Role of oxidized low density lipoprotein in atherogenesis

Role of oxidized low density lipoprotein in

atherogenesis.

J L Witztum, D Steinberg

J Clin Invest. 1991;88(6):1785-1792. https://doi.org/10.1172/JCI115499.

Evidence to support an important role of oxidative modification in mediating the

atherogenicity of LDL continues to grow. New hypotheses suggest mechanisms by which Ox-LDL or products of Ox-LDL can affect many components of the atherogenic process, including vasomotor properties and thrombosis, as well as lesion initiation and progression itself. These ideas suggest new approaches, that in combination with lowering of plasma cholesterol, could lead to the prevention of atherosclerosis and its complications.

Research Article

Perspectives

Role of

Oxidized

Low

Density Lipoprotein

in

Atherogenesis

Joseph L. Witztum and Daniel Steinberg

DivisionofEndocrinology and Metabolism, DepartmentofMedicine, UniversityofCalifornia, SanDiego,LaJolla, California 92093

Overview

of

the concept

of

oxidative

modification

ofLDL

There can be no doubt now that thereisacontinuum of

in-creasingrisk forcomplications of atherosclerosiswhen plasma cholesterol levels exceed 160-180mg/dl. Manytypesof

ex-perimentalandclinical evidence substantiatethe "cholesterol

hypothesis." Manyprimary and secondaryprevention trials, includingthe recentangiographic trials (CLAS and FATS) doc-ument that reduction of plasmacholesterolisaspowerful as hasbeen predictedinslowingtheprogression and clinical

ex-pression of coronary atherosclerosis. However,thecellularand molecular mechanisms linking hypercholesterolemia to

ath-erogenesisand itssequelaeremain unclear.

Iflowering ofLDLisefficacious in amelioratingthe

athero-genicprocess, why then shouldonebotherto understand the mechanisms? SimplybecauseloweringLDLwillnotbe a total

solution. Although itmay be truethat if cholesterollevels were reduced to < 150 mg/dl there would be littleifanycoronary

artery disease(CAD),' it isnotlikely that this will occur any

timesoon. At anygiven level ofLDLthereisgreatvariability in theclinical expression ofthedisease. Patients with heterozy-gousfamilial hypercholesterolemia exhibitastriking diversity in theage atwhichthey develop CAD, and even patientswith homozygous familial hypercholesterolemia, inwhom plasma

cholesterollevelsexceed 800 mg/dl,maydiffer byasmuch as 30 yr in the age at which CAD is expressed clinically. This striking diversity in the expression of CAD undoubtedly

re-flects the complex and multifactoral events involved in the

reactions of the artery tohypercholesterolemia that result in

atherosclerosis. In other words, there must be intervening

eventslinkinghypercholesterolemiatomorbidityand

mortal-ity. Whilethediscussionbelowwillfocuson the role that

oxi-dative modification ofLDLplays inthis process,otherfactors

mayalsoplayacrucial, perhaps even adominantrole in the

ultimate expression of CAD inanyindividual.

Theearlyfattystreakistheprecursorlesionwhich

subse-quently leadstodevelopment oftheintermediateandthefinal complicated lesion of atherosclerosis. Consequently,much

at-Address correspondencetoJoseph L.Witztum,M.D., UCSD Depart-mentofMedicine 0613-D,9500GilmanDrive, La Jolla, CA

92093-0613.

Receivedfor publication 19 June 1991.

1.Abbreviations used in this paper: CAD, coronary artery disease; EDRF,endothelial-dependent relaxing factor; MDA-LDL,

malodial-dehyde-conjugated LDL; Ox-LDL, oxidizedLDL.

tention is nowfocusedon understandingtheetiology of the

fattystreakandthe mechanisms bywhichmonocyte-derived macrophages,thechief cell type ofthislesion,accumulate cho-lesterol from LDL(1).

The concept that modification of LDL isaprerequisitefor

macrophageuptake and cellular accumulation of cholesterol

has been reviewedindetail elsewhere (2).ItwasGoldsteinand Brown who firstproposedthat modification of LDLwas a

pre-requisiteformacrophage uptake (3). Theyshowed that a chemi-cal derivatization, acetylation, led to enhanced macrophage

uptakebyanovel receptor, termed the "acetyl LDL" receptor

(to distinguishit from the LDLreceptor).Othersimilarly

chem-icallymodifiedforms of LDL such as malondialdehyde-conju-gatedLDL(MDA-LDL)wererecognizedbythe samereceptor (4). Studies in this laboratory showed that incubation of LDL with cultured endothelialcells, or smooth muscle cells,

con-verted it to a form taken up much more rapidly and in a

satura-blemannerby macrophages,in partbyway of theacetylLDL receptor (5). Subsequently, the modification induced was

shown to be due to oxidation (6), a finding confirmed for smooth muscle cell-induced modification as well (7). In im-portantparallel studies, it was shown that LDL was cytotoxic

toendothelial cells and smooth muscle cells and that the cyto-toxicitydepended on oxidation of the LDL during the incuba-tion (8). Many studies have now documented that oxided LDL

(Ox-LDL), produced by a variety of different techniques, shows enhanced uptake inmacrophages,and can lead to cho-lesteryl ester accumulation and foam cell formation (1, 2). Al-though it is beyond the scope of this review, it should be noted that a number of other modifications of LDL convert it to forms taken up more rapidly by macrophages. These include: other types of chemical modifications, self-aggregation, com-plex formation with avariety of other macromolecules, includ-ingproteoglycansand various matrix proteins, and immune complex formationleadingtomacrophage uptakeviaFc recep-torpathways (see reference 2).

What is Ox-LDL?

Oxidation ofLDL canbeinducedbyincubationwith cellsin

culture (endothelial cells, smooth muscle cells, or

macro-phages),or byincubationwith a heavy metal ion such as cop-per(Fig. 1). Inaddition, LDL can be oxidized byincubation

with crude soybeanlipoxygenase(9).Several lines of evidence suggest that oxidation inducedbyendothelialcells(10) or by

macrophages(11) dependsonlipoperoxides generated

intracel-lularlyandthentransferredtothe LDL. Cellularlipoxygenases,

especially 15-lipoxygenase,appear to beinvolved(10, 1 1).

Al-ternatively,reactive oxygenspecies,such assuperoxideanion, maybesecretedinto the medium,leading toinitiationoflipid

peroxidation inthe LDL. Thismechanism maypredominate

in smooth muscle cells (7). Once the LDL is "seeded" with

J.Clin. Invest.

©TheAmericanSocietyforClinical Investigation,Inc.

0021-9738/91/12/1785/08 $2.00

OPERATIONAL METHODS

* ANTIOXIDANTSINLDL

(Vit.E,_0-carotene,etc.)

