HDL and the Inflammatory Response Induced by LDL-Derived Oxidized Phospholipids

Brian J. Van Lenten, Deven Vora and Alan M. FogelmanLusis, Lawrence W. Castellani, Srinivasa Reddy, Diana Shih, Weibin Shi, Andrew D. Watson, Mohamad Navab, Judith A. Berliner, Ganesamoorthy Subbanagounder, Susan Hama, Aldons J.HDL and the Inflammatory Response Induced by LDL-Derived Oxidized Phospholipids

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HDL and the Inflammatory Response Induced byLDL-Derived Oxidized Phospholipids

Mohamad Navab, Judith A. Berliner, Ganesamoorthy Subbanagounder, Susan Hama, Aldons J. Lusis,Lawrence W. Castellani, Srinivasa Reddy, Diana Shih, Weibin Shi, Andrew D. Watson,

Brian J. Van Lenten, Deven Vora, Alan M. Fogelman

Abstract—Oxidation of low density lipoprotein (LDL) phospholipids containing arachidonic acid at thesn-2 positionoccurs when a critical concentration of “seeding molecules” derived from the lipoxygenase pathway is reached in LDL.When this critical concentration is reached, the nonenzymatic oxidation of LDL phospholipids produces a series ofbiologically active, oxidized phospholipids that mediate the cellular events seen in the developing fatty streak. Normalhigh density lipoprotein (HDL) contains at least 4 enzymes as well as apolipoproteins that can prevent the formation ofthe LDL-derived oxidized phospholipids or inactivate them after they are formed. In the sense that normal HDL canprevent the formation of or inactivate these inflammatory LDL-derived oxidized phospholipids, normal HDL isanti-inflammatory. HDL from mice that are genetically predisposed to diet-induced atherosclerosis became proinflam-matory when the mice are fed an atherogenic diet, injected with LDL-derived oxidized phospholipids, or infected withinfluenza A virus. Mice that were genetically engineered to be hyperlipidemic on a chow diet and patients with coronaryatherosclerosis, despite normal lipid levels, also had proinflammatory HDL. It is proposed that LDL-derived oxidizedphospholipids and HDL may be part of a system of nonspecific innate immunity and that the detection ofproinflammatory HDL may be a useful marker of susceptibility to atherosclerosis.(Arterioscler Thromb Vasc Biol.2001;21:481-488.)

Key Words: HDL n LDL n atherosclerosisn oxidized phospholipids

The events involved in fatty streak formation resemblethose elicited by mycobacteria.1 Over the past decade,

there has been increasing evidence that this inflammatoryresponse may, in part, be elicited by the oxidation ofphospholipids contained in LDL.2,3 Several oxidized phos-pholipids that are able to induce the genes and proteinsnecessary for the cellular response seen in the fatty streakhave been identified in mildly oxidized LDL and in lesions ofanimal models of atherosclerosis.4–9 Two of these oxidizedphospholipids, 1-palmitoyl-2(5-oxovaleroyl)-sn-glycero-3-phosphorylcholine (POVPC) and 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphorylcholine (PGPC), both induced mono-cytes to bind to endothelial cells.10 However, PGPC but notPOVPC also induced neutrophils to bind to endothelialcells.10 Indeed, POVPC strongly inhibited lipopolysaccha-ride-mediated induction of neutrophil binding and expressionof E-selectin protein and mRNA.10 This inhibition by POVPCwas mediated by a protein kinase A–dependent pathway thatresulted in downregulation of nuclear factor-kB–dependenttranscription.10 PGPC, on the other hand, induced bothE-selectin and vascular cell adhesion molecule-1 (VCAM-1)expression on endothelial cells.10 On the basis of studies in

Xenopus laevisoocytes, Leitinger et al10 concluded thatPOVPC and PGPC bound to different receptors. Furthermore,they demonstrated that at concentrations equal to thosepresent in mildly oxidized LDL, POVPC prevented theinduction of neutrophil binding and E-selectin expression inendothelial cells despite the presence of PGPC.10 Thus, wehypothesized that the relative concentrations of POVPC andPGPC will determine whether an acute (neutrophilic) orchronic (monocytic) inflammation would result in any giventissue. A third group of oxidized phospholipids that alsoinduce monocyte binding to endothelial cells was identifiedas 1-palmitoyl-2(5,6-epoxyisoprostane E2)-sn-glycero-3-phosphorylcholine (PEIPC).7,11

