Modified HDL: Biological and physiopathological consequences

Nutrition, Metabolism & Cardiovascular Diseases (2006) 16, 371e386

www.elsevier.com/locate/nmcd

REVIEW ARTICLE

Modified HDL: Biological and physiopathologicalconsequences

Giuseppe Danilo Norata a,b,1, Angela Pirillo a,1,Alberico Luigi Catapano a,b,*

a Department of Pharmacological Sciences, University of Milan, Italyb Center for the Prevention and Therapy of Global Cardiovascular Risk,Italian Society for the Study of Atherosclerosis, Bassini Hospital, Cinisello Balsamo, Italy

Received 14 December 2005; accepted 3 January 2006

KEYWORDSModified HDL;Myeloperoxidase;Glycation;Paraoxonase

Abstract Epidemiological and clinical studies have demonstrated the inverse as-sociation between HDL cholesterol levels (HDL-C) and the risk of coronary heart dis-ease (CHD). This correlation is believed to relate to the ability of HDL to promotereverse cholesterol transport. Remodeling of HDL due to chemical/physical modifi-cations can dramatically affect its functions, leading to dysfunctional HDL thatcould promote atherogenesis. HDL modification can be achieved by differentmeans: (i) non-enzymatic modifications, owing to the presence of free metal ionsin the atherosclerotic plaques; (ii) cell-associated enzymes, which can degradethe apoproteins without significant changes in the lipid moiety, or can alternativelyinduce apoprotein cross-linking and lipid oxidation; (iii) association with acutephase proteins, whose circulating levels are significantly increased during inflam-mation which may modify HDL structure and functions; and (iv) metabolic modifi-cations, such as glycation that occurs under hyperglycaemic conditions. Availabledata suggest that HDL can easily be modified losing their anti-atherogenic activi-ties. These observation results mainly from in vitro studies, while few in vivo data,are available. Furthermore the in vivo mechanisms involved in HDL modification areill understood. A better knowledge of these pathways may provide possible thera-peutic target aimed at reducing HDL modification.ª 2006 Elsevier B.V. All rights reserved.

* Corresponding author. Department of PharmacologicalSciences, Via Balzaretti, 9, 20133 Milano, Italy. Tel.: þ39 0250318302; fax: þ39 02 50318386.

E-mail address: [email protected] (A.L. Catapano).1 These authors equally contributed to the work.

0939-4753/$ - see front matter ª 2006 Elsevier B.V. All rights resedoi:10.1016/j.numecd.2006.01.012

Introduction

Epidemiological and clinical studies have demon-strated the inverse association between HDL cho-lesterol levels (HDL-C) and the risk of coronary

rved.

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372 G.D. Norata et al.

heart disease (CHD) [1e3]. Low HDL-C is the mostfrequent dyslipoproteinemia in patients with pre-mature myocardial infarction [4] and is an inde-pendent predictor of recurrent coronary events[5,6]. Furthermore raising HDL, decreases the inci-dence of coronary artery disease [7]. However, inaddition to plasma levels, the composition andthe structure of HDL play a key role in the anti-atherosclerotic activities. The various HDL sub-classes vary in quantitative and qualitative contentof lipids, apolipoproteins, enzymes and lipid trans-fer proteins, resulting in differences in shape,density size, charge and, eventually, function. Infact remodeling of HDL due to chemical/physicalmodifications can dramatically affect its functions,leading to dysfunctional HDL that could promoteatherogenesis. The purpose of this review is topresent and discuss recent evidence regardingthe modifications of HDL and how they can affectthe anti-atherosclerotic properties of HDL. Physio-logical function(s) of HDL will only be outlined andfor a more comprehensive review the reader isreferred to other publications [8,9].

Biological functions of HDL

The cardiovascular protective effects of HDL havebeen mainly attributed to its role in reverse

cholesterol transport; however a number of ‘‘pleio-tropic’’ atheroprotective effects of HDL areemerging.

HDL and reverse cholesterol transport(Fig. 1)

The inverse correlation between HDL cholesterollevels and the risk for coronary heart disease isoften explained by the ability of HDL to removecholesterol from the periphery for delivery to theliver and excretion into the bile, a process namedreverse cholesterol transport [10]. The concept ofreverse cholesterol transport provides the theoret-ical framework for understanding body cholesterolhomeostasis [8].

Lipid-free Apo A-I and/or lipid-poor pre-b-HDLparticles are produced either in the intestine orliver or shed from the surface of triglyceride-richlipoproteins (TGRL) during lipolysis. These parti-cles initiate efflux of phospholipids and cholesterolfrom cell membranes [8]. This process is facili-tated by the adenosine triphosphate-binding cas-sette transporter 1 (ABCA1) that moves cellularlipids across the bilayer in a process requiring hy-drolysis of ATP. As ABCA1 does not bind cholesterol[11] it is believed to induce translocation of phos-pholipids to lipid poor Apo A-I, thus triggering

LargeHDL2

Lipid-free A-I

Nascent HDL

MediumHDL3

Small

Peripheral tissues

LCAT

Cholesterol and

Phospholipid

ABCA-1

Cholesterol and

Phospholipid

SR-B1HL

ABCA-1

SR-B1

ABCA-1 or SR-B1

Figure 1 HDL formation and reverse cholesterol transport. After secretion from liver or intestine Apo A-I is lipidatedvia the interaction with ABCA1, generating nascent pre-b-HDL. Pre-b-HDL acquires free cholesterol from peripheralcells and is converted to mature HDL, enriched in cholesteryl esters through the action of LCAT. Mature HDL are takenup by the liver through SR-BI receptor.

