inhypoxia,which isdependentonHIF-1αactivation inhepatocytes,

thatpromotesaninflammatoryphenoptypeinNASHthoughtinflammasomeactivation.

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Figure3.SilencingofHIF-1αpreventsinflammasomeactivationinKCs.Culturemediumofhypoxicfat-laden

HepG2cellstransfectedwithnegativecontrolsiRNA(sieGFP)orHIF-1αsiRNA(siHIF-1α)wasreplacedafter24

hoursoftreatmentbyfreshmediumwithoutFFAandCoCl2.Cellswereculturedforanother24hoursandthe

conditionedmedium(CM)wascollected.Kupffercellswere treated for24hourswithCMfromHepG2cells.

mRNAlevelsofNLRP3,Caspase-1,ASCandIL-1βweremeasuredasdescribedinMaterialsandMethods.Data

wereshownasmean±SEM(n≥3)*indicatesP<0.05;**indicatesP<0.01.

3.3ExtracellularvesiclesfromsteatoticandhypoxicHepG2cellscontainpromotesinflammasome

activationand inflammatoryphenotype inKupffer cells:Ourprevious studieshavedemonstrated

that conditioned medium from hypoxic fat-laden HepG2 cells increase the expression of pro-

inflammatory cytokines in Kupffer cells (Chapter 3). According with that, we next investigated

whetherEVfromhypoxicandsteatotichepatocytespromotethepro-inflammatoryphenotypeinKC.

EV were isolated from conditioned medium of HepG2 cells (HepG2-EV) following different

treatments.KCsweretreatedwith15μg/mlofHepG2-EVfromeachtreatmentandgeneexpression

of inflammasome components was evaluated. As shown in Figure 4, HepG2-EV from steatotic

hypoxicconditionsignificantlyincreasedgeneexpressionoftheinflammasomecomponentsNLRP3,

ASC, Caspase-1 and IL-1β in KC (Figure 4). HepG2-EV from steatotic and hypoxic conditions also

significantly increased gene expression of IL-6, iNOS and TNF-α (Figure 5). These results suggest a

synergisticeffectof steatosis andhypoxiaon thepro-inflammatoryeffectofHepG2-derivedEVon

KCs.

Figure4.EVfromhypoxic,fat-ladenHepG2cellsincreasetheexpressionofinflammasomecomponentsand

cytokinesinKupffercells.Kupffercellswereexposedto15μgofEVisolatedfromHepG2cellsthathadbeen

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treated with CoCl2, FFA and CoCl2 + FFA for 24h. mRNA levels of NLRP3, ASC, Caspase-1 and IL-1β were

measuredasdescribedinMaterialandMethods.Datawereshownasmean±SEM(n≥3)*indicatesP<0.05;

**indicatesP<0.01;***indicatesP<0.005;****indicatesP<0.001.

Figure5. EV fromhypoxic, fat-ladenHepG2cells increase theexpressionofpro-inflammatory cytokines in

Kupffercells.Kupffercellswereexposedto15μgofEVisolatedfromHepG2cellstreatedwithCoCl2,FFAand

CoCl2 + FFA for 24h. mRNA levels of IL-6, iNOS and TNF-α were measured as described in Material and

Methods.Datawereshownasmean±SEM(n≥3)*indicatesP<0.05;**indicatesP<0.01;***indicatesP<

0.005;****indicatesP<0.001.

3.4ExtracellularvesiclesfromsteatoticandhypoxicHepG2cellscontainincreasedlevelsofactive

caspase-1:WenextinvestigatedwhetherEVobtainedfromhypoxicandfat-ladenHepG2cellsare

enrichedin(active)caspase-1.Proteinlevelsofpro-caspase-1andcleavedcaspase-1(p10andp20)

andtheactivityofcaspase-1weredeterminedinpurifiedEV.EVisolatedfromhypoxicfat-laden

HepG2cellscontainsignificantlyincreasedlevelsofactivecaspase-1comparedtoallothergroups

(Figure6).

a)

b)

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Figure6.EVfromsteatoticandhypoxicHepG2cellshaveincreasedlevelsofcleavedcaspase-1andcaspase-1

activity.a)Western Blot analyses of pro-caspase-1 and cleaved caspase-1 (sumof p10 and p20) and b) the

activityofcaspase-1frompurifiedEVweremeasuredasdescribedinMaterialandMethods.Datawereshown

asmean±SEM(n≥3)*indicatesP<0.05;***indicatesP<0.005;****indicatesP<0.001.

3.5 Intermittent hypoxia in an experimentalmodel of diet-induced non-alcoholic steatohepatitis

increases caspase-1 in circulating EV levels: In order to validate our in vitro findings, we tested

wheterintermittenthypoxiafor12weeksinananimalmodelofCDAAdiet-inducedNASHincreases

caspase-1 activation in circulating EV. First, we characterized EVwith positivemarkers, CD63 and

CD81 (Figure 7) and thenwe analyzed the caspase-1 contain EV.Wedemonstrate that IH inmice

withNASH significantly increased caspase-1 levels andactivity in circulatingEV isolated frommice

subjectedtoIHandNASHcomparedtoEVisolatedfromallothergroups(Figure8).Thesefindingare

correlatingwith our previous studies which Intermittent hypoxia inmicewith CDAA-induced liver

injury increases pro-inflammatory signaling, caspase-1 protein levels and caspase-1 activity in the

liver(Chapter3)andincreasescirculatingEV(Chapter4).Takentogether,theseresultsconfirmthat

hypoxiaaggravatesinflammationinaninvivomodelofNASHinapossiblemechanismthatinvolves

circulatingcaspase-1-EV.

