Modulation of oxidative stress, inflammation, and atherosclerosis by

This article is available online at http://www.jlr.org Journal of Lipid Research Volume 53, 2012 1767

Copyright © 2012 by the American Society for Biochemistry and Molecular Biology, Inc.

Supplementary key words PAF-AH, PLA2G7 • darapladib • cardiovascu-lar