Autoantibodies specific for products of oxidized1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine(Ox-PAPC), including POVPC, have been identified in apoE-deficient mice and have been shown to inhibit macrophageuptake of oxidized LDL.12 These antibodies have also beenshown to bind to apoptotic cells and inhibit their phagocytosisby macrophages, indicating that these oxidation-specificepitopes mediate macrophage recognition of apoptotic cells.13

These antibodies, which have been found in atherosclerotic

Received October 30, 2000; revision accepted January 8, 2001.From the Departments of Medicine (M.N., J.A.B., G.S., S.H., A.J.L., L.W.C., S.R., D.S., W.S., A.D.W., B.J.V.L., D.V., A.M.F.); Pathology (J.A.B.);

Microbiology, Immunology, and Molecular Genetics (A.J.L.); and Human Genetics (A.J.L.), UCLA School of Medicine, Los Angeles, Calif.Correspondence to Alan M. Fogelman, MD, Department of Medicine, UCLA School of Medicine, 10833 Le Conte Ave, Los Angeles, CA 90095-1736.

E-mail [email protected]© 2001 American Heart Association, Inc.

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lesions, were found to be structurally and functionally iden-tical to classic “natural” T15 anti-phosphorylcholine antibod-ies that are of B-1 cell origin and that have been reported toprovide protection against virulent pneumococcal infection.14

The inflammatory response elicited by the oxidized phos-pholipids (eg, Ox-PAPC) found in mildly oxidized LDL ismediated in part by the induction in endothelial and smoothmuscle cells of monocyte chemoattractant protein-1 (MCP-1)15; macrophage colony stimulating factor (M-CSF),16 amember of the GRO family of chemokines17; P-selectin18;and interleukin 8 (IL-8).19 Additionally, these oxidized phos-pholipids induce the accumulation of connecting segment-1of fibronectin on the apical surface of endothelial cells byactivating endothelialb1 integrins, particularly those thatassociate witha5 integrins.20 Connecting segment-1 serves asthe endothelial ligand that binds toa4b1 (very late antigen-4,VLA4) on monocytes, thus promoting adhesion of the mono-cytes to activated endothelial cells.20 The induction of mono-cyte binding to endothelial cells exposed to mildly oxidizedLDL also involves lipoxygenase (LO) metabolites.21 Themechanism for the induction of MCP-1 and IL-8 in endothe-lial cells exposed to mildly oxidized LDL, Ox-PAPC,POVPC, or PGPC appears to involve the lipid-dependenttranscription factor peroxisome proliferator–activatedreceptor-a.19

The response of endothelial cells to these oxidized phos-pholipids appears to be genetically determined.22–25 Using anovel explant technique, Shi et al22 isolated endothelial cellsfrom the aortas of inbred mouse strains with differentsusceptibilities to diet-induced atherosclerosis. The responseof these endothelial cells to mildly oxidized LDL wasdetermined by measuring levels of mRNA for inflammatorygenes, including MCP-1, M-CSF, and the oxidative stressgene, heme oxygenase-1.22 Endothelial cells derived from theatherosclerosis-susceptible mouse strain C57BL/6J (B6) ex-hibited dramatic inductions of mRNA for MCP-1, M-CSF,and heme oxygenase-1.22 In contrast, endothelial cells de-rived from the atherosclerosis-resistant strain C3H/HeJ(C3H) showed little or no induction.22 The authors concludedthat the genetic difference between the 2 strains for thedevelopment of diet-induced atherosclerosis was determinedat the level of the vessel wall.22 In other studies, these authorsstudied a congenic strain of C3H mice carrying an apoE-nullallele (apoE–/–).24 Although the C3H.apoE–/– mice had higherplasma cholesterol levels, they developed much smallerlesions than did their B6.apoE–/– counterparts on either chowor Western diets.24 Reciprocal bone marrow transplantationbetween the strains, with congenics carrying the same H-2haplotype, was performed to determine the role of mono-cytes.24 Atherosclerosis susceptibility was not altered in therecipient mice, indicating that variations in monocyte func-tion were not involved.24 In a set of recombinant inbredstrains derived from the B6 and C3H parental strains, endo-thelial cell responses to mildly oxidized LDL cosegregatedwith aortic lesion size, providing strong genetic evidence thatthe endothelial cells, but not monocytes or plasma lipidlevels, accounted for the susceptibility to atherosclerosisbetween these 2 mouse strains.24 These data do not excludethe possibility that differences in monocytes and lipid levelsmay be responsible for susceptibility to atherosclerosis inother mouse models.