Biological and physiopatological consequences of HDL 373

cholesterol efflux to the Apo A-I-phospholipidscomplex. The nature of the preferred phospholipidsubstrate for ABCA1 remains a matter of debate,phosphatidylserine (PS) and phosphatidylcholine(PC) are major candidates [12]. Of note patientswith dysfunctional ABCA-1 present a severe anal-phalipoproteinemia [13e15].

Cholesterol in nascent discoidal HDL is thenesterified by the action of lecithin-cholesterolacyltransferase (LCAT). Cholesteryl esters readilymove to the core of HDL particles, producinga steady gradient of free cholesterol and enablingHDL to further accept cholesterol from variousdonors [8]. The reciprocal exchange of cholesterylester for triglycerides mediated by cholesteylester transfer protein (CETP) moves the bulk ofthe cholesteryl esters to Apo B-containing lipopro-teins, which are eventually cleared by the liver. Atthe same time, HDL becomes enriched with triglyc-erides, which are substrate for CETP and HL. Theconcerted action of CETP-mediated cholesterylester transfer and HL-mediated hydrolysis of tri-glycerides and phospholipids leads to smaller HDLparticles that are the preferred binding partnersfor scavenger receptor type BI (SR-BI), themajor HDL receptor in hepatocytes. The bindingof HDL to SR-BI mediates the selective uptake ofcholesteryl esters. Lipid-free apolipoproteins orlipid-poor pre-b-HDL are also formed during the re-modeling by phospholipid transfer protein (PLTP),CETP, and hepatic lipase (HL). The endocytic re-ceptors, cubulin and megalin, act in concert tomediate the endocytosis and catabolism of lipidpoor Apo A-I by the kidney.

The vast majority of available evidence suggeststhat impairment of the reverse cholesterol trans-port may favor deposition of cholesterol in thearterial wall and thereby contribute to the de-velopment of arteriosclerosis [8].

‘‘Pleiotropic’’ effects of HDL (Table 1)

Evidence is accumulating suggesting that, in addi-tion to their role in reverse cholesterol transport,

HDLs positively influence vascular functions includ-ing endothelial responses during atherogenesis,coagulation, fibrinolysis, platelet adhesion, adhe-sion molecules and protease expression, and exertantioxidant activity [9,16]. HDLs promote endo-thelial proliferation and decrease endothelial apo-ptosis [17,18]. HDLs also play a key role invasorelaxation by increasing the release of nitricoxide and prostacyclin through the induction ofthe expression and the activity of endothelial ni-tric oxide synthase [19] and the coupling of cyclo-oxygenase 2 and prostacyclin synthase [20]. HDLsdecrease the expression of adhesion molecules,platelet-activating factor and Von Willebrand factor[16,21]; several of these effects may contribute tothe anti-inflammatory effect of HDL. Theseeffects are achieved at the gene expression leveland are dependent on the activation of severalintracellular signaling pathways, including PI3K/Akt, ERK1/2, PKC, p38MAPK [16,22]. The complex-ity of the signaling pathways modulated by HDLmost likely depends on the balance among thebiological activities of the components of this classof lipoproteins, such as apolipoproteins or lipids,on cellular gene expression.

HDL inhibits the oxidation of LDL by transitionmetal ions, and also prevents 12-lipoxygenase-mediated formation of lipid hydroperoxides [23].Inhibition of LDL oxidation by HDL is usually attri-buted to the high content of antioxidants in thislipoprotein, to antioxidative properties of Apo A-I,and to the association with HDL of several enzy-mes, such as paraoxonase (PON), platelet activatingfactor acetylhydrolase (PAF-AH), and glutathioneperoxidase (GPX), which prevent LDL oxidationand/or contribute to the degradation of bioactiveproducts that are formed during oxidation. Apo A-Ireduces phospholipids and cholesteryl ester perox-ides present in copper oxidized LDL and removeshydroperoxyeicosatetraenoic acid (HPETE) andhydroperoxyoctadecadienoic acid (HPODE), in 12-lipoxygenase modified LDL [24,25]. Both HPETEand HPODE are examples of so-called ‘‘seeding mol-ecules,’’ compounds necessary for the induction of

Table 1 Pleiotropic effects of HDL

HDL Key references

Cell proliferation [ proliferation, Y apoptosis [17,18]Vascular tone [ NO, [ PGI2 [19,20]Inflammation Y VCAM-1, Y ICAM-1, Y E-selectin, [ TGF-b2 [9,16,21,22]Coagulation, fibrinolysis Y TF, Y PAF, [ activated protein C, [ protein S [9]LDL oxidation Y metal ions mediated oxidation [24e26]

Y 12-Lox mediated oxidationScavenge of lipid peroxides and LPCPON, PAF-AH and GPX


the non-enzymatic oxidation of lipoprotein phos-pholipids [26].

All together the ability of HDL to act as a ‘‘de-pot’’ explains the antioxidant properties of HDL,but, at the same time, renders HDL itself moresusceptible to oxidation: The delicate relationbetween the modification and the impairment ofHDL function probably plays a role in determiningthe anti-atherogenic potential of HDL.