Figure 7. Extracellular vesicles in a model of diet-induced NASH and

intermittenthypoxia.a)CirculatingEVwerecharacterizedbydetection

of EV-positive marker CD63 and hepatic positive marker

asialoglycoprotein receptor (ASGRP1) by Western blot as described in

MaterialandMethods.

a)

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b)

Figure8. Extracellular vesicles fromCDAAdiet-fedmice subjected to IntermittentHypoxiaareenriched in

caspase-1.a)WesternBlotanalysisofpro-caspase-1andcleavedcaspase-1(p10andp20)andb)theactivityof

caspase-1 from purified circulating EV were measured as described in Materials and Methods. Data were

shownasmean±SEM(n≥3)**indicatesP<0.01;****indicatesP<0.001.

4.-DISCUSSION

Theprincipalfindingofthisstudyisthatextracellularvesciclesfromhepatocytesplayanimportant

roleinintercellularcommunicationinthecontextofNAFLD.Specifically,wehaveshownthatEVfrom

fat-laden and hypoxic hepatocytes induce a pro-inflammatiory phenotype in Kupffer cells, which

contributes to the initiation and progression of inflammation in NASH. The induction of this pro-

inflammatory phenotype is at least in part mediated by active caspase-1, and possibly other

componentsoftheinflammasome,containedwithinEV.

In recent years, different studies have provided evidence that hepatocyte injury induces a pro-

inflammatoryresponse inmacrophagesviathereleaseofhepatocyte-derivedEV,thuscontributing

to the development and progression of NAFLD (10, 47). The progression from simple steatosis to

NASH isnot completelyunderstood.TheonsetandprogressionofNAFLD fromsimple steatosis to

inflammation(NASH)ismostlikelytheresultofmultiplefactorsactinginparallelorsequentially(48).

Therecentclinicalobservationthatobstructivesleepapneasyndrome(OSAS) isapredictive factor

fortheprogressionandaggravationofNAFLD,promotedustoconsiderhypoxiathroughHIF-1α,the

hallmarkofOSAS,asan important trigger forhepatocellulardamage,exarcebating lipotoxicityand

inflammationandincreasingthereleaseofEV.Previously,wedemonstratedthatchemicalhypoxia,

mimicked by the HIF-1α stabilizer CoCl2, further increases hepatocellular TG content, pyroptosis-

induced cells death and mitochondrial ROS generation in FFA-treated primary hepatocytes and

HepG2 cells (10, 11). Now, we complement these finding, adding also that chemical hypoxia

increasesmRNA levelsof inflammasomecomponents inHepG2cells.These resultsare in linewith

our previous studies that hypoxia exacerbates apoptotic cell death determined by caspase-3 and

promotes disruption of cellular membranes in fat-laden hepatocytes (10). Interestingly, we

94

determinatedcellularcrosstalkmechanism-dependentofHIF-1alphainhypoxicfat-ladenHepG2cells

onexpressionof inflammasome-relatedgenes inKCs. Thisparacrineeffect is correlatedwithour

previous studies between hepatocytes with KCs and stellate cells (10, 11). Moreover, we

demonstratedanincreaseincaspase-1levelscontaininEVfromconditionedmediumobtainedfrom

hypoxicfat-ladenhepatocytes.TheseEVpromotedaninflammatoryphenotypeinKCsasevidenced

by increasedmRNA expression of inflammasome components in EV-treated Kupffer cells. Via this

mechanism,Caspase-1-EVmaycontributetotheinflammationassociatedwithNASH(12-49,50).The

inflammasome is an intracellularpro-inflammatory structure thatpromotes inflammatory signaling

and metabolic disruption (51). The activation of inflammasomes involves activation of caspase-1

which in turn cleaves pro-IL-1β into its active secreted form, thus amplifying the inflammatory

response(52). Interestingly,weobservedincreasedcaspase-1activity inEVfromfat-laden,hypoxic

hepatocytes.Inthecurrentstudywealsodemonstrateincreasedcaspase-1activityincirculatingEV

inmicewithNASHsubjectedtointermittenthypoxia(IH).Previousstudieshavedemonstratedthat

circulatingEVlevelsandinflammasomeactivationcorrelatestronglywithpathophysiologicalchanges

observedduringNASHdevelopment(4,27,28,53).Inourpreviousstudies,wealsoconfirmedthatIH

promotes NLRP3 inflammasome activation and pro-inflammatory and pro-fibrotic signaling in the

contextofNASH(10,11). In linewiththesepreviousresults,wehavenowdemonstrated increased

levelsofcirculatingEVinmicewithNASHandsubjectedtoIHandwealsodemonstratethattheseEV

containahigheractivityofcaspase-1comparedtocirculatingEVfromcontrolmice.Ourresultsare

supported by studies reporting that EV released by hepatocytes exposed to lipotoxic stress may

actively contribute to pro-inflammatory responses by activating macrophages in a NLRP3

inflammasome-dependent, paracrine pathway (54-56). In particular, a recent study demonstrated

the presence of the inflammasome components NLRP3, pro-caspase-1, pro-IL-1β and ASC in

macrophage-derivedEV(57).Takentogether,ourdataextendtheseobservationsbydemonstrating

thatEVfromhypoxicfat-ladenhepatocytes,andin invivomodelofhypoxiainexperimentalNASH,

containactivecaspase-1thatcouldinducesandextensepro-inflammatorysignaling.