Formation of LDL-Derived OxidizedPhospholipids That Induce an

Inflammatory ResponseLDL is usually thought of as the major source of extracellularcholesterol. However, LDL is also a major source of extra-cellular phospholipid. As noted above, some of these phos-pholipids can yield oxidized phospholipids that induce aninflammatory response. Subbanagounder and colleagues11

found that the major structural determinant of the biologicalactivity of oxidized phospholipids was at thesn-2 position.Substituting stearoyl for palmitoyl at thesn-1 position orethanolamine for choline at thesn-3 position did not alterbioactivity.11 All oxovaleroyl phospholipids studied stimu-lated monocyte binding and inhibited lipopolysaccharide-induced expression of E-selectin.11 All oxovaleroyl phospho-lipids but not the glutaroyl phospholipids induced monocytebinding without increasing VCAM-1.11 Glutaroyl phospho-lipids but not oxovaleroyl phospholipids stimulatedE-selectin and VCAM-1.11 However, intact phospholipidmolecules were required for bioactivity, since activity wasdestroyed after treatment of the phospholipids with phospho-lipase (PL) A1, PLA2, or PLC.11 The levels of POVPC, PGPC,and PEIPC were increased 3- to 6-fold in rabbit atheroscle-rotic lesions and corresponded to'116, 62, and 85mg/mLPOVPC, PGPC, and PEIPC, respectively.11 These levels were'10 to 20 times higher than those needed to activateendothelial cells in culture.11

The concept that LDL must be “primed” for oxidation hasemerged from the work of many laboratories. Sevanian andcolleagues26 described a subpopulation of freshly isolatedLDL that was enriched in lipid hydroperoxides, which theynamed LDL–. Parthasarathy,27,28 Witztum and Steinberg,29

Witztum,30 Chisolm,31 Thomas and Jackson,32 Frei and col-leagues (Shwarery et al33 and Polidori et al34), and Thomas etal35 all studied LDL oxidation by metal ions in vitro and, onthe basis of their findings, concluded that LDL must be“seeded” with reactive oxygen species before it can beoxidized. Thomas and Jackson32 and Parthasarathy28 sug-gested that LOs might play a role in this seeding of LDL.

Watson et al4 used defatted albumin to remove the inflam-matory lipids from mildly oxidized LDL. Because the lipid-binding properties of apoA-I36–39 are greater than those ofdefatted albumin, Navab et al40 reasoned that if freshlyisolated LDL contained seeding molecules, incubating theLDL with apoA-I and then separating the apoA-I from theLDL might result in a transfer of the seeding molecules fromLDL to apoA-I. They hypothesized that this simple strategycould result in the concentration of seeding molecules onapoA-I, from which they could be extracted, identified, andcharacterized. When freshly isolated LDL was incubated withapoA-I and then separated from apoA-I, the resulting LDLcould not be oxidized by human artery wall cells, nor could itinduce human artery wall cells to produce monocyte chemo-tactic activity. However, when the lipids that transferred fromLDL to apoA-I were extracted from the apoA-I and subse-quently added back to the treated LDL, the reconstituted LDLwas readily oxidized and induced monocyte chemotacticactivity.40 Similar results were obtained with an apoA-Imimetic peptide.40 Analysis revealed that the apoA-I–associated seeding molecules removed from freshly isolated

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LDL included hydroperoxyoctadecadienoic acid (HPODE),hydroperoxyeicosatetraenoic acid (HPETE), and cholesterollinoleate hydroperoxide. These results were not in vitroartifacts, because freshly isolated LDL taken from 7 of 7normal volunteers were found to contain HPODE andHPETE.40 The levels of HPODE and HPETE in the LDLremained the same or declined after incubation for 2 hoursunder conditions identical to those used to transfer the lipidsto apoA-I, indicating that HPODE and HPETE were presentin the LDL in vivo and were not formed in vitro.40 Treatmentof LDL with apoA-I reduced the levels of seeding moleculesin LDL by approximately two thirds.40 Thus, approximatelyone third of the seeding molecules remained in the apoA-I–treated LDL. However, as noted above, the apoA-I–treatedLDL was resistant to oxidation by human artery wall cellsand did not induce monocyte chemotactic activity, and addingback the lipids that had transferred from LDL to apoA-Irestored these properties. It was concluded that there must bea threshold concentration of seeding molecules required forLDL oxidation and LDL-induced monocyte chemotacticactivity.40