HDL modification (Fig. 2 and Table 2)

HDL modification can be achieved by differentmeans: (i) non-enzymatic modifications, owing tothe presence of free metal ions in the atheroscle-rotic plaques [27]; (ii) cell-associated enzymes,which can degrade the apoprotein componentswithout significant changes in the lipid moiety, orcan alternatively induce apoproteins cross-linkingand lipid oxidation; (iii) acute phase proteins,whose circulating levels are significantly increasedduring inflammation and, by interacting with HDLmay modify their structure and functions; (iv) met-abolic modifications, such as glycation that occursunder hyperglycemic conditions.

Non-enzymatic modifications

Human atherosclerotic plaques contain free metalions [27e29]. Metal ion-induced production ofoxygen radical has been suggested to play a role incardiovascular disease [30], probably due to the ox-idation of low density lipoproteins [31,32]. Themodification of HDL by copper induces lipid peroxi-dation and oxidation of apoproteins, thus increasingthe negative charge of the particles [33] and deter-mining the appearance of cross-linked apoproteins(Fig. 3A) (Pirillo et al., unpublished observations).During the early stages of oxidation, three oxidizedspecies of Apo A-I can be detected [34], showing anincreased mass due to the modification of two

ModifiedHDL

RCT Oxidative stress

NF-kB activation

PAI-1 production

apoptosis

eNOS expression

NO production

Figure 2 Biological effects of modified HDL.

critical methionine residues (Met86 and Met112);Apo A-II can also be converted to a species with in-creased mass due to oxidation of the single methio-nine residue (Met26). These modifications of HDLreduce their ability to stimulate cholesterol effluxfrom lipid-laden macrophages [33] (Fig. 3B) (Pirilloet al., unpublished observations), and increasecellular free cholesterol content [35]. Moreover,copper oxidized HDLs are cytotoxic to macrophagesin a dose-dependent manner [36].

Copper oxidized HDLs also bind endothelial cells[37], promoting the activation of a network of intra-cellular kinases including ERK1/2 and p38 MAPK [16]in a time- and dose-dependent manner, an effectalso observed in HepG2 cells [16]. Furthermore, in-cubation of endothelial cells with oxidized-HDLelicits a marked increase in the activation of theNF-kB pathway [38], a key player in the inflamma-tory response [39]. In addition, Ox-HDL inducesa dose-dependent increase in reactive oxygen spe-cies (ROS) production, that is inhibited in the pres-ence of probucol or diphenylene iodonium (aninhibitor of NADPH oxidase) [38]. Of note, pretreat-ment with probucol or diphenylene iodonium alsoinhibited Ox-HDL induced IkBa phosphorylation[38], suggesting that increased generation of ROSby Ox-HDL may be associated with NF-kB activation.The effects of Ox-HDL on the endothelium are notrestricted to NF-kB activation and ROS generation.Ox-HDL3, but not native HDL3, increases PAI-1mRNA expression and protein synthesis in endothe-lial cells [40]. Moreover incubation of EC with spe-cific inhibitors of p38 MAPK suppresses theOx-HDL3-dependent PAI-1 induction [40]. Transienttransfection experiments suggested that none ofthe response elements in the proximal promoter(�804 to 17) are involved in Ox-HDL3 mediatedPAI-1 expression, instead Ox-HDL3 increases thePAI-1 mRNA half-life [16]. Also Cox-2 has been shownto be induced upon Ox-HDL incubation via NF-kB andcEBP activation (Callegari et al., unpublishedobservations).

Myeloperoxidase-mediated HDLmodification

Myeloperoxidase (MPO) is a heme protein secretedby activated phagocytes, and is expressed inhuman atherosclerotic lesions [41]. MPO utilizeshydrogen peroxide and a variety of low-molecularweight organic and inorganic substances as sub-strates to generate reactive oxidant species as in-termediates. The major product of MPO activityis hypochlorous acid (HOCl), formed from H2O2

and chloride, which can modify different biomole-cules by chlorination and/or oxidation [42,43].


Table 2 Structural and biological effects of HDL modification

Type of modification Effects on HDL particles [ref.] Biological effects [ref.]

Non-enzymatic modificationCopper Apoprotein cross-linking [33];

lipid peroxidation [33];[ negative charge [33];Y PON1 activity [126];Y PLTP activity [136];Y LCAT activity [139,140]

Y RCT [33];[ cellular FC content [35]; cytotoxicity [36];NF-kB activation [38]; [ ROS production [38];[ PAI-1 (mRNA and protein);[ COX-2 expression(Callegari et al, unpublished)

Cell-associated enzymesMyeloperoxidase:

hypoclorous acidApoprotein cross-linking [44];Y PLTP activity [137]

Y RCT (SR-BI- and ABCA1-mediated) [44e46];Y uptake of CE [45];Y eNOS expression and activity [47]

Myeloperoxidase: tyrosylradicals

Apoprotein cross-linking [53,54];lipid peroxidation [54,55]

[ RCT from human skin fibroblasts [56e58];Y atherosclerosis in apoE KO mice [59];Y RCT (J774) [60]

Chymase-tryptase Apoprotein degradation(pre-b-particles) [65e67,70]

