Oxidized phospholipids and lipoprotein-associated phospholipase A2as important determinants of Lp(a)functionality and pathophysiological role

Alexandros D.Tselepis

Atherothrombosis Research Centre/Laboratory of Biochemistry,Department of Chemistry,University of Ioannina,45110 Ioannina,Greece.

Introduction

Lipoprotein(a)[Lp(a)]consists of a low-density lipoprotein(LDL)-like particle to which a large,highly glycosylatedapolipoprotein(a)[apo(a)]is covalentlybound to the apoB-100 moiety of LDL via a single disul fide bridge[1-3].Apo(a)is highly homologous to the plasma protease zymogen,plasminogen,which contains five tri-loop structures stabilized by three disul fide bonds,named as kringles(K),and a protease domain;thus,it can be activated to plasmin.Apo(a)contains only KIV and KV and has an inactive protease-like domain[1-5].This domain is catalytically inactive,despite having an intact Ser-His-Asp catalytic triad[6].A Ser561-Ile562substitution for Arg561-Val562has been proposed to render the protease-like domain in apo(a)inactive[6,7].Importantly,apo(a)contains 10 subtypes of KIV(KIV-1 to KIV-10);the KIV-2 subtype being present in variable numbers(5 to 50)of identically-repeated copies.KIV-9 contains an additional cysteine residue(Cys4057),which is attached by a disul fide bond to a cysteine residue(Cys4326)of apoB-100,located near the binding site of LDL to its receptor[8-10].Apo(a)is highly polymorphic in length due to the number variation of the KIV-2 copies;thus,the molecular mass of apo(a)isoforms can range between 200 and 800 kDa[1].Apo(a)is encoded by the LPA gene,which contains a 5.6 kb segment existing in multiple repeats(KIV-2 repeat pol-ymorphism)that is responsible for the apo(a)isoform variation[7].Plasma Lp(a)levels vary widely among individuals,are inversely correlated with the apo(a)size and are primarily genetically determined by variation in the LPA gene coding for apo(a)[11].

Lp(a)is considered a causal risk factor for ischemic cardiovascular disease(CVD)[12,13].The evidence for a causal role of Lp(a)in CVD is based on data from large epidemiological databases,mendelian randomization studies,and genome-wide association studies linking genetically determined Lp(a)levels to CVD events.In this regard,epidemiological studies suggest a log-linear relationship between circulating Lp(a)levels and CVD risk that is independent of other lipid measures and conventional risk factors.Mendelian randomization studies demonstrate a causal,multivariable-adjusted,linear association between genetically determined Lp(a)levels and CVD risk.Finally,genome-wide association studies show that the LPA variants are strongly associated with reduced copy numbers of KIV-2 repeats,increased Lp(a)levels,and higher CVD risk[12-13].Despite the well-established role of Lp(a)as a causal risk factor for ischemic CVD,the underlying mechanisms by which it mediates atherogenicity are not well understood[12].The present review article focuses on the pathophysiological role of Lp(a)and particularly discusses new data on the potential role of two Lp(a)components,the oxidized phospholipids(OxPL)and the lipoprotein-associated phospholipase A2(Lp-PLA2),in its functionality.

Oxidized phospholipids:Formation and association with Lp(a)

A key role in the Lp(a)functionality may be played by its OxPL content[14].OxPL are generated by the oxidation of polyunsaturated fatty acid residues,which are usually esteri fied at the sn-2 position of phospholipids[15-17].Oxidation of such phospholipids is initiated either enzymatically by lipoxygenases or by reac-tive oxygen species and propagates via the classical mechanism of lipid peroxidation chain reaction.This implies that the production of OxPL cannot be regulated by adjusting the amount or activity of enzymes;hence,there is an uncontrolled generation of OxPL during oxidative stress[15-17].OxPL can be formed on cell membranes under oxidative stress conditions or during apoptosis and cell death or on LDL during its oxidative modi fication.The major bioactive lipids in oxidatively modi fied LDL(OxLDL)are derived from oxidation of sn-2 arachidonoyl phospholipids[18-19].Three bioactive OxPL derived from oxidation of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine have been primarily identi fied as 1-palmitoyl-2-oxovaleroyl-sn-glycero-3-phosphorylcholine(POVPC),1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphoryl choline(PGPC),and 1-palmitoyl-2-(5,6-epoxyisopros-tane E2)-sn-glycero-3-phosphorylcholine(PEIPC)[20-21].POVPC and PGPC are typically present in OxLDL,can induce various cellular responses or cell death and have been detected in atherosclerotic lesions[20-21].