Inconclusion,ourdataclearlyindicatetheimportanceofcrosstalkbetweenhepatocytesandKupffer

cells under steatotic and hypoxic conditions. Moreover, our results suggest that this crosstalk is

mediated via activated caspase-1 contained in EV and that this is essential for inflammasome

activationinmacrophages.Ourdataprovidenewinsightsinintercellularcommunicationandtherole

ofinflammasomesinhepatocyte-derivedEVinthecontextofNAFLD.

Our findingscouldhave implications in thedevelopmentofEV-basedbiomarkers,deliveryvehicles

andtherapeuticsforthetreatmentofliverdiseases.

95

5.-SUPPLEMENTARYFIGURES

FigureS1.SilencingofHIF-1α inHepG2cells . CulturemediumofhypoxicHepG2-transfectedwithnegative

control siRNA (sieGFP) orHIF-1αsiRNA (siHIF-1α).a)Protein levelsandb)mRNA levelsofHIF-1αdecreased

comparewithtreatmentofCoCl2 orCoCl2 +sieGFP.Valuesrepresentmean±standarddeviationfromthree

independentexperiments.Datawereshownasmean±SEM(n≥3)*indicatesP<0.05.

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99

Chapter6

1.-GENERALDISCUSSIONANDCONCLUSION

NAFLDreferstoaspectrumofhistologicalabnormalitiesintheliver,rangingfromisolatedsteatosis

tonon-alcoholicsteatohepatitis(NASH),theaggressiveformofNAFLD,whichischaracterizedbythe

presence of necro-inflammatory and fibrotic phenomena in the liver (1,2). Understanding the

transitionfromsimplesteatosistoNASHisakeyissueintheNAFLDfield.Currently,severalstudies

have shown thatOSAS,mainly via the occurrence of intermittent hypoxia (IH), predisposes to the

development of NASH (3). Recent studies have also demonstrated that hypoxia can aggravate

hepatocellular damage by triggering pro-inflammatory and pro-fibrotic signals (4,5). However, the

pathwaysunderlyingtheseeffectsarestill largelyunexplored.Thepresentdoctoralthesisproposes

an important role for extracellular vesicles in the hypoxia-induced exarcebation of hepatocellular

damage. We used in vitro models of NAFLD/NASH to explore whether hypoxia induces

hepatocellular damage, hepatic inflammation and fibrosis via mechanisms that involve cellular

crosstalkmediatedbyextracellularvesicles.

IntheChapter2,wereviewedthemostrecentfindingsconcerningtheroleofextracellularvesicles

(EV) inmediating autocrine and paracrine intercellular communication in both ALD andNAFLD as

wellastheirpotentialuseasbiomarkersofdiseaseseverityandprogression(15-17).Consideringour

resultsonthecrosstalkbetweenhepatocytesandnon-parenchymalcells,weproposearoleofEVin

intercellularcommunicationandtheaggravationofdamageinourNAFLDmodels

Theprincipal findings inChapter 3 suggest thathypoxiapromotes lipidsdroplet accumulation and

cell death via apoptosis, necrosis and pyroptosis in fat-laden primary rat hepatocytes. These

phenomena are associated with increased oxidative stress and inflammatory signals including

inflammasome/caspase-1 activation and contribute to cellular crosstalk between hepatocytes and

Kupffercells throughtEV,thusaggravatingthepathologicalphenomenaofNASH(6,7).Our invitro

findingscorrelatedwithour findings inananimalmodelofNASH inducedby feedingaCDAAdiet.

Ourresultsarealsoinlinewithpreviousstudiesthatindicatethathypoxia,viaHIF-1α,promoteslipid

accumulationinhepatocytesandcontributestoliver inflammationandfibrosis inrodentmodelsof

NAFLD (8-10).Our studiesare the first to investigate theeffectsofhypoxia inan invitromodelof

NAFLD/NASH that involves EV (11). Interestigly, we found a synergistic effect of hypoxia and fat

accumulationinhepatocytesontheincreasedexpressionofinflammasomecomponents,suggesting

that pyroptosis is the major form of cell death observed in these conditions. Moreover, we

investigatedtheeffectofhypoxia inan invivomicemodelofNASH(CDAAdiet). In linewithour in

vitroresults,intermittenthypoxiaincreasessteatosis,pro-inflammatorygeneexpressionandhepatic

100

inflammasome/caspase-1activationinmicewithNASH.Ithasbeenshownthatcaspase-1activation

contributestoliverdamageinNASH(12,13).

In Chapter 4 we demonstrated that hypoxia exarcebates hepatocellular damage in HepG2 cells.