Y ABCA1-mediated RCT [68,70]

Matrix metalloproteinases Apoprotein degradation [74,75];pre-b-particles depletion [80]

Y ABCA1-mediated RCT [75]

PMN-associated enzyme Apoprotein degradation [80,82] Y RCT [81]; [ modified HDLinternalization [82]

Endothelial lipase Y PC content ([ lysoPC andnon-esterified FA) [90]

Y SR-BI-mediated RCT [90]

Acute phase proteinsSerum amyloid A protein Apo A-I displacement [94];

pre-b particles generation [97];[ lipoprotein size [93];Y LDL protection from oxidation [95];Y PON1 activity [95];Y PAF-AH activity [95];Y LCAT activity [95]

Y RCT [96]

Secretory phospholipaseA2;group II

[ Exposure of apolipoproteindomains [103];[ HDL catabolism [106]

[ Binding to the cells [103,106,107];[ sterols and apoproteins transfer [104,105]

Groups V and X PL hydrolysis without Apo A-Imodification [108]

Y RCT [108]

Metabolic modificationsGlycation [ Lipid peroxidation [111,112];

[ HDL susceptibility tooxidation [115,118,119];[ HDL catabolism [113];Y PON1 activity [111,120];Y LCAT activity [141]

Y Binding to human skin fibroblasts [114];Y RCT [115]

Glycoxidation [ EC apoptosis [121];[ H2O2 release from EC [122];Y catalase and CuZn-superoxide dismutase [122];Y NO production [122]

HOCl-mediated modification of HDL results in theformation of high molecular weight Apo A-I ad-ducts [44], similar to those observed after Cu2þ-mediated oxidation. The ability of MPO-modifiedHDL to promote cholesterol efflux from J774 mac-rophages is impaired, and this effect is dependent

upon the apoprotein modification [44]. Methioninemodification appears to be responsible for thesignificant decrease in cholesterol efflux, as itsoxidation modifies the conformation of lipid-freeor lipid-associated Apo A-I [44]. The preferentialamino acids targeted by HOCl modification of


HDL3 20’ 1h 4h

CuSO4

AI-AI-AI

AI-AI

AI-AII

AI

µg lipoprotein/ml

0 100 200 300 400

dp

m/m

g cell p

ro

tein

0

100000

200000

300000

HDL3Cu++ 20µM, 20'Cu++ 20µM, 4hCu++ 20µM, 24h

A B

Figure 3 Effect of Cuþþ-mediated oxidation of HDL on Apo A-I structure (A) and on cholesterol efflux in macro-phages (B).

Apo A-I are methionine residues, which are de-stroyed at low oxidant/protein ratio; at a higher ra-tio, lysine and arginine are partially modified, whiletyrosine, phenylalanine and histidine are lost [44].

The modification of HDL by HOCl increases theaffinity of these particles for SR-BI, as theselipoproteins associate with SR-BI much fasterthan native HDL. However, this increased associa-tion is ‘‘non-productive’’, since cholesterol effluxis actually decreased [45]. Furthermore, the selec-tive uptake of cholesteryl esters from HOCl-HDL isalso impaired, suggesting that this modificationleads to a ‘‘defective’’ binding of HDL to SR-BI un-able to support efficiently the bi-directional trans-fer of cholesterol mediated by this receptor [45].Moreover, both HDL and Apo A-I modified by HOCllose their ability to promote ABCA1-dependentcholesterol efflux [46].

Incubation of endothelial cells with HOCl-HDLattenuates the expression and the activity ofvasculoprotective endothelial NOS (eNOS) [47].NO biosynthesis critically depends on the correctcompartmentalization of eNOS in specific intracel-lular membrane domains, i.e., Golgi complex andplasmalemmal caveolae [48]. Incubation withHOCl-HDL leads to changes of intracellular eNOSdistribution including translocation from theplasma membrane and disruption of the perinu-clear location [47]. The active component mediat-ing this effect is 2-chlorohexadecanal [47] that isformed during HOCl-mediated oxidative cleavageof HDL-associated plasmalogen [47].

MPO can also produce the nitrogen dioxideradical, a species which can convert tyrosine to3-nitrotyrosine [49]; HDL can be nitrated in vitroby MPO [50]. Tyrosine 192 is the major site forMPO-induced chlorination and nitration of Apo A-I[51]; however, while chlorination reduces ABCA1-mediated cholesterol efflux, nitration minimally

affects this process [51,52], suggesting that thesetwo modifications differently affect conformationof Apo A-I.