The ability of apoA-I to render LDL resistant to oxidationby human artery wall cells was also demonstrated in mice.Injection of apoA-I (but not apoA-II) into mice resulted inLDL that, when isolated, was resistant to oxidation by humanartery wall cells.40 Similar results were seen in humans afterinfusion of apoA-I/phosphatidylcholine discs.40 Pretreatmentof artery wall cells with apoA-I or an apoA-I mimetic peptide(but not apoA-II) or with LO inhibitors also prevented LDLoxidation and LDL-induced monocyte chemotactic activity. Itwas concluded that the artery wall cells needed to provideadditional seeding molecules to those already present incirculating LDL to reach the critical threshold concentrationnecessary for phospholipid oxidation.41 The human arterywall cells were found to contain 12-LO protein, and transfec-tion with antisense (but not sense) to 12-LO eliminated the12-LO protein and inhibited LDL-induced monocyte chemo-tactic activity.41 In contrast, enriching the human artery wallcells with linoleic acid (but not oleic acid) promoted LDLoxidation by the artery wall cells and promoted LDL-inducedmonocyte chemotactic activity. HPODE and HPETE werefound to dramatically enhance the nonenzymatic formation ofPOVPC, PGPC, and PEIPC fromL-a-1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine (ie, PAPC) and togreatly enhance the nonenzymatic formation of cholesteryllinoleate hydroperoxide. On a molar basis, HPODE and HPETEwere'2 orders of magnitude greater in potency than hydrogenperoxide in causing the nonenzymatic formation of POVPC,PGPC, and PEIPC.41 A scheme for the formation of theseLDL-derived oxidized phospholipids is shown in Figure 1.

The results of the studies by Navab et al41 were consistentwith those of Cyrus et al,42 who reported that disruption of the12/15-LO gene diminished atherosclerosis in apoE–/– mice. Inconsidering possible mechanisms for their findings, Cyrus etal42 favored 1 in which “. . . lipoxygenase-derived hydroper-oxides or secondary reactive lipid species may be transferredacross the cell membrane to ‘seed’ the extracellular LDL,which would then be more susceptible to a variety ofmechanisms that could promote lipid peroxidation.” Navab etal41 proposed a 3-step model for the mild oxidation of LDLby artery wall cells. In the first step, LDL is seeded. In the

second step, the seeded LDL is trapped in the artery wall andreceives further seeding molecules derived from the LOpathway(s) of nearby artery wall cells. In the third step, acritical level of seeding molecules relative to phospholipids isreached in the LDL, and a nonenzymatic oxidation processgenerates POVPC, PGPC, PEIPC, and other similar mole-cules.11,41 Many factors likely determine the critical level ofseeding molecules needed relative to the phospholipids inLDL to generate the inflammatory oxidized phospholipids.These include the concentration of antioxidants in LDL, theconcentration of phospholipids in LDL with arachidonic acidat the sn-2 position, and the content of platelet-activatingfactor acetylhydrolase (PAF-AH) in LDL.3

The Role of HDL in Modulating theInflammatory Response Induced by

LDL-Derived Oxidized PhospholipidsAs noted above, the major apolipoprotein of HDL, apoA-I(but not apoA-II), prevented the formation of LDL-derivedoxidized phospholipids by removing seeding molecules fromLDL and/or from artery wall cells. However, apoA-I wasactive only in a preincubation step: adding apoA-I in acoincubation together with LDL did not prevent LDL oxida-tion or LDL-induced monocyte chemotactic activity.43 ApoJis an acute-phase reactant that associates with HDL.44 Incontrast to apoA-I, apoJ was effective in preventing bothLDL oxidation and LDL-induced monocyte chemotacticactivity during coincubation with LDL.45