Furthermore, hypoxia promotes pro-fibrotic signaling that correlates with increased release of EV

from hepatocytes in our experimental models of NASH. Morever, EV from hypoxic fat-laden

hepatocytesevokepro-fibroticresponsesinLX-2cells.Theseresultsareinagreementwithprevious

studies demonstrating that EV from lipotoxic hepatocytes induce pro-fibrogenic signals in stellate

cells(18,19).Furthermore,wedemonstratedincreasedlevelsofEVinserumfromCDAA-fedanimals

exposedtohypoxiaandthese increased levelscorrelatewithhistologicallydeterminedfibrosisand

pro-fibroticgeneexpressionintheliver.InChapter5weinvestigatedtheparacrineeffectofEVfrom

fat-ladenhypoxichepatocytesonKupffercellsthroughtCaspase-1likecargointoEV.Theexpression

ofpro-inflammatoryandinflammasome-relatedgeneswasincreasedinKupffercellstreatedwithEV

from hypoxic fat-laden hepatocytes compare to EV from non-treated hepatocytes, similar finding

thatwedescribesinChapter3.Theseresultscorrelatewellwithpreviousresultsindicatingcrosstalk

betweeninjuredhepatocytesandKupffercellsthatinvolvestheparticipationofEV(20-23).Wealso

performedexperiments to silenceHIF-1α inhypoxic fat-ladenHepG2 cells, to establish the roleof

HIF-1α in thepro-inflammatorycrosstalkbetweenhypoxic fat-ladenhepatocytes (HepG2cells)and

Kupffer cells. We demonstrated that conditioned medium from hypoxic fat-laden HepG2 cells

induced an increase in the expression of inflammasome-related genes in Kupffer cells via a

mechanismthatrequiresactivationofHIF-1α.Thesenovelresultsareinlinewitharecentstudythat

proposes a paracrine crosstalk between Kupffer cells and hepatocytes that promotes pro-

inflammatory signaling and leads to inflammatory injury (14). Taken together,we identify hypoxia

andHIF-1αactivationasapotentialaggravatingfactorinthedevelopmentofNASHinamechanism

thatinvolvescaspase-1/inflammasomeactivationandcellularcrosstalk.

Consideringthepro-infammatoryroleofEV(24-27),wealsoevaluatedthecontentofEV.Wecould

demonstratethepresenceofcleavedandactivecaspase-1inEVfromhypoxicfat-ladenhepatocytes

andEVfromserumofCDAA-fedmiceexposedtoIH.Collectively,thesenovelfindingsindicatethat

EV fromhypoxic fat-ladenhepatocytes inducepro-inflammatory andpro-fibrotic signals in Kupffer

cells and LX-2 cells, respectively via a mechanism that involves activation of caspase-1 and

inflammasomes.

In summary, this doctoral thesis identifies hypoxia as an aggravating factor in thepathogenesis of

NASH.ThisaggravatingeffectisdependentonintactHIF-1αsignalinginhepatocytesandintercellular

crosstalk between hepatocytes on the one hand and Kupffer cells and stellate cells on the other

101

hand. This crosstalk is dependentonextracellular vesicles released fromhepatocytes and involves

active caspase-1 and inflammasomes. We demonstrate that fat-laden hepatocytes are more

susceptible to the damaging effects of hypoxia than non-steatotic hepatocytes and that hypoxia

increasestheextentoffataccumulation,inflammationandfibrosisbothinvitroandinvivo.Further

characterizationofEVreleasedunderhypoxicconditionswillallowthedetaileddelineationoftheir

roleinthepromotionofsteatosis,inflammationandfibrosisinthecontextofNAFLDandOSAS.

Some limitations of our studies include the possible contamination of the EV fraction with

lipoproteinsduetosimilaritiesinsizeandcontamination,intheinvivostudies,withnon-hepaticEV.

Also,thesmallnumberofmicepergroupaswellastheuseofonly1modelofNASHarelimitations.

FuturestudiesshouldincludedifferentmodelsofNAFLD,e.g.highfatdietandalsohumanstudies,to

confirm our findings. Also, the cargo of EV should be further analyzed for the presence of

components,inadditiontoinflammasomecomponents,thatmaymodulatetheresponseofKupffer

cells and stellate cells to EV.Our findings couldhavepotential implications in thedevelopmentof

novel biomarkers that involve EV or selected components of EV, like caspase-1 and provide novel

therapeutictargetsforthetreatmentofNAFLDorliverdamageinOSAS.

2.-PERSPECTIVES

The information generated in this thesismay contribute to a better understanding of intercellular

communicationinthepathogenesisofNAFLDandthedevelopmentofnovel(EV-based)biomarkers

andtherapies.Itwillalsobeinterestingtoperformsimilarstudiesinotherliverdiseases.

AnexhaustivecharacterizationofthecontentofEVisolatedfrominvivoandinvitromodelsofNASH,

using genomic, proteomic and metabolomic techniques, will allow us to identify hypoxia-specific

mechanisms related to hepatocellular damage that occurs in NASH. In addition, our experimental

approach should be extended to clinical samples of patients with OSA and/or NASH, in order to

generate translational data. These studies should include characterization of EV obtained from

patients from blood or other bodily fluids such as urine or saliva. This non-invasive approachwill

allowtheidentificationandvalidationofnovelbiomarkersandimprovediagnosisandpredictionof

prognosis of NAFLD/NASH. Finally, non-pharmacological or pharmacological management of the

levelsofcirculatingEVcanbecomeanewtherapeutictargetinthecontextofNAFLDandOSAS.