Other reactive intermediates formed by MPOare tyrosyl radicals; the exposure of lipoproteins totyrosyl radicals induces protein cross-linking[53,54] and lipid peroxidation [54,55]. HDL modifi-cation by tyrosyl radicals induces cross-linking ofapoproteins, with formation of Apo A-I dimersand trimers and Apo A-I/Apo A-II complexes, dueto phenolic coupling of tyrosines [56]. The tyrosy-lation of HDL increases their ability to inhibit cho-lesterol esterification in human skin fibroblast andto deplete cholesterol-loaded mouse peritonealmacrophages of esterified cholesterol [56], sug-gesting that tyrosylated HDLs efficiently reducethe pool of cellular cholesterol available for ester-ification, thus increasing cellular free cholesterol,which becomes available for removal by an exter-nal acceptor, such as Apo A-I [57]. Apo A-I/Apo A-IIheterodimers are responsible for the enhancedcholesterol efflux induced by tyrosylated-HDLfrom human skin fibroblasts [58]. ApoE-deficientmice administered with tyrosylated HDL for8 weeks showed a marked increase in endogenousHDL-cholesterol and a decreased formation of ath-erosclerotic lesions, probably due to the mobiliza-tion of cholesterol from peripheral cells [59].Opposing data have been obtained in a differentstudy, in which tyrosylated-HDLs were shown tobe a lesser efficient cholesterol acceptor in J774murine macrophages [60]. The interaction of tyro-sylated-HDL with SR-BI was not compromised, theaffinity for SR-A was increased, as was the abilityto transfer cholesteryl esters to the cells, whilethere was a decrease in the activity of neutral cho-lesterol ester hydrolase, an enzyme which controlsthe formation of free cholesterol for cellular re-lease, thus explaining the decrease in cholesterol


efflux [60]. These discrepancies can be related toa different degree of oxidative tyrosylation or tothe different cell lines used in these studies.

Mast cell derived enzymes(chymase and tryptase)

Mast cells are a component of the immune systemand are present in the human arterial intima [61,62],localized close to foam cells [63]. After activation,mast cells degranulate and release a number ofbiologically active components, including proteo-lytic enzymes such as tryptase and chymase [63].

When HDL3 is incubated with mast cells stimu-lated to degranulate, the ability of lipoprotein topromote cholesterol efflux from macrophages issignificantly reduced [64]; this effect is related tothe proteolytic activity of chymase, the main prote-ase present in the granules, which degrades HDL3

apoproteins [65], mainly those present in the pre-b-migrating particles [66]. As a consequence, pre-b-migrating particles are lost, while a-migratingparticles are spared [67]. Chymase-modified HDL3

shows an impairment of the ABCA1-mediated cho-lesterol efflux, without affecting SR-BI-mediatedcholesterol efflux, owing to the disappearance ofpre-b-migrating particles [68].

Granules secreted by mast cells contain alsotryptase, a serine protease which requires glycos-aminoglycans to be enzymatically active [69]. Tryp-tase degrades apolipoproteins of HDL3, in particularlipid free or lipid poor Apo A-I and Apo A-IV presentin the pre-b-particles [70]. This results in the reduc-tion of the high affinity component of cholesterolefflux probably ABCA1-mediated [70]. The limitedproteolysis of Apo A-I in a-migrating particles doesnot induce changes in their size or integrity; infact, no effect on the a-HDL3-mediated cholesterolefflux was observed [70]. Altogether these observa-tions suggest that, in the atherosclerotic lesion,chymase and tryptase secreted by mast cells inthe extracellular space near foam cells, might im-pact the high-affinity component of the cholesterolefflux process by degrading specific epitopes of ApoA-I critical for cholesterol efflux activity of HDL.

Matrix metalloproteinases

Matrix metalloproteinases (MMPs) are a family ofdistinct but structurally related proteases involvedin the degradation of extracellular matrix compo-nents [71]. MMPs activity is implicated in vascularremodeling in both physiological and pathologicalconditions [72]; MMPs are thought to weaken thearterial wall, thus contributing to destabilizationand rupture of atherosclerotic plaques [73].

Beside, MMPs can degrade Apo A-I [74,75], thusreducing the activity of HDL3 as cholesterol accep-tor [75]. In fact, incubation of HDL3 with differentmetalloproteinases (MMP-3, MMP-7 and MMP-12)causes the complete loss of pre-b-migrating parti-cles, thus significantly reducing the high affinitycomponent of cholesterol efflux from macrophages[75]. On the contrary, MMP-1 causes a slight reduc-tion of cholesterol efflux and MMP-9 does not havean effect, reflecting a slight or absent ability todegrade Apo A-I [75]. These data suggest thatMMPs, besides their role in increasing plaquevulnerability, may contribute to atherogenesis byimpairing the atheroprotective role of HDL.

PMN-associated enzymes

Polymorphonuclear leukocytes (PMNs) are presentduring the development of early atheroscleroticlesions [76,77], and are believed to play a role in themodification of LDL [78,79] by producing largeamounts of reactive oxygen species and/or proteo-lytic enzymes. HDL can be modified by the contactwith PMN [80], resulting in a reduced protein con-tent due to a complete degradation of Apo A-II andto a partial loss of Apo A-I. PMN-induced modifica-tion of HDL3 impairs their ability to trigger choles-terol efflux from macrophages [81]. Among theprotease produced by PMN, elastase is able to de-grade Apo A-II and, to a lesser extent, Apo A-I [82].Elastase-modified HDL, which is recognized by SR-BI, is more efficiently internalized by macrophagesthan native HDL [82]. ApoA-I is the main plasma fac-tor regulating the secretion of elastase from PMNsand smooth muscle cells [83,84]; elastase releasein the arterial lesion, by enhancing uptake of modi-fied HDL3 by macrophages, may be involved in theformation of dysfunctional lipoprotein.

