Science in medicine

Molecular regulation of HDL metabolism and function: implications for novel therapies
Daniel J. Rader
Institute for Translational Medicine and Therapeutics, Cardiovascular Institute, and Institute for Diabetes, Obesity, and Metabolism, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, USA.

HDL metabolism represents a major target for the development of therapies intended to reduce the risk of atherosclerotic cardiovascular disease. HDL metabolism is complex and involves dissociation of HDL apolipoprotein and HDL cholesterol metabolism. Advances in our understanding of the molecular regulation of HDL metabolism, macrophage cholesterol efflux, and HDL function will lead to a variety of novel therapeutics.
Historical perspective HDLswerefirstcharacterizedbyusingultracentrifugationof plasmatoseparatelipoproteinsofdifferentdensities.Severalstudieswerepublishedinthe1970sreportinganinverseassociation betweenplasmaHDLcholesterol(HDL-C)levelandcoronaryheart disease(1),confirmedsincethenbynumerousadditionalstudies throughouttheworld.HDL-Cwasfirstendorsedasaformalindependentriskfactorforcoronaryheartdiseaseinthe1980sandhas sinceevolvedintooneofthefewtraditionalriskfactorsusedby clinicianstoassesscardiovascularrisk.Theguidelines(2)definea lowHDL-Clevelaslessthan40mg/dlandalsoidentifylowHDL-C asoneofthecorecomponentsofthemetabolicsyndrome. HDL-Clevelsvarywidelyovermorethananorderofmagnitude inhumans.Thisvariationisatleast50%,andasmuchas70%, geneticallydetermined.Onlyahandfulofgeneshavebeenproven tocontributetothegeneticvariationofHDL-Cinhumans,and theydosoprimarilybyinfluencingtherateofHDLcatabolism. Furthermore,theconditionmostcommonlyassociatedwithlow HDL-C,theinsulin-resistantmetabolicsyndrome,isalsoassociatedwithincreasedcatabolismofHDL(3).Thus,HDLcatabolism isatargetfordevelopmentofnewtherapiestoraiseHDL. AnimportantdebateovertheyearshasbeenwhetherlowHDL-C issimplyamarkerofcardiovascularriskoriscausallyassociated withincreasedrisk.WhilelowHDL-Cisoftenseeninassociation withotherconditionsthatalsoincreasecardiovascularrisk,itis widelybelieved,basedinlargepartonanimalstudies,thatHDL candirectlyinhibitatherogenesis.Themostpopularmechanistic explanationhasbeenthatHDLfacilitatesuptakeofperipheral cholesterolanditsreturntotheliverforexcretioninthebileand feces,aconceptfirstintroducedbyGlomsetin1968(4)andsubsequentlytermedreversecholesteroltransport(RCT).LaterRoss andGlomsetsuggestedthatRCTcouldbeaprotectivemechanism
Nonstandard abbreviations used:CE,cholesterylester;CETP,CEtransferprotein; CVD,cardiovasculardisease;EL,endotheliallipase;HDL-C,HDLcholesterol;HL, hepaticlipase;LCAT,lecithin:cholesterolacyltransferase;LDLR,LDLreceptor;LXR, liverXreceptor;PL,phospholipid;PLTP,PLtransferprotein;RCT,reversecholesterol transport;SR-BI,scavengerreceptorclassBI;TG,triglyceride. Conflict of interest:Theauthorhasreceivedresearchsupport,consultingfees,or honorariafromAbbott,AstraZeneca,BoehringerIngelheim,Bristol-MyersSquibb, BruinPharma,GlaxoSmithKline,Johnson&Johnson,KOSPharmaceuticalsInc., Merck,Merck/Schering-Plough,Pfizer,ReliantPharmaceuticals,Sanofi-Aventis, Schering-Plough,TakedaPharmaceuticalsNorthAmerica,andWyeth. Citation for this article:J. Clin. Invest.116:30903100(2006).doi:10.1172/JCI30163.
3090

againstatherosclerosis(5),andMillerandMillersuggestedthat HDLmightprotectagainstatherosclerosisbypromotingRCT (1).WhiletheabilityofHDLtoprotectagainstatherosclerosisby promotingRCTmostlyremainsahypothesis,promotionofRCT isnonethelessaprimarytargetforthedevelopmentofnoveltherapeuticstargetedtowardHDL.Morerecently,avarietyofother functionsofHDLhavebeendescribed,primarilybasedoninvitro assays,includingantiinflammatory,antioxidant,antithrombotic, andnitricoxideinducingmechanisms(6,7).Theirrelevanceto humanphysiologyremainsuncertain.However,thesefindings haveraisedtheimportantquestionofwhetherasubstantialportionoftheprotectiveeffectofHDLmaybeduetofunctions beyondRCTandwhetherthesefunctionsofHDLcanbeenhanced withoutincreasingplasmalevelsofHDL-Cperse. It remains to be definitively proven that raising HDL-C or improving its function will reduce the risk of cardiovascular events.ResultsofsomeclinicaltrialshavedemonstratedthattreatingpatientswhohavelowHDL-CwiththerapiesthatraiseHDL-C canreducemajorcoronaryevents(2).However,newtherapeutic approachestoraisingHDL-Clevelsand/orimprovingHDLfunctionarerequiredtoconclusivelyprovethishypothesis.Thisreview focusesonrecentdiscoveriesandremainingquestionsregardingthemolecularregulationofHDLmetabolismandfunction (includingRCT)andthestatusofnoveltherapeuticsbeingdevelopedbasedonthesediscoveries. HDL biosynthesis Apolipoprotein production.ThebiosynthesisofHDL(Figure1)is complexandinvolvesthesynthesisandsecretionofthemajor proteincomponentsofHDLfollowedbythelargelyextracellular acquisitionoflipid(phospholipids[PLs]andcholesterol)and theassemblyandgenerationofthematureHDLparticle.The majorHDLapolipoproteinsareapoA-IandapoA-II,andbothare requiredfornormalHDLbiosynthesis.ApoA-Iconstitutesapproximately70%ofHDLproteinandispresentonvirtuallyallHDL particles(3).Thus,itisnotsurprisingthatgenedeletionofApoa1/ APOA1resultsinextremelylowlevelsofHDL-Cinmice(8)and inhumans(9).Atherosclerosis-pronemicelackingapoA-Idevelop significantlyincreasedatherosclerosis(10). ApoA-Iissynthesizedinboththeintestineandtheliver(Figure1), buttherelativecontributionoftheintestineandthelivertothe plasmaapoA-Ipoolinhumansremainsunknown.Hepaticoverex-

The Journal of Clinical Investigation http://www.jci.org Volume116 Number12 December2006

science in medicine

Figure 1
HDL biosynthesis. Enterocytes and hepatocytes synthesize apoA-I, which is secreted in a lipid-poor form and then immediately recruits additional PLs and free cholesterol (FC) via the ABCA1 pathway, forming nascent HDL. Nascent HDL acquires more lipid from other peripheral tissues and from lipoproteins, and LCAT generates CE, forming mature HDL. The liver also synthesizes apoA-II, which results in a subclass of HDL containing both apoA-I and apoA-II.

pressionofapoA-IsignificantlyraisesHDL-Clevelsandinhibitsthe progressionofandevenregressesatherosclerosisinmice(1113). Thus,upregulationofendogenousapoA-Iexpressioniswidely consideredoneofthemostpromisingapproachestothedevelopmentofnewtherapiestargetedtoHDL.Muchhasbeenlearned abouttheregulationofapoA-Itranscription(14).PPARagonists, suchasfibrates,havebeenshowntoupregulatetranscriptionof thehumanAPOA1geneinhumanAPOA1transgenicmiceinvivo (15).However,fibratesarerelativelyweakPPARagonists,have modesteffectsonapoA-Iconcentrationsinhumans,andhave notbeenconclusivelydemonstratedtoincreaseapoA-Iproductioninhumans;morepotentPPARagonistscouldpotentially haveagreatereffect.Theorphannuclearreceptorliverreceptor homolog-1(LRH-1)wasshowntoupregulateapoA-Itranscription throughdirectbindingtotheapoA-Ipromoter(16),whereasthe transcriptionalrepressorsmallheterodimerpartnersuppresses apoA-ItranscriptionbyinhibitingtheactivityofLRH-1.While knowledgeregardingthetranscriptionalregulationofapoA-Ihas notyetproducedsmallmoleculesthathaveadvancedinclinical development,thisremainsanareaofactiveinterest. ApoA-IIconstitutesapproximately20%ofHDLprotein,ispresentonabouttwo-thirdsofHDLparticlesinhumans,andissynthesizedonlyintheliver.GenedeletionofApoa2 inmicemarkedly reducesHDL-Clevels(17),suggestingthatapoA-IIisalsorequired fornormalHDLbiosynthesisandmetabolism.WhileoverexpressionofapoA-IIinmiceraiseslevelsofHDL-C,itincreasesatherosclerosis(18,19).Inbothmiceandhumans,quantitativetraitloci

linkagestudiessuggestthattheApoa2/APOA2genelocusisadeterminantofHDL-Clevels(20).UnlikeapoA-I,plasmaapoA-IIlevels aredeterminedprimarilybytherateofproduction(21).Interestingly,fibrates(PPARagonists),thiazolidinediones(PPARagonists),andalcoholallsignificantlyincreaseplasmaapoA-IIlevels inconcertwithincreasedHDL-Clevels.Thus,increasingAPOA2 genetranscriptioncouldbeastrategyforraisingHDL-Clevels; however,theimplicationsofthisstrategyforatherosclerosisin humansareuncertain. Lipid acquisition and maturation.NewlysecretedHDLapolipoproteinsmustacquirelipid(PLsandcholesterol)inordertogenerate HDLparticles.Recentstudieshaveestablishedthatthelipidation ofHDLapolipoproteinsoccursprimarilyaftertheirsecretionand thatABCA1isacriticalparticipantintheearlylipidationofnewly secretedapoA-I.GeneticabsenceofABCA1formsthemolecular basisofTangierdisease(2224),whichisassociatedwithextremelylowlevelsofHDL-CandapoA-I.Abca1-knockoutmicehavea phenotypesimilartothatofTangierdiseasepatients(25).The productionrateofapoA-IinTangierdiseaseisnormal,butapoA-I isextremelyrapidlycatabolized(26).Althoughitisubiquitouslyexpressed,ABCA1intheliverandtheintestineappearstobe responsibleforthevastmajorityoftheinitiallipidationoflipidpoorapoA-I(Figure1).MicethatlackABCA1specificallyinthe liverhaveHDL-Clevelsthatarereducedby80%(27),andmice thatlackABCA1intheintestinehavea30%reductioninHDL-C (28). Upregulation of hepatic and/or intestinal ABCA1 could potentiallybeatherapeuticstrategyforraisingHDL-Clevels.
3091

The Journal of Clinical Investigation http://www.jci.org Volume116 Number12 December2006

science in medicine

Figure 2
Pathways of HDL cholesterol uptake by the liver. HDL-CE and HDL free cholesterol can be directly and selectively taken up by the liver via SR-BI. Alternatively, HDL-CE can be transferred to apoB-containing lipoproteins by CETP and then taken up by liver via the LDLR. In the hepatocyte, CE is hydrolyzed to free cholesterol, which is either excreted directly into the bile or converted to bile acid (BA) and excreted into the bile.