102

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104

Appendices

EnglishSummary

Background:Nonalcoholic fatty liverdisease (NAFLD) isconsideredthemostcommon liverdisease

worldwide.Transitionfromsteatosistonon-alcoholicsteatohepatitis(NASH)isakeyissueinNAFLD.

Observations in patientswithobstructive sleep apnea syndrome (OSAS),which is characterizedby

occurrenceof intermittenthypoxia (IH),suggest thathypoxiacontributestodevelopNASH.Among

othermechanisms,releaseofextracellularvesicles(EV)byinjuredhepatocyteshasbeenimplicated

inNAFLDprogression.Aim: In this thesiswe aimed to investigate the roleof hypoxia condition in

modulation of steatosis and liver injury in both in vitro and in vivo models of NAFLD. Also we

evaluated the cellular crosstalk between hypoxic and steatotic hepatocytes and non-parenchymal

cellsthroughEV.Methods:PrimaryrathepatocytesandhepatomacelllineHepG2treatedwithfree

fatty acids (FFA) were subjected to chemically induced hypoxia (CH) using the hypoxia-inducible

factor-1alpha (HIF-1α)stabilizercobaltchloride (CoCl2).Triglyceride (TG)content,oxidativestress,

cell death rates, pro-inflammatory and pro-fibrotic cytokines, inflammasome components gene

expressionandproteinlevelsofcleavedcaspase-1wereassessed.Also,Kupffercells(KC)andhuman

stellate cells (LX-2) were treated with conditioned medium (CM) and EV from hypoxic fat-laden

hepatocytes. The choline deficient L-amino acid diet (CDAA)-fed mice model used to assess the

effects of IH on experimental NAFLD. Results: Hepatocytes exposed to FFA and CoCl2 exhibited

increased TG content and higher cell death rates aswell as increased, oxidative stress andmRNA

levelsofpro-inflammatory,pro-fibroticcytokinesandinflammasome-componentscomparedtonon-

treatedhepatocytes.Protein levelsof cleaved caspase-1 increased inCH-exposedhepatocytesand

from EV-hepatocytes. CM and EV from hypoxic fat-laden hepatic cells evoked a pro-inflammatory

and pro-fibrotic phenotype in KC and LX-2 respectively. Livers from CDAA-fedmice exposed to IH

exhibitedincreasedofsteatosis,portalinflammation,fibrosis,mRNAlevelsofpro-inflammatory,pro-

fibrotic and inflammasome genes as well as increased levels of cleaved caspase-1 that correlated

with an increase of circulating EV-caspase-1.Conclusion: Our findings in both in vivo and in vitro

models of NAFLD/NASH indicate that hypoxia may increase liver injury and promote disease

progression through amplification of inflammatory and fibrotic signals including

inflammasome/caspase-1 activation. Hypoxia also promotes the release of EV from hepatocytes

contributing to cellular crosstalkwith non-parenchymal cells by EV-caspase-1-relatedmechanisms.

TheseresultssuggestEVandtheircontent(caspase-1)asapotentialnovelbiomarkerinNAFLDand

OSAS. Further characterization of EV released under hypoxic conditions will allow the detailed

delineationof their role in thepromotionof steatosis, inflammation and fibrosis in the context of

NAFLDandOSAS.

105

Resumen

Antecedentes: El hígado graso no alcohólico (HGNA) es actualmente la enfermedad hepáticamás

prevalente en todo elmundo. Losmecanismos que subyacen a la transición de la esteatosis a la

esteatohepatitis no alcohólica (EHNA) no está del todo comprendida. Observaciones clínicas en

pacientesconelsíndromedelaapneaobstructivadelsueño(SAOS),caracterizadapor lapresencia

de hipoxia intermitente (HI), sugieren que esta condición contribuye al desarrollo de EHNA.

Recientemente, el rol de las vesículas extracelulares (EV) desde hepatocitos esteatóticos ha sido

estudiado en la progresión del daño en el HGNA. Objetivo general: Investigar si modelos

experimentalesdeEHNAsonmássusceptiblesalosefectosdehipoxia,ysitalcondiciónpromueveel

aumento de EV que señalizan a células no parenquimatosas. Métodos: Cultivo primario de

hepatocitosyuna líneacelularHepG2fuerontratadosconácidosgrasos libres(FFA)ysometidosa

hipoxia inducidaquímicamente (CH) usando clorurode cobalto (CoCl2). Se evaluóel contenidode

triglicéridos (TG), el estrés oxidativo, muerte celular, la expression génica de citoquinas pro-

inflamatorias,pro-fibróticas,ydeloscomponentesdelinflamasoma,incluídalacaspasa-1escindida.