By using the murine monoclonal antibody E06,an IgM natural antibody that speci fically binds to the phosphorylcholine(PC)head group of oxidized but not native phospholipids,it was possible to detect the content of PC-OxPL per apoB-100 particle(OxPL/ApoB)in plasma and lipoprotein subspecies[22-23].E06 recognizes several PC-OxPL molecules,primarily POVPC,but not PGPC[24].Importantly,most of circulating OxPL/ApoB are associated with Lp(a)with only small amounts found on LDL and high-density lipoprotein(HDL)[25].Although the underlying mechanisms leading to this preferential sequestration of OxPL on Lp(a)over the other lipoproteins are mostly unknown,in vitro experiments demonstrated that the more polar OxPL can exchange from the OxLDL donor to the Lp(a)particle in a manner independent on the lipid transfer proteins,which usually mediate the transfer of lipids among the lipoprotein particles in plasma[25].In support of this hypothesis,it has been reported that immediately after iatrogenic plaque rupture due to percutaneous coronary intervention(PCI),plasma levels of Lp(a)and OxPL/apoB increased by 65%and 35%,respectively,the 50%of OxPL detected in plasma being associated with Lp(a),whereas the other 50%were detected on other apoB lipoproteins.However,by 6h,nearly all of the OxPL were associated with Lp(a)[23].Lp(a)contains OxPL,such as POVPC,which are recognized by E06,as well as other OxPL molecules such as PGPC,which are not recognized by E06[24-25].These results suggest that the ability of Lp(a)to bind various OxPL is a generalized property of this lipoprotein and it is not lim-ited only to E06-recognized OxPL[25].A proportion of Lp(a)-associated OxPL are covalently bound to apo(a),primarily to its KIV10 domain.In general,candidate amino acids for OxPL binding are cysteines,lysines,and histidines.However,all six cysteines are occupied in disul fide bonds and there are no lysines in KIV10 of human apo(a).Therefore,it appears that the 3 histidines present in KIV10,are likely the sites in KIV10 that bind OxPL.In particular,His31and His33,which flank Arg32,are strong candidates for OxPL binding[26].A role in the association of OxPL with apo(a)may be also played by β-2 glycoprotein I(β2GPI),which can bind to apo(a)[27],as well as to anionic phospholipids and OxPL[28-29].However the contribution of β2GPI-mediated bridging of OxPL and apo(a)in the overall association of OxPL with apo(a)needs to be further established[30].OxPL do not only bind to apo(a)but are also present in the lipid phase of Lp(a)[25].The factors that determine the distribution of OxPL between apo(a)and lipid phase of Lp(a),the biological meaning of this distribution in terms of Lp(a)functionality and pathophysiological role as well as possible variations in the relative amounts of OxPL on apo(a)versus the lipid phase of Lp(a)in various disease states,remains to be elucidated.In this regard,it was demonstrated that the amount of OxPL/ApoB in organic extracts of human Lp(a)varied considerably,with about 30%–70%of them in various subjects being lipid soluble[25].

Roleof OxPL onLp(a)-mediatedcardiovascularrisk

Several studies in vitro have demonstrated that OxPL interact with speci fic binding sites and various signal transduction receptors as well as pattern recognition receptors present on the cell surface,including CD36,SRB1,EP2,VEGFR2 and the platelet activating factor(PAF)receptor[16,31].Interaction of distinct OxPL species with the above receptors leads to the activation of indi-vidual signaling pathways in various cell types.Many cellular events are initiated and modulated by biologi-cally active OxPL such as the binding of leukocytes to endothelial cells,upregulation of expression,produc-tion and secretion of various cytokines and chemokines in vitro and in animal models ex vivo and in vivo[32-38],modulation of expression of a number of genes related to atherosclerosis,angiogenesis,in flammation and wound healing in various cell types in vitro[39-40].OxPL also play important role in the recognition and uptake of OxLDL by macrophages.Increased levels of OxPL have been detected in apoptotic cells in vitro[41-42]and cells stimulated with in flammatory agonists in vitro[32],as well as in various organs and tissues under patholog-ical conditions,including plasma of patients with cor-onary artery disease[14],and advanced atherosclerotic lesions in vitro[43-44]in animal models in vivo[21,44].Upon formation,OxPL are mainly associated with Lp(a)invivo and several studies support the hypothesis that the risk of Lp(a)is primarily driven by its OxPL content[45].Lp(a)at very high plasma levels promotes atheroscle-rosis and has been hypothesized to exert anti fibrino-lytic/prothrombotic activities and to contribute to wound healing.Epidemiological studies have shown a remarkable correlation of plasma levels of OxPL/apoB and Lp(a)[45-47].Importantly,OxPL are mainly present on small apo(a)isoforms and their plasma levels are strongly correlated with Lp(a)particles having the low-est number of kringle repeats,this correlation being weakest for the largest Lp(a)isoforms[45-47].The stronger association of oxPL with small Lp(a)isoforms may at least partially explain their enhanced atherogenicity as well as their association with higher CVD risk as com-pared with large ones[46-48].High plasma OxPL/apoB levels independently predict the presence and extent of angiographically determined coronary artery disease,OxPL levels also identify the presence and progres-sion of carotid and femoral atherosclerosis,and pre-dict CVD events over a 10 year interval[45-47].Recent studies have demonstrated that Lp(a)may also play a causal role in the pathophysiology of calci fic aortic valve stenosis(CAVS),a chronic disorder characterized by pathological mineralization and remodeling[49].A genome-wide association study has identi fied a genetic variant(rs10455872)in the LPA gene locus,determin-ing plasma levels of Lp(a),to be causally related to CAVS[50].A recent prospective study involving 77,680 persons(from 2 large prospective studies of the Danish general population,the CCHS;Copenhagen City Heart Study and the CGPS;Copenhagen General Population Study),demonstrated that increased Lp(a)levels and corresponding 3 LPA genetic variants(all associated with Lp(a)levels)were associated with increased risk of aortic stenosis in the general population,with lev-els＞90 mg/dL predicting a threefold increased risk[51].The results of these studies suggest that the association between Lp(a)and aortic stenosis may be causal[50-51].Importantly,the recent ASTRONOMER(Aortic Stenosis Progression Observation:Measuring Effects of Rosuvastatin)trial analysis demonstrated that ele-vated OxPL/apoB and Lp(a)levels are independently associated with an increased risk of echocardiograph-ically determined aortic stenosis progression rate[52].Furthermore,this faster progression rate translated to a higher need for aortic valve replacement,which was accentuated in younger patients with elevated OxPL/apoB or Lp(a)levels[52].The risk of Lp(a)as an inde-pendent predictor of the progression of aortic stenosis could be explained by OxPL/apoB or OxPL/apo(a)lev-els.These findings support the hypothesis that aortic stenosis progression and need for aortic valve replace-ment are mediated by the OxPL content of Lp(a)[52-53].Overall,the results of the above studies provide strong evidence that Lp(a)and its OxPL content may mediate a common biological in fluence on atherosclerosis and CVD as well as on CAVS.