Whiletheliverandintestinearecriticalforinitiallipidationof apoA-IviaABCA1,HDLderivesmuchofitslipidmassfromother sources.Thepotentialadditionalsourcesincludeothertissuesand otherlipoproteins(Figure1).Inmice,about90mgcholesterolper kilogramofbodyweightiseffluxeddailyfromextrahepatictissuestoHDL(29).VerylittleisknownaboutthesourcesormechanismsofextrahepaticlipidationofHDL.Macrophages,themost importantcelltypetoeffluxcholesterolfromthepointofviewof atherosclerosis,almostcertainlydonotcontributemuchcholesterolmasstoHDL.Forexample,whenwild-typebonemarrowwas transplantedintoABCA1-knockoutmice,therewaslittleincrease inplasmaHDL-Clevels(30).Becauseallextrahepaticcellsrequire cholesterolandyetcannotmetabolizeit,theyneedtoeffluxcholesteroltoHDL.Thus,itstandstoreasonthatthemostimportantcontributorstotheHDLcholesterolpooloutsidetheliver andintestinewouldbelargeorganbedssuchasskeletalmuscle, adiposetissue,andskin.Indeed,skeletalmyocytes,adipocytes,and skinfibroblastshaveallbeenshowntohavetheabilitytoefflux cholesteroltoacceptorssuchasapoA-IandHDLinvitro.More researchisneededtodeterminetherelativecontributionofthese andotherorganstothelipidationofHDLandthepathwaysand molecularmechanismsbywhicheffluxofPLsandcholesterolfrom thesetissuesoccurs.Thisissueisnotpurelyacademic,aspharmacologicupregulationofeffluxpathwaysoflipidsfromextrahepatic tissuestoHDLcouldresultinincreasedlevelsofHDL-C. HDLalsoderiveslipids,particularlyPLs,fromotherlipoproteins (Figure1).Whentriglyceride-rich(TG-rich)lipoproteinsundergo hydrolysisoftheTGcore,surfacePLs(andapolipoproteins)are shedandacquiredbyHDL.Thus,theactivityoflipoproteinlipase isinverselyassociatedwithHDL-Clevels(3).Lipoprotein-derived PLsaretransferredtoHDLbythePLtransferprotein(PLTP)(31).
3092

MicelackingPLTPhaveasignificantreductioninHDL-Clevels (32),andmiceoverexpressingPLTPhaveincreasedlevelsofHDL-C (33).TheroleofPLTPinhumans,andwhetherPLTPisatargetfor therapeuticdevelopment,remaintobedetermined. ThedevelopmentofmatureHDLrequirestheesterificationof cholesteroltoformcholesterylester(CE)andthehydrophiclipid coreofHDL.HDL-CEisformedbytheactionoflecithin:cholesterolacyltransferase(LCAT),anHDL-associatedenzymethat catalyzesthetransferofafattyacidfromPLtofreecholesterol (Figure1).LCATiscriticalforthemaintenanceofnormalHDL metabolism.LCATdeficiencyinhumans(34)andinmice(35) causesmarkedlyreducedlevelsofHDL-Candrapidcatabolism ofapoA-IandapoA-II(36).Conversely,overexpressionofLCAT inmiceresultsinsubstantiallyincreasedHDL-Clevels(37).LCAT hasbeenconsideredtobeimportantintheprocessofRCTbygeneratingagradientoffreecholesterolfromcellstoHDL,butthe impactofLCATactivityonRCTinvivohasnotbeenexamined.In humans,freecholesterolinHDLcanbedirectlytransferredtothe liverandsecretedinbile(38).Nevertheless,upregulationofLCAT activitywouldbeexpectedtoincreaseHDL-Clevelsandtherefore isapotentialgoalforthedevelopmentofnewtherapies. HDL catabolism HDL cholesterol catabolism.AdetailedunderstandingofthepathwaysforHDLcholesterolcatabolismisextremelyimportantfor developmentofnoveltherapeutics.Thefluxofcholesterolthrough theseHDLpathwaysmaybemoreimportantthanthesteady-state HDL-Clevel.ThemajorsiteofHDLcholesteroluptakeistheliver (Figure2).Thebest-understoodmechanismofdirectuptakeof HDLcholesterolbytheliveristhatmediatedbythescavenger receptorclassBI(SR-BI).Importantly,SR-BIpromoteshepatic

The Journal of Clinical Investigation http://www.jci.org Volume116 Number12 December2006

science in medicine
uptakeofHDLcholesterol(bothesterifiedandunesterified)withoutmediatingdegradationofHDLapolipoproteins,aprocess knownasselectiveuptake(Figure2).StudiesofSR-BIinpolarizedhepatocytessuggestthatSR-BImediatesinternalizationof wholeparticlesofHDL,withsubsequentremovalofcholesterol andresecretionofsmallercholesterol-depletedHDLparticles(39). AbundantdataindicatethatinrodentsSR-BIisacriticalregulatorofHDLmetabolism.HepaticoverexpressionofSR-BIinmice markedlyincreaseshepaticHDLcholesteroluptakeandreduces plasmaHDL-Clevels(40).Conversely,deletionoftheSR-BIgene Scarb1inmiceslowshepaticHDLcholesteroluptakeandincreases plasmaHDL-Clevels(41,42).Inmice,hepaticSR-BIexpression appearstobeanimportantoverallregulatorofRCT:SR-BIoverexpressionincreasedRCT,andSR-BIdeficiencyreducedRCT(43). ThisisconsistentwiththeobservationsthathepaticSR-BIoverexpressionreducedatherosclerosisdespitereducingHDL-Clevels (4446)andthatScarb1-knockoutmicehavemarkedlyincreased atherosclerosisdespiteincreasedHDL-Clevels(4749). Overall,thisbodyofdataisprobablythebestexampleofthe conceptthatfluxofHDLcholesterolismoreimportantthan steady-stateconcentrationswithregardtoatherosclerosis.These observationshaveledtotheconceptthatupregulationofhepatic SR-BIcouldbeantiatherogenicdespitethereducedplasmaHDL-C. However,thereremainquestionsregardingthephysiologicimportanceofthehepaticSR-BIpathwayforuptakeofHDLcholesterol inhumans.Studiesinhealthy,normolipidemichumanshave suggestedthatrelativelylittleHDL-CEisdirectlytakenupbythe liverandtargetedtobile(38).StudiesofSCARB1geneticpolymorphismsinhumanshaveindicatedonlyamodestassociationwith variationinplasmaHDL-Clevels(50),andnoSR-BIdeficient patientshavebeendefinitivelyreportedtodate.Basedonresults ofmurinestudies,onemightpredictthatthephenotypeofSR-BI deficiencyinhumansiselevatedHDL-Clevelsaccompaniedby increasedriskofatheroscleroticcardiovasculardisease(CVD). Inhumans,thereisclearlyatleastonealternativepathwayby whichHDLcholesterolismetabolizedandultimatelytransportedtotheliver,apathwaymediatedbytheCEtransferprotein (CETP).CETPtransfersTGfromapoB-containinglipoproteins inexchangeforHDL-CE,thusresultinginCEdepletionandTG enrichmentofHDL(Figure2).RodentsnaturallylackCETP,and whenengineeredtoexpressit,theyexperiencesubstantialreductioninHDL-Clevels(51).TheproofthatCETPisimportantfor humanHDLmetabolismcamefromthediscoveryofhumans geneticallydeficientinCETP(52,53).Theseindividuals,whohave loss-of-functionmutationsinbothallelesoftheCETPgeneand arefoundalmostexclusivelyinJapan,haveextremelyhighlevels ofHDL-C.Inaddition,theirHDLisexceptionallylarge,andthe turnoverofapoA-Iissubstantiallyslowed(54).Confirmationthat theCETPpathwayisquantitativelyimportantforhepaticuptake ofHDL-CEinhumanswasobtainedinastudyinwhichinjection ofHDLlabeledwithaCEtracerdemonstratedthatthelabeled cholesterolthatwasexcretedintobilewasfirstlargelytransferred toapoB-containinglipoproteins(38). ThediscoveryofCETPdeficiencyraisedthequestionofwhetherpharmacologicinhibitionofCETPmightbeanovelapproach to raise plasma HDL-C levels. CETP inhibitors are now the mostadvancedclassofnovelHDLlevelraisingdrugs(55,56). TheCETPinhibitorJTT-705wasshowntoraiseHDL-Clevels asmonotherapy(57)andincombinationwithpravastatin(58). AnotherCETPinhibitor,torcetrapib,increasedHDL-Clevelsin