Las células de Kupffer (KC) y las células estrelladas humanas (LX-2) se trataron con medio

condicionado (CM) y con EV aisladas de hepatocitos. El modelo de ratones alimentados con L-

aminoácidodefinidocondeficienciadecolina(CDAA)seusóparaevaluarlosefectosdeIHenHGNA

experimental.Resultados:LoshepatocitosexpuestosaFFAyCoCl2exhibieronunmayorcontenido

de TG, muerte celular, estrés oxidativo y un aumento en los niveles de la expression génica de

citoquinas proinflamatorias, pro-fibróticas y de los componentes de inflamasoma en comparación

con loshepatocitosnotratados.Losnivelesdecaspasa-1escindidaaumentaronen loshepatocitos

expuestos a CH, así comoen las EV de los hepatocitos hipóxicos y estetaóticos. El CM y elmayor

número de EV de las células hepáticas hipóxicas tratadas con FFA promovieron un fenotipo pro-

inflamatorio ypro fibróticoen las KC y LX-2 respectivamente. Loshígadosde ratones alimentados

conCDAAexpuestosaIHmostraronunaumentoenlaesteatosis,enlainflamaciónyfibrosisquese

correlacionó con un aumento de las EV circulantes con cargo de caspasa-1.Conclusión: Nuestros

hallazgosindicanquelosmodelosexperimentalesdeHGNAsonmássusceptiblesalosefectosdela

hipoxiaquesecorrelacionanconelaumentodelasEV.Lahipoxiapromovióseñalesinflamatoriasy

fibróticas, incluida la activación del inflamasoma/caspasa-1 en hepatocitos cargados de grasa y en

nuestromodeloinvivodeEHNA,contribuyendoaladiafoníacelularconcélulasnoparenquimatosas

por EV-caspasa-1. Estos resultadosproponena las EV, y su contenido (caspasa-1), comounnuevo

biomarcadorpotencialenHGNAySAOS.

106

Nederlandsesamenvatting

Achtergrond:Niet-alcoholische leververvetting (NAFLD)wordtwereldwijdbeschouwdalsdemeest

voorkomende leverziekte. Overgang van steatose naar niet-alcoholische steatohepatitis (NASH) is

een belangrijk probleem bij NAFLD. Waarnemingen bij patiënten met obstructief

slaapapneusyndroom(OSAS),datwordtgekenmerktdoorhetoptredenvanintermitterendehypoxie

(IH),suggererendathypoxiebijdraagtaandeontwikkelingvanNASH.Naastanderemechanismenis

de afgifte van extracellulaire blaasjes (EV) door beschadigde hepatocyten betrokken bij NAFLD-

progressie. Doel: In dit proefschrift wilden we de rol van hypoxie bij modulatie van steatose en

leverbeschadiging in zowel invitroals invivomodellenvanNAFLDonderzoeken.Ookevalueerden

wedecellulairesamenwerkingtussenhypoxischeensteatotischehepatocytenenniet-parenchymale

cellenviaEV.Methoden:PrimairerattenhepatocytenenhepatoomcellijnHepG2behandeldmetvrije

vetzuren (FFA)werdenonderworpenaan chemisch geïnduceerdehypoxie (CH)metbehulp vande

hypoxie-induceerbare factor-1 alfa (HIF-1α) stabilisator kobaltchloride (CoCl2). Triglyceride (TG) -

gehalte, oxidatieve stress, celsterftecijfers, pro-inflammatoire en pro-fibrotische cytokines,

genexpressie van ontstekingscomponenten en eiwitniveaus van gesplitst caspase-1 werden

beoordeeld. Ook werden Kupffer-cellen (KC) en humane stellaatcellen (LX-2) behandeld met

geconditioneerdmedium(CM)enEVvanmethypoxischevetgeladenhepatocyten.Hetmetcholine-

deficiënteL-aminozuurdieet(CDAA)gevoedemuizenmodeldatwerdgebruiktomdeeffectenvanIH

op experimentele NAFLD te beoordelen. Resultaten:Hepatocyten blootgesteld aan FFA en CoCl2

vertoonden een verhoogd TG-gehalte en hogere celsterftecijfers, evenals verhoogde oxidatieve

stress en mRNA-niveaus van pro-inflammatoire, pro-fibrotische cytokines en

inflammasoomcomponenten in vergelijking met niet-behandelde hepatocyten. Eiwitniveaus van

gesplitst caspase-1namen toe inCH-blootgesteldehepatocytenenvanEV-hepatocyten.CMenEV

van met hypoxisch vet geladen levercellen wekten respectievelijk een pro-inflammatoire en pro-

fibrotischefenotypeopinKCenLX-2.LeversvanCDAA-gevoedemuizendiewarenblootgesteldaan

IH vertoonden verhoogde steatose, portale ontsteking, fibrose, mRNA-niveaus van pro-

inflammatoire, pro-fibrotische en inflammasoomgenen, evenals verhoogde niveaus van gesplitst

caspase-1 die correleerden met een toename van circulerende EV-caspase-1. Conclusie: Onze

bevindingen in zowel in vivo als in vitro modellen van NAFLD/NASH geven aan dat hypoxie

leverbeschadiging kan vergroten en ziekteprogressie kan bevorderen door versterking van

inflammatoire en fibrotische signalen, waaronder de activering van inflammasoom/caspase-1.