Endothelial lipase

Endothelial cell-derived lipase (EL) is a member ofthe lipase family which exhibits primarily a phos-pholipase activity [85,86], with HDL as the pre-ferred substrate [87]. The expression of EL isupregulated during inflammation [88,89], raisingthe possibility that EL overexpression may be in-volved in atherogenesis. The in vitro incubationof HDL with EL results in a decreased content inphosphatidylcholine (PC) and a corresponding in-crease in lysoPC and non-esterified fatty acids[90], while no significant changes can be detectedin total cholesterol, free cholesterol or triacylgly-cerol content of the lipoproteins. As the phospho-lipid composition is one of the main factorsdetermining SR-BI-dependent cholesterol efflux


[91], EL-modified HDLs show a decreased ability toassociate with SR-BI and the SR-BI-dependent cho-lesterol efflux is impaired, while ABCA1-dependentlipid efflux is not affected [90]. In fact, the PL hy-drolysis induced by EL can generate lipid poor ApoA-I, supporting the ABCA1-mediated cholesterolefflux. These findings suggest that EL affects dif-ferently the ability of HDL to promote cholesterolefflux and that the overall balance may also de-pend upon local expression of ABCA1 and SR-BI.

Serum amyloid A protein (SAA)

Serum amyloid A protein (SAA) is an acute-phaseprotein synthesized by the liver during inflamma-tion [92]. Within plasma SAA associates with HDL,mainly with subfraction 3 (HDL3) [93], altering itsphysicochemical composition. In fact, SAA dis-places Apo A-I [94], induces an enlargement ofthe lipoprotein size [93] and alters its ability toprotect LDL from oxidation [95], probably due toimpaired activities of HDL-associated enzymes,such as paraoxonase and PAF-acetylhydrolase[95]. In vitro, acute-phase HDLs are less effectivethan native HDLs in removing excess cholesterolfrom mouse macrophages, while the selectiveuptake of cholesteryl esters was significantlyhigher [96]. On the other hand, the displacementof Apo A-I by SAA generates pre-b-migrating parti-cles from a-HDL [97]. No data are available regard-ing the activity of SAA-induced pre-b-particles inthe cholesterol efflux from cells.

Secretory phospholipase A2

Secretory type II phospholipase A2 (sPLA2) enzymeis among the inflammatory mediators likely to beinvolved in atherogenesis. In fact, its circulatinglevels are elevated in atherosclerosis and havebeen detected in early and in more advancedatherosclerotic lesions [98,99] and in ischemic car-diomyocytes [100]. Both serum amyloid protein A(SAA) and sPLA2 are expressed during inflamma-tion. In acute-phase HDL (which contain 27% SAA)sPLA2 activity is enhanced, while normal HDL re-duces it [101]. In vitro modification of HDL byPLA2 does not change their Apo A-I content[102,103], and no proteolytic fragmentation ofApo A-I can be detected [103]. However, sPLA2treatment of HDL results in an increased presenta-tion of specific apolipoprotein domains on the par-ticle surface as a consequence of phospholipidhydrolysis [103], which may be responsible for anelevated binding capacity of modified HDL to thecells. Further, different studies have shown thatfollowing phospholipid hydrolysis by sPLA2, HDLs

display an increased uptake of free and esterifiedcholesterol by different cell types [103e105]. Astudy with transgenic mice showed that overex-pression of sPLA2 reduced plasma HDL cholesterollevels, due to a rapid catabolism of sPLA2-modifiedHDL [106]. The cholesteryl ester component of HDLisolated from sPLA2 transgenic mice was more rap-idly catabolized, compared to the protein moiety,as a result of an increase in the SR-BI-mediatedselective uptake [106,107].

Recently it has been reported that sPLA2(groups V and X) can induce an efficient hydrolysisof HDL phospholipids, and an increase in thenegative charge without Apo A-I modification[108]. Cholesterol efflux to sPLA2-X or -V-treatedHDL is significantly decreased compared to nativeHDL [108], suggesting that the modification ofphospholipid content and composition by the ac-tivity of these enzymes reduces the capacity ofHDL to mediate cholesterol efflux.

Glycation

Individuals with type 2 diabetes present low HDLlevels, this is believed to contribute to the in-creased cardiovascular risk in these patients[109]. Type 1 diabetic subjects develop severeatherosclerosis in spite of normal or elevatedHDL levels [110]; one possible explanation isthat hyperglycemia triggers functional alterationsof HDL that may contribute to accelerate athero-sclerosis. In fact, in these subjects HDL apopro-teins are highly glycated compared to thosefrom normoglycemic individuals. In vitro glycationof HDL increases lipid peroxidation [111,112], ac-celerates its catabolism [113], reduces its bindingaffinity to human skin fibroblasts [114] and de-creases the ability of HDL to promote reverse cho-lesterol transport [115]. Different results havebeen obtained by Rashduni et al., who showedthat glycation of HDL does not impair its functionas cholesterol acceptor [116], and by Passarelliet al. who demonstrated that HDLs isolated fromdiabetic patients exhibit a lower ability to removecholesterol from cells, yet this event is not re-lated to the glycation degree of HDL [117], andmust be attributed to other types of lipoproteinmodification, such as oxidation or desialylation,that occur in diabetes.

Glycation increases protein susceptibility tooxidation [115,118] and in vitro glycated HDLsare more sensitive to the prooxidant effect ofaluminum [119]. A combination of glycative andoxidative stress may thus impair biological func-tions of HDL. As an example, the in vitro glycoxida-tion reduces the ability of HDL to remove lipid


peroxides from oxidized membranes while preserv-ing PON1 activity [120]. This effect is also observedusing HDL isolated from subjects with type 1 diabe-tes [120].