healthyvolunteers(59)andinsubjectswithlowbaselineHDL-C levels (60). In subsequent phase II studies, dose-dependent increasesinHDL-Clevelswereseenwithtorcetrapibaloneaswell asincombinationwithatorvastatin(61).ModestLDL-Clowering hasalsobeenreportedwithCETPinhibitionthatwasproportionaltotheincreaseinHDL-C(5861).Finally,incontrastto thesmall-moleculeapproach,astrategyofgeneratinganimmune responsetoCETPusingaCETP-basedpeptideforimmunization isinclinicaldevelopment(62). WhetherCETPinhibitionreducesCVDremainsanopenquestion.TherelationshipofCETPdeficiencytoCVDisuncertain: therearearelativelysmallnumberofCETP-deficienthomozygotes,andpopulation-basedattemptstodefinitivelyaddressthis questioninhomozygoteshavenotbeencarriedout.Individuals heterozygousforCETPmutationshaveonlyamodestincrease inHDL-Clevelsandhavenomajordifferenceincardiovascular risk(63).AdditionalgeneticstudiesofCETPpolymorphismsin otherpopulations(64)andmeasurementofCETPinlargeprospectiveobservationalstudies(65)areconflictingbutsuggest thatincreasedCETPmaybeaCVDriskfactorinhumans.There isapossibilitythatCETPinhibitionmaynotpromoteandcould actuallyimpairRCT,giventheapparentimportanceoftheCETP pathwayintransportingHDL-CEtotheliver(38).Administrationoftorcetrapibresultedinsignificantlyslowercatabolism ofapoA-Ibuthadnosignificanteffectonfecalneutralsterol excretion(66).WhileanearlyreportsuggestedthatHDLfrom CETP-deficientsubjectswasdysfunctionalinitsabilitytopromotemacrophagecholesterolefflux(67),resultsofarecentstudy indicatethatCETP-deficientHDLismoreeffectivethancontrol HDLinpromotingmacrophagecholesteroleffluxviatheABCG1 pathway(seebelow)(68).DeterminingtheeffectsofCETPinhibitiononatherosclerosisisthemostdirectmethodofproviding insightintotheeffectsofthisstrategy.StudiesofCETPinhibitioninrabbitshaveconsistentlyshownareductioninatherosclerosis(56).Atherosclerosisimagingtrialswithtorcetrapibusing intravascularultrasoundofthecoronariesandmeasuringintimamediathicknessofthecarotids,aswellasalargecardiovascular outcometrialwithtorcetrapib,areinprogress(69).Thequestion ofwhetherCETPinhibitionreducesCVDisacriticallyimportant issueforthefieldofHDLtherapeutics. HDL apoA-I catabolism. Results of kinetic studies in humans havedemonstratedthattheturnoverrateofapoA-Iismuchmore variablethanitsproductionrateandisamoreimportantdeterminantofplasmaapoA-IandHDL-Cconcentrations(3).Thus, understandingthesitesandmechanismsofapoA-Icatabolismis important(Figure3).Studiesinanimalsusingtrappedligands (70)establishedthatasubstantialportion(approximatelyonethird)ofapoA-Iiscatabolizedbythekidneys,andtherest(greater thanhalf)iscatabolizedbytheliver.NotethatthesitesofapoA-I catabolisminhumansareassumedtobesimilarbuthavenever beenformallystudied. MoreisunderstoodregardingthemechanismsofapoA-Icatabolismbythekidneythanbytheliver.Lipid-poorapoA-Icanbefilteredattheleveloftheglomerulusandthencatabolizedbyproximal renaltubularepithelialcells(Figure3).Cubilinisanextracellular proteinsynthesizedbyproximalrenaltubularcellsandlocalizedto theapicalsurfaceanchoredbyanotherproteincalledamnionless (71).CubilinbindsHDLandapoA-Iwithhighaffinity(72,73)and theninteractswithacoreceptorcalledmegalin,amemberofthe low-density lipoprotein receptor(LDLR)genefamily,tomediateuptake
3093

The Journal of Clinical Investigation http://www.jci.org Volume116 Number12 December2006

science in medicine

Figure 3
Pathways of apoA-I catabolism. Lipid-poor apoA-I can be filtered through the renal glomerulus and be degraded by the renal tubular cell via the cubulin-megalin pathway. Lipid-poor apoA-I is generated by the action of lipases such as HL and EL on mature HDL, and the action of these lipases is enhanced by the CETP-mediated transfer of TG into HDL. Mature HDL can also be catabolized by the hepatocyte through binding of HDL-apoE to hepatic receptors or through a poorly characterized mechanism of direct interaction with the hepatocyte. AMN, amnionless.

anddegradationofapoA-I(74).However,functionalcubilindeficiencyinanimalsorhumansisnotassociatedwithchangesinplasmaHDL-CorapoA-Iconcentrations(75).Thus,itisdoubtfulthat thisrenaltubularpathwayisresponsibleforvariationinHDL-C levelsinhumansorisatargetforthedevelopmentoftherapeutic approachestoraiseHDL. Therate-limitingstepintherenalcatabolismofapoA-Iisprobablyattheglomerularfiltrationlevelratherthanatthelevelof tubularcatabolism.Therefore,thereismajorinterestinthefactors thatregulatetherateofglomerularfiltrationofapoA-I.Mature HDListoolargetobefiltered,andprobablyonlyapoA-Ithatis relativelylipidpoorcanbefilteredtoanyextent.Theextentof apoA-IlipidationthusplaysamajorroleintherateofrenalapoA-I catabolism.ApoA-Ilipidationisdeterminedbybothacquisition andmaturationoflipidaswellasbyremodelingofthemature HDLparticle.ThereareseveralexamplesfromhumanpathophysiologyofimpairedlipidacquisitionbyapoA-Ileadingtoincreased apoA-Icatabolism.Asnotedabove,thebestexampleisthatof Tangierdisease,inwhichtheinabilityoftheliverandtheintestinetolipidatenewlysynthesizedapoA-IviatheABCA1pathway resultsinapoorlylipidatedapoA-Ithatisrapidlycatabolized(26) primarilybythekidneys(27).Importantly,evenABCA1heterozygoteshaveincreasedapoA-Icatabolism(26),demonstratingagene doseeffectofABCA1withregardtoapoA-Iturnover.Furthermore, inLCATdeficiencythelackofLCAT-mediatedcholesterolesterificationresultsinacceleratedapoA-Icatabolism(36),presumably becausetheformationoftheCEcoreinHDLhelpstogenerate particlesoflargeenoughsizetoescaperenalglomerularfiltration. However,heterozygotesforLCATmutationshavenormalplasma
3094

apoA-IlevelsandthuspresumablynormalapoA-Iturnover,suggestingthatLCATisnotadeterminantofapoA-Iturnoveracross arangeofLCATactivity. MatureHDLisremodeledbyseveralfactors,resultinginthe generationofsmallerHDLparticlesandlipid-poorapoA-Ithatis subjecttoglomerularfiltrationandcatabolism.HDLremodeling consistsofremovalofCEaswellashydrolysisofPLsandTGs; theseprocessesareoftenlinked.The2mostimportantprocesses involvingremovalofHDL-CEaretransfertootherlipoproteins viaCETPandselectiveuptakeviaSR-BI.Botharediscussedin detailabove,butitisworthnotingherethatbothinfluencethe turnoverofapoA-I.Forexample,CETPdeficiencyinhumansis associatedwithreducedapoA-Iturnover(54),asispharmacologic inhibitionofCETP(66).Furthermore,overexpressionofCETP (76)andSR-BI(77)inmiceincreasestheturnoverofapoA-I. Thus,factorsthatinfluencetheamountofCEinHDLmodulate therateofapoA-Icatabolism. ThemodificationofHDLbylipolyticenzymesisanotherimportantdeterminantofapoA-Iturnover.Thebest-studiedenzyme withregardtoHDLmetabolismishepaticlipase(HL).HLhasthe abilitytohydrolyzebothTGandPLinHDL(Figure3).Aseriesof studieshavedemonstratedthatHLismosteffectiveinhydrolyzingHDLiftheHDLisTGenriched(3),aprocessthatoccurswith increasedCETP-mediatedexchangeofHDL-CEforTGfromTGrichlipoproteins.TheactionofHLonTG-enrichedHDLresults inthesheddingoflipid-poorapoA-IfromHDLandincreased apoA-Icatabolism.Indeed,apoA-IassociatedwithTG-enriched HDLiscatabolizedsignificantlyfasterthannonTG-enriched HDL(78).Insulin-resistantstates,whichareknowntobeassoci-

The Journal of Clinical Investigation http://www.jci.org Volume116 Number12 December2006

science in medicine
atedwithincreasedratesofapoA-Icatabolism,arealsoassociated withincreasedHLactivity(3).Thus,inhibitionofHLwouldbe expectedtoreduceHDLremodeling,slowapoA-Icatabolism,and increaseapoA-IandHDL-Clevels.However,becauseHLmayplay aroleintheclearanceofapoB-containingremnantparticles,inhibitionofHLfortherapeuticpurposesisnotsostraightforward. Morerecently,endotheliallipase(EL),whichiscloselyrelatedto HL,hasbeendescribedandcharacterized(79,80).Incontrastto HL,ELappearstohaverelativelymorephospholipaseactivitythan TGlipaseactivityandappearstohaveagreaterpreferenceforHDL overapoB-containinglipoproteins.OverexpressionofELinmice causesareductioninHDL-Clevels(79,81)andalsoreducesapoA-I levelsbecauseofincreasedcatabolismprimarilyviathekidneys (82).Conversely,antibodyinhibition(83)orgenedeletion(81,84) ofELresultsinincreasedHDL-CandapoA-Ilevels.Subjectswith highHDL-ClevelsmaybemorelikelytohavepotentiallyfunctionalmutationsintheELgeneLIPG(85),butassociationstudies ofcommonpolymorphismsinELhavenotyettoldacompelling story.PlasmaELlevelsinhumanswerefoundtobesignificantly inverselyassociatedwithHDL-Clevelsandpositivelyassociated withavarietyofmeasuresofthemetabolicsyndrome(86),which suggeststhatELcontributestothelowHDL-Clevelsthatoccur withinsulinresistance.Whileresultsofatherosclerosisstudiesin ELknockoutmicehavebeenconflicting(87,88),plasmaELlevels inhumanswerefoundtobesignificantlyassociatedwithcoronary atherosclerosis(86).InhibitionofELisanattractivetherapeutic approachthatmightbeexpectedtoreduceapoA-Icatabolismand increaseplasmaapoA-IandHDL-Clevels. In animals, the liver is responsible for substantial degradationofapoA-I(70).ThemechanismsofhepaticuptakeandHDL apolipoproteindegradationremainpoorlyunderstood.OnemechanismthatlikelyplaysatleastsomeroleisrelatedtoapoE.Aportion ofHDLparticlescontainapoE,andapoE-richHDLisknowntobe aligandfortheLDLreceptor(LDLR)andotherapoEreceptors(89). ThusthispathwaymaycontributetotheuptakeofsomeHDLby theliver(Figure3).However,HDLdepletedofapoEstillhastheabilitytobetakenupanddegradedbyhepatocytes,andothermechanismsmustalsoexist.Therehasbeensubstantialeffortinvestedin identifyingotherhepaticHDL-bindingproteins(90),butnonehave beenproventobebonafideHDLreceptorsinvivo.Thechainof ATPsynthasewasreportedtobeectopicallylocalizedtothehepatocyteplasmamembrane,whereitmayactasahigh-affinityreceptorforHDLapoA-ItotriggertheendocytosisofHDLparticlesin anADP-dependentprocess(91).ThenucleotideGproteincoupled receptorP2Y13wassuggestedtoactasaparticipantinthisprocess(92).Gain-andloss-of-functionstudiesinanimalsandhuman geneticstudiesarerequiredtoassesstherealphysiologicroleofthis pathwayandofotherhepaticHDL-bindingproteins. AthoroughunderstandingofthepathwaysandmolecularregulationofhepaticapoA-Icatabolismiscriticallyimportanttothe developmentofnewtherapeuticapproaches.Inconcept,ifthe hepaticcatabolismofapoA-Icouldbeslowedwithoutaffecting therateofHDLcholesterolturnoverorRCT,thiscouldbeanovel approachtoraisingapoA-Iconcentrationsandthusinhibiting atherosclerosis.Infact,themosteffectivepharmacologicmethodforraisingHDLcurrentlyavailableisnicotinicacid(niacin), andstudiesinhumanshavesuggestedthatniacinadministrationreducesthecatabolicrateofapoA-I(93).Whilethemolecular mechanismunderlyingthisobservationremainsunknown,niacin hasbeenshowntoreduceuptakeofHDLapoA-Iinhepatocytes