CHEMILUMINESCENCE; IODOMETRY

(methodsmay notbe sensitive enoughatthis stage)

4ABSORBANCE AT 234nm

4 CHEMILUMINESCENCE 4PEROXIDES BY IODOMETRY

$ POLYUNSATURATED

FATTYACID CONTENT

4FLUORESCENCE

4TBARS

4IMMUNOREACTIVITY

(e.g. anti-MDA-LDL)

*RATEOF DEGRADATION

BYFIBROBLASTS

4RATEOFDEGRADATION

BYMACROPHAGES

CHEMICAL EVENTS

Cu++orcells

\.. Lipoperoxides

02,H202,OH NATIVE from cells LDL

INITIATION

"SEEDED"LDL (containinglimited number oflipoperoxides)

PROPAGATION | (phospholipase A2

+ activity)

AMPLIFIEDNUMBEROF LIPOPEROXIDESINLDL,

REARRANGEMENT OF DOUBLEBONDS DECOMPOSITION

ALDEHYDES,KETONES FROMFATTYACIDFRAGMENTATION

CONJUGATIONOFALDEHYDES TO apoBandPHOSPHOLIPIDS

~1

LDLNOLONGER RECOGNIZED

BY NATIVE LDL RECEPTOR

LDLRECOGNIZEDBY

SCAVENGER RECEPTORS ("BIOLOGICALLY MODIFIEDLDL")

Figure1.

lipoperoxides, copper ion in the medium will then generate peroxyradicals, leadingto achain reaction whichresults in an amplifiednumberoflipoperoxides,accompanied by

rearrange-mentof thefattyacid double bonds,yieldingthecharacteristic 234nmabsorptionof"conjugateddienes."Oxidative modifi-cation ofLDLinvitro isabsolutely dependentonlow

concen-trations ofcopper

ion;

itiscompletelyinhibited by metal

chela-torssuchasEDTA.Duringoxidative modification there is

ex-tensive conversion of lecithin to lysolecithin catalyzed by a phospholipaseA2activitypresent intheLDLandapparently intrinsic to apo B itself(12). Inhibitors ofphospholipase A2

block both thegenerationoflysolecithinand thepropagation

of

lipid peroxides,

and thuspreventoxidative modification of

LDLcompletely (2). Consequenttothepropagationreactions

fatty acid fragmentation occurs, leadingtothe formation of

highlyreactiveintermediates, suchasaldehydesandketones, whichcanthencomplex with theadjacentapo B(13),aswellas

withphospholipids. Thelysineresidues ofapo B arerequired for theinteraction ofLDLwith theLDL receptor. As

increas-ing

numbersoflysineresiduesarederivatized by the

fragmen-tation

products,

LDL

recognition

bythe LDL receptoris de-creased. Haberlandetal.(14)showedthatacriticalnumberof

lysine residues must be conjugated with MDA before the

MDA-LDLcanbe

recognized by

theacetylLDL receptoron

the

macrophage. Presumably

acritical numberof

lysine

resi-dues mustalso reactwith the various

aldehydic

fragments

of

oxidized

fatty

acidstogeneratethe

epitope(s)

onOx-LDL that

reactwith the

acetyl-LDL

receptor

(and possibly

other

recep-tors) on macrophages. Parthasarathy et al. (15) have shown

that it isamodifiedfragment ofapoproteinBthat isrecognized

by the macrophage acetyl LDL receptor.

Thechemical events accompanying oxidation of LDL are schematized inFig. 1 in what might be called "a probable

se-quence" but itismorelikelythatthesecomplex reactions

oc-curinavariable andevenchaoticmanner.Thereareprobably

manyfactors thatinfluencethe events resultingin amodified LDLrecognized by the scavenger receptors. It should be clear that Ox-LDL is not asingle, homogeneous entity. There are many oxidation products that form from both the peroxidation andfragmentation of the lipidcomponents ofLDL, andthe

modificationandoxidative degradation ofapoprotein B. The rapid propagation phase amplifies in a dramatic fashion the numberof oxygen and carbon centered free radicals formed,

resultingin alterations in all of the components of LDL,

in-cludingsterols,fatty acids, phospholipids, and theapoprotein

Bitself.

Inpractical terms, there is considerable variability in prod-uctsformed from preparation to preparation even when efforts are made to hold conditions constant. These differences may depend on the cell type used toinitiatetheoxidation,themetal

ion concentration in the medium, the composition of the me-diumitself, the incubation conditions, andofcourse,

differ-encesin the inherent susceptibility to modification of different LDLpreparations (asdiscussed further below). Many

investi-gators are now oxidizing LDL and using them inbiological

studies, in cellculture or in vivo. The variabilityinsuch prepa-rations must be appreciated and thefact that varying

"opera-tional methods" to measure the extent of LDL modification may reflect different chemical events, or stages ofoxidative

modification. In Fig. 1 we have also listed the operational methods in common use for following LDL oxidation. For example, it is likely that decreases in vitamin E and,8-carotene areearly events reflecting the initial stages of lipid peroxida-tion. Increasedabsorbance at 234 nm (indicative ofconjugated

dienes), increasedchemiluminescence and increased peroxides

determinedby iodometry are all indicative of lipoperoxide

for-mationand the accompanying rearrangement ofdouble bonds. Asfatty acidsare oxidized,the content of intact polyunsatu-ratedfatty acidsdecreases and asconjugation of aldehyde frag-ments to apo B occurs,immunoreactivitywithantibodies

spe-cific forsuchadducts can then be found. As lysine residues are blocked, LDL uptake by fibroblasts is decreased and finally, as

the epitopes on Ox-LDL that are recognized by the macro-phage receptors areformed,enhanced uptakeoftheOx-LDL

by macrophageswilloccur.Thus, a commonly usedlaboratory

technique, suchas measurement of TBARS, will be a reason-able(albeit nonspecific) indication of lipid peroxidation, yet notnecessarilybeindicative of increasedmacrophage uptake. Inthis laboratory we haveoperationally defined "biologically modifiedLDL" as anOx-LDL thatis takenup via the scaven-ger receptors on macrophages. However, as seen in Fig. 1, many changesinthe LDLparticlemay haveoccurred before

reaching thatstage and thosechangesmay haveimportant

bio-logical consequences. Some laboratorieshave used the term

"minimally modified LDL" todesignateapartiallyOx-LDL madeeitherbyallowing nativeLDLto agefor months,orby

incubatingLDL inthe presenceof iron forvarying periodsof

time (16). Such partiallyoxidized LDLparticleshave anumber

with specificcellsin culture(as notedbelow). Itislikelythat one or moresoluble, oxidized products have been created by suchapartial oxidation.

Evidencefor the presencein vivoofOx-LDL

Theevidence for the presence in vivo of Ox-LDL hasbeen reviewed elsewhere insomedetail and willnotbeelaborated extensively (1, 2).Itshould be noted thattodate theevidence

forthe presenceof Ox-LDL is confinedtoatherosclerotic

tis-sue. Immunocytochemical techniques have demonstratedthe presence inarteriallesions, butnotinnormalarteries, of

epi-topes foundin Ox-LDL. LDL extracted from arterial lesions

resemblesinvitro Ox-LDL withrespect tophysical properties, immunoreactivity with antibodiestoepitopes of Ox-LDLand byits enhanced uptake in macrophages mediated byone or more scavenger receptor pathways. Finally, convincing evi-dencefortherole ofoxidative modification in lesion formation

comes from studies demonstrating thatantioxidants such as

probucol(17, 18)orbutylatedhydroxytoluene (19)caninhibit lesionformation inWHHLrabbits and cholesterol-fed rabbits, respectively. In the case of the probucolexperiments, itwas specificallydemonstrated thatprobucol inhibited the uptake anddegradationof LDL inmacrophage-rich lesions,whilenot

inhibiting such uptake in normalarterialtissue(17). Recently,

aprobucolanalogue that retains its antioxidantactivitybut has

noeffectonplasmacholesterol levelswasalso showntohave prominentantiatherogenic potential inagenetically hypercho-lesterolemicrabbit(20).