HypoxiebevordertookdeafgiftevanEVuithepatocytendiebijdragenaancellulaireoverspraakmet

niet-parenchymalecellendoorEV-caspase-1-gerelateerdemechanismen.Dezeresultatensuggereren

EV en hun inhoud (caspase-1) als een potentiële nieuwe biomarker in NAFLD en OSAS. Verdere

107

karakterisering van EV die vrijkomt onder hypoxische omstandigheden zal de gedetailleerde

afbakening van hun rol bij de bevordering van steatose, ontsteking en fibrose in de context van

NAFLDenOSASmogelijkmaken.

108

ACKNOWLEDGEMENTS

ThisdoctoralthesiswassupportedbytheChileangovernmentthroughFondoNacionaldeDesarrollo

Científico y Tecnológico (FONDECYT 1150327 and 1119145) and PhD fellowship from Comisión

NacionalparalaInvestigaciónenCienciayTecnología(CONICYT)andVicerrectorateofResearchand

School of Medicine, PUC. Also, the support from the Abel Tasman Talent Program (ATTP) of the

GraduateSchoolofMedicalSciencesandtheUniversityMedicalCenterGroningen(UMCG)fromThe

Netherlandsisgratefullyacknowledged.

109

ABBREVIATIONS

OSAS:obstructivesleepapneasyndrome

IH:intermittenthypoxia

NAFLD:non-alcoholicfattyliverdisease

NAFL:non-alcoholicfattyliver

NASH:non-alcoholicsteatohepatitis

CDAA:choline-deficientaminoacid-defined

NLRP3:NOD-likereceptorPyrinDomainContaining3

GSDMD:gasderminD

KC:Kupffercells

TG:triglyceride

IL:interleukin

TNF-α:tumornecrosisfactor-alpha

HIF-1α:hypoxiainduciblefactor1alpha

EV:extracellularvesicles

FBS:fetalbovineserum

FFA:freefattyacids

CM:conditionedmedium

AUF:Arbitraryunitsoffluorescence

PBS:phosphate-bufferedsaline

LDH:Lactatedehydrogenase

NTA:nanoparticletrackinganalysis

ASC:Apoptosis-AssociatedSpeck-LikeProteinContainingCARD

110

Gene Primers SpecieNLRP3 Sense 5`-GTGCGTGGGACTGAAGCAT-3` Rattusnorvegicus

Anti-sense 5`-CTGACAACACGCAGATGTGAGA-3`Probe 5`-AGCAGCTGACCAACCAGAGTTTCTGCA-3`

ASC Sense 5`-AGCTGTGGCTACTGCAACCA-3` RattusnorvegicusAnti-sense 5`-GACCCTGGCAATGAGTGCTT-3`Probe 5`-ACAAAATGTTCTGTTCTGGCTGTGCCCT-3`

Caspase-1 Sense 5`-CGGAGTTTCCTACTGAATCTTTTAACA-3 RattusnorvegicusAnti-sense 5`-GAAAGACAAGCCCAAGGTTATCA-3`Probe 5`-ACCACTCCTTGTTTCTCTCCACGGCA-3`

IL-1β Sense 5’-ACCCTGCAGCTGGAGAGTGT-3’ RattusnorvegicusAnti-sense 5`-TTGACTTCTATCTTGTTGAAGACAAACC-3`Probe 5`-CCCAAACAATACCCAAAGAAGAAGATGGAAAAG-3`

IL-6 Sense 5`-CCGGAGAGGAGACTTCACAGA-3 RattusnorvegicusAnti-sense 5`-AGAATTGCCATTGCACAACTCTT-3`Probe 5`-ACCACTTCACAAGTCGGAGGCTTAATTACA-3`

TNF-α Sense 5`-GTAGCCCACGTCGTAGCAAAC-3` RattusnorvegicusAnti-sense 5`-AGTTGGTTGTCTTTGAGATCCATG-3`Probe 5`-CGCTGGCTCAGCCACTCCAGC-3`

iNOS Sense 5`-CTATCTCCATTCTACTACTACCAGATCGA-3` RattusnorvegicusAnti-sense 5`-CCTGGGCCTCAGCTTCTCAT-3`Probe 5`-CCCTGGAAGACCCACATCTGGCAG-3`

IL-10 Sense 5`-TGCAGGACTTTAAGGGTTACTTGG-3` RattusnorvegicusAnti-sense 5`-CAGGGAATTCAAATGCTCCTTG-3`Probe 5`-TCTCTGCCTGGGGCATCACTTCTACCA-3`

Arg-1 Sense 5`-AGCTGGGAATTGGCAAAGTG-3` RattusnorvegicusAnti-sense 5`-TCCAGTCCATCAACATCAAAACTC-3`Probe 5`-AATGGGCCTTTTCTTCCTTCCCAGCAG-3`

18S Sense 5`-CGGCTACCACATCCAAGGA-3` RattusnorvegicusAnti-sense 5`-CCAATTACAGGGCCTCGAAA-3`Probe 5`-CGCGCAAATTACCCACTCCCGA-3`

IL-1β Sense 5`-ACAGATGAAGTGCTCCTTCCA-3`Homo SapiensAnti-sense 5`-GTCGGAGATTCGTAGCTGGAT-3`Probe 5`-CTCTGCCCTCTGGATGGCGG-3`