invitro(94).ItispossiblethatniacinactsonahepaticHDL receptororonanas-yet-unknownpathwaytoslowHDLapoA-I catabolism.AGproteincoupledreceptorcalledGPR109Ahas beendescribedasaniacinreceptor(95),butisnotthoughttobe highlyexpressedbyhepatocytes.Instead,itisprimarilyexpressed byadipocytesanditsactivationresultsinreducedreleaseoffatty acidsfromadipose(94),whichmayexplainniacinseffectsonplasmaTGsbutdoesnotobviouslyexplainitseffectsonHDL.The molecularmechanismsbywhichniacinraisesHDL-Clevelshave yettobeestablished.Nevertheless,niacinremainsausefulexampleofhowreducinghepaticHDLapoA-Icatabolismmightbea viablestrategyforincreasingHDL-CandapoA-Ilevels.Whether compoundsspecificallytargetedtowardactivatingGPR109Awill haveeffectsinreducinghepaticapoA-Icatabolism,oranyHDL-C raisingpropertiesatall,isacriticallyimportantquestionforthe field.Furtherinvestigationofthepathwaysofhepaticcatabolism ofapoA-Icouldyieldnewtargetsfordrugdiscovery. HDL function Theabovediscussionfocusedonthemolecularmechanismsthat regulate plasma concentrations of HDL-C and apoA-I. These mechanismshavetherapeuticimplications,becauseincreasing theirproductionorreducingtheircatabolismhasthepotential toincreaselevelsofHDL-CandapoA-I,andplasmalevelsarethe mostdirectbiomarkerofaneffectonHDL.Thesimplestparadigm fornewtherapeuticdevelopmentwouldbeoneinwhichraising plasmaHDL-C/apoA-Iconcentrationsbyanymechanismreliably translatedintoreducedatheroscleroticvasculardisease,similarto thatofreducingplasmaLDL-Cconcentrations.However,thiscannotbeassumed:intheory,certainmethodsofraisingHDLlevel couldimpairitsfunctionandnottranslateintovascularbenefit (e.g.,SR-BIdeficiencyinmice).Thus,fortheforeseeablefuture anynewtherapythatraisesHDL-Clevelswillneedtodemonstrate actualcardiovascularbenefit,eitherthroughimagingatherosclerosisand/orthroughreductioninhardcardiovascularevents. Nevertheless,thedevelopmentofanewtherapeuticapproachthat actuallyraisesHDL-Clevelsprovidesausefulearlybiomarkerand alsoraiseshopethattheincreaseinHDL-Cwillbebeneficial. Ontheotherhand,itisalsorelativelyeasytoimaginethepossibilitythatcertainapproachescouldimprovethefunctionofHDL withoutnecessarilyincreasingplasmaHDL-Cconcentrations. Forexample,fluxthroughtheRCTpathwaycouldtheoretically beincreasedwithoutincreasingthesteady-statelevelofHDL-C. TherateofRCTcannotbeinferredfromtheplasmaHDL-Clevel, butratherrequiressophisticatedkineticmeasuresofcholesterol flux(96).Inthissection,themolecularregulationofthefunctionsofHDLthatmaycontributetoitsantiatherogeniceffects andsubsequentinsightsintothedevelopmentofnoveltherapeuticapproachesarereviewed. Promoting macrophage cholesterol efflux and RCT.Therehasbeena majoremphasisoverthelast2decadesinattemptingtounderstand themolecularmechanismsbywhichtheeffluxofcholesterolfrom macrophagestoHDLisregulated.Macrophagesareprofessional phagocytesthattakeupdeadanddyingcellsaswellasaggregated andmodifiedlipoproteins,allofwhichcontainabundantcholesterol.Becausetoomuchcholesterolcanbetoxic,theyhaveevolvedseveralpathwaystoeffluxcholesteroltotheextracellularenvironment (Figure4).Themost-studiedpathwayformacrophagecholesterol effluxisthepathwaybywhichtheABCA1transporterpromotes cholesteroleffluxtolipid-poorapoA-Iasanacceptor.Macrophages
3095

The Journal of Clinical Investigation http://www.jci.org Volume116 Number12 December2006

science in medicine
oleffluxtobothlipid-poorapoA-IandmatureHDL.Furthermore, asyntheticLXRagonistwasshowntosignificantlypromotemacrophagecholesteroleffluxandRCTinvivodespitehavinglittle effectonplasmaHDL-Clevels(104).SyntheticLXRagonistshave alsobeenshowntoinhibittheprogression(105,106),andeven promotetheregression(107),ofatherosclerosisinmicedespite havinglittleeffectonplasmaHDL-Clevels.Thus,administration ofLXRagonistsareagoodexampleoftheconceptthatRCTcan besubstantiallypromoted(andatherosclerosisinhibited)without actuallyraisingplasmalevelsofHDL-C.Asaresult,LXRagonists havebeenviewedasanattractivenoveltherapeuticapproachfor atherosclerosis.However,somenonselectiveLXRagonistshave beenfoundtocausehepaticsteatosisandhypertriglyceridemia inanimals,whichisbelievedtobeduetoinductionofhepatic expressionofSREBP1candfattyacidsyntheticgenes(108).Furthermore,someLXRagonistsincreasedLDL-Clevelsinanimals thatexpressCETP(109).Nevertheless,LXRremainsaprimetherapeutictarget,andintheorymoreselectiveLXRmodulatorsmaybe foundthathavethebeneficialeffectsinmacrophageswithoutthe adverseeffectsintheliver. Alternativeapproachestoupregulatingmacrophagecholesterol effluxarebeingactivelyexplored.BothPPARandPPARagonistshavebeenshowntoupregulateABCA1,promotemacrophage cholesteroleffluxinvitro(110,111),andreducetheformationof foamcellsinvivo(112).IthasbeensuggestedthatPPARagonists mayupregulateLXRsinmacrophages(110,111),andaPPAR agonistwasshowntoinhibitfoamcellformationinanLXRdependent(butABCA1-independent)manner(112).Itisinterestingtoconsiderthatfibrates,relativelyweakPPARagonists, mayreducecardiovascularriskinpartbypromotingmacrophage cholesteroleffluxandthatmorepotentPPARagonistscouldbe evenmoreeffective.IthasalsobeensuggestedthatPPARagonists mayupregulateLXRsinmacrophages,butaPPARagonistinhibitedfoamcellformationinanLXR-independentmanner(112). PPAR/agonistshavebeensuggestedtopromotemacrophage cholesterolefflux(113),butthishasnotbeendefinitivelyestablished.Studiesareneededtoformallytestwhetheragonistsofany ofthePPARsarecapableofpromotingmacrophageRCTinvivo. ItmaybepossibletofunctionallyincreasemacrophageABCA1 andABCG1throughapproachesotherthantranscriptionalregulation.Inaddition,thereremainssubstantialinterestinidentifyingotherpathwaysbywhichmacrophageseffluxcholesterol.For example,SR-BIisexpressedinmacrophagesandcanpromotecholesteroleffluxtomatureHDL(114).ItisuncertainwhethermacrophageSR-BIquantitativelyparticipatesinmacrophagecholesterol effluxandunderwhatconditions.However,bonemarrowtransplantationfromSR-BIdeficientmiceintoLDLR-deficient(49)or apoE-deficient(115)miceresultedinincreasedatherosclerosis, consistentwithanatheroprotectiveroleofmacrophageSR-BI. Theremaybeotherregulatedeffluxpathwaysinthemacrophage aswell.Itisanticipatedthattherapiesthatpromotemacrophage cholesteroleffluxbyupregulatingoneormoreoftheeffluxpathwayswilleventuallybedevelopedforclinicaluse. Becauseeffluxisinpartdependentontheconcentrationofthe acceptor,itisalsologicaltoattempttoincreasetheconcentration oftheacceptor.Asnotedabove,increasingendogenousproductionofapoA-Imightbeconsideredtheidealapproach.However, exogenousapproacheshavealsobeenexplored;particularattentionhasbeengiventolipid-poorapoA-Iinthisregard.IntravenousbolusinfusionofapoA-Iinhumansresultsinasubstantial,

Figure 4
Pathways of cholesterol efflux from macrophages. Macrophages have several pathways for efflux of cholesterol. They efflux free cholesterol to lipid-poor apoA-I via the ABCA1 pathway and to mature HDL via the ABCG1 pathway. Both ABCA1 and ABCG1 are regulated by the nuclear receptor LXR, which is activated by binding of oxysterols, derived from free cholesterol. SR-BI also has the capacity to efflux free cholesterol to mature HDL.