We have assumed that theoxidative modificationoccurs

primarily intheintima, in microdomainssequesteredfromthe manyplasmaantioxidants. Wehavereasoned thatsignificant degrees of oxidative modification ofLDL do nottake place in plasmabecauseof its high antioxidant content. Furthermore, our invivo studies inguinea pigsshowed that even veryslight degrees of oxidation ofLDL(notsufficienttoincrease in vitro uptake in macrophages) nevertheless led to more rapid

re-movalof theLDLfrom plasma, presumably byscavenger re-ceptors present on sinusoidal cellsoftheliver(2). However, plasma LDL could undergolimiteddegreesofoxidation, that would have no consequences while still in the vascular space; yet, when such LDL entered the intima, it might then be "primed" formorerapid oxidative modification mediated by cells. Infact,there are numerous reportsintheliterature

sug-gesting differences inplasma LDL that could beattributableto

such early changes of oxidation. For example, LDLisolated

fromdiabetic rats showed enhanced TBARS and cytotoxicity

TableI.Potential Mechanisms by which Ox-LDL May BeAtherogenic

1. Ithasenhanced_{uptake by macrophagesleading}to_cholesterylester

enrichment.

2. It is chemotactic for_circulatingmonocytes.

3. Itinhibits the_motilityof tissue_macrophages.

4. Itis_cytotoxic.

5. Itcan_{alter geneexpression}of_neighboringcells suchasinduction of MCP-Iand

colony-stimulating

factors.

6. It is

_immunogenic

andcanelicit_autoantibodyformation. 7. Itcan_adverselyalter_{coagulation pathways.}

8. Itcanadverselyaltervasomotorpropertiesof coronary arteries.

(21) and others have reportedthe isolation of a small

subfrac-tion of LDL withadifferent charge (22). Severalgroups have

reportedthat LDL from smokers is moresusceptibleto

oxida-tive modification and that this could be prevented byvitamin C(23).If one could measure suchearly changesinplasmait

mighteven predict which LDLwere more atriskandmight

proveuseful as anepidemiologic and clinical tooltopredict

differingrisks for oxidative modification. It will be ofconsider-able interesttodetermine ifanyof the"operationalmethods"

noted inFig. 1 to measurechanges in LDL during the oxidative

process canbe usedtoreliably distinguishthe LDL of popula-tions withcoronary artery disease or thoseat increased risk for it.

Potential mechanisms

by

whichOx-LDL

maybeatherogenic

Interest instudyingOx-LDL stemmedoriginallyfrom observa-tions that this modification of LDL ledtoits enhanced uptake inmacrophages. However, the ability of Ox-LDLto cause cho-lesteryl esteraccumulation may inpart also berelated toan inabilityof macrophagestodegrade Ox-LDLasreadilyas

na-tive LDL, resultingin the accumulation ofundegraded Ox-LDL(24).Therecentdemonstration that arterial wall

macro-phages, isolated from cholesterol-fed rabbits, can contain as

muchas600

.g

ofcholesterolper mgcellproteinand that they also stain forepitopesofOx-LDLevenafter being in culture for severaldaysis consistent with thissuggestion (25).Inaddition,

the various oxidized products, suchasoxidized sterols, may

interfere witha variety ofintracellularenzymes(26, 27), for

example, inhibitingthe normal cellularprocessing of ingested

lipidsandproteins,andpossiblyevenleadingtothe accumula-tion within the cellof toxic intermediates. It isimportantfor futurestudiestoinvestigateindetailtheconsequences to the macrophage of uptake of Ox-LDL.

The residence time ofLDL in theintima appears to be selectivelyincreasedatcertainlesion-susceptiblesiteseven be-fore lesion formation (28). ByincreasingthetimeLDLis

ex-posedtopro-oxidant conditions,this might be enoughto

initi-ateoxidative modificationyet notleadtothestrikingchanges that result in macrophage uptake. Nevertheless, such mini-mally modified (oxidized) LDL differs fromnative LDL in a

numberof additionalwaysthat makeitpotentially more ath-erogenic(TableI).Itis chemotactic for circulatingmonocytes,

which is attributable inpart to the lysolecithin generated dur-ing the oxidation ofLDL (2). Thus, as minimally Ox-LDL

accumulates ina localized subintimal regionof the artery, it mayrecruitfurthermonocytesintothat same area. In turn, the

further accumulation ofmonocytes and their differentiation

intotissue macrophages, which can oxidize LDL, leads to a viciouscycle, generatingever more Ox-LDL and the macro-phagesresponsible forLDLoxidationand uptake. Presumably

thenormalfunctionofatissuemacrophage, after it has

accu-mulatedlocalized debris,would be to exit the given tissue space

andthus help remodelthe damaged area. However, Ox-LDL actuallyinhibits the motility of tissuemacrophages, thus lead-ingtotheir "trapping"within the intima (2). In addition, Ox-LDLiscytotoxicto avarietyofcells in culture (8). In the artery

wall, localizedaccumulations of Ox-LDL may play a role in disrupting a variety ofcellular processes, including macro-phage motility.Asnoted above, during the unregulated

includ-ingpolar sterols, are generated and some of these are likely to

be agentsthat mediate this cytotoxicity (8). In experimental

atherosclerosis caused by cholesterol feeding the initial

accu-mulation offoam cells occurs under an intact endothelium.

Subsequently, the endotheliummay be damaged and loss of

endotheliummaythenoccur. However, even before frank cell

death one could imagine that normal endothelial function could be disrupted. For example, minimally modified (oxi-dized)LDLhas been showntoinduce expressionon

endothe-lialcells ofalteredsurface moleculesthat could also play an

importantroleinmediatingtheinflux into the artery of mono-cytes(16).

Aresearch area ofpotentially great importance has been openedrecently bythedemonstrationthat Ox-LDL may alter geneexpression in arterialwall cells. For example, Ox-LDL has

been reported to inhibit the expression and production of

PDGFby culturedbovine aorticendothelial cells (29), as well astoinhibittheexpressionof the PDGF-B chainandsecretion by cultured human monocyte-derived macrophages (30).

Also, Ox-LDL inhibits theexpression ofTNF-a mRNAin

mu-rineperitoneal macrophages (31).Incontrast,minimally Ox-LDLstimulatestheexpressionandsecretion ofM-CSF,

GM-CSF, and G-CSF by human aortic endothelialcells(32), and wheninjected in vivo in mice,caused a 7- to 26-fold increase in serum M-CSF activity (33). Of particularrelevance was the

demonstration that incubation of minimally Ox-LDL with rabbit (or human) endothelial cells stimulatedthe adherenceof

monocytes totheendothelium, presumablythroughincreased

expressionofmonocyte-specificadherence molecules(16).