Exposure of human aortic endothelial cells toglycated-oxidized-HDL induces apoptosis via in-creased expression of Bax, Bad, active caspase 3and 9 [121]. These features are associated withrelease of cytochrome c, cell shrinkage, membraneblebbing and concentration and fragmentation ofnucleus [121]. Glycated-oxidized-HDLs also inducesignificant release of H2O2 and a marked down-regulation of catalase and CuZn-superoxide dismu-tase [122], suggesting that H2O2 formation bymodified HDL is due to a disturbance of oxidantand antioxidant enzymes in the cell. Also nitricoxide production is impaired upon incubation withglycated-HDL and this is dependent mainly ona reduced expression of endothelial NOS whileinducible NOS expression is not affected [122].

Effect of modifications on the activityof HDL-associated enzymes

HDLs protect LDLs from oxidation, and reduce theinflammatory response induced by oxidized LDL.These positive effects are due to the activity ofenzymes associated with plasma HDL, such asparaoxonase or platelet-activating factor acetyl-hydrolase. The activity of these enzymes may bealtered during modification of HDL, raising thepossibility that, besides the impairment of reversecholesterol transport, HDL modification can re-duce their protective role in atherosclerosis viadifferent mechanisms.

Paraoxonase

Paraoxonase (PON) is a calcium-dependent enzymeassociated with HDL [123], which plays a role ininhibiting lipoprotein oxidation [124,125]. HDLsusceptibility to oxidation is related to PONactivity [125], suggesting that individuals with lowPON activity are exposed to increased oxidativestress. Moreover, paraoxonase activity is reducedduring cell-mediated or copper-mediated HDLoxidation [126]; the extent of PON inactivation isstrictly related to the extent of HDL oxidation[126]. Under hyperglycemic conditions, HDL aremodified and the glycation of HDL reduces PONactivity [111]. As native phospholipids stabilizePON activity and are essential for PON binding atthe lipoprotein surface, the enzyme inactivationcan be due to an altered interaction of PONwith PL or apoproteins upon HDL glycation.

Finally, acute-phase HDLs, which possess lowerPON activity, lose the ability to protect LDL from ox-idation [95].

Of note, a polymorphism of PON1 (Q192R) in-fluences the lag time of HDL oxidation. A negativecorrelation between Q192R (QQ ¼ 1, QR ¼ 2,RR ¼ 3) and maximum diene formation is present;this suggests that the PON1 R allozyme may bemore efficient than the PON1 Q allozyme[127,128]. Similarly, the correlation between theL55M polymorphism and the parameters of HDLoxidation (LL ¼ 1, LM ¼ 2, MM ¼ 3) is consistentwith the lower serum concentrations of the PON-1M allozyme in comparison with the PON-1 Lallozyme [129]. Subjects with the LL/QQ genotypealso display a significant increased IMT [130],although not all papers are consistent with thisfinding [131].

PAF-AH

Platelet activating factor acetylhydrolase (PAF-AH) is a lipoprotein-associated member of thephospholipase A2 family [132]. In normolipemicsubjects, PAF-AH associates mainly with apoB-con-taining lipoproteins, while less than 20% associateswith HDL. In hypercholesterolemia, the plasma en-zyme activity is increased, and the distribution ofenzyme activity between LDL and HDL is altered[133]. As a consequence, the ratio of HDL-associ-ated PAF-AH to total plasma enzyme activity isdecreased. PAF-AH activity is required for thehydrolysis of bioactive lipids (such as PAF or oxi-dized phospholipids) involved in the pathophysiol-ogy of atherosclerosis. Thus, HDL-associatedPAF-AH may contribute to the protective role ofHDL by reducing LDL oxidation. However, duringinflammation, SAA associates with acute-phaseHDL, determining a significant decrease of PAF-AHactivity compared to normal HDL [95], suggestingthat, during the acute phase response, pro-inflammatory HDLs are generated.

PLTP

The plasma phospholipid transfer protein (PLTP)plays a key role in the transfer of PL betweenlipoproteins [134]; this protein is also involved indetermining the levels of plasma HDL-cholesterol,with the release of small Apo A-I-PL-rich particles(i.e. pre-b-lipoproteins), which act as first accep-tor of free cholesterol during reverse cholesteroltransport [135]. In vitro copper-mediated oxida-tion of HDL reduces PLTP-mediated PL transfer tothe oxidized lipoprotein [136], but this effectoccurs only after extensive HDL apoproteins


modification. This finding suggests that, followingin vivo oxidative modification of HDL, PL transferto the lipoprotein might not be impaired. More-over, the decreased PL transfer is not related toa decreased binding of PLTP to oxidized HDL [136].

Hypochlorite-induced modification of HDL3 re-duces its ability to act as phospholipid acceptorcompared to native HDL3, despite similar bindingof PLTP to native or modified HDL [137]; moreover,PLTP induces the release of Apo A-I from HOCl-modified HDL3; the resulting particles, however,do not display a pre-b-mobility [137].