fromABCA1-knockoutmicehavesubstantiallyreducedcholesterol effluxtolipid-poorapoA-Iasanacceptor(30).Indeed,transplantationofbonemarrowfromABCA1-knockoutmiceintoatherosclerosis-pronemiceresultedinsignificantlyincreasedatherosclerosis eventhoughplasmaHDL-Clevelswerevirtuallyunchanged(30). Conversely,overexpressionofABCA1incellsofmice,includingbut notlimitedtomacrophages,wasassociatedwithasignificantreductioninatherosclerosis(97).Thus,itispossiblethatupregulation ofABCA1inmacrophagescouldbeapowerfulantiatherogenic strategy.ThemajorregulatorsofABCA1geneexpressionarethe nuclearreceptorliverXreceptorsand(LXRandLXR),which actasheterodimerswiththeirpartnertheretinoidXreceptor(98). LXRsareactivatedendogenouslybyoxysterols,whicharegenerated enzymaticallyfromcholesterol.BothoxysterolsandsyntheticLXR agonistsupregulateABCA1transcriptioninmacrophagesandcause increasedcholesteroleffluxtolipid-poorapoA-I. However,invivomostofapoA-Iisnotlipidpoor(thepreferred acceptorforABCA1)butinlarge,matureHDLparticles.Infact, mature HDL is also capable of promoting cholesterol efflux frommacrophages.IthasrecentlybeenestablishedthatanothermemberoftheABCtransporterfamily,ABCG1,iscapableof promotingcholesteroleffluxfrommacrophagestomatureHDL (99,100)byanunknownmechanism.Inmacrophages,ABCG1is transcriptionallyupregulatedaswellastranslocatedtotheplasma membranebycholesterolloadingorLXRagonists(101).ABCG1deficientmacrophageshaveimpairedcholesteroleffluxtomature HDL,andABCG1-knockoutmiceaccumulatecholesterolandTGs inmacrophages,particularlyinthelung(100).Theimportanceof ABCG1incholesteroleffluxfrommacrophagesinvivoremains uncertain. Somewhat paradoxically, macrophage deficiency ofABCG1wasshowntoresultinreducedatherosclerosis(102, 103),possiblyduetocompensatoryupregulationofmacrophage ABCA1andapoE(102)orincreasedsusceptibilityofABCG1-deficientmacrophagestooxidizedLDLinducedapoptosis(103). Treatment of macrophages with LXR agonists results in upregulationofbothABCA1andABCG1andincreasedcholester3096

The Journal of Clinical Investigation http://www.jci.org Volume116 Number12 December2006

science in medicine
buttransient,increaseinlipid-poorapoA-I(116).Asinglebolusof proapoA-IdidnotraiseHDL-Clevelsbutresultedinasignificant increaseinfecalsterolexcretion,suggestingpromotionofRCT (117).InfusionofapoA-IMilano/phosphotidylcholinecomplexes weeklyfor5weekswasassociatedwithmodestregressionofcoronaryatheromavolumefrombaselineasmeasuredbyintravascular ultrasound(118),althoughtheseresultsmustbeinterpretedwith caution(119).SelectivedelipidationofHDLexvivo,whichgenerateslipid-poorapoA-I,mightbeabletobereinfusedinanautologousfashion(120).Finally,theconceptofusingapoA-Imimetic peptidesratherthanfull-lengthapoA-Iisbeingactivelyexplored (121).Amphipathicpeptidesof1822aminoacidshavebeendevelopedthathavepropertiessimilartothoseofapoA-I,includingthe abilitytopromotecholesterolefflux,activateLCAT,andreduce inflammation.RepeatedinjectionofthepeptideL-5Freducedthe progressionofatherosclerosisinmice(122).Oraladministration ofrelatedpeptideD-4Falsoreducedprogressionofatherosclerosis (123).D-4Fwasshowntopromotemacrophagecholesterolefflux invitroandRCTinvivo(124)andisinearlyclinicaldevelopment. TheapoA-ImimeticpeptideETC-642(alsoknownasRLTpeptide) promotesLCATactivationandisalsoinclinicaldevelopment.SeveralotherapoA-Imimeticpeptideshavebeendevelopedandtested incellandanimalmodels(121).ApoA-Imimeticpeptidesmayultimatelyprovetobeaneffectivemethodofpromotingmacrophage cholesterolefflux,improvingotheraspectsofHDLfunction(see below),andinhibitingatherosclerosisinhumans. Promoting antiinflammatory and other beneficial effects of HDL.In addition to cholesterol efflux promotion, several additional, potentially antiatherogenic properties of HDL have been described.TheseincludeinhibitionofLDLoxidation,inhibitionof endothelialinflammation,promotionofendothelialnitricoxide production,promotionofprostacyclinbioavailability,andinhibitionofplateletaggregationandcoagulation(6,7).Themolecular mechanismsoftheseeffectshaveyettobefullyelucidated,andthe importanceofthesemechanismstotheatheroprotectiveeffects ofHDLremainsuncertain.However,inconcepttheyarepotentiallyquiteimportant.First,strategiesthatraisethelevelofHDL throughinhibitingitscatabolismmaybeantiatherogeniceven iftheydonotpromotecholesteroleffluxandRCTbyworking throughtheseothermechanisms.Second,therapiesmaybedevelopedthatenhancethesefunctionsofHDLevenwithoutincreasingHDL-Clevelsperse.Third,oncethemolecularmechanisms arebetterunderstood,therapiesmaybedevelopedthatdirectly mimictheseHDLeffectsonthevasculature. Perhapsthebestcurrentexampleofanapproachthatmay increaseHDLfunctionistheapoA-ImimeticpeptideD-4Fmentionedabove.D-4FiscomposedofallDaminoacidsandtherefore isnotrecognizedbygutpeptidasesandispartiallyabsorbedafter oraldosing.SomedatasuggestthatD-4FenhancestheantiinflammatoryfunctionofHDLinmicewithoutincreasingHDL-C levels(121),althoughthemechanismofthiseffectisuncertain andthisfindingmustbeconfirmedinhumans.D-4FmaypermitthefirsttestofthehypothesisthatenhancingHDLfunction withoutincreasingplasmaHDL-Clevelscanreduceatherosclerosisorcardiovascularrisk. Future directions AggressivereductionofLDL-Cisacornerstoneofpreventivecardiovascularcare,butadditionaltherapeuticapproachestoreduce atherosclerosisprogression(orinduceitsregression)andprevent

cardiovasculareventsarestillneeded.HDLisanobviousand attractivetarget,butcurrenttherapeuticapproachestotargeting HDLareinadequate.Therearerelativelyfewvalidatedtargetsfor developingnoveltherapeuticapproachestargetedtowardHDL. The most advanced, CETP inhibition, is in late-stage clinical development,andtheoutcomemayhaveatremendousimpacton thefield.IfCETPinhibitorsareshowntobeeffectiveinreducing atheroscleroticCVDandapproved,theywilllikelybecomewidely usedclinically.Inthissetting,thepotentialforadditionalHDLtargetedtherapieswillneedtobecarefullyassessed.TheprobabilityisthatadditionalHDL-basedtherapieswillbecomplementary toCETPinhibitionandfindtheirrole,muchasadditionalLDLloweringapproachesbeyondstatinsarestillneededformany patients.Forexample,itmaystillbeveryusefultoupregulatemacrophageeffluxpathways(i.e.,LXRagonists)inordertomoreeffectivelypromotecholesteroleffluxtotheincreasedHDLacceptors inducedbyinhibitionofCETP,oritmaybepossibletofurther improvethefunctionoftheHDL(i.e.,apoA-Imimeticpeptides) inthesettingofCETPinhibition. Ontheotherhand,ifCETPinhibitorsareineffectiveinreducingatherosclerosis,itwillnotmeanthattheoverallconceptof targetingHDLfortherapeuticpurposesisflawed.Asdiscussed above,CETPinhibitioniscomplexbecauseitraisesHDL-Clevels byfundamentallyslowingthemetabolismofHDLcholesterol.In theeventthatCETPinhibitorsarefoundtobeineffective,theneed todevelopotherHDL-basedtherapeuticsthatworkthroughdifferentmechanismswillbeevenmoreimperative.Whilesomeof thecurrenttargetsforsmallmoleculedevelopmentsuchasLXR, EL,andtheniacinreceptorhavepotential,thereisstilladearth ofvalidatedtargetsforHDLtherapeutics.Additionaleffortsare neededtobetterunderstandthemolecularregulationofHDL metabolismaswellastoidentifyadditionalgenescontributing tovariationofHDL-Clevelsinhumans.Indeed,someofthemost importantinsightsregardingmoleculartargetsforHDL-based therapieshavebeenderivedfromstudiesofhumangeneticsyndromes(e.g.,TangierdiseaseandCETPdeficiency).Genome-wide studiesinmiceandhumans(20)promisetoyieldnew,previously unsuspectedgenesinvolvedinHDLmetabolism,someofwhich willbecometargetsfornewtherapeuticdevelopment. Theconceptthatacuteparenteraladministrationofasynthetic formofHDLtopatientspresentingwithacutecoronarysyndromesmayreducethehighshort-termriskofrecurrentevents isattractiveandplausible.Resultsofanimalstudiesindicatethat regressionandchangesinplaquemorphologyconsistentwith stabilizationcanoccurrapidlyaftertheintroductionofanHDLbasedtherapeutic,andatleastoneclinicaltrialisconsistentwith thisnotion.Multipleapproachestotheconceptarebeingtaken, andoneormoreoftheseapproachesmayultimatelyproveto effectivelyreducecardiovasculareventsafteracutecoronarysyndromesandbeintroducedtothemarket.Acuteinductiontherapy forunstablecoronarydiseasewouldthenbecomestandardin inpatientsettings,withmaintenancetherapyinoutpatientsettingscontinuingforweeksormonthsafterdischarge.Thiswould bearadicalchangeintheparadigmoftreatingcoronarydisease. ForallnovelHDL-basedtherapeutics,thechallengeisdeterminingearlyindevelopmentwhetheraparticularstrategyislikelytobe effectivewithregardtoatherosclerosis.RaisingplasmaHDL-ClevelsisneitheradequatenornecessaryforasuccessfulHDL-targeted therapy.Thereremainstremendousneedfornewinvivokinetic approachesandnovelbiomarkerstoreliablyassesstheeffects
3097

The Journal of Clinical Investigation http://www.jci.org Volume116 Number12 December2006

science in medicine
ofnewtherapiesonRCTandHDLfunctionandpredicttheir effectsonatherosclerosis.Closecollaborationbetweencompanies developingHDL-basedtherapeuticsandacademiciansexpertin thecomplexassessmentofRCTandHDLfunctioninvivowill berequiredtodevelopthenecessarytoolsforearlyassessmentof promisingtherapies.Insummary,theeffortsofthebasicscience communitytounderstandthemolecularbasisofHDLmetabolismandfunctionhaveyieldedimportantinsightsthatareleading tonewtherapeuticadvances.Thenextfewdecadeswilllikelybeto HDL-basedinterventionwhatthelastcoupleofdecadeshavebeen toLDL-basedintervention:atimeofdefinitiveproofoftheHDL hypothesisandanexpansionofthetherapeuticarmamentarium formanipulatingHDLintheinterestoffurtherreducingtherisk andburdenofatheroscleroticCVD.
1.Miller,G.J.,andMiller,N.E.1975.Plasma-highdensity-lipoproteinconcentrationanddevelopmentofischaemicheart-disease.Lancet.1:1619. 2.ExpertPanelonDetection,Evaluation,andTreatmentofHighBloodCholesterolinAdults(Adult TreatmentPanelIII).2001.Executivesummaryof thethirdreportoftheNationalCholesterolEducationProgram(NCEP).JAMA.285:24862497. 3.Lewis,G.F.,andRader,D.J.2005.Newinsightsinto theregulationof HDLmetabolismand reverse cholesteroltransport.Circ. Res.96:12211232. 4.Glomset,J.A.1968.Theplasmalecithins:cholesterolacyltransferasereaction.J. Lipid Res.9:155167. 5.Ross,R.,andGlomset,J.A.1973.Atherosclerosis andthearterialsmoothmusclecell:proliferation ofsmoothmuscleisakeyeventinthegenesisofthe lesionsofatherosclerosis.Science.180:13321339. 6.Barter,P.J.,etal.2004.AntiinflammatorypropertiesofHDL.Circ. Res.95:764772. 7.Mineo,C.,Deguchi,H.,Griffin,J.H.,andShaul, P.W.2006.Endothelialandantithromboticactions ofHDL.Circ. Res.98:13521364. 8.Williamson,R.,Lee,D.,Hagaman,J.,andMaeda, N.1992.Markedreductionofhighdensitylipoproteincholesterolinmicegeneticallymodifiedto lackapolipoproteinA-I.Proc. Natl. Acad. Sci. U. S. A. 89:71347138. 9.Schaefer, E.J., Heaton, W.H., Wetzel, M.G., and Brewer,H.B.,Jr.1982.PlasmaapolipoproteinA-1 absence associated with a marked reduction of highdensitylipoproteinsandprematurecoronary arterydisease.Arteriosclerosis.2:1626. 10.Moore,R.E.,etal.2005.Increasedatherosclerosis inmicelackingapolipoproteinA-Iattributableto bothimpairedreversecholesteroltransportand increasedinflammation.Circ. Res.97:763771. 11.Rubin,E.,Krauss,R.,Spangler,E.,Verstuyft,J.,and Clift,S.1991.Inhibitionofearlyatherogenesis intransgenicmicebyhumanapolipoproteinAI. Nature.353:265267. 12.Plump,A.,Scott,C.,andBreslow,J.1994.Human apolipoproteinA-Igeneexpressionincreaseshigh densitylipoproteinandsuppressesatherosclerosis intheapolipoproteinE-deficientmouse.Proc. Natl. Acad. Sci. U. S. A.91:96079611. 13.Tangirala,R.K.,etal.1999.Regressionofatherosclerosis induced by liver-directed gene transfer of apolipoprotein A-I in mice. Circulation. 100:18161822. 14.Mooradian,A.D.,Haas,M.J.,andWong,N.C.2004. TranscriptionalcontrolofapolipoproteinA-Igene expressionindiabetes.Diabetes.53:513520. 15.Berthou, L., et al. 1996. Opposite regulation of human versus mouse apolipoprotein A-I by fibratesinhumanapolipoproteinA-Itransgenic mice.J. Clin. Invest.97:24082416. 16.Delerive,P.,Galardi,C.M.,Bisi,J.E.,Nicodeme,E., andGoodwin,B.2004.Identificationofliverreceptor homolog-1asanovelregulatorofapolipoproteinAI
3098

Acknowledgments TheauthorhasbeensupportedbyNationalHeart,Lung,and BloodInstitutegrantsHL55323,HL22633,HL62250,HL59407, andHL70128;NationalInstituteofDiabetesandDigestiveand KidneyDiseasesgrantsDK59533andDK06990;NationalCenter forResearchResourcesgrantM01RR00040;theBurroughsWellcomeFundClinicalScientistAwardinTranslationalResearch; andaDorisDukeCharitableFoundationDistinguishedClinical ScientistAward. Addresscorrespondenceto:DanielJ.Rader,UniversityofPennsylvaniaMedicalCenter,654BRBII/IIILabs,421CurieBoulevard, Philadelphia,Pennsylvania19104-6160,USA.Phone:(215)5734176;Fax:(215)573-8606;E-mail:rader@mail.med.upenn.edu.
lipidtransferprotein(PLTP)onHDLmetabolism. Atherosclerosis.155:269281. 32.Jiang, X.C., et al. 1999. Targeted mutation of plasmaphospholipidtransferproteingenemarkedlyreduceshigh-densitylipoproteinlevels.J. Clin. Invest.103:907914. 33.Jiang,X.,etal.1996.Increasedpre-highdensity lipoprotein,apolipoproteinAI,andphospholipid inmiceexpressingthehumanphospholipidtransferproteinandhumanapolipoproteinAItransgenes.J. Clin. Invest.98:23732380. 34.Kuivenhoven,J.A.,etal.1997.Themolecularpathologyoflecithin:cholesterolacyltransferase(LCAT) deficiencysyndromes.J. Lipid Res.38:191205. 35.Ng,D.S.2004.InsightintotheroleofLCATfrom mousemodels.Rev. Endocr. Metab. Disord.5:311318. 36.Rader,D.J.,etal.1994.Markedlyacceleratedcatabolism of apolipoprotein A-II (ApoA-II) and high densitylipoproteinscontainingApoA-IIinclassic lecithin:cholesterolacyltransferasedeficiencyand fish-eyedisease.J. Clin. Invest.93:321330. 37.Francone,O.L.,Gong,E.L.,Ng,D.S.,Fielding,C.J., andRubin,E.M.1995.Expressionofhumanlecithin-cholesterolacyltransferaseintransgenicmice. EffectofhumanapolipoproteinAIandhuman apolipoproteinallonplasmalipoproteincholesterolmetabolism.J. Clin. Invest.96:14401448. 38.Schwartz,C.C.,VandenBroek,J.M.,andCooper, P.S.2004.Lipoproteincholesterylesterproduction, transfer, and output in vivo in humans. J. Lipid Res.45:15941607. 39.Silver,D.L.,Wang,N.,Xiao,X.,andTall,A.R.2001. Highdensitylipoprotein(HDL)particleuptake mediated by scavenger receptor class B type 1 resultsinselectivesortingofHDLcholesterolfrom proteinandpolarizedcholesterolsecretion.J. Biol. Chem.276:2528725293. 40.Kozarsky,K.F.,etal.1997.Overexpressionofthe HDLreceptorSR-B1altersplasmaHDLandbile cholesterollevels.Nature.387:414417. 41.Rigotti,A.,etal.1997.Atargetedmutationinthe murinegeneencodingthehighdensitylipoprotein (HDL)receptorscavengerreceptorclassBtypeI revealsitskeyroleinHDLmetabolism.Proc. Natl. Acad. Sci. U. S. A.94:1261012615. 42.Brundert,M.,etal.2005.ScavengerreceptorclassB typeImediatestheselectiveuptakeofhigh-density lipoprotein-associatedcholesterylesterbytheliver inmice.Arterioscler. Thromb. Vasc. Biol.25:143148. 43.Zhang,Y.,etal.2005.HepaticexpressionofscavengerreceptorclassBtypeI(SR-BI)isapositiveregulatorofmacrophagereversecholesteroltransport invivo.J. Clin. Invest.115:28702874.doi:10.1172/ JCI25327. 44.Kozarsky,K.F.,Donahee,M.H.,Glick,J.M.,Krieger, M.,andRader,D.J.2000.Genetransferandhepatic overexpressionoftheHDLreceptorSR-BIreduces atherosclerosisinthecholesterol-fedLDLreceptor-deficientmouse.Arterioscler. Thromb. Vasc. Biol.

genetranscription.Mol. Endocrinol.18:23782387. 17.Weng, W., and Breslow, J.L. 1996. Dramatically decreased high density lipoprotein cholesterol, increasedremnantclearance,andinsulinhypersensitivityinapolipoproteinA-IIknockoutmice suggestacomplexroleforapolipoproteinA-IIin atherosclerosissusceptibility.Proc. Natl. Acad. Sci. U. S. A.93:1478814794. 18.Schultz,J.R.,Verstuyft,J.G.,Gong,E.L.,Nichols, A.V.,andRubin,E.M.1993.Proteincomposition determinestheanti-atherogenicpropertiesofHDL intransgenicmice.Nature.365:762764. 19.Warden,C.H.,Hedrick,C.C.,Qiao,J.-H.,Castellani, L.W.,andLusis,A.J.1993.AtherosclerosisintransgenicmiceoverexpressingapolipoproteinA-II.Science.261:469472. 20.Wang,X.,andPaigen,B.2005.GeneticsofvariationinHDLcholesterolinhumansandmice.Circ. Res.96:2742. 21.Ikewaki,K.,Zech,L.A.,Kindt,M.,Brewer,H.B.,Jr., andRader,D.J.1995.ApolipoproteinA-IIproductionrateisamajorfactorregulatingthedistributionofapolipoproteinA-IamongHDLsubclasses LpA-IandLpA-I:A-IIinnormolipidemichumans. Arterioscler.Thromb. Vasc. Biol.15:306312. 22.Rust,S.,etal.1999.Tangierdiseaseiscausedby mutationsinthegeneencodingATP-bindingcassettetransporter1.Nat. Genet.22:352355. 23.Bodzioch,M.,etal.1999.ThegeneencodingATPbindingcassettetransporter1ismutatedinTangierdisease.Nat. Genet.22:347351. 24.Brooks-Wilson,A.,etal.1999.MutationsinABC1 inTangierdiseaseandfamilialhigh-densitylipoproteindeficiency.Nat. Genet.22:336345. 25.McNeish,J.,etal.2000.Highdensitylipoprotein deficiencyandfoamcellaccumulationinmicewith targeteddisruptionofATP-bindingcassettetransporter-1.Proc. Natl. Acad. Sci. U. S. A.97:42454250. 26.Schaefer,E.J.,etal.1978.Metabolismofhigh-densitylipoproteinapolipoproteinsinTangierdisease. N. Engl. J. Med.299:905910. 27.Timmins,J.M.,etal.2005.Targetedinactivation ofhepaticAbca1causesprofoundhypoalphalipoproteinemia and kidney hypercatabolism of apoA-I.J. Clin. Invest.115:13331342.doi:10.1172/ JCI200523915. 28.Brunham,L.R.,etal.2006.IntestinalABCA1directlycontributestoHDLbiogenesisinvivo.J. Clin. Invest.116:10521062.doi:10.1172/JCI27352. 29.Dietschy,J.M.,andTurley,S.D.2002.Controlof cholesterolturnoverinthemouse.J. Biol. Chem. 277:38013804. 30.Haghpassand,M.,Bourassa,P.A.,Francone,O.L., and Aiello, R.J. 2001. Monocyte/macrophage expressionofABCA1hasminimalcontributionto plasmaHDLlevels.J. Clin. Invest.108:13151320. doi:10.1172/JCI200112810. 31.Huuskonen,J.,Olkkonen,V.M.,Jauhiainen,M., andEhnholm,C.2001.Theimpactofphospho-

The Journal of Clinical Investigation http://www.jci.org Volume116 Number12 December2006

science in medicine
20:721727. 45.Arai, T., Wang, N., Bezouevski, M., Welch, C., andTall,A.R.1999.Decreasedatherosclerosisin heterozygous low density lipoprotein receptordeficientmiceexpressingthescavengerreceptorBI transgene.J. Biol. Chem.274:23662371. 46.Ueda, Y., et al. 2000. Relationship between expressionlevelsandatherogenesisinscavenger receptorclassB,typeItransgenics.J. Biol. Chem. 275:2036820373. 47.Trigatti,B.,etal.1999.InfluenceofthehighdensitylipoproteinreceptorSR-BIonreproductiveand cardiovascularpathophysiology.Proc. Natl. Acad. Sci. U. S. A.96:93229327. 48.Braun,A.,etal.2002.LossofSR-BIexpression leadstotheearlyonsetofocclusiveatherosclerotic coronaryarterydisease,spontaneousmyocardial infarctions,severecardiacdysfunction,andprematuredeathinapolipoproteinE-deficientmice. Circ. Res.90:270276. 49.Covey,S.D.,Krieger,M.,Wang,W.,Penman,M.,and Trigatti,B.L.2003.ScavengerreceptorclassBtype I-mediatedprotectionagainstatherosclerosisin LDLreceptor-negativemiceinvolvesitsexpression inbonemarrow-derivedcells.Arterioscler. Thromb. Vasc. Biol.23:15891594. 50.Osgood,D.,etal.2003.Geneticvariationatthe scavengerreceptorclassBtypeIgenelocusdetermines plasma lipoprotein concentrations and particlesizeandinteractswithtype2diabetes: theframinghamstudy.J. Clin. Endocrinol. Metab. 88:28692879. 51.Agellon,L.B.,etal.1991.Reducedhighdensity lipoproteincholesterolinhumancholesterylester transfer protein transgenic mice. J. Biol. Chem. 266:1079610801. 52.Brown, M.L., et al. 1989. Molecular basis of lipidtransferproteindeficiencyinafamilywith increased high-density lipoproteins. Nature. 342:448451. 53.Inazu,A.,etal.1990.Increasedhigh-densitylipoproteinlevelscausedbyacommoncholesteryl-ester transfer protein gene mutation. N. Engl. J. Med. 323:12341238. 54.Ikewaki,K.,etal.1993.Delayedcatabolismofhigh densitylipoproteinapolipoproteinsA-IandA-IIin humancholesterylestertransferproteindeficiency. J. Clin. Invest.92:16501658. 55.DeGrooth,G.J.,etal.2004.AreviewofCETP and its relation to atherosclerosis. J. Lipid Res. 45:19671974. 56.Rader,D.J.2004.Inhibitionofcholesterylester transfer protein activity: a new therapeutic approachtoraisinghigh-densitylipoprotein.Curr. Atheroscler. Rep.6:398405. 57.DeGrooth,G.J.,etal.2002.Efficacyandsafetyof anovelcholesterylestertransferproteininhibitor, JTT-705,inhumans:arandomizedphaseIIdoseresponsestudy.Circulation.105:21592165. 58.Kuivenhoven, J.A., et al. 2005. Effectiveness of inhibitionofcholesterylestertransferproteinby JTT-705incombinationwithpravastatinintypeII dyslipidemia.Am. J. Cardiol.95:10851088. 59.Clark,R.W.,etal.2004.Raisinghigh-densitylipoproteininhumansthroughinhibitionofcholesterylestertransferprotein:aninitialmultidose studyoftorcetrapib.Arterioscler. Thromb. Vasc. Biol. 24:490497. 60.Brousseau,M.E.,etal.2004.Effectsofaninhibitor ofcholesterylestertransferproteinonHDLcholesterol.N. Engl. J. Med.350:15051515. 61.Davidson,M.H.,McKenney,J.M.,Shear,C.L.,and Revkin,J.H.2006.Efficacyandsafetyoftorcetrapib,anovelcholesterylestertransferproteininhibitor,inindividualswithbelow-averagehigh-density lipoproteincholesterollevels.J. Am. Coll. Cardiol. 48:17741781. 62.Davidson,M.H.,etal.2003.ThesafetyandimmunogenicityofaCETPvaccineinhealthyadults. Atherosclerosis.169:113120. 63.Curb,J.D.,etal.2004.AprospectivestudyofHDL-C andcholesterylestertransferproteingenemutationsandtheriskofcoronaryheartdiseaseinthe elderly.J. Lipid Res.45:948953. 64.Boekholdt,S.M.,andThompson,J.F.2003.Naturalgeneticvariationasatoolinunderstandingthe roleofCETPinlipidlevelsanddisease.J. Lipid Res. 44:10801093. 65.Boekholdt,S.M.,etal.2004.Plasmalevelsofcholesterylestertransferproteinandtheriskoffuturecoronaryarterydiseaseinapparentlyhealthymenand women:theprospectiveEPIC(EuropeanProspective InvestigationintoCancerandnutrition)-Norfolk populationstudy.Circulation.110:14181423. 66.Brousseau,M.E.,etal.2005.Effectsofcholesteryl estertransferproteininhibitiononhigh-density lipoproteinsubspecies,apolipoproteinA-Imetabolism,andfecalsterolexcretion.Arterioscler. Thromb. Vasc. Biol.25:10571064. 67.Ishigami, M., et al. 1994. Large and cholesteryl ester-richhigh-densitylipoproteinsincholesteryl estertransferprotein(CETP)deficiencycannot protectmacrophagesfromcholesterolaccumulationinducedbyacetylatedlow-densitylipoproteins.J. Biochem. (Tokyo).116:257262. 68.Matsuura,F.,Wang,N.,Chen,W.,Jiang,X.C.,and Tall,A.R.2006.HDLfromCETP-deficientsubjects shows enhanced ability to promote cholesterol effluxfrommacrophagesinanapoE-andABCG1dependentpathway.J. Clin. Invest.116:14351442. doi:10.1172/JCI27602. 69.Bays,H.,McKenney,J.,andDavidson,M.2005. Torcetrapib/atorvastatin combination therapy. Expert Rev. Cardiovasc. Ther.3:789820. 70.Glass, C., Pittman, R.C., Weinstein, D.B., and Steinberg,D.1983.Dissociationoftissueuptake ofcholesterolesterfromthatofapoproteinA-Iof ratplasmahighdensitylipoprotein:selectivedeliveryofcholesterolestertoliver,adrenal,andgonad. Proc. Natl. Acad. Sci. U. S. A.80:54355439. 71.Moestrup,S.K.,andNielsen,L.B.2005.Theroleof thekidneyinlipidmetabolism.Curr. Opin. Lipidol. 16:301306. 72.Hammad,S.M.,etal.1999.Cubilin,theendocytic receptorforintrinsicfactor-vitaminB12complex, mediates high-density lipoprotein holoparticleendocytosis.Proc. Natl. Acad. Sci. U. S. A. 96:1015810163. 73.Kozyraki, R., et al. 1999. The intrinsic factorvitaminB12receptor,cubilin,isahigh-affinity apolipoproteinA-Ireceptorfacilitatingendocytosis ofhigh-densitylipoprotein.Nat. Med.5:656661. 74.Hammad,S.M.,Barth,J.L.,Knaak,C.,andArgraves, W.S.2000.Megalinactsinconcertwithcubilinto mediateendocytosisofhighdensitylipoproteins. J. Biol. Chem.275:1200312008. 75.Christensen,E.I.,andGburek,J.2004.Proteinreabsorptioninrenalproximaltubule-functionand dysfunctioninkidneypathophysiology.Pediatr. Nephrol.19:714721. 76.Hayek,T.,etal.1992.Aninteractionbetweenthe humancholesterylestertransferprotein(CETP) andapolipoproteinA-Igenesintransgenicmice resultsinaprofoundCETP-mediateddepression of high density lipoprotein cholesterol levels. J. Clin. Invest.90:505510. 77.Webb,N.R.,etal.2002.OverexpressionofSR-BI byadenoviralvectorpromotesclearanceofapoA-I, butnotapoB,inhumanapoBtransgenicmice. J. Lipid Res.43:14211428. 78.Lamarche, B., et al. 1999. Triglyceride enrichmentofHDLenhancesinvivometabolicclearanceofHDLapoA-Iinhealthymen.J. Clin. Invest. 103:11911199. 79.Jaye,M.,etal.1999.Anovelendothelial-derived lipasethatmodulatesHDLmetabolism.Nat. Genet. 21:424428. 80.Hirata,K.,etal.1999.Cloningofauniquelipase fromendothelialcellsextendsthelipasegenefamily.J. Biol. Chem.274:1417014175. 81.Ishida,T.,etal.2003.Endotheliallipaseisamajor determinantofHDLlevel.J. Clin. Invest.111:347355. doi:10.1172/JCI200316306. 82.Maugeais,C.,etal.2003.Dose-dependentaccelerationofhigh-densitylipoproteincatabolismby endotheliallipase.Circulation.108:21212126. 83.Jin,W.,Millar,J.S.,Broedl,U.,Glick,J.M.,andRader, D.J.2003.Inhibitionofendotheliallipasecauses increasedHDLcholesterollevelsinvivo.J. Clin. Invest.111:357362.doi:10.1172/JCI200316146. 84.Ma,K.,etal.2003.Endotheliallipaseisamajor geneticdeterminantforhigh-densitylipoprotein concentration,structure,andmetabolism.Proc. Natl. Acad. Sci. U. S. A.100:27482753. 85.deLemos,A.S.,Wolfe,M.L.,Long,C.J.,Sivapackianathan,R.,andRader,D.J.2002.Identification ofgeneticvariantsinendotheliallipaseinpersons withelevatedhigh-densitylipoproteincholesterol. Circulation.106:13211326. 86.Badellino,K.O.,Wolfe,M.L.,Reilly,M.P.,andRader, D.J.2006.Endotheliallipaseconcentrationsare increasedinmetabolicsyndromeandassociated withcoronaryatherosclerosis.PLoS Med.3:e22. 87.Ishida,T.,etal.2004.Endotheliallipasemodulates susceptibilitytoatherosclerosisinapolipoproteinE-deficientmice.J. Biol. Chem.279:4508545092. 88.Ko,K.W.,Paul,A.,Ma,K.,Li,L.,andChan,L.2005. Endothelial lipase modulates HDL but has no effectonatherosclerosisdevelopmentinapoE/ andLDLR/mice.J. Lipid Res.46:25862594. 89.Mahley,R.W.,Huang,Y.,andWeisgraber,K.H.2006. Puttingcholesterolinitsplace:apoEandreverse cholesteroltransport.J. Clin. Invest.116:12261229. doi:10.1172/JCI28632. 90.Fidge,N.H.1999.Highdensitylipoproteinreceptors, binding proteins, and ligands. J. Lipid Res. 40:187201. 91.Martinez,L.O.,etal.2003.Ectopicbeta-chainof ATPsynthaseisanapolipoproteinA-Ireceptorin hepaticHDLendocytosis.Nature.421:7579. 92.Jacquet,S.,etal.2005.Thenucleotidereceptor P2Y13isakeyregulatorofhepatichigh-density lipoprotein(HDL)endocytosis.Cell. Mol. Life Sci. 62:25082515. 93.Carlson,L.A.2005.Nicotinicacid:thebroad-spectrumlipiddrug.A50thanniversaryreview.J. Intern. Med.258:94114. 94.Meyers,C.D.,Kamanna,V.S.,andKashyap,M.L. 2004.Niacintherapyinatherosclerosis.Curr. Opin. Lipidol.15:659665. 95.Tunaru,S.,etal.2003.PUMA-GandHM74are receptorsfornicotinicacidandmediateitsanti- lipolyticeffect.Nat. Med.9:352355. 96.Cuchel, M., and Rader, D.J. 2006. Macrophage reversecholesteroltransport:keytotheregression ofatherosclerosis?Circulation.113:25482555. 97.VanEck,M.,etal.2006.MacrophageATP-bindingcassettetransporterA1overexpressioninhibits atheroscleroticlesionprogressioninlow-density lipoproteinreceptorknockoutmice.Arterioscler. Thromb. Vasc. Biol.26:929934. 98.Repa,J.J.,andMangelsdorf,D.J.2002.TheliverX receptorgeneteam:potentialnewplayersinatherosclerosis.Nat. Med. 8:12431248. 99.Wang,N.,Lan,D.,Chen,W.,Matsuura,F.,andTall, A.R.2004.ATP-bindingcassettetransportersG1 andG4mediatecellularcholesteroleffluxtohighdensitylipoproteins.Proc. Natl. Acad. Sci. U. S. A. 101:97749779. 1 00.Kennedy,M.A.,etal.2005.ABCG1hasacritical roleinmediatingcholesteroleffluxtoHDLand preventingcellularlipidaccumulation.Cell Metab. 1:121131. 1 01.Wang,N.,Ranalletta,M.,Matsuura,F.,Peng,F.,

The Journal of Clinical Investigation http://www.jci.org Volume116 Number12 December2006

3099

science in medicine
andTall,A.R.2006.LXR-inducedredistribution ofABCG1toplasmamembraneinmacrophages enhancescholesterolmasseffluxtoHDL.Arterioscler. Thromb. Vasc. Biol.26:13101316. 1 02.Ranalletta,M.,etal.2006.Decreasedatherosclerosisinlow-densitylipoproteinreceptorknockout micetransplantedwithAbcg1/bonemarrow. Arterioscler. Thromb. Vasc. Biol.26:23082315. 1 03.Baldan,A.,etal.2006.ImpaireddevelopmentofatherosclerosisinhyperlipidemicLdlr/andApoE/ micetransplantedwithAbcg1/bonemarrow. Arterioscler. Thromb. Vasc. Biol.26:23012307. 1 04.Naik,S.U.,etal.2006.Pharmacologicalactivation ofliverXreceptorspromotesreversecholesterol transportinvivo.Circulation.113:9097. 1 05.Terasaka,N.,etal.2003.T-0901317,asynthetic liverXreceptorligand,inhibitsdevelopmentof atherosclerosis in LDL receptor-deficient mice. FEBS Lett.536:611. 1 06.Joseph, S.B., et al. 2002. Synthetic LXR ligand inhibits the development of atherosclerosis in mice.Proc. Natl. Acad. Sci. U. S. A.99:76047609. 1 07.Levin,N.,etal.2005.MacrophageliverXreceptor isrequiredforantiatherogenicactivityofLXRagonists.Arterioscler. Thromb. Vasc. Biol.25:135142. 1 08.Li,A.C.,andGlass,C.K.2004.PPAR-andLXRdependentpathwayscontrollinglipidmetabolism andthedevelopmentofatherosclerosis.J. Lipid Res. 45:21612173. 1 G 09. root, P.H., et al. 2005. Synthetic LXR agonistsincreaseLDLinCETPspecies.J. Lipid Res. 46:21822191. 1 10.Chawla,A.,etal.2001.APPARgamma-LXR-ABCA1 pathwayinmacrophagesisinvolvedincholesterol effluxandatherogenesis.Mol. Cell.7:161171. 1 11.Chinetti,G.,etal.2001.PPAR-alphaandPPARgammaactivatorsinducecholesterolremovalfrom humanmacrophagefoamcellsthroughstimulationoftheABCA1pathway.Nat. Med.7:5358. 1 L 12. i, A.C., et al. 2004. Differential inhibition of macrophagefoam-cellformationandatherosclerosisinmicebyPPAR,/,and.J. Clin. Invest. 114:15641576.doi:10.1172/JCI200418730. 1 13.Oliver, W.R., Jr., et al. 2001. A selective peroxisomeproliferator-activatedreceptordeltaagonist promotesreversecholesteroltransport.Proc. Natl. Acad. Sci. U. S. A.98:53065311. 1 14.Rothblat,G.H.,etal.1999.Cellcholesterolefflux: integrationofoldandnewobservationsprovides newinsights.J. Lipid Res.40:781796. 1 15.Zhang,W.,etal.2003.Inactivationofmacrophage scavengerreceptorclassBtypeIpromotesatheroscleroticlesiondevelopmentinapolipoproteinEdeficientmice.Circulation.108:22582263. 1 16.Nanje,M.N.,etal.1996.Effectsofintravenous infusionoflipid-freeapoA-Iinhumans.Arterioscler. Thromb. Vasc. Biol.16:12031214. 1 17.Eriksson,M.,Carlson,L.A.,Miettinen,T.A.,and Angelin,B.1999.StimulationoffecalsteroidexcretionafterinfusionofrecombinantproapolipoproteinA-I:potentialreversecholesteroltransportin humans.Circulation.100:594598. 1 18.Nissen, S.E., et al. 2003. Effect of recombinant ApoA-I Milano on coronary atherosclerosis in patientswithacutecoronarysyndromes:arandomizedcontrolledtrial.JAMA.290:22922300. 1 19.Rader,D.J.2003.High-densitylipoproteinsasan emergingtherapeutictargetforatherosclerosis. JAMA.290:23222324. 1 20.Brewer,H.B.,Jr.2004.Focusonhighdensitylipo proteinsinreducingcardiovascularrisk.Am. Heart J. 148(Suppl.):S14S18. 1 21.Navab,M.,Anantharamaiah,G.M.,Reddy,S.T.,and Fogelman,A.M.2006.ApolipoproteinA-Imimetic peptidesandtheirroleinatherosclerosisprevention.Nat. Clin. Pract. Cardiovasc. Med.3:540547. 1 22.Garber,D.W.,etal.2001.AnewsyntheticclassA amphipathicpeptideanalogueprotectsmicefrom diet-inducedatherosclerosis.J. Lipid Res.42:545552. 1 23.Navab, M., et al. 2002. Oral administration of an Apo A-I mimetic peptide synthesized from D-aminoacidsdramaticallyreducesatherosclerosis inmiceindependentofplasmacholesterol.Circulation.105:290292. 1 24.Navab,M.,etal.2004.OralD-4Fcausesformation ofpre-betahigh-densitylipoproteinandimproves high-density lipoprotein-mediated cholesterol effluxandreversecholesteroltransportfrommacrophagesinapolipoproteinE-nullmice.Circulation. 109:32153220.

3100

The Journal of Clinical Investigation http://www.jci.org Volume116 Number12 December2006