Lipoprotein-associated phospholipase A2:enzymatic properties and binding to lipoproteins

Detoxi fication of reactive OxPL comprises the mechanisms that terminate peroxidation chain reaction and inactivate chemically reactive toxic groups produced by oxidation[54].OxPL subspecies can also be detoxi fied through enzymatic degradation catalyzed by several enzymes such as glutathione peroxidases[55]and aldo-keto reductases[56].An important enzyme that cata-lyzes the degradation of OxPL is lipoproteinassociated phospholipase A2(Lp-PLA2).Lp-PLA2,also named as platelet-activating factor(PAF)-acetylhydrolase,exhib-its a Ca2+-independent phospholipase A2activity and catalyzes the hydrolysis of the ester bond at the sn-2 position of PAF and OxPL[57].OxPL are hydrolyzed by Lp-PLA2into oxidized free fatty acid(OxFFA)and lysophosphatidylcholine(lyso-PC)[57].Like OxPL,both products of Lp-PLA2activity manifest pro-in flammatory and proatherogenic effects.However,it has not been established which is more clinically rele-vant[57-58].Lp-PLA2circulates in plasma in active form,the vast majority of plasma enzyme being associated with LDL while a smaller amount is associated with high-density lipoprotein(HDL)[57-58].Lp-PLA2is also bound to Lp(a)[59].Interestingly,it was demonstrated that Lp(a)is enriched in Lp-PLA2since it contains 1.5 to 2-folds higher enzyme mass and a 7-fold higher speci fic activity compared with LDL when assayed at equimolar protein concentrations[30,60].The binding of Lp-PLA2on LDL is primarily mediated through the enzyme α-helix(114-126).Particularly,residues Trp-115,Leu-116 and Tyr-205 of Lp-PLA2,and to lesser extent Met-117,are critical for enzyme association with LDL[61].Lp-PLA2is bound primarily on apoB-100 and especially on its carboxyl terminus(residues 4119–4279)[61].The binding of Lp-PLA2on HDL is mediated through its C-terminal residues His-367,Met-368,Leu-369,Lys-370[62].Met-368 and Leu-369 residues play a prominent role for this binding,whereas a moderate contribution may have His-367 and Lys-370 residues.Met-368 and Leu-369 are necessary,but not suf ficient for binding to HDL,suggesting that His-367 and Lys-370 either directly participate in Lp-PLA2association with HDL or contribute to the formation of a binding pocket that optimizes interaction of Met-368 and Leu-369 with the lipoprotein[62].Furthermore,it has been demonstrated that regions 107–120,192–204 and 360–368 of Lp-PLA2contribute to enzyme association with HDL,the region 192–204 being particularly important for Lp-PLA2interaction with apoA-I[63].The major role in the attachment of Lp-PLA2on Lp(a)is played by its apoB-100 moiety,whereas the enzyme does not bind to apo(a)[59-60].Importantly,there are marked differences in the enzyme catalytic properties among the various Lp(a)isoforms,the small isoforms exhibiting higher apparent Km values,ie being less active compared to large ones,suggesting that the apo(a)may in fluence the association of Lp-PLA2with Lp(a),although it does not bind the enzyme itself[59].The factors that contrib-ute to the differences among the small and high Lp(a)isoforms in the enzyme catalytic ef ficiency remain still unknown.However,the low catalytic ef ficiency of the Lp-PLA2associated with the small apo(a)isoforms could be one of the factors that favor the sequestration of plasma oxPL on these isoforms[30,59],and consequently the strong correlation between small Lp(a)isoforms and oxPL/apoB levels in plasma[45-47].However other,unknown yet,factors may contribute to this phenomenon,which needs to be further investigated.

Lipoprotein-associated phospholipase A2as an important determinant of Lp(a)functionality