TNF-α Sense 5`-CCCTGGTATGAGCCCATCTATC-3`Homo SapiensAnti-sense 5`-AAAGTAGACCTGCCCAGACTCG-3`Probe 5`-ATCAATCGGCCCGACTATCTCGACTTT-3`

NLRP3 Sense 5`-GGGATTCGAAACACGTGCAT-3` Homo SapiensAnti-sense 5`-CAGGAGAGACCTTTATGAGAAAGCA-3`Probe 5`-ATCTGAACCCCACTTCGGCTCATCTCTTT-3`

TGF-β1 Sense 5`-GGCCCTGCCCCTACATTT-3’ Homo SapiensAnti-sense 5`-CCGGGTTATGCTGGTTGTACA-3`Probe 5`-ACACGCAGTACAGCAAGGTCCTGGC-3`

CTGF Sense 5`-TGTGTGACGAGCCCAAGGA-3` Homo SapiensAnti-sense 5`-TCTGGGCCAAACGTGTCTTC-3`Probe 5`-CCTGCCCTCGCGGCTTACCG-3`

α-SMA Sense 5`-GGGACGACATGGAAAAGATCTG-3` Homo SapiensAnti-sense 5`-CAGGGTGGGATGCTCTTCA-3`Probe 5`-CACTCTTTCTACAATGAGCTTCGTGTTGCCC-3

Collagen-I Sense 5`-GGCCCAGAAGAACTGGTACATC-3` Homo SapiensAnti-sense 5`-CCGCCATACTCGAACTGGAA-3Probe 5`-CCCCAAGGACAAGAGGCATGTCTG-3`

18S Sense 5`-CGGCTACCACATCCAAGGA-3` Homo SapiensAnti-sense 5`-CCAATTACAGGGCCTCGAAA-3`Probe 5`-CGCGCAAATTACCCACTCCCGA-3

IFN-γ Sense 5`-ACTGTCGCCAGCAGCTAAAA-3` Homo SapiensAnti-sense 5`-TATTGCAGGCAGGACAACCA-3`

IL-18 Sense 5`-AAGATGGCTGCTGAACCAGT-3` Homo SapiensAnti-sense 5`-GAGGCCGATTTCCTTGGTCA-3

TIMP-1 Sense 5`-CTTCTGGCATCCTGTTGTTG-3`Homo SapiensAnti-sense 5`-GGTATAAGGTGGTCTGGTTG-3`

TGF-β1 Sense 5`-CTCCCGTGGCTTCTAGTGC-3` Mus musculusAnti-sense 5`-GCCTTAGTTTGGACAGGATCTG-3`

CTGF Sense 5`-GGGCCTCTTCTGCGATTTC-3` Mus musculusAnti-sense 5`-ATCCAGGCAAGTGCATTGGTA-3`

Collagen-I Sense 5`-GCTCCTCTTAGGGGCCACT-3` Mus musculusAnti-sense 5`-CCACGTCTCACCATTGGGG-3`

α-SMA Sense 5`-GTCCCAGACATCAGGGAGTAA-3` Mus musculusAnti-sense 5`-TCGGATACTTCAGCGTCAGGA-3`

TIMP-1 Sense 5`-GCAACTCGGACCTGGTCATAA-3` Mus musculusAnti-sense 5`-ACTGTTCCTGAACTCAACT-3`

HIF-1α Sense 5`-AGGATGAGTTCTGAACGTCGAAA-3` Mus musculusAnti-sense 5`-CTGTCTAGACCACCGGCATC-3`

TNF-α Sense 5`-CCCTCACACTCAGATCATCTTCT-3`Mus musculusAnti-sense 5`-GCTACGACGTGGGCTACAG-3`

IL-1β Sense 5`-ACTGTTCCTGAACTCAACT-3` Mus musculusAnti-sense 5`-ATCTTTTGGGGTCCGTCAACT-3`

IL-18 Sense 5`-GACTCTTGCGTCAACTTCAAGG-3` Mus musculusAnti-sense 5`-CAGGCTGTCTTTTGTCAACGA-3`

NLRP3 Sense 5`-ATTACCCGCCCGAGAAAGG-3` Mus musculusAnti-sense 5`-TCGCAGCAAAGATCCACACAG-3`

Caspase-1 Sense 5`-ACAAGGCACGGGACCTATG-3` Mus musculusAnti-sense 5`-TCCCAGTCAGTCCTGGAAATG-3`

IL-6 Sense 5`-TAGTCCTTCCTACCCCAATTTCC-3` Mus musculusAnti-sense 5`-TTGGTCCTTAGCCACTCCTTC-3`

IFN-γ Sense 5`-ATGAACGCTACACACTGCATC-3` Mus musculusAnti-sense 5`-CCATCCTTTTGCCAGTTCCTC-3`

MCP-1 Sense 5`-TTAAAAACCTGGATCGGAACCAA-3` Mus musculusAnti-sense 5`-GCATTAGCTTCAGATTTACGGGT-3`

18S Sense 5’-CGGCTACCACATCCAAGGA-3’ Mus musculusAnti-sense 5’-CCAATTACAGGGCCTCGAAA-3’

SUPPLEMENTARYMATERIAL

TableS1:SequencesofprimersandprobesusedforquantitativerealtimePCR

111

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