Paraoxonase (PON) is a component of HDL that has beendemonstrated both to prevent the formation of mildly oxi-dized LDL and to inactivate LDL-derived oxidized phospho-lipids once they are formed.5,40,41 Mackness et al46 firstdescribed the role of PON in preventing metal ion oxidation

Figure 1. Formation of LDL-derived oxidized phospholipids. LDLcontains PAPC that has a mass to ion ratio (m/z) of 782.4. Ara-chidonic acid is shown in the diagram at the sn-2 position ofPAPC. The 12-LO pathway generates HPETE and HPODE,which directly associate with LDL or interact with cholesteryllinoleate (Chol.18:2) to form cholesteryl linoleate hydroperoxide(CE-OOH), which then associates with LDL. Although the dia-gram depicts CE-OOH as being formed and then associatingwith LDL, the CE-OOH could also be formed within LDL afterHPODE and HPETE are associated with LDL. When a criticalconcentration of HPETE, HPODE, and CE-OOH is reached inLDL, PAPC is oxidized forming the pro-inflammatory oxidizedphospholipids found in mildly (MM) oxidized LDL. The 3 oxi-dized phospholipids depicted in MM-LDL are POVPC (m/z594.3), PGPC (m/z 610.2), and PEIPC (m/z 828.6). See text forexplanation of abbreviations.

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of LDL. Aviram and colleagues47–49reported that PON has aperoxidase activity that may explain its ability to renderfreshly isolated LDL resistant to oxidation by human arterywall cells.40 PAF-AH, another enzyme associated with someHDL particles, has also been shown to be able to inactivateLDL-derived oxidized phospholipids.4 A third enzyme asso-ciated with HDL that may play a role in preventing theformation of and in inactivating LDL-derived oxidized phos-pholipids is lecithin:cholesterol acyltransferase.50–54A fourthHDL-associated enzyme that reduces organic hydroperoxidesand is inhibited by physiological concentrations of homocys-teine is plasma reduced glutathione selenoperoxidase.55 Thus,normal HDL contains several enzymes that can potentiallyprevent the formation of and inactivate the inflammatoryLDL-derived oxidized phospholipids. Except for PAF-AH,the other 3 enzymes are associated exclusively with HDL.Whereas PAF-AH is associated with both LDL and HDL inhuman plasma, Stafforini and colleagues56 have suggestedthat for the prevention of LDL oxidation, PAF-AH transfersto HDL where it functions more efficiently.

Direct proof of a role for 1 of these HDL-associatedenzymes in the development of atherosclerosis has beenprovided in mouse models. Shih et al57 demonstrated thatmice lacking the serum PON gene were susceptible toorganophosphate toxicity and diet-induced atherosclerosis. Inother studies, these authors demonstrated that combinedserum PON-knockout/apoE-knockout mice exhibited in-creased lipoprotein oxidation, with higher levels of POVPC,PGPC, and PEIPC in their IDL and LDL fractions and thatthe double-knockout mice had significantly more atheroscle-rosis compared with apoE-knockout mice.58 This finding isespecially interesting because the apoE-knockout mice atbaseline had low levels of PON,45 yet removal of theirresidual PON activity clearly added to lipoprotein oxidationand the development of atherosclerosis,58 indicating theimportance of PON to these processes.

LDL-Derived Oxidized Phospholipids andHDL as Components of a System of

Nonspecific Innate ImmunitySepsis is a major cause of fetal wastage and infant death,especially during the first year of life when the infant’sspecific immune system is immature. It is likely that a systemof nonspecific innate immunity resulted, in part, in responseto this evolutionary pressure. We propose here that LDL-derived oxidized phospholipids and HDL may be part of asystem of nonspecific innate immunity. As noted above,natural antibodies to LDL-derived oxidized phospholipidshave been identified.14 Napoli and colleagues59 reported thepresence of fatty streaks in human fetal aortas. In that study,59

serial sections of the arch, thoracic, and abdominal aortaswere immunostained for recognized markers of atherosclero-sis, including macrophages, apoB, and the oxidation-specificepitopes malondialdehyde and 4-hydroxynonenal-lysine. Theauthors concluded that because LDL and oxidized LDL werefrequently found in the absence of monocyte-macrophages,while the opposite was rare, suggested that intimal LDLaccumulation and oxidation contributed to monocyte recruit-ment in vivo.59 Naturally occurring antibodies to the LDL-derived oxidized phospholipids have been viewed as a com-