Due to degradation and inactivation of the proin flammatory phospholipid PAF,initial data supported an anti-in flammatory role of Lp-PLA2(Table 1)[57].However,the concept on the pathophysiological role of Lp-PLA2signi ficantly changed when it was demonstrated that Lp-PLA2also hydrolyzes OxPL into OxFFA and lyso-PC.These products are accumulated in the artery wall and display a wide range of pro-in flammatory,proapoptotic and proatherogenic effects[57].More recent studies provided evidence that the role of plasma Lp-PLA2in atherosclerosis may depend on the type of lipoprotein particle with which this enzyme is associated[57-58].In this regard,anti-in flammatory and antiatherogenic effects of the LDL-Lp-PLA2attributed to the catabolism and inactivation of PAF and OxPL molecules exhibiting pro-in flammatory and proatherogenic activities have been described in Table 1[57-58].In addition,pro-in flammatory and proatherogenic effects of the LDL-Lp-PLA2attributed to the hydrolysis of OxPL and generation of OxFFA and lyso-PC have been described in Table 1[57-58].LDL-Lp-PLA2is increased in patients with primary hypercholesterolemia and combined hyperlipidemia and data from large Cauca-sian popu-lation studies have supported plasma Lp-PLA2(pri-marily LDL-associated Lp-PLA2)as a cardiovascular risk marker,independent of and additive to traditional risk factors(Table 1)[64-65].On the contrary,the HDL-associated Lp-PLA2may express anti-in flammatory,antioxidative and antiatherogenic activities.It also enhances the HDL-induced cholesterol ef flux in vitro(Table 1)[58,65].HDL-Lp-PLA2is reduced in patients with combined hyperlipidemia,primary hypertriglyc-eridemia,pre-diabetes and metabolic syndrome[58,65]and is also independently associated with lower risk for cardiac death(Table 1)[66].The role of Lp(a)-associated Lp-PLA2(Lp(a)-Lp-PLA2)has not been adequately studied.In subjects exhibiting high Lp(a)plasma lev-els,the Lp(a)-Lp-PLA2may play a role similar to that observed for the LDL-Lp-PLA2in the artery wall.Lp(a)can be accumulated preferentially to LDL within lesions to an extent proportional to its plasma levels where it binds very tightly to lesion components[67,68].In the intima,Lp(a)may transfer preformed OxPL,and it can undergo oxidative modi fication(Fig.1A)thus further enriched in OxPL[59].Lp(a)-bound OxPL are then hydro-lyzed by the Lp(a)-Lp-PLA2to OxFFA and lyso-PC[59],which signi ficantly contribute to plaque formation[57,69].Consequently,through this mechanism,the Lp(a)-Lp-PLA2may signi ficantly in fluence the biological activities of oxidized Lp(a)in the artery wall[70,71],thus promoting atherogenesis(Fig.1A)[17,58].

Table 1 Physiological and pathophysiological functions of Lp-PLA2associated with different lipoprotein particles

In contrast to the above pathophysiological role of Lp(a)-Lp-PLA2in the artery wall,this enzyme may confer Lp(a)with a physiological role in the circulation.Indeed,in contrast to the other plasma lipoproteins for which the physiological role in lipid metabolism has been well established,the physiological role of Lp(a)is still unknown.The existence of Lp-PLA2on Lp(a)in concert with the preferential accumulation of OxPL on circulating Lp(a)may confer this lipoprotein with a physiological role as a scavenger of oxPL in human plasma[72].Thus,under normal conditions(low oxidative stress,absence or low in flammation),Lp(a)may be bene ficial by collecting OxPL from various sources,which are then degraded by Lp(a)-Lp-PLA2.Under these conditions,the pro-in flammatory lyso-PC,deriving from the degradation of OxPL,could then be transferred in the circulation from Lp(a)to albumin,which represents the major carrier and inhibitor of lyso-PC in plasma(Fig.1B)[73].Importantly,we had previ-ously shown that the Lp(a)of patients with coronary artery disease carries signi ficantly lower amounts of Lp-PLA2mass that expresses lower catalytic ef ficiency compared with controls,a phenomenon which is not observed for the LDL-Lp-PLA2[30].The low catalytic ef ficiency of the Lp(a)-Lp-PLA2in these patients may be due to the sequestration of oxPL on the apo(a)moi-ety of Lp(a)and may represent an important defect of Lp(a)in these patients since it exhibits a diminished capability to degrade and detoxify OxPL(Fig.1A)[30].

Fig.1 Roles of different concentrations of Lp(a)-Lp-PLA2in plasma.A:Pathophysiological role of Lp(a)-Lp-PLA2in atherogenesis.At high plasma levels,Lp(a)can be accumulated preferentially to LDL in the artery wall to an extent proportional to its plasma levels where it may transfer preformed OxPL and can undergo oxidative modi fication.During Lp(a)oxidation,more OxPL are formed and be hydrolyzed by the Lp(a)-Lp-PLA2to OxFFA and lyso-PC,which signi ficantly contribute to atherogenesis.B:Physiological role of low Lp(a)levels as a scavenger of OxPL in the circulation.Under normal conditions(low oxidative stress,absence or low in flammation),Lp(a)collects OxPL from various sources,which are then degraded by Lp(a)-Lp-PLA2.Under these conditions,the pro-in flammatory lyso-PC,deriving from the degradation of OxPL,could then be transferred in the circulation from Lp(a)to albumin,which represents the major carrier and inhibitor of lyso-PC in plasma.Lp-PLA2:Lipoprotein-associated phospholipase A2;LDL:low density lipoprotein;Lyso-PC:lysophosphatidylcholine;Lp(a):lipoprotein(a),OxFFA;oxidized free fatty acid;OxPL:oxidized phospholipids

Overall,the OxPL and Lp-PLA2components of Lp(a)are important determinants of its functionality.However,this Lp(a)functionality may be in fluenced by various factors such as the Lp(a)plasma levels,the extent of OxPL production(low or high oxida-tive stress,in flammation,apoptosis,tissue injury)as well as the levels of Lp(a)-Lp-PLA2activity.In this regard,it should be pointed out that despite the con-sistent findings of many clinical trials showing that the total plasma Lp-PLA2,which represents primarily the LDL-associated enzyme,is an independent risk factor for cardiovascular disease,the results of two recent phase 3 clinical trials(STABILITY;Stabilization of Atherosclerotic Plaque by Initiation of Darapladib Therapy,and SOLID-TIMI 52;the Stabilization of Plaques Using Darapladib-Thrombolysis in Myocardial Infarction 52)aiming to determine whether the inhi-bition of plasma Lp-PLA2with its speci fic inhibitor darapladib has any clinical bene fit in the primary and secondary prevention of cardiovascular disease,were negative[74-75].Since the inhibitory effect of darapladib on Lp-PLA2has been studied in total plasma and on LDL[76],it is not known whether and to which extent darapladib inhibits Lp-PLA2associated with the anti-atherogenic HDL as well as with Lp(a).Thus,we may speculate that any possible inhibitory effect of darap-ladib on circulating Lp(a)-Lp-PLA2may represent an adverse effect of this agent,thus partially explaining its failure to show a clinical bene fit in the above men-tioned phase 3 clinical trials.