LCAT

Lecithin-cholesterol acyl transferase (LCAT) isa plasma glycoprotein that catalyzes the esterifi-cation of cholesterol, a process in which pre-b-migrating particles are converted to a-migratingHDL [138]. Aldehydes that are generated duringlipid peroxidation can modify HDL structure and in-hibit LCAT activity [139], and this may explain whyLCAT activity is decreased in copper-modified HDL[140]. LCAT activity is also decreased in HDL iso-lated from diabetic subjects and in in vitro gly-cated HDL, owing to the glycation of lysineresidues in Apo A-I [141]. In addition, the displace-ment of Apo A-I by SAA during acute phase reactioninhibits LCAT activity [95].

Evidence for HDL modification in vivoand antioxidant intervention

While several papers reported the presence ofmodified LDL in atherosclerotic plaques fromexperimental animals and human tissues [142e146], evidence for the presence of modified HDLin atherosclerotic tissues has been reported onlyrecently.

Artola et al. [147] first reported the presence ofcross-linked apoproteins in the absence of ele-vated lipid peroxidation products in HDL isolatedfrom hypercholesterolemic chickens and Niuet al. [148] have shown that HDL and LDL isolatedfrom human endarterectomy specimens containsimilar levels of oxidized lipids, but did not addressthe presence of changes in HDL proteins.

As more accurate methods to detect oxidized-HDL are available [149], these lipoproteins weredetected in atheromatous plaques of the abdominalaorta [37] and in the sera from patients with chronicrenal failure [150]. Nakajima et al. [151], usinga monoclonal antibody based ELISA, has shown thatthe concentration of oxidized-HDL in plasma from23 healthy controls (127 � 50 mg/mL; mean � SD)

is significantly different from that observed in 30patients with non-insulin dependent diabetesmellitus (191 � 65 mg/mL) or in 25 patients withcoronary artery disease (200 � 87 mg/mL) Althoughinteresting, these findings should be confirmed inlarger studies.

Modified HDL colocalize with endothelial cells innon-diseased vessel [45] and bind endothelial cells[37] via interaction with LOX-1 or SR-B1 [38,45]. Inatherosclerotic lesions from human samples, HOCl-modified epitopes colocalize with Apo A-I in areascontaining either lipid droplets or cholesterol crys-tals [45]. More recently two independent groupshave shown that chlorotyrosine (a specific productof HOCl)-modified HDL (ClTyr-HDL) is present inhuman atherosclerotic tissue and human plasma[46,52]. The same groups have shown also thatplasma levels of ClTyr-HDL are increased in sub-jects with cardiovascular disease, suggesting thatcirculating levels of modified HDL representa unique marker for clinically significant athero-sclerotic disease. Also nitrotyrosine modified HDL(NO2Tyr-HDL) levels are increased in subjectswith cardiovascular disease [52]. In both casesApo A-I has been identified as a selective targetfor myloperoxidase-catalyzed oxidative modifica-tion in human atheroma [46,52]. These observa-tions suggest that dysfunctional HDL can begenerated in vivo and can support atherogenesis.As less expensive and time-consuming methods todetect these Apo A-I modifications become avail-able, the clinical relevance of these findings willbe evaluated.

To date the few data available show that in ex-vivo studies HDL isolated from volunteers afterconsumption of a vitamin E supplement are moreresistant to Cu2þ-mediated oxidation assessed bymonitoring the formation of either lipid dienes[152,153], lipid peroxides [152] or reactive lipid al-dehydes [154]. Vitamin C also inhibits lipid oxida-tion in human HDL [155].

A 12-week treatment of 22 hypertriglyceridemicpatients with gemfibrozil resulted in an increaseresistance of HDL to oxidation [156]. Several fac-tors, including Apo A-I and Apo A-II content, theHDL2/HDL3 subfraction ratio and the fatty acidcomposition [156], could account for this effect;further investigation however is warranted to clar-ify this issue. Also the role of estrogen replace-ment on HDL oxidation has been investigated.Zago et al. [157] have shown that HDL resistanceto oxidation is impaired in healthy postmenopausalwoman compared to healthy premenopausal inde-pendently of a-tocopherol content, CETP and pa-roxonase activity. In vitro raloxifene decreasesHDL oxidation and in vivo the combination of


estrogen replacement and exercise protectsagainst HDL oxidation in postmenopausal women.

Conclusion

Available data suggest that HDL can easily bemodified, losing their anti-atherogenic activities.These observations result mainly from in vitrostudies, while fewer in vivo data are available.Furthermore the in vivo mechanisms involved in HDLmodification are ill understood. A better knowledgeof these pathways may provide possible therapeutictargets aimed at reducing HDL oxidation.

The findings summarized in this review suggestthat in addition to HDL plasma levels, also thequality of HDLs contribute to their anti-athero-genic effects. This is the case in subjects carryingthe Apo A-I variant, Apo A-I Milano, who, in spite ofvery low levels of circulating HDL, are protectedfrom cardiovascular events [158]. Similarly sub-jects with high levels of HDL that are prone tomodification could be less protected than subjectswith average levels of HDL that are more resistantto modification.

Future studies are clearly warranted to investi-gate the in vivo predisposition of HDL to modifica-tion in relation to cardiovascular disease and toidentify nutritional and therapeutic approachesaimed at controlling HDL modification and howthey may improve clinical outcome.

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Modified HDL: Biological and physiopathological consequences

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