ponent of the innate immune system.60 We hypothesize thatthe oxidative environment created by the inflammatory re-sponse to LDL-derived oxidized phospholipids may also bepart of a system of innate immunity that evolved to protect thefetus and infant against sepsis. Napoli and colleagues59 foundthat maternal hypercholesterolemia enhanced fatty streakformation in fetal aortas. If our hypothesis is correct, theremay also have been evolutionary pressure to select formaternal hypercholesterolemia and this may, in part, explainwhy hypercholesterolemia is common among many humanpopulations.

Further evidence to support a role for LDL-derived oxi-dized phospholipids as part of a system of nonspecific innateimmunity comes from studies of HDL and LDL during anacute-phase response. Van Lenten et al61 reported that HDL,during an acute-phase response in humans (induced bysurgery) or in rabbits (induced by injection of croton oil), lostPON and PAF-AH activities and gained the pro-oxidantsceruloplasmin and serum amyloid A. As a result of thesechanges, HDL was converted from an anti-inflammatory to aproinflammatory particle, as judged by its ability to protectagainst or enhance LDL oxidation by artery wall cells or toprotect against or enhance LDL-induced monocyte chemo-tactic activity.61

More recently, Memon et al62 demonstrated that LDL takenfrom Syrian hamsters after they had been injected withbacterial lipopolysaccharide, zymosan, or turpentine con-tained increased amounts of conjugated dienes and lipidhydroperoxides as well as lysophosphatidylcholine, and theacute-phase LDL had a shorter lag phase when oxidized withmetal ions in vitro. On the basis of these studies, Hajjar63

raised the question as to whether oxidized lipoproteins andinfectious agents are in collusion to accelerate atherosclero-sis. Although the answer to Hajjar’s question63 may well beyes, the reason that these systems evolved could not havebeen to accelerate atherosclerosis. More likely they evolvedas part of a system of nonspecific innate immunity.

Van Lenten et al64 sacrificed B6 mice either before or 2, 3,5, 7, or 9 days after intranasal infection with 105 plaque-forming units of influenza A. Peak infectivity in the lung wasreached by 72 hours and returned to baseline by 9 days.64 Noviremia was observed at any time. PON and PAF-AHactivities in HDL decreased after infection, reaching theirlowest levels 7 days after inoculation.64 The ability of HDLfrom infected mice to inhibit LDL oxidation and LDL-induced monocyte chemotactic activity in human artery wallcell cocultures decreased with time after inoculation.64 As theinfection progressed, LDL more readily induced monocytechemotaxis. Peak IL-6 and serum amyloid A plasma levelswere observed 2 and 7 days after inoculation. HDL apoA-Ilevels did not change, but apoJ and ceruloplasmin levels inHDL peaked 3 days after infection. Ceruloplasmin markedlyincreased and remained elevated throughout the time course,whereas apoJ levels decreased toward baseline after the thirdday. It was concluded that alterations in the relative levels ofPON, PAF-AH, ceruloplasmin, and apoJ in HDL occurredduring acute influenza infection and caused HDL to lose itsanti-inflammatory properties.64

In other studies, Van Lenten et al65 found that a keycytokine in the acute-phase response, IL-6, was required forshort-term regulation of PON but not of MCP-1 and was not

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required for the long-term downregulation of PON by anatherogenic diet in susceptible B6 mice. In short-term feedingexperiments (1 to 7 days), Hedrick et al66 found that there wasa dramatic decrease in HDL cholesterol, apoA-I, and PON insusceptible B6 LDL receptor–knockout mice that was asso-ciated with a rapid increase in HDL lipid hydroperoxides andformation of high-molecular-weight forms of apoA-I thatcontained an epitope recognized by a monoclonal antibodythat recognizes POVPC. Measurement of the levels of apoA-Icomplexes associated with immunoglobulins, together withthe time course of events, suggested that preformed antibod-ies to oxidized lipid–apoA-I complexes were present beforethe atherogenic diet was administered.66 It was concluded thaton feeding the atherogenic diet, the number of epitopesincreased to a critical threshold, and this resulted in theclearance of the immune complexes.66 Ox-PAPC inducedIL-6, a potent acute-phase response mediator, when injectedinto B6 LDL receptor–knockout mice.65 HDL from B6 miceon a chow diet inhibited LDL oxidation, whereas HDL fromthe same mice on an atherogenic diet promoted oxidation.67

The latter was enriched in apoJ, which is a marker of theacute-phase response.44 In contrast, HDL from C3H mice thatwere resistant to diet-induced atherosclerosis protected LDLfrom oxidation, whether the mice were maintained on a chowor an atherogenic diet,67 and HDL from C3H mice on theatherogenic diet did not have increased levels of the acute-phase reactant apoJ.45 These studies suggest a link betweenHDL and LDL-derived oxidized phospholipids and a nonspe-cific innate immune system that probably evolved to protectagainst infection, particularly in the young, and that appearsto be activated in susceptible mouse strains by an atherogenicdiet.

Proinflammatory HDL as a Potential Markerof Susceptibility to Atherosclerosis

HDL has been previously described as a “chameleon-like”lipoprotein,3 being anti-inflammatory in the basal state andproinflammatory during an acute-phase response. As notedabove, LDL-derived oxidized phospholipids were found toinduce IL-6 in hepatocytes and to repress PON mRNAlevels.65 A number of mouse models that exhibit susceptibil-ity to atherosclerosis have been found to have proinflamma-tory HDL and decreased PON activity.8,45,67–69Leitinger etal8 found that an atherogenic diet resulted in the formation ofoxidized phospholipids in the livers of mice that weregenetically susceptible to diet-induced atherosclerosis, andthese oxidized phospholipids were increased further in micetransgenic for secretory PLA2.

8 Presumably, these oxidizedphospholipids induced an acute-phase response, which re-sulted in proinflammatory HDL in mice susceptible to diet-induced atherosclerosis.8,45,67ApoE–/– mice also had evidenceof proinflammatory HDL and low PON activity.45 HDL fromtransgenic mice overexpressing apoA-II had proinflammatoryHDL and developed atherosclerosis on a chow diet.68,69TotalHDL concentrations in the transgenic mice overexpressingapoA-II were elevated but the PON activity was not, resultingin a concentration of PON in HDL of approximately halfnormal.69 Addition of exogenous PON to the HDL of thetransgenic mice overexpressing apoA-II converted the HDLfrom proinflammatory to anti-inflammatory, suggesting that

in this model the PON activity was responsible for theproinflammatory HDL.69

PON has also been associated with atherosclerosis inhumans. James and colleagues70 reported that smoking wasindependently associated with significant decreases in serumPON activities and concentrations in patients with coronaryartery disease and that cessation of smoking led to an increase

Figure 2. The acute-phase (AP) reaction favors the formation ofproinflammatory HDL and mildly oxidized LDL. A, In the basalstate, HDL contains apoA-I and apoJ as well as 4 enzymes,PON, PAF-AH, lecithin:cholesterol acyltransferase (LCAT), andplasma reduced glutathione selenoperoxidase (GSH peroxidase)that can prevent the formation of or inactivate the inflammatoryLDL-derived oxidized phospholipids found in mildly oxidizedLDL. As a result, in the basal state, HDL may be consideredanti-inflammatory. During the acute-phase reaction, A-I may bedisplaced by the pro-oxidant acute-phase reactant SAA.Another pro-oxidant acute-phase reactant, ceruloplasmin, asso-ciates with HDL as does the anti-oxidant acute phase reactantapoJ. PON, PAF-AH, and LCAT decrease in HDL during theacute-phase reaction, and the lipid hydroperoxides HPETE,HPODE, and cholesteryl linoleate hydroperoxide (CE-OOH)increase in HDL. A-II and GSH peroxidase are shown asunchanged during the acute-phase reaction although there areno data on the latter. The net effect of the changes in HDL dur-ing the acute-phase reaction is the production of pro-oxidant,proinflammatory HDL particles (AP-HDL). B, In the basal state,HDL prevents the formation of and inactivates the LDL-derivedoxidized phospholipids shown in Figure 1. As a result, HDLfavors the maintenance of noninflammatory LDL and the conver-sion of the proinflammatory, mildly oxidized LDL (MM-LDL) to anoninflammatory state. In contrast, during an acute-phase reac-tion, AP-HDL favors the conversion of LDL to the proinflamma-tory MM-LDL. As discussed in the text, the acute-phase reac-tion can be truly acute, as in the case of a viral infection, or itmay become chronic, as in mice that are genetically susceptibleto diet-induced atherosclerosis when they are fed an athero-genic diet or in some patients with normal blood lipids andatherosclerosis.

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in serum PON within months. This group of patients hadmildly elevated LDL cholesterol levels on average andnormal HDL cholesterol levels that were lowest in thesmokers.70 The relationship of PON to coronary heart diseaseis complicated by the polymorphisms present in humans.71

However, the concentration of PON and its activity weresignificantly lower in patients immediately after myocardialinfarction compared with those in age- and sex-matchedcontrols.72 Forty-two days after infarction, PON activity hadincreased but was still lower than in controls.72 Analysis ofPON1 genotypes did not discriminate between patients andcontrols.72

Navab and colleagues41 found that PON activity in HDLwas significantly lower in 24 patients with angiographicallydocumented coronary artery disease who were normolipi-demic and who were neither diabetic nor taking hypolipi-demic medications. However, there was an overlap betweenpatients and 29 age- and sex-matched controls.41 As notedabove, PON is just 1 of at least 4 enzymes and 2 apolipopro-teins associated with HDL that can potentially modulate theformation of or inactivate LDL-derived oxidized phospholip-ids. Therefore, it was not surprising that there was overlap inPON activities between patients and controls. To focus onwhole HDL rather than on PON exclusively, Navab et al41

studied a subset of the patients to determine whether theirHDL was anti-inflammatory or proinflammatory.41 HDLfrom 10 of 10 of the patients not only failed to inhibit LDLoxidation by artery wall cells and the biological activity ofOx-PAPC but, on average, also actually increased LDLoxidation and enhanced the biological activity of Ox-PAPC.41

HDL from 10 of 10 age- and sex-matched controls inhibitedboth LDL oxidation and the biological activity of Ox-PAPC.41 These data suggest that some patients with coronaryartery disease and normal HDL cholesterol levels haveproinflammatory HDL. These data also suggest that proin-flammatory HDL may be a marker of susceptibility toatherosclerosis in humans as it appears to be in mice.However, large-scale studies will be required to determine thetrue predictive value of such tests. Such studies would have totake into consideration the age of the subject. At birth, humanHDL cholesterol levels are equal to or greater than adultlevels. However, PON activity at term is approximately halfof what it is at age 2 years.73 Low PON activity relative toHDL cholesterol levels at birth may be part of a nonspecificinnate immune system that protects the infant against sepsisas discussed above. However, thepersistenceof proinflam-matory HDL into adulthood may predispose and predictsusceptibility to atherosclerosis.

There is increasing evidence that markers of inflamma-tion74 and the acute-phase response, including induction ofC-reactive protein, predict susceptibility to risk for coronarysyndromes.75–78 It has been suggested that the acute-phaseresponse can become chronic79 and that a state of low-gradesystemic inflammation is a consequence of being over-weight.80 It may well be that the changes seen in HDL in micewith diet-induced atherosclerosis and in some patients withnormal blood lipid levels represent a chronic acute-phaseresponse. This chronic acute-phase response could be perpet-uated, in part, by LDL-derived oxidized phospholipids andmay be exacerbated by the infections and stresses (eg,surgeries) that humans endure in modern society. Figure 2A

summarizes the changes that occur in HDL during an acute-phase response, and Figure 2B indicates what the impact ofthese changes in HDL would be on the balance between LDLand mildly oxidized LDL in the artery wall. If the concen-tration of LDL-derived oxidized phospholipids determinesthe intensity of the inflammatory response in the artery wall,then the balance between noninflammatory LDL (ie, LDLthat does not induce artery wall cells to make proinflamma-tory molecules such as MCP-1) and mildly oxidized LDL,which is proinflammatory, would in part determine plaquevulnerability and hence, susceptibility to heart attack andstroke.81–84As indicated in Figure 2, HDL likely plays a keyrole in modulating these processes and hence, in preventingor promoting heart attack and stroke.

AcknowledgmentsThis work was supported by US Public Health Service grantHL-30568 and the Laubisch, Castera, and M.K. Grey funds atUCLA.

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