Conclusions and perspectives

OxPL may signi ficantly contribute or even pri-marily account for the atherogenicity of Lp(a)and its increased risk for cardiovascular disease.OxPL may be also signi ficantly involved in the causal role that Lp(a)plays a in the pathophysiology of CAVS.In addition,abnormal function of Lp(a)-Lp-PLA2may signi ficantly in fluence Lp(a)functionality.However,further studies are required to assess the coordinated role of OxPL and Lp-PLA2on Lp(a)as well as their pathophysiological and clinical relevance in large data sets.Furthermore,the hypothesis drawn in the present review for a pathogenic role of Lp(a)at high concentrations driven mainly by its OxPL and Lp-PLA2content should be established by clinical studies using speci fic Lp(a)lowering therapies.Up to date,there are data from new therapies that reduce Lp(a),such as the cholesteryl ester trans-fer protein inhibitors[77],the antisense oligonucleotide mipomersen[78],and the proprotein convertase subtilisin/kexin type 9(PCSK9)inhibitors[79];however,such therapies in fluence other lipid components in tandem.Thus,from the results of studies evaluating the above drugs,it is not possible to draw safe conclusions on the clinical importance for reducing Lp(a)as well as for the pathophysiological role of this lipoprotein and especially of its OxPL and Lp-PLA2content.Recently,the pharmacokinetics,pharmacodynamics and safety of a second-generation antisense drug(ISIS-APO(a)Rx)designed to reduce the synthesis of apo(a)in the liver were evaluated in a randomised,double-blind,placebocontrolled,phase 1 study performed in healthy adults with plasma Lp(a)levels≥25 nmol/L(100 mg/L).The results showed that ISIS-APO(a)Rx,selectively and potently reduces plasma Lp(a)concentrations as well as OxPL/apoB and OxPL/apo(a)levels in a dose dependent manner without affecting the plasma levels of other lipoproteins[80].No serious or severe adverse events were recorded[80].These results provide the basis for future clinical trials to test whether lowering Lp(a)plasma concentrations and speci fically in fluencing the Lp(a)-Lp-PLA2-OxPL axis will reduce the risk of CVD and CAVS.

[1] Hobbs HH,White AL.Lipoprotein:intrigues and insights[J].Curr Opin Lipidol,1999,10(3):225–236.

[2] Scanu AM,Nakajima K,Edelstein C.Apolipoprotein(a):structure and biology[J].Front Biosci,2001,6:D546–D554.

[3] Utermann,G.The mysteries of lipoprotein(a)[J].Science,1989,246(4932):904–910.

[4] Kostner KM,Kostner GM.Lipoprotein(a):still an enig-ma?[J].Curr Opin Lipidol,2002,13(4):391–396.

[5] Berglund L,Ramakrishnan R.Lipoprotein(a):An elusive cardiovascular risk factor[J].Arterioscler Thromb Vasc Biol,2004,24(12):2219–2226.

[6] Gabel BR,Koschinsky MI.Analysis of the proteolytic activity of a recombinant form of apolipoprotein(a)[J].Biochemistry,1995,34(48):15777–15784.

[7]McLean JW,Tomlinson JE,Kuang WJ,et al.cDNA sequence of human apolipoprotein(a)is homologous to plasminogen[J].Nature,1987,330(6144):132–137.

[8]Dubé,JB,Boffa MB,Hegele RA,Koschinsky ML.Lipoprotein(a):more interesting than ever after 50 years[J].Curr Opin Lipidol,2012,23(2):133–140.

[9] Kronenberg,F,Utermann G.Lipoprotein(a):resurrected by genetics[J].J Intern Med,2013,273(1):6–30.

[10]Hoover-PlowJ,Huang M.Lipoprotein(a)metabolism:potential sites for therapeutic targets[J].Metabolism,2013,62(4):479–491.

[11]Utermann G.Lipoprotein(a).In:Scriver CR,Beaudet AL,Sly WS,Valle D,eds.The Metabolic and Molecular Bases of Inherited Disease.New York,NY:McGraw-Hill,Medical Publishing Division.2006:2753–87.

[12]Clarke R,Peden JF,Hopewell JC,et al.Genetic variants associated with Lp(a)lipoprotein level and coronary dis-ease[J].N Engl J Med,2009,361(26):2518–2528.

[13]Tsimikas S,Hall JH.Lipoprotein(a)as a potential causal genetic risk factor of cardiovascular disease:a rationale for increased efforts to understand its pathophysiology and develop targeted therapies[J].J Am Coll Cardiol,2012,60(8):716–721.

[14]Tsimikas,S,Brilakis ES,Miller ER,et al.Oxidized phospholipids,Lp(a)lipoprotein,and coronary artery dis-ease[J].N Engl J Med,2005,353(1):46–57.