Fur-thermore,minimallyOx-LDL stimulatedtranscriptionand

se-cretion ofmonocyte chemotactic protein-l (MCP-1)by cul-tured humanaortic endothelial and smooth muscle cells (34) and wheninjectedinvivoinmice,increasedmRNA expres-sion ofJE(themousehomologue ofMCP- 1) in liver and other

tissues (33).Usinginsituhybridization, wedemonstrated the

presence of

MCP-l

mRNA in macrophage-rich regions of

plaques in both rabbit and humanaortas(butnotin normal aortas) and demonstrated by Northern blottingthatthere is

significant expression of MCP-1 mRNA in

macrophage-de-rived foam cells isolatedfromballoonedaortasof cholesterol-fedrabbits(35). Thus, it islikelythatOx-LDLmayhave pro-foundeffectsontheabilityof the cells within thearterywallto

produce various potent chemotactic factors, cytokines, and growthfactorsthatplayanimportantrole in thedevelopment andprogressionof theatherosclerotic plaque. Currentlywedo

notknowwhich ofthemanydifferent products generated

dur-ingtheoxidation ofLDLisresponsiblefor thesevarying effects

andthis will beanimportantareaof future research.

Wehavepreviously demonstrated thatavariety of

autolo-gous modifications of LDL render it immunogenic. This provedtobe thecasealso withchanges thatoccurwith oxida-tivemodification. Antibodiestoepitopesof Ox-LDL,suchas

MDA-lysineand4-hydroxynonenallysine,werereadily gener-atedbyimmunizinganimals with their autologousLDL modi-fied byMDAor4-HNE,respectively (36). Similarly,when

au-tologous Ox-LDLwasusedastheimmunogen, antiserawere

generatedthat reactedwiththe

modified,

butnot

native,

LDL (36). These various antibodies have been used in studies to

demonstrate thepresenceofOx-LDL in

vivo,

asnoted above. Furthermore, antibodiesagainst Ox-LDL,orepitopespresent

inOx-LDL,have been demonstrated in bothhumanand

rab-bitsera(37, 38).

Preliminary

data suggest that

antigen-anti-bodycomplexes

involving

suchantibodiesandoxidizedLDL

exist withinplaquesof the artery wall

(S.

Yla-Herttuala and

J. L.Witztum, unpublished data).Itis

likely

thata

variety

of modifications ofproteinstructure occurwithin thearterywall,

inpartmediatedbyoxidative modification.Forexample,

gen-eration ofhighlyreactiveMDA and4-HNE, mayleadto ad-duct formation with matrix

proteins

and cellular

proteins

as

wellaswithapo B. Thesemodifiedproteinsmayalso be

im-munogenicandform immunecomplexeswithspecific antibod-ies. In

addition,

terminalC5b-9 complement complexeshave beenobserved in theplaque

(39),

consistent with

complement

activation,which could be the result of

antigen-antibody

com-plexes.Complement activationmayinitiate and further

am-plify inflammatory responses,becauseproducts generated by

thispathwayareknowntopromoteleukocytechemotaxis and adhesiontoendothelial cells.In

addition, macrophage uptake

of such immunecomplexes

containing

modifiedLDLwould lead to further deposition of cholesterol within the

macro-phage.

Severalgroupshavealso

reported

thatOx-LDLcan stimu-lateplateletaggregation (40)orpromote

procoagulant activity

on the surface of human monocyte

macrophages by

an

in-creasein tissue

thromboplastin activity (41)

or

by stimulating

the

expression

andsecretionof tissue factor

by

monocytesor

aorticendothelial cells(42).

Finally,

an

increasing body

of

evi-dence has been

accumulating

suggesting

arolefor Ox-LDL in

mediating

someof the alteredvasomotor

properties

knownto

accompanytheatheroscleroticstate.Acharacteristicofthe ath-eroscleroticcoronary artery appearstobeareduced

responsive-ness to endothelial-dependent

relaxing

factor

(EDRF)-me-diatedvasodilation

(43).

Manystudies havenowdemonstrated that Ox-LDL can inhibit such EDRF-mediated vasodilation

although

the mechanismsremain controversial.Evidence has been

presented

thatthis effect is dueto

lysolecithin-mediated

alteration of endothelial membranes

resulting

in

disruption

of

receptors

specific

for

vasodilatory

agonists (44).

Others have

suggested

other

mechanisms,

such as direct inactivation of

EDRF

(i.e.,

nitric

oxide)

after its release fromtheendothelial cells

(45),

or adirect actiononthevascularsmoothmusclecells themselves

(46).

While thereseemslittle doubt that Ox-LDL has the

ability

toinhibit EDRF-mediated

vasodilation,

the

ex-actmechanisms remaintobeworkedout.

Itis

likely

thatan

increasing

numberof studies will be

re-ported

looking

attheeffects of Ox-LDLon a

variety

of

biologi-calprocesses, both in vitro and in vivo.Asafinal caveat,we

wouldurgethe readertotakenoteof thediscussionabout the

variability in preparingOx-LDL noted above and thateven

nativeLDLisreadily

oxidized,

especially

when incubated inan

oxygen-containing physiological

buffer.Itis

likely

thatsomeof the

_{discrepancies}

and controversythat will arisemayin fact derive from the use of "oxidizedLDL" at

_differing

stages of oxidation and

containing

different oxidation

products.

The emergence

of

a consensus

hypothesis

of

atherogenesis

Atherosclerosisis

unlikely

tobea

_single

disease

entity

and there

maybea

variety

of

pathways

leading

tolesions thatat

_endpoint

lookmuch alike. Our focushere has beenonwhatwe

might

designate

as

_{"lipoprotein-induced}

atherosclerosis." Until

re-cently

investigators

interested in

atherogenesis

havetendedto

prolifera-tion, on the onehand,and thoseemphasizing lipidinfiltration, on the other (1, 47). The former focused on"injury" and re-sponses to it thatmight account for cellular proliferation.

Lipo-proteins and thedeposition oflipidwerebarely givena nod. The latter, in contrast, tended to gloss over the fact that there

wascellularproliferation andinjuryandfocusedonthefact thathypercholesterolemia alone could induce the disease in

experimental animalsand inpatientsand thatlipid deposition

was ahallmark of the lesion.Fromtheexamplescited above, we can see that Ox-LDL can play a partinatherogenesis in ways that need not bedirectlyrelated toitsroleinlipid

deposi-tion. Forexample, through its cytotoxic effects on endothelial

cells(48) it couldactually induce thesequenceofevents origi-nally envisioned intheendothelialinjury hypothesis. Its

che-motactic activity, direct andindirect, could playarole in the

recruitmentofmonocytes, a veryearlyeventin experimental atherosclerosisinducedbycholesterolfeeding (34,49). By

stim-ulatingrelease of M-CSF(32) itmayaffectgrowthand

matura-tion of macrophages and subsequent release of other cytokines andgrowth factors.Allof these phenomena would beexpected tobe moreevident whenthe concentration ofLDL is high because therelativeconcentration of Ox-LDL would then also

behigh. Thus, there would beanexcellent correlationbetween thedepositionoflipid in the lesion,onthe onehand,andthese cellulareffects of Ox-LDL,ontheother. The accumulation of lipidper seneednotnecessarily be ofcentralimportancein the proliferativeaspectsof the disease.In fact, it has been

specu-lated that the uptakeof Ox-LDL by the macrophage in early

stagesof atherosclerosismaybebeneficial rather than harmful (50). Bytakingupandsequestering Ox-LDL itmayactually

protectcells,including endothelial cells, from its cytotoxic ef-fects. Later, when the macrophages have become excessively loadedwith lipid anddie, the neteffect maybe harmful

be-causeof the release ofcytotoxiccomponentsfromlipid-laden

macrophages.Inotherwords, themacrophage may play a

pro-tective role early butapathogenetic role later in thecourseof

atherogenesis.In part,thetransition fromfattystreaktomore complicatedlesionsmightbeaconsequenceof the necrosis of oldercholesterol-loaded macrophages in the intima.

Thus, we now canunderstandhowhyperlipoproteinemia couldof itself beasufficient basisfor initiatingthe atheroscle-rotic process in all its manifestations, including the cellular

proliferationand matrixdeposition.Asrecentlyshown in vi-tro, componentsof oxidized lipidcanalsostimulatecollagen

geneexpression, atleast infibroblasts(51). Thus,there could be a link between matrix formation and oxidation as well. As already mentioned, many additional factors undoubtedly af-fect the rate and extent of lesion progression at any level of

hyperlipoproteinemiaand much remains to be learned in this connection. Finally,we can nowseethat the accumulation of

lipidper se, whilecertainly contributingtotheplaqueand the narrowing of the vessel's lumen, nevertheless need not be di-rectlycontributingin amajor way to the early "pathogenesis" of thefatty streak. The damage may be doneprimarilyby oxi-dized LDL (or its oxidation products) before or coincident with thedeliveryoflipidtoarterialcells.

Factorspotentially affecting the oxidation ofLDL invivo While evidence continues to accumulate supporting an impor-tant role for oxidative modification in atherogenesis, ulti-mately a clinical test ofthis hypothesis in man will be needed to demonstrate thatinhibition ofoxidation of LDL will inhibit theatherogenicprocess. Todesign effective clinicaltrials,one must understandthosefactorsresponsible forLDLoxidation in vivo. Conceptually, one can group such factors into two classes: first, those intrinsic to LDL, i.e., compositional and structural factors increasingor decreasing susceptibility of a

givenLDL tooxidation; and second, those extrinsictoLDL, i.e., factors inplasma ortissues thatpromote orinhibit

oxida-tion of LDL(Table II).

The nature of the substrate forlipidperoxidation, mainly thepolyunsaturated fatty acidsinlipidestersandcholesterol,is adominantinfluenceindeterminingsusceptibility. As noted

by Esterbaueretal.(52), there isa vast excessof

polyunsatu-ratedfatty acidsin LDL, inrelationshiptothecontentof

natu-ral, endogenousantioxidants.Theimportance ofthefatty acid compositionwasimpressively demonstratedby ourrecent

stud-iesof rabbits fedadiet high inlinoleic acid (18:2)orin oleic acid(18:1) foraperiod of10wk.LDLisolated from the

ani-mals onoleic acid-rich dietweregreatlyenrichedinoleate and lowinlinoleate. ThisLDL wasremarkably resistant to

oxida-tivemodification,measuredeitherbydirectparametersoflipid

peroxidation (i.e., TBARS and conjugated dienes) orby the indirect criterion ofuptake by macrophages (53). (Whether

TableII. FactorsPotentially AffectingOxidation ofLDL In Vivo

A. FactorsintrinsictoLDL

1. Fatty acid composition (polyunsaturated fatty acid content in particular)

2. Content of antioxidants: endogenous (e.g., ,-carotene, vitamin E,ubiquinol-1O);exogenous (e.g., probucol) 3. PhospholipaseA2activity

4. Others, includingsizeof particle, inherent properties of apo B, location of fatty acids (e.g., on surface phospholipids or in core triglycerides

orcholesteryl esters)

B. Factorsextrinsic to LDL

1. Potential variation in cellular prooxidant activity (e.g., genetic variation in macrophage expression of 15-lipoxygenase activity or cellular

superoxideanion secretion)

2. Concentration of plasma and extracellular fluid prooxidant components (e.g., trace metal concentrations) 3. Concentrationsof plasma and extracellular fluid antioxidant components (e.g., ascorbate, urate)

4. Concentrationsof other factors influencing LDL oxidation (e.g.,HDL)

5. Factorsinfluencingresidence time of LDL in intima (e.g., factors that increase binding such as Lp[a]; nonenzymatic glycosylation of LDL

these dramatic effects were due to simple depletion of the lin-oleic acid or enrichment of the monounsaturated acid, or a combination, is as yet unknown). In a recent study, human volunteers were fed a similar oleic acid-rich diet. When their LDL wastestedforsusceptibility to oxidative modification, it was reduced albeit to a lesser degree than that noted in the

rabbit studies(54). These studies demonstrate the feasibility of

dietary modification ofLDLfatty acidcontent in order to

re-duceits susceptibilitytomodification.

Theantioxidantcontent of LDL obviously plays an impor-tantrole inprotectingLDLfromoxidation. In the most

dra-matic example,the use ofthe lipophilic agent, probucol, which

istransportedwithintheLDL particle and has an antioxidant

potentialatleastfour timesthatofvitamin E, can dramatically protect LDL from in vitro-mediated oxidative modification

(55) andcanameliorate atherosclerosisin

hypercholesterole-mic rabbits(17, 18). In man, vitamin E, ,8-carotene, and

ubi-quinol-10 all appear tobe endogenous antioxidants in LDL (52, 56).Epidemiologicdata suggest a negative correlation be-tween coronary disease and levels of vitamin E (57), and a

preliminary report suggested that daily p3-carotene ingestion

might beofimportance inthe secondary prevention of CAD

(58).Clinical studiesareneededrelating dietarycontentof

vita-minEand,8-carotene totheircontentinplasma LDL and their

abilitytoprotect LDLfrom oxidativestress.

Athird potential variable determining the susceptibilityof LDLtooxidative modification is variabilityin intrinsic

phos-pholipaseA2activity (2, 12).Asdiscussedabove,thisactivity is

required fortheoxidative modification ofLDL. Variation in

thisactivitycould thus leadtodifferences insusceptibility of

LDL tomodification. Finally,avariety ofotherfactorssuch as the sizeof theparticle, intrinsic properties ofapoB, and the

location of the susceptible fatty acids(on thesurfaceorinthe

core) could theoretically influence the susceptibility ofagiven

LDL tooxidative modification.

Asdiscussed above, it ismostlikely that oxidative modifica-tion ofLDL occursprimarilyin theintimainmicrodomains protected from the various antioxidants found in plasma and in the bulk extracellularspace.Underappropriateinvitro

con-ditions all ofthe celltypesfoundinthearterycanoxidizeLDL. Inpartthismaybemediated bytheabilityof cellsto secrete

reactiveoxygen species, such assuperoxide anion. However,

15-lipoxygenase activity in culturedendothelial and smooth muscle cellsappears toplayadominant rolein theabilityof these cellsto oxidatively modify LDL(10), and recent data

supports the presence of thisactivity in macrophagesas well (11). Wehaverecentlyreportedthepresenceof1 5-lipoxygen-ase mRNAandproteininmacrophage-richareas ofatheroscle-rotic lesions in rabbits (59)andman (60)anddemonstrated

that thiscolocalized to areas

staining

for oxidized-LDL

epi-topes. Variation in 15-lipoxygenase activity, possibly due to

genetic or localized factors, could

explain

differences in the

abilityofdifferentsubjectstomodifyLDL.Inhibitors of lipox-ygenase areavailable and studiesareunder wayin animalsthat mayhelp demonstrate the importanceof thisactivity. Some

antioxidants,suchasprobucolcaneffectivelyinhibitthe

ability

of cellstomodifyLDL(Parthasarathy,

S.,

submittedfor

publi-cation).

Variation intheintimal extracellularcontentof free metal ions, suchascopperoriron, perhapsduetovariations in metal

bindingproteins, could also

theoretically

influence the

ability

to

oxidatively

modify

LDL.

Ascorbate,

which isawater-soluble

antioxidant, mayalso playan importantrole. In anin vitro setting,ascorbatecanprotect LDLfromoxidation,apparently by maintaining vitaminEand

_#-carotene

in areduced, antioxi-dant state(52, 61). Presumably this occursbyinteraction of vitamin C with theselipophilicantioxidants at a lipid-water

interface. In fact, administration of vitamin C has been

re-portedtodecrease the enhanced susceptibilitytooxidationof LDLisolated fromsmokers (22). Theeffect of vitaminC on

established experimentalmodelsofatherosclerosisneeds to be

ascertained.

In vitro, HDLcan partiallyprotect LDL from oxidation mediated bycells orbycopper(62). Little is knownaboutthe

rates offlux ofHDL oritsresidence time inthe artery wall.

Variations in localized concentrations ofHDLcouldplayan

important role in preventingtheformation of Ox-LDLandin

part thiseffect ofHDLcould explain the protective role of

HDLthat has been demonstrated by epidemiologicstudies. Finally,factorsthatprolongtheresidence time ofLDLin the artery wall mayallowoxidative modificationtoproceedtoa

greater extent. LDL has ahigh affinity for proteoglycansand

localized differences in theircontent could be an important factorinthe localization of lesion formation (63).Similarly,

Lp(a), whichmay haveenhancedbindingtosuchcomplexes,

maythusbe moresusceptibleaswell(63). Other factors, such as nonenzymatic glycation ofLDL (or the matrix proteins) mayalsoserve toenhanceentrapmentofLDL,prolongation of

itshalf-life, and thuspromoteoxidation(Palinski,W., T. Kos-chinsky, S. W.Butler, M. E. Rosenfeld, H. Vlassara, A.

Cer-ami,andJ. L.Witztum, submittedforpublication). Measurestoinhibitoxidative

modification

in man

Fromthediscussion above it followsthatinterventionsto

in-hibit oxidativemodificationcanbefocused intwo areas: mea-sures toprotect LDLitself, andmeasures toreduce those

fac-torsresponsiblefor the oxidantstress to LDL. Atpresent, pro-bucol is the most potent lipophilic antioxidant capable of

protectingLDL, butitsotherproperties,such asitspotent

abil-itytolower HDLlevels, raisequestionsabout itsuseina clini-cal trial to test theantioxidant hypothesis. Preliminary data suggestthatlimitation ofdietary unsaturated fattyacidsand

replacement with monounsaturatedfattyacids isaneffective

way toprotect LDLfrom oxidativestress.Dietary supplemen-tation with vitamin E,

fl-carotene

(and presumablyother

carot-enoids)seemsreasonable, particularlybecausetherewould be no concerns abouttoxicity or sideeffects,but few dataas to

their effectivenessinvivo areyetavailable.Finally, combina-tionsof these interventions couldprovesynergistic, aswould thedietary

supplementation

of ascorbic acid.Allofthese inter-ventionsarecurrently feasibleandintensivestudyis under way

todeterminetheeffectiveness ofsuchmodificationsto_protect

LDLfromoxidative modification.Datashouldbecome avail-able inthenextfewyearstodeterminethemosteffective

ap-proach for an intervention trial in man.

Experimental

ap-proachesdesignedtoinhibittheabilityof cellsto

modify

LDL,

asforexample,byinterventionstoinhibit 1

_{5-lipoxygenase}

ac-tivity, awaitthe resultsof

experiments

inanimal modelsand proofofsafety,butultimatelymayofferalternative

approaches

aswell.

Summary

Evidencetosupportan

important

roleofoxidative

New hypotheses suggest mechanisms by which Ox-LDL or products of Ox-LDLcanaffect manycomponentsof the ath-erogenicprocess,includingvasomotorproperties and thrombo-sis,aswellaslesion iniationandprogression itself. These ideas

suggestnewapproaches, that in combination with lowering of plasmacholesterol, could leadtotheprevention ofatherosclero-sis and itscomplications.

References

1.Steinberg, D., and J. L.Witztum. 1990.Lipoproteinsandatherogenesis:

currentconcepts.JAMA(J.Am.Med.Assoc.).264:3047-3052.

2.Steinberg,D., S. Parthasarathy,T. E.Carew,J.D.Khoo,and J.L. Witztum.

1989.Beyond cholesterol: modifications oflowdensity lipoproteinthatincrease

itsatherogenicity.N.Engl. J. Med. 320:915-924.

3. Goldstein,J.L.,Y. K.Ho,S. K.Basu,and M.S. Brown. 1979.Bindingsite

onmacrophagesthat mediatesuptakeanddegradationofacetylatedlowdensity lipoprotein, producing massive cholesterol deposition.Proc. Natl. Acad. Sci. USA.76:333-337.

4.Fogelman,A.M.,J. S.Schechter,M. Hokom,J. S.Child,and P. A. Ed-wards. 1980. Malondialdehyde alteration of low density lipoprotein leadsto

cho-lesterol accumulationinhumanmonocyte-macrophages.Proc.Nail. Acad. Sci. USA.77:2214-2218.

5. Henriksen, T., E. M. Mahoney, andD.Steinberg. 1981.Enhanced

macro-phagedegradation of low density lipoprotein previously incubatedwithcultured endothelial cells:recognition byreceptorsforacetylated low density lipoproteins.

Proc. Natl.Acad. Sci. USA.78:6499-6503.

6.Steinbrecher, U. P.,S. Parthasarathy,D.S. Leake, J.L.Witztum,and D.

Steinberg. 1984. Modification of low density lipoprotein by endothelialcells in-volves lipid peroxidation and degradation of low density lipoprotein

phospho-lipids. Proc. Natl. Acad. Sci. USA. 81:3883-3887.

7.Heinecke, J. W.,H.Rosen, andA.Chait. 1987.Ironandcopperpromote

modificationof low-densitylipoprotein by human arterialsmooth musclecellsin

culture. J.Clin. Invest. 74:1890-1894.

8.Morel,D.W.,P. E.DiCorleto, andG.M.Chisolm. 1984.Endothelial and

smoothmuscle cells alter lowdensity lipoproteininvitro by freeradical

oxida-tion.Arteriosclerosis. 4:357-364.

9.Sparrow,C.P., S. Parthasarathy,and D.Steinberg.1988.Enzymatic

modifi-cation of lowdensity lipoprotein by purified lipoxygenase plus phospholipaseA2 mimicscell-mediated oxidativemodification. J.LipidRes.29:745-753.

10.Parthasarathy, S.,E.Wieland, and D. Steinberg. 1989.Arolefor

endothe-lial cell lipoxygenaseintheoxidative modification oflowdensity lipoprotein.

Proc. Natl.Acad. Sci. USA.86:1046-1050.

1 1.Rankin, S.M.,A.Parthasarathy, andD.Steinberg. 1991. Evidence fora

dominant roleoflipoxygenase(s)intheoxidation ofLDLbymouseperitoneal macrophages. J. Lipid Res. 32:449-456.

12.Parthasarathy, S., and J. Barnett. 1990. PhospholipaseA2activityof low

densitylipoprotein: evidenceforanintrinsicphospholipaseA2activity of

apopro-teinB-100. Proc.Natl. Acad. Sci.USA. 87:9741-9745.

13.Jurgens, G.,H. F.Hoff, G.M.Chisolm III, and H. Esterbauer. 1987.

Modification of humanserumlow density lipoproteinbyoxidation:

characteriza-tion andpathophysiological implications.Chem.Phys.Lipids. 45:315-316.

14.Haberland,M.E.,A.M.Fogelman, andP. A.Edwards. 1982.Specificity

ofreceptor-mediated recognition of malondialdehyde-modified low density lipo-proteins.Proc. Natl.Acad. Sci. USA. 79:1712-1716.

15.Parthasarathy, S.,L.G. Fong, D. Otero, and D. Steinberg.1987.

Recogni-tion ofsolubilizedapoproteins from delipidated, oxidizedlowdensity lipoprotein (LDL) by the acetyl-LDLreceptor.Proc.Natl. Acad.Sci. USA. 84:537-540.

16.Berliner,J. A., M. C. Territo,A.Sevanian, S. Ramin,J. A. Kim, B.

Bamshad, M. Esterson, and A. M. Fogelman. 1990. Minimally modifiedlow

densitylipoprotein stimulatesmonocyteendothelial interactions.J.Clin.Invest. 85:1260-1266.

17.Carew,T. E., D. C. Schwenke, and D.Steinberg.1987. Antiatherogenic effect ofprobucol unrelatedtoits hypocholesterolemic effect:evidence that

an-tioxidantsinvivocanselectively inhibitlowdensitylipoprotein degradation in

macrophage-rich fattystreaks slowing the progression of atherosclerosisin the

WHHLrabbit.Proc.Natl. Acad.Sci. USA. 84:7725-7729.

18.Kita, T., Y.Nagano, M. Yokode, K.Ishii, N. Kume,A.Ooshima,H.

Yoshida, and C. Kawai. 1987.Probucolpreventsthe progression of

atherosclero-sis inWatanabeheritablehyperlipidemic rabbit,ananimalmodelfor familial

hypercholesterolemia.Proc. Natl.Acad. Sci. USA. 84:5928-5931.

19. Bjorkham, I.,A.Henriksson-Freyschuss, 0.Breuer, U. Diczfalusy, L.

Berglund,and P.Henriksson. 1991. Theantioxidantbutylated hydroxytoluene

protectsagainst atherosclerosis. Arteriosclerosis Thromb.11:15-22.

20.Mao,S.J. T.,M. T.Yates,R. A. Parker, E. M. Chi, andR.L. Jackson.

1991.Attenuationofatherosclerosis inhypercholesterolemicWatanaberabbits

usingaprobucol analog (MDL 29,31 1) thatdoes not lower cholesterol.

Arterio-sclerosis. In press.

21.Morel,D.W.,andG.M.Chisolm. 1989. Antioxidant treatmentof

dia-beticratsinhibitslipoproteinoxidation andcytotoxicity. J. LipidRes.

30:1827-1834.

22. Avogaro, P.,G. B.Bon,andG.Cassalato. 1988. Presenceofamodified low density lipoprotein in humans. Arteriosclerosis. 8:79-87.

23. Harats, D., M. Ben-Naim, Y. Dabach, G. Hollander, E. Havivi, 0. Stein, andY.Stein. 1990. Effectof vitamin C andE supplementationonsusceptibility ofplasma lipoproteinstoperoxidation induced byacutesmoking. Atherosclero-sis. 85:47-54.

24.Sparrow,C. P., S. Parthasarathy, and D. Steinber. 1989. A macrophage receptor that recognizes oxidizedLDLbutnotacetylated LDL. J. Biol. Chem. 264:2599-2604.

25.Rosenfeld,M.E., J. C. Khoo,E.Miller, S.Parthasarathy,W.Palinski,and

J. L. Witztum. 1991. Macrophage-derived foam cells freshly isolated from rabbit atherosclerotic lesions degrade modified lipoproteins, promote oxidation ofLDL, andcontain oxidation specific lipid-protein adducts. J. Clin. Invest. 87:90-99.

26.Zhang, H., H. S. K. Basra, and U.P.Steinbrecher.1990.Effects of oxida-tively modified LDLon cholesterol esterification in cultured macrophages. J. Lipid Res. 31:1361-1369.

27.Jialal, I., and A. Chait. 1989. Differences in the metabolism of oxidatively modified lowdensity lipoproteinandacetylated lowdensity lipoproteinby

hu-manendothelial cells: inhibition ofcholesterol esterification by oxidatively

modi-fied lowdensity lipoprotein.J.LipidRes.30:1561-1568.

28. Schwenke, D. C., andT. E. Carew. 1989. Initiation of atherosclerotic

lesionsincholesterol-fedrabbits.II.Selective retention ofLDL vs.selective

in-creases in LDLpermeability in susceptible sites of arteries. Arteriosclerosis.

9:908-918.

29. Fox, P. L., G.M.Chisolm, andP.E. DiCorleto.1987. Lipoprotein-me-diated inhibition ofendothelial cellproductionofplatelet-derived growth

factor-like protein dependsonfree radicallipidperoxidation.J.Biol.Chem.

262:6046-6054.

30.Malden, L. T., R. Ross, andA.Chait. 1990.Oxidativelymodified low

density lipoproteinsinhibitexpression of platelet-derived growthfactorby hu-manmonocyte-derivedmacrophages. Clin.Res.38:291-A.(Abstr.)

31. Hamilton, T. A., G. P. Ma, and G. M. Chisolm. 1990. Oxidized low

density lipoproteinsuppresses theexpressionoftumor necrosisfactor-amRNA

in stimulated murineperitonealmacrophages. J.Immunol. 144:2343-2350.

32.Rajavashisth,T.B.,A.Andalibi,M.C.Territo,J.A.Berliner,M.Navab,

A.M.Fogelman, andA.J.Lusis. 1990. Induction ofendothelial cellexpressionof

granulocyte andmacrophage colony-stimulatingfactorsbymodified lowdensity lipoproteins.Nature(Lond.).344:254-257.

33.Liao, F., J.A.Berliner,M.Mehrabian,M.Navab,L. L.Demer,A.J.

Lusis,andA. M.Fogelman. 1991.Minimallymodified lowdensity lipoproteinis biologically active invivo.J.Clin. Invest. 87:2253-2257.

34.Cushing,S. D.,J. A.Berliner,A.J.Valente, M.Navab,F.Parhami,R.

Gerrity, C. J. Schwartz, andA.M.Fogelman. 1990.Minimallymodified low densitylipoproteininduces monocyte chemotacticproteinIin human endothe-lial cells and smooth muscle cells. Proc.Natl.Acad.Sci. USA. 87:5134-5138.

35.Yla-Herttuala,S.,B.A.Lipton,M.E.Rosenfeld,T.Sarkioja, T. Yoshi-mura, E. J. Leonard, J. L. Witztum, andD.Steinberg. 1991. Macrophages express monocytechemotactic protein(MCP-1) in human and rabbitatherosclerotic lesions.Proc.Natl.Acad.Sci. USA. 88:5252-5256.

36. Palinski,W., S. YlS-Herttuala, M. E.Rosenfeld, S. W. Butler, S. A.

Socher,S.Parthasarathy,L.K.Curtiss,and J. L.Witztum. 1990. Antisera and monoclonalantibodiesspecificforepitopes generated during oxidative modifica-tion of lowdensity lipoprotein. Arteriosclerosis. 10:325-335.

37.Palinski, W., M. E. Rosenfeld, S. Yla-Herttuala, G. C. Gurtner, S. A.

Socher,S. W.Butler,S. Parthasarathy,T. E. Carew,D. Steinberg,and J. L. Witztum. 1989. Low density lipoprotein undergoes oxidative modification in vivo. Proc.Natl. Acad.Sci. USA. 86:1372-1376.

38.Parums,D.V.,D.L.Brown, andM. J.Mitchinson. 1990. Serum antibod-iestooxidized lowdensity lipoprotein and ceroid in chronic periaortitis. Arch.

Pathol. Lab. Med. 114:383-387.

39. Vlaicu, R., F. Niculesco, H. G. Rus, and A. Cristea. 1985. Immunohisto-chemical localization of the terminal C5b-9 complement complex in human aorticfibrous plaques. Atherosclerosis. 57:163-177.

40.Aviram,M.1989.Modifiedforms oflow density lipoprotein affect platelet

aggregationin vitro.Thromb. Res. 53:561-567.

41. Schuff-Werner,P., G. Claus, V. W. Armstrong, H. Kostering, and D. Seidel. 1989. Enhanced procoagulatory activity (PCA) of human

monocytes/ma-crophagesafterinvitrostimulation with chemically modified LDL.

Atherosclero-sis.78:109-112.

42. Drake, T. A., K. Hannai, H. Fei, S. Lavi, and J. A. Berliner. 1991. Mini-mally oxidizedLDLinduces tissue factor expression in cultured human endothe-lial cells.Am.J.Pathol.138:601-607.

43.Bossaller, C., G. B. Habib,H.Yamamoto, C. Williams, S. Wells, and P. D.

guanosine 5'-monophosphate formation in atherosclerotic human coronary ar-tery andrabbit aorta. J. Clin. Invest. 79:170-174.

44.Kugiyama, K., S. A.Kerns, J. D. Morrisett, R. Roberts, and P. D. Henry. 1990. Impairment ofendothelium-dependent arterial relaxation by lysolecithin inmodified low-density lipoproteins. Nature (Lond.). 344:160-162.

45.Galle, J., A.Miklsch,R.Busse, and E. Bassenge. 1991. Effects of native and oxidized low density lipoproteins on formation and inactivation ofendothelium-derivedrelaxing factor. Arteriosclerosis Thromb. 11:198-203.

46.Galle, J., R. Bassenge, and R. Busse. 1990. Oxidized low density lipopro-teins potentiate vasoconstrictions to various agonists by direct interaction with vascular smooth muscle. Circ.Res. 66:1287-1293.

47. Ross, R.1986. Thepathogenesis of atherosclerosis: an update. N. Engl. J. Med. 314:488-500.

48.Hessler,J.R.,A.L.Robertson, Jr., and G.M.Chisolm. 1979. LDL-in-ducedcytotoxicityandits inhibition byHDLin human vascular smooth muscle andendothelial cells in culture.Atherosclerosis. 32:213-219.

49. Quinn, M.T., S. Parthasarathy, L. G. Fong,and D.Steinberg. 1987.

Oxidativelymodified lowdensitylipoproteins:apotentialrole in recruitment and

retention ofmonocyte/macrophagesduring atherogenesis.Proc. Natl.Acad.Sci.

USA.84:2995-2998.

50.Steinberg, D. 1990.Arterial metabolismof lipoproteins in relationto

atherogenesis.Ann.NYAcad. Sci. 598:125-135.

51.Chojkier,M., K.Houglum, J.A.Solis-Herruzo,and D.A.Brenner. 1989. Stimulation ofcollagen geneexpressionby ascorbic acid in cultured human fibro-blasts.Aroleforlipid peroxidation. J. Biol. Chem. 264:16957-16962.

52. Esterbauer, H., G. Striegl, H. Puhl, and M. Rotheneder. 1990. Continuous monitoring of in vitro oxidation of human low density lipoprotein. Free Radical Res.Commun. 6:67-75.

53. Parthasarathy, S., J. C.Khoo, E. Miller, J. Barnett, J. L. Witztum, and D. Steinberg. 1990. Low density lipoprotein enriched in oleic acid is protected against oxidative modification: implications for dietary prevention of atheroscle-rosis. Proc.Natl. Acad. Sci. USA. 87:3894-3898.

54.Reaven, P., S. Parthasarathy, B. J. Grasse, E. Miller, F. Almazan, F. H. Mattson, J. C. Khoo, D. Steinberg, and J.L.Witztum. 1991.Feasibility of an oleate-richdiet to reducethesusceptibility oflowdensity lipoproteintooxidative modification in man. Am. J. Clin. Nutr. 54:701-706.

55.Parthasarathy,S., S. G. Young, J.L.Witztum, R. C. Pittman, and D. Steinberg. 1986. Probucol inhibits oxidative modification of lowdensity lipopro-tein.J. Clin.Invest. 77:641-644.

56. Frei, B., M. C. Kim, andB.N. Ames. 1990.Ubiquinol-10 isan effective

lipid-solubleantioxidantatphysiologicalconcentrations. Proc.Natl. Acad.Sci.

USA. 87:4879-4883.

57.Gey,K.F., and P. Puska. 1989. Plasma vitaminsEandAinversely

corre-lated to mortality from ischemic heart diseasein cross-culturalepidemiology. Ann.NYAcad. Sci. 570:268-282.

58.Gaziano, J. M., J. E. Manson, P.M. Ridker, J.E. Buring, and C.H.

Hennekens. 1990.Beta carotenetherapy for chronic stableangina. Circulation. 82:III-201.

59.Yla-Herttuala, S., M. E.Rosenfeld, S. Parthasarathy, C. K. Glass,E.Sigal, J. L.Witztum, and D. Steinberg. 1990. Colocalization of 15-lipoxygenasemRNA

andoxidized lowdensity lipoprotein in macrophage-richareasofatherosclerotic lesions. Proc. Natl. Acad. Sci. USA. 87:6959-6963.

60.Yla-Herttuala, S., M.E.Rosenfeld, S. Parthasarathy,E.Sigal,T.Sirkioja, J. L.Witztum, and D. Steinberg. 1991. Geneexpression inmacrophage-rich humanatherosclerotic lesions:15-lipoxygenase and acetylLDLreceptormRNA

colocalize withoxidation-specific lipid-proteinadducts. J. Clin. Invest. 87:1146-1152.

61.Jialal, I., and S.M.Grundy.1991.Preservation ofthe endogenous antioxi-dantsinlowdensity lipoprotein byascorbate but notprobucolduringoxidative

modification. J. Clin. Invest. 87:597-601.

62.Parthasarathy,S.,J.Barnett, andL.G.Fong. 1990. Highdensity lipopro-tein inhibits the oxidative modification of lowdensity lipoprotein. Biochim. Biophys. Acta. 1044:275-283.

63.Scanu,A.M., and G. M. Fless. 1990.Lipoprotein(a).Heterogeneityand