The effect of ABCG1 deficiency on atherosclerotic lesion development in LDL receptor knockout mice depends on the stage of atherogenesis

The effect of ABCG1 deficiency on atherosclerotic lesion development in LDL receptor knockout mice depends on the stage of atherogenesis

Meurs, I.; Lammers, B.; Zhao, Y.; Out, R.; Hildebrand, R.B.; Hoekstra, M.; ... ; Eck, M. van

Citation

Meurs, I., Lammers, B., Zhao, Y., Out, R., Hildebrand, R. B., Hoekstra, M., … Eck, M. van.

(2012). The effect of ABCG1 deficiency on atherosclerotic lesion development in LDL receptor knockout mice depends on the stage of atherogenesis. Atherosclerosis, 221(1), 41-47. doi:10.1016/j.atherosclerosis.2011.11.024

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License: Leiden University Non-exclusive license Downloaded from: https://hdl.handle.net/1887/46569

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Atherosclerosis

j ou rn a l h o m e pa g e:w w w . e l s e v i e r . c o m / l o c a t e / a t h e r o s c l e r o s i s

The effect of ABCG1 deficiency on atherosclerotic lesion development in LDL receptor knockout mice depends on the stage of atherogenesis

IllianaMeurs^∗,BartLammers,YingZhao,RuudOut,ReeniB.Hildebrand,MennoHoekstra, TheoJ.C.VanBerkel,MirandaVanEck

DivisionofBiopharmaceutics,Leiden/AmsterdamCenterforDrugResearch,GorlaeusLaboratories,LeidenUniversity,Leiden,TheNetherlands

a r t i c l e i n f o

Articlehistory:

Received14December2010

Receivedinrevisedform25October2011 Accepted17November2011

Available online 7 December 2011

Keywords:

ABCG1deficiency Atherosclerosis Atherogenesis WTD Mice

a b s t r a c t

Objective:AsABCG1playsaroleincholesterolefflux,macrophageABCG1expressionhasbeensuggested toprotectagainstatherosclerosis.However,weandothersobservedvaryingeffectsofABCG1deficiency onatheroscleroticlesionsize.TheobjectiveofthisstudywastodefinetheeffectofABCG1deficiency duringatheroscleroticlesionprogressioninLDLreceptorknockout(LDLr^−/−)mice.

Methodsandresults:ABCG1^−/−/LDLr^−/−andABCG1^+/+/LDLr^−/−littermateswerefedaWestern-typediet for10and12weeksinordertostudytheeffectofABCG1 deficiencyintheexponentialphaseof atheroscleroticlesionformation.At10weeksofdietfeeding,asignificant1.5-foldincreaseinearly atheroscleroticlesionsize(130±12×10³␮m²)wasobservedinABCG1^−/−/LDLr^−/−micecomparedto ABCG1^+/+/LDLr^−/−mice(88±11×10³␮m²;p<0.05).Interestingly,inmoreadvancedlesions,inducedby 12weeksofWTDfeeding,ABCG1^−/−/LDLr^−/−miceshowedasignificant1.7-folddecreaseinatheroscle- roticlesionsize(160±20×10³␮m²vs273±19×10³␮m²incontrolmice;p<0.01),indicatingthatin theABCG1^−/−/LDLr^−/−miceprogressionoflesionformationisretardedascomparedtoABCG1^+/+/LDLr^−/−

mice.Inaddition,correlationanalysisperformedon7independentpublishedstudiesandthecurrent studyconfirmedthatABCG1isatheroprotectiveinearlylesions,whilethedevelopmentofadvanced lesionsisstimulated.

Conclusions:ItappearsthattheeffectofABCG1deficiency onlesiondevelopmentinLDLr^−/− mice dependsonthestageofatherogenesis,wherebytheabsenceofABCG1leadstoincreasedlesionsat sizes<167×10³␮m²whileinmoreadvancedstagesofatherosclerosisenhancedapoptosisand/orcom- pensatorymechanismsleadtoretardedlesionprogression.

© 2011 Elsevier Ireland Ltd.

1. Introduction

Reversecholesteroltransport (RCT),defined asthetransport ofexcesscholesterolfromperipheraltissuesbacktotheliverfor biliaryexcretion,playsanimportantprotectiveroleinthedevelop- mentofatherosclerosis[1,2].Previously,theATP-bindingcassette (ABC) transporter A1 has been reported to play an important role in theprevention of atherosclerosis by facilitating choles- terolandphospholipideffluxfrommacrophagestolipid-pooror lipid-freeapolipoproteinAI(apoAI)[3–6].SimilartoABCA1,ABCG1 hasbeenimplicatedinmacrophagelipidhomeostasisbyactively effluxingcellularcholesteroltomatureHDL[7,8].Ascholesterol effluxfrommacrophagesis animportantprotectivemechanism to prevent excessive cellular lipid accumulation, macrophage

∗ Correspondingauthorat:DivisionofBiopharmaceutics,GorlaeusLaboratories, Einsteinweg55,2333CCLeiden,TheNetherlands.Tel.:+31715276238;

fax:+31715276032.

E-mailaddress:m.eck@lacdr.leidenuniv.nl(M.VanEck).

ABCG1expressionwasexpectedtoprotectagainstatherosclero- sis.However,independentgroupshaveshownthatABCG1might be pro-atherogenic as well as anti-atherogenic. Previously, we reportedthatbothtotalbodyandmacrophageABCG1deficiency ledeithertoasignificantlyincreasedsusceptibilitytoatheroscle- rotic lesion development [9,10] or to no change in lesion size [11,12].Incontrast,thegroupofEdwardsetal.[13,14]andTalletal.

[15] reported decreased atherosclerosis in LDL receptor knock- out (LDLr^−/−) mice transplanted with ABCG1^−/− bone marrow cells,whichwasexplainedbyacceleratedapoptosisofABCG1^−/−

macrophagesorcompensatoryupregulationofABCA1expression andapoEsecretioninmacrophageslackingABCG1.Thus,therole ofmacrophageABCG1inthedevelopmentofatherosclerosisstill remainsuncertain.

TheaimofthisstudywastoassesstheeffectofABCG1defi- ciencyondifferentstagesofatheroscleroticlesiondevelopment andespecially duringtheexponentialphaseoflesionformation in ordertounravelthemechanism bywhich ABCG1deficiency affects atherogenesis.Uponatherogenicdiet feeding,total body

0021-9150/© 2011 Elsevier Ireland Ltd.

doi:10.1016/j.atherosclerosis.2011.11.024

Open access under the Elsevier OA license.

Open access under the Elsevier OA license.

42 I.Meursetal./Atherosclerosis221 (2012) 41–47

ABCG1^−/− mice develop only modest atherosclerotic lesions, therefore, we generated ABCG1/LDLr double knockout (ABCG1^−/−/LDLr^−/−)micetoperformthislesionstagedependent study.

2. Materialsandmethods

Fordetailedmethodology,pleaserefertothedatasupplement.

ABCG1^+/− mice, obtained from Deltagen Inc., San Carlos, CA, were cross-bred with single LDLr^−/− mice to generate ABCG1^−/−/LDLr^−/−miceonaC57Bl/6background.

To induce atherosclerosis development, the mice were fed Western-type diet (WTD), containing 15% (w/w) cocoa butter and 0.25% (w/w) cholesterol (Diet W, Ab Diets, Woerden, The Netherlands)for10and12weeksafterwhichthemicewereeuth- anizedandatheroscleroticlesiondevelopmentwasquantifiedin oilredO-stainedcryosections. Furthermore,bone marrowcells wereisolatedfromABCG1^+/+/LDLr^−/−andABCG1^−/−/LDLr^−/− and differentiatedintobonemarrow-derivedmacrophagestoevaluate macrophagecholesterolefflux.

3. Results

3.1. EffectofABCG1deficiencyonserumlipidlevelsandlipid homeostasisintissues

Onregularchowdiet,containing4.3%fatandnoaddedcholes- terol,no significantdifferencein total serum cholesterol levels wereobservedbetweenABCG1^+/+/LDLr^−/−andABCG1^−/−/LDLr^−/−

mice (Table 1). Fractionation of serum lipoproteins, however, showed a moderate shift of HDL cholesterol to the LDL and VLDL fraction in ABCG1^−/−/LDLr^−/− mice (HDL: 75±4 com- paredto90±3mg/dLforABCG1^+/+/LDLr^−/−mice,p<0.05;VLDL:

36±3 compared to 19±3mg/dL, p<0.001; and LDL: 123±5 comparedto108±4mg/dL for ABCG1^+/+/LDLr^−/− mice,p<0.05) (Table1 and Fig.1A).Toinduceatheroscleroticlesiondevelop- ment,ABCG1^−/−/LDLr^−/−andcontrolmicewerefedaWTDfor10 and12weeks,whichinducedapproximatelya5-foldincreasein serumcholesterol concentrationsin bothABCG1^+/+/LDLr^−/− and ABCG1^−/−/LDLr^−/− mice.Both after10 weeks and 12 weekson theWTD,totalserumcholesterollevelsdidnotdifferbetweenthe groups.LipoproteinprofilesofmicefedtheWTDfor10and 12 weekswereessentiallyidentical.Therefore,therepresentative12 weeksWTDprofileofABCG1^−/−/LDLr^−/−andcontrolmiceonWTD isshowninFig.1A.AmoderateincreaseinVLDL(∼25%)andLDL (∼23%)cholesterollevelswasobservedinABCG1^−/−/LDLr^−/−mice comparedtocontrolanimals,whichonlyreachedsignificancefor LDLafter12weeksWTDfeeding(p<0.01)(Table1).Thisincrease inVLDLandLDLwasassociatedwithamoderatenon-significant decreaseinHDL(∼16%)levelsinABCG1^−/−/LDLr^−/−.Furthermore, duringthecourseoftheexperiment,theweightgain curvedid notshowsignificantdifferencesbetweenABCG1^−/−/LDLr^−/− and ABCG1^+/+/LDLr^−/−micebothafter10or12weeksonWTD(data notshown).

ABCG1deficiencyhasbeenshowntocoincidewithincreased secretionofapoEbymacrophagesandelevatedserumapoElev- els[15].ImmunoblottingforapoEwasperformedtoanalyzethe associationof ABCG1 deficiency withserum apoE levelsof the LDLr^−/−micefedWTDfor10and12weeks.Nosignificanteffectof ABCG1deficiencywasobservedonserumapoElevelsbetweenthe ABCG1^−/−/LDLr^−/−andABCG1^+/+/LDLr^−/−micefedWTD(Fig.1B).

Inaddition,ABCG1deficiencyinmacrophageshasbeenshowntobe correlatedwithaninductionofABCA1expression[15].However, immunohistochemicalstainingofaorticrootcryosectionsshowed noapparentincreaseinABCA1expressioninmacrophageslocated

inatheroscleroticlesionsofABCG1^−/−/LDLr^−/−mice,eitherafter10 or12weeksWTDfeeding(datanotshown).

AbnormallungmorphologywasobservedinABCG1^−/−/LDLr^−/−

micecompared withcontrolmice.Bothafter 10and 12 weeks of diet feeding, ABCG1 deficiency resulted in accumulation of large amounts of lipids in the subpleuralregions of the lungs (SupplementalFig.IA).Inaddition,spleensofABCG1^−/−/LDLr^−/−

showedlipidaccumulationintheredpulpregions,whilenoaccu- mulationwasobservedinspleensofcontrolmice(Supplemental Fig.IB).

3.2. EffectofABCG1disruptiononatheroscleroticlesion formation

TodefinetheroleofABCG1intheexponentialphaseofathero- genesis,atheroscleroticlesiondevelopmentwasanalyzedinthe aorticrootofABCG1^+/+/LDLr^−/−andABCG1^−/−/LDLr^−/−miceafter 10 and 12 weeks of WTD feeding.Representative photomicro- graphsoftheaorticrootofcontrolmiceand micedeficientfor ABCG1areshowninFig.2A.After10weeksWTDfeeding,asignif- icant1.5-foldincreaseinatheroscleroticlesionsizewasobserved intheaorticrootofABCG1^−/−/LDLr^−/−mice(130±12×10³␮m² [n=8]comparedto88±11× 10³␮m²[n=7]forABCG1^+/+/LDLr^−/−

mice; p<0.05). In vitro studies using bone marrow-derived macrophages of ABCG1^+/+/LDLr^−/− and ABCG1^−/−/LDLr^−/− mice showed that disruptionof ABCG1 resultsin a 15% decrease in cholesteroleffluxtoHDL(p<0.001),whereasthecholesterolefflux toApoAI wasunaffected (Fig. 2B). Additional2 weeks of WTD feeding resulted in rapid progression of atherosclerotic lesion development in control mice (3.1-fold) (Fig. 2C). Interestingly, atherogenesisintheABCG1-deficientmiceappearedtobeatten- uatedfrom10till12weeksandonlya1.2-foldincreaseinlesion sizeisnoticedoverthisperiod.Asaresult,ABCG1-deficientmice showeda1.7-foldsmalleratheroscleroticlesionsascomparedto control mice after 12 weeks WTD feeding(160±20×10³␮m² [n=8] compared to 273±19×10³␮m² [n=9]; p<0.01, respec- tively)(Fig.2A).

Disruption of ABCG1 in LDLr^−/− mice also affected the composition of atherosclerotic lesions. Immunostaining for macrophages showed less staining in atherosclerotic lesions of ABCG1^−/−/LDLr^−/− mice fed WTD for 10 weeks (65±2% of atherosclerotic area compared with 81±4% in control mice;

p<0.01)(Fig.3A).Thelowermacrophagecontent,coincidedwith analmostsignificantlylargerpercentualnecroticcoreareaafter 10 weeks on WTD (22±5% of atherosclerotic area compared with 8±3% in control mice; p=0.06). Additional 2 weeks of WTDfeedingresultedinanincreaseinabsolutemacrophagearea and necroticcorearea(Fig.3A).However,nosignificantdiffer- encescouldbeobservedbetweenlesionsofABCG1^+/+/LDLr^−/−and ABCG1^−/−/LDLr^−/−mice.Furthermore,MassonTrichrome-staining showednosignificantdifferencesincollagenaccumulationinthe atheroscleroticplaques betweenthe ABCG1-deficient mice and controlmiceafter10weeksand12weeksofWTDfeeding(data notshown).

Asinvitrostudieshaverecentlydemonstratedthatmacrophage ABCG1deficiencyisassociatedwithincreasedsusceptibilitytoapo- ptosisinresponsetothealteredcellularlipidhomeostasis[13], weexaminedtheapoptoticmacrophagecontentofthelesionsby TUNELstaining.LesionsofABCG1-deficientmicefedtheWTDfor 10weeksshowednodifferencesinTUNEL-positivemacrophages comparedwithlesionsofcontrolmice(Fig.3B).After12weeksof WTDfeeding,a2.5-foldincreaseinTUNEL-positivemacrophages was observed in lesions of ABCG1^−/−/LDLr^−/− mice compared withABCG1^+/+/LDLr^−/−animals(p<0.05,n=7–8).Thedecreasein atheroscleroticlesionsizeobservedinABCG1^−/−/LDLr^−/−micefed

Table1

SerumlipidlevelsinABCG1^−/−/LDLr^−/−andcontrolmiceonchowandWTD.

Mice Time(weeks) Diet Freecholesterol(mg/dL) Totalcholesterol(mg/dL) VLDL-C(mg/dL) LDL-C(mg/dL) HDL-C(mg/dL)

ABCG1^+/+/LDLr^−/− 0 Chow 96±4 230±12 19±3 108±4 90±3

10 WTD 289±32 1030±36 361±82 369±42 58±4

12 WTD 289±16 1010±82 441±53 374±24 65±6

ABCG1^−/−/LDLr^−/− 0 Chow 91±4 233±9 36±3^*** 123±5^* 75±4^*

10 WTD 292±23 1206±107 492±81 451±33 58±6

12 WTD 332±20 1047±111 536±57 467±19^** 54±5

Datarepresentthemeans±SEMof8mice.

Abbreviations:WTD,Western-typediet;VLDL,very-low-densitylipoprotein;LDL,low-densitylipoprotein;HDL,high-densitylipoprotein;C,cholesterol.

*Statisticalsignificanceofp<0.05comparedwithABCG1^+/+/LDLr^−/−mice.

**Statisticalsignificancep<0.01comparedwithABCG1^+/+/LDLr^−/−mice.

***Statisticalsignificancep<0.001comparedwithABCG1^+/+/LDLr^−/−mice.

WTDfor12weeks,mightthereforebearesultofincreasedsus- ceptibilitytoapoptosisofABCG1-deficientmacrophagesinsidethe lesions.

Overall,thesefindingsindicatethattheeffectofABCG1defi- ciency on atherosclerotic lesion development depends on the stageofatherogenesis. ABCG1expressionprotectsagainst early atherosclerotic lesion development, by facilitating cholesterol effluxfrommacrophagestoHDL.In themoreadvanced lesions, however,accumulationofcholesterolduetoimpairedcholesterol effluxfromABCG1-deficientmacrophages,eventually,willleadto increasedmacrophageapoptosisandareducedfurtherprogression ofatherogenesis.

4. Discussion

Severalpathwaysareinvolvedintheeffluxofcholesterolfrom macrophages,includingaqueousdiffusion,SR-BImediatedcholes- terolefflux,effluxdependentonmacrophageapoEsecretion,and

activecholesteroleffluxmediatedbyABCA1[16–18].AlsoABCG1 has been implicated in cellular lipid homeostasis by its prop- ertytoactivelyeffluxcholesteroltomatureHDL[8,19].Studies usinggeneticallyengineeredmicehaveestablishedthephysiolog- icalimportanceofABCG1.TargeteddisruptionofABCG1inmice resultedinage-relatedprogressivepulmonarylipidosiswhenfeda regularchowdiet[20–22].Inaddition,overexpressionofABCG1 protected against diet-induced lipid depositionwithin multiple tissues[8].ThesefindingsimplyacriticalroleforABCG1inmain- tainingnormallipidmetabolisminthelung,therebypreventing inflammatoryresponsestriggeredbymassivecholesterol and/or cholesterolmetaboliteaccumulation.

Although thecritical role of ABCG1 in lung lipid homeosta- sis is clearly established, contradictory findings on the role of macrophageABCG1 inthedevelopmentof atherosclerosishave beenreportedbydifferentgroups/laboratories[9–15,23–25].

TransgenicmiceoverexpressinghumanABCG1showedeither no effect [24] or increased atherosclerosis [25]. In contrast,

Fig.1.TheeffectofABCG1deficiencyonserumcholesteroldistributionandapoElevelsinLDLr^−/−mice.(A)Bloodsamplesweredrawnafteranovernightfastingperiod whilefeedingaregularchowdietandafter12weeksonWTD.SerafromindividualmicewereloadedontoaSuperose6column,andfractionswerecollected.Fractions2–5 representVLDL,fractions6–14representLDL,andfractions15–20representHDL.ThedistributionofcholesteroloverthedifferentlipoproteinsinABCG1^+/+/LDLr^−/−()and ABCG1^−/−/LDLr^−/−()miceisshown.Valuesrepresentthemean±SEMof8micepergroup.(B)ArepresentativeimmunoblotofapoEinserumofABCG1^+/+/LDLr^−/−and ABCG1^−/−/LDLr^−/−miceafter12weeksofWTDfeeding.

44 I.Meursetal./Atherosclerosis221 (2012) 41–47

Fig.2.TheeffectofABCG1deficiencyonatheroscleroticlesionformationandcholesteroleffluxinLDLr^−/−mice.(A)Atheroscleroticlesionformationwasdeterminedinthe aorticrootatthelevelofthetricuspidvalvesofABCG1^+/+/LDLr^−/−andABCG1^−/−/LDLr^−/−micefedaWTDfor10and12weeks(separatedbythedottedline).Meanlesion areaofeachindividualmouseisshown.Thehorizontaldottedlinesrepresentthemeansofeachgroupof7–9mice.RepresentativephotomicrographsofoilredO-stained lesionsareshown(magnification50×).(B)CholesteroleffluxtoHDLisimpairedinABCG1-deficientmacrophages.ApoA-I(10␮g/mL)andHDL(50␮g/mL)inducedcellular cholesteroleffluxfrom³H-cholesterol-labeledbonemarrow-derivedmacrophagesofABCG1^+/+/LDLr^−/−andABCG1^−/−/LDLr^−/−mice.BasaleffluxtoBSA(intheabsenceof addedacceptors)hasbeensubtractedfromthedatashown.Valuesrepresentthemean±SEMof4animals.(C)ProgressionofatheroscleroticlesionsofABCG1^+/+/LDLr^−/−and ABCG1^−/−/LDLr^−/−micefrom10to12weeksWTDfeedingisshown.Valuesrepresentthemean±SEMof7–9mice.Statisticallysignificantdifference*p<0.05and***p<0.001 ascomparedtoABCG1^+/+/LDLr^−/−controls.

Westerterpetal.[26]recentlyreportedanatheroprotectiveroleof vascularABCG1,whichislikelyrelatedtoitsroleinthepreserva- tionofendothelialNOsynthaseactivity.Furthermore,independent studies,usingABCG1-deficientmiceorLDLr^−/−micetransplanted withbonemarrowcellsofABCG1-deficientmice,haveshownthat macrophageABCG1mightbeproatherogenic[9,10,12]aswellas antiatherogenic[13–15].Inthepresentstudy,weshowthatABCG1 deletioninLDLr^−/−micecanbothinduceandattenuateatheroscle- roticlesiondevelopment.ABCG1deficiencyledtoasignificant48%

increaseinatheroscleroticlesionsizeafteronly10weeksWestern- typedietfeeding,whileasignificant32%decreaseinlesionsizewas observedafter12weeksWTDfeeding.Thesedataimplythatthe effectofABCG1deficiencyonatheroscleroticlesiondevelopment inLDLr^−/−micedependsonthestageofatherogenesis.

ThereducedatherosclerosisinLDLr^−/−micetransplantedwith ABCG1^−/− bone marrow was suggested to be a result of com- pensatoryinductionofapoEsecretionandABCA1expressionin

ABCG1-deficientmacrophages[15].Inthisstudy,however,both at 10 weeks and 12 weeks of WTD feeding,ABCG1^−/−/LDLr^−/−

mice showed no compensatory increase in serum apoE lev- els,although ABCG1^−/−/LDLr^−/− mice didexhibit a decrease in atherosclerotic lesion development at 12 weeks of WTD feed- ing.These findings arein agreementwithourpreviousstudies showing that apoE mRNA and protein expressions were not affectedupondeletion ofABCG1[10,12].In addition,noappar- entincreaseinABCA1expressionwasobservedinABCG1-deficient macrophagesin atheroscleroticlesionsofLDLr^−/−.Furthermore, accelerated apoptosis was proposed as a mechanism for the reduced atherosclerosis susceptibility of LDLr^−/− mice lacking ABCG1inmacrophages[13,14].Inagreement,inthepresentstudy, themoreadvanced atheroscleroticlesionsofABCG1^−/−/LDLr^−/−

micefedWTDfor12weeksweredecreasedinsizeandshowed a significant increase in TUNEL-positive macrophages as com- pared to control mice. Macrophage apoptosis is an important