[15]Bochkov VN,Oskolkova OV,Birukov KG,et al.Generation and Biological Activities of Oxidized Phospholipids[J].Antioxidants Redox Signaling,2010,12(8):1009–1059.

[16]Bochkov VN.In flammatory pro file of oxidized phospho-lipids[J].Thromb Haemost,2007,97(3):348–354.

[17]Leitinger N.Oxidized phospholipids as modulators of in flammation in atherosclerosis[J].Curr Opin Lipidol,2003,14(5):421–430.

[18]Watson AD,Berliner JA,Hama SY,et al.Protective effect of high density lipoprotein associated paraoxonase:inhibition of the biological activity of minimally oxidized low density lipoprotein[J].J Clin Invest,1995,96(6):2882–2891.

[19]Watson AD,Navab M,Hama SY,et al.Effect of plate-let activating factor-acetylhydrolase on the formation and action of minimally oxidized low density lipoprotein[J].J Clin Invest,1995,95(2):774–782.

[20]Watson AD,Subbanagounder G,Welsbie DS,et al.Structural identi fication of a novel pro-in flammatory epoxyisoprostane phospholipid in mildly oxidized low density lipoprotein[J].J Biol Chem,1999,274(35):24787–2479

[21]Watson AD,Leitinger N,Navab M,et al.Structural identi fication by mass spectrometry of oxidized phospholip-ids in minimally oxidized low density lipoprotein that induce monocyte/endothelial interactions and evidence for their presence in vivo[J].J Biol Chem,1997,272(21):13597–13607.

[22]Tsimikas S,Bergmark C,Beyer RW,et al.Temporal increases in plasma markers of oxidized low-density lipo-protein strongly re flect the presence of acute coronary syn-dromes[J].J Am Coll Cardiol,2003,41(3):360–370.

[23]Tsimikas S,Lau HK,Han K-R,et al.Percutaneous Coronary Intervention Results in Acute Increases in Oxidized Phospholipids and Lipoprotein(a).Short-Term and Long-Term Immunologic Responses to Oxidized Low-Density Lipoprotein[J].Circulation,2004,109(25):3164–3170.

[24]Friedman P,Horkko S,Steinberg D,et al.Correlation of antiphospholipid antibody recognition with the structure of synthetic oxidized phospholipids.Importance of Schiff base formation and aldol condensation[J].J Biol Chem,2002,277(9):7010–7020.

[25]Bergmark C,Dewan A,Orsoni A,et al.A novel function of lipoprotein[a]as a preferential carrier of oxidized phospholipids in human plasma[J].J Lipid Res,2008,49(10):2230–2239.

[26]Leibundgut G,Scipione C,Yin H,et al.Determinants of binding of oxidized phospholipids on apolipopro-tein(a)and lipoprotein(a)[J].J Lipid Res,2013,54(10):2815–2830.

[27]Kochl S,Fresser F,Lobentanz E,et al.Novel interaction of apolipoprotein(a)with β-2 glycoprotein I mediated by the kringle IV domain[J].Blood,1997,90(4):1482–1489.

[28]McNeil PH,Simpson RJ,Chesterman CN,Krilis SA.Antiphospholipid antibodies are directed against a com-plex antigen that includes a lipid-binding inhibitor of coag-ulation:β2-Glycoprotein I(apolipoprotein H)[J].Proc Natl Sci USA,1990,87(11):4120–4124.

[29]Hasunuma Y,Matsuura E,Makita Z,et al.Involvement of beta 2-glycoprotein I and anticardiolipin antibodies in oxidatively modi fied low-density lipoprotein uptake by macrophages[J].Clin Exp Immunol,1997,107(3):569–73.

[30]Tsironis LD,Katsouras CS,Lourida ES,et al.Reduced PAF-acetylhydrolase activity associated with Lp(a)in patients with coronary artery disease[J].Atherosclerosis,2004,177(1):193–201.

[31]Zimman A,Mouillesseaux KP,Le T,et al.Vascular endothelial growth factor receptor 2 plays a role in the activation of aortic endothelial cells by oxidized phospholipids[J].Arterioscler Thromb Vasc Biol,2007,27(2):332–338.

[32]Subbanagounder G,Wong JW,Lee H,et al.Epoxyisoprostane and epoxycyclopentenone phospholip-ids regulate monocyte chemotactic protein-1 and interleu-kin-8 synthesis.Formation of these oxidized phospholipids in response to interleukin-1 beta[J].J Biol Chem,2002,277(9):7271–7281.

[33]Furnkranz A,Schober A,Bochkov VN,et al.Oxidized phospholipids trigger atherogenic in flammation in murine arteries[J].Arterioscler Thromb Vasc Biol,2005,25(3):633–638.

[34]Lee H,Shi W,Tontonoz P,et al.Role for peroxisome proliferator-activated receptor alpha in oxidized phospholip-idinduced synthesis of monocyte chemotactic protein-1 and interleukin-8 by endothelial cells[J].Circ Res,2000,87(6):516–521.

[35]Reddy ST,Grijalva V,Ng C,et al.Identi fication of genes induced by oxidized phospholipids in human aortic endothelial cells[J].Vascul Pharmacol,2002,38(4):211–218.

[36]Kadl A,Huber J,Gruber F,et al.Analysis of in flamma-tory gene induction by oxidized phospholipidsin vivoby quantitative real-time RT-PCR in comparison with effects of LPS[J].Vascul Pharmacol,2002,38(4):219–227.

[37]Gargalovic PS,Gharavi NM,Clark MJ,et al.The unfolded protein response is an important regulator of in flammatory genes in endothelial cells[J].Arterioscler Thromb Vasc Biol,2006,26(11):2490–2496.

[38]Huo Y,Weber C,Forlow SB,et al.The chemokine KC,but not monocyte chemoattractant protein-1,triggers mono-cyte arrest on early atherosclerotic endothelium[J].J Clin Invest,2001,108(9):1307–1314.

[39]Berliner JA,Gharavi NM.Endothelial cell regulation by phospholipid oxidation products[J].Free Radic Biol Med,2008,45(2):119–123.

[40]Gargalovic PS,Imura M,Zhang B,et al.Identi fication of im flammatory gene modules based on variations of human endothelial cell responses to oxidized lipids[J].Proc Natl Acad Sci USA,2006,103(34):12741–12746.

[41]Huber J,Valves A,Mitulovic G,et al.Oxidized membrane vesicles and blebs from apoptotic cells contain biologi-cally active oxidized phospholipids that induce monocyte endothelial interactions[J].Arterioscler Thromb Vasc Biol,2002,22(1):101–107.

[42]Chen R,Yang L,MsIntyre TM.Cytotoxic phospholipid oxidation products.Cell death from mitochondrial damage and the intrinsic caspase cascade[J].J Biol Chem,2007,282(34):24842–24850.

[43]Subbanagounder G,Leitinger N,Schwenke DC,et al.Determinants of bioactivity of oxidized phospholipids.Speci fic oxidized fatty acyl groups at the sn-2 position[J].Arterioscler Thromb Vasc Biol,2000,20(10):2248–2254.

[44]Podrez EA,Poliakov E,Shen Z,et al.A novel family of atherogenic oxidized phospholipids promotes macrophage foam cell formation via the scavenger receptor CD36 and is enriched in atherosclerotic lesions[J].J Biol Chem,2002,277(41):38517–38523.

[45]Taleb A,Witztum JL,Tsimikas S.Oxidized phospholipids on apoB-100-containing lipoproteins:a biomarker predict-ing cardiovascular disease and cardiovascular events[J].Biomark Med,2011,5(5):673–694.

[46]Kiechl S,Willeit J,Mayr M,et al.Oxidized phospholipids,lipoprotein(a),lipoprotein-associated phospholipase A2 activity,and 10-year cardiovascular outcomes:prospective results from the Bruneck study[J].Arterioscler Thromb Vasc Biol,2007,27(8):1788–1795.

[47]Tsimikas S,Willeit P,Willeit J,et al.Oxidation-speci fic biomarkers,prospective 15-year cardiovascular and stroke outcomes,and net reclassi fication of cardiovascular events[J].J Am Coll Cardiol,2012,60(21):2218–2229.

[48]Erqou S,Thompson A,Di Angelantonio E,et al.,Apolipoprotein(a)Isoforms and the Risk of Vascular Disease.Systematic Review of 40 Studies Involving 58,000 Participants.J Am Coll Cardiol,2010;55(19):2160–2167.

[49]Rajamannan NM,Evans FJ,Aikawa E,et al.Calci fic aortic valve disease:not simply a degenerative process:a review and agenda for research from the National Heart and Lung and Blood Institute Aortic Stenosis Working Group.Executive summary:calci fic aortic valve disease-2011 update[J].Circulation,2011,124(16):1783–1791.

[50]Thanassoulis G,Campbell CY,Owens DS,et al.Genetic associations with valvular calci fication and aortic steno-sis[J].N Engl J Med,2013,368(6):503–512.

[51]Kamstrup PR,Tybjærg-Hansen A,Nordestgaard BG.Elevated Lipoprotein(a)and Risk of Aortic Valve Stenosis in the General Population[J].J Am Coll Cardiol,2014,63(5):470–477.

[52]Capoulade R,Chan KL,Yeang C,et al.Oxidized Phospholipids,Lipoprotein(a),and Progression of Calci fic Aortic Valve Stenosis[J].J Am Coll Cardiol,2015,66(11):1236–1246.

[53]Hung M-Y,Witztum JL,Tsimikas S.New Therapeutic Targets for Calci fic Aortic Valve Stenosis.The Lipoprotein(a)-Lipoprotein-Associated Phospholipase A2-Oxidized Phospholipid Axis[J].J Am Coll Cardiol,2014,63(5):478–480.

[54]Jira W,Spiteller G,Richter A.Increased levels of lipid oxidation products in low density lipoproteins of patients suffering from rheumatoid arthritis[J].Chem Phys Lipids,1997,87(1):81–89.

[55]Savaskan NE,Ufer C,Kuhn H,Borchert A.Molecular biology of glutathione peroxidase 4:From genomic struc-ture to developmental expression and neural function[J].J Biol Chem2007,388(10):1007–1017.

[56]Jin Y,Penning TM.Aldo-keto reductases and bioactiva-tion/detoxication[J].Annu Rev Pharmacol Toxicol,2007,47:263–292.

[57]Tselepis AD,Chapman MJ.In flammation,bioactive lipids and atherosclerosis:potential roles of a lipoprotein-associ-ated phospholipase A2,platelet activating factor-acetylhy-drolase[J].Atherosclerosis,2002,Suppl 3(4):57–68.

[58]Tellis CC,Tselepis AD.The role of lipoprotein-associ-ated phospholipase A2 in atherosclerosis may depend on its lipoprotein carrier in plasma[J].Biochim Biophys Acta,2009,1791:327–38.

[59]Karabina SA,Elisaf MC,Goudevenos J,et al.PAF-acetylhydrolase activity of Lp(a)before and during Cu(2+)-induced oxidative modi fication in vitro[J].Atherosclerosis,1996,125(1):121–134.

[60]Blencowe C,Hermetter A,Kostner GM,Deingner HP.Enhanced association of platelet-activating factor acetylhydrolase with lipoprotein(a)in comparison with low density lipoprotein[J].J Biol Chem,1995,270(52):31151–31157.

[61]Stafforini DM,Tjoelker LW,McCormick SP,et al.Molecular basis of the interaction between plasma plate-let-activating factor acetylhydrolase and low density lipo-protein[J].J Biol Chem,1999,274(11):7018–7024.

[62]Gardner AA,Reichert EC,Topham MK,Stafforini DM.Identi fication of a domain that mediates association of platelet-activating factor acetylhydrolase with high density lipoprotein[J].J Biol Chem,2008,283(25):17099–17106.

[63]Cao J,Hsu YH,Li S,et al.Structural basis of speci fic interactions of Lp-PLA2 with HDL revealed by hydrogen deuterium exchange mass spectrometry[J].J Lipid Res,2013,54(1):127–133.

[64]Moutzouri E,Tsimihodimos V,Tselepis AD.In flammatory biomarkers and cardiovascular risk assessment.Current knowledge and future perspectives[J].Curr Pharm Des,2013,19(21):3827–3840.

[65]Tellis CC,Tselepis AD.Pathophysiological role and clin-ical2signi ficance2of lipoprotein-associated phospholipase A(Lp-PLA)bound to LDL and HDL[J].Curr Pharm Des,2014,20(40):6256–6269.

[66]Rallidis LS,Tellis CC,Lekakis J,et al.Lipoprotein-associated phospholipase A(2)bound on high-density lipoprotein is associated with lower risk for cardiac death in stable coronary artery disease patients:a 3-year fol-low-up[J].J Am Coll Cardiol,2012,60(20):2053–2060.

[67]Cushing GL,Gaubatz JW,Nava ML,et al.Quantitation and localization of apolipoproteins[a]and B in coro-nary artery bypass vein grafts resected at re-operation[J].Arteriosclerosis,1989,9(5):593–603.

[68]Pepin JM,O’Neil JA,Hoff HF.Quanti fication of apo(a)and apoB in human atherosclerotic lesions[J].J Lipid Res,1991,32(2):317–327.

[69]Berliner JA,Subbanagounder G,Leitinger N,et al.Evidence for a role of phospholipid oxidation products in atherogenesis[J].Trends Cardiovasc Med,2001;11(3-4):142–147.

[70]Wei DH,Zhang XL,Wang R,et al.Oxidized lipoprotein(a)increases endothelial cell monolayer permeability via ROS generation[J].Lipids,2013,48(6):579–586.

[71]Morishita R,Ishii J,Kusumi Y,et al.Association of serum oxidized lipoprotein(a)concentration with coronary artery disease:potential role of oxidized lipoprotein(a)in the vascular wall[J].J Atheroscler Thromb,2009,16(4):410–418.

[72]Tsimikas S,Tsironis LD,Tselepis AD.New insights into the role of lipoprotein(a)-associated lipoprotein-associated phospholipase A2 in atherosclerosis and cardiovascular disease[J].Arterioscler Thromb Vasc Biol,2007,27(10):2094–2099.

[73]Kim YL,Im YJHa NC,Im DS.Albumin inhibits cytotoxic activity of lysophosphatidylcholine by direct binding[J].Prostaglandins Other Lipid Mediat.2007,83(1-2):130–138.

[74]STABILITY Investigators,White HD,Held C,Stewart R,et al.Darapladib for preventing ischemic events in stable coronary heart disease[J].N Engl J Med,2014,370(18):1702–1711.

[75]O’Donoghue ML,Braunwald E,White HD,et al.Effect of darapladib on major coronary events after an acute coro-nary syndrome:the SOLID-TIMI 52 randomized clinical trial[J].JAMA,2014,312(10):1006–1015.

[76]Mohler ER 3rd,Ballantyne CM,Davidson MH,et al.The effect of darapladib on plasma lipoprotein-associated phospholipase A2 activity and cardiovascular biomarkers in patients with stable coronary heart disease or coronary heart disease risk equivalent:the results of a multicenter,randomized,doubleblind,placebo-controlled study[J].J Am Coll Cardiol,2008,51(17):1632–1641.

[77]Bochem AE,Kuivenhoven JA,Stroes ES.The promise of cholesteryl ester transfer protein(CETP)inhibition in the treatment of cardiovascular disease[J].Curr Pharm Des,2013,19(17):3143–3149.

[78]Santos RD,Raal FJ,Catapano AL,et al.Mipomersen,an antisense oligonucleotide to apolipoprotein B-100,reduces lipoprotein(a)in various populations with hypercholesterolemia:results of 4 phase III trials[J].Arterioscler Thromb Vasc Biol,2015,35(3):689–699.

[79]Raal FJ,Giugliano RP,Sabatine MS,et al.Reduction in lipoprotein(a)with PCSK9 monoclonal antibody evolo-cumab(AMG 145):a pooled analysis of more than 1,300 patients in 4 phase II trials[J].J Am Coll Cardiol,2014,63(13):1278–1288.

[80]Tsimikas S,Viney NJ,Hughes SG,et al.Antisense therapy targeting apolipoprotein(a):a randomised,double-blind,placebo-controlled phase 1 study[J].Lancet,2015,386(10002):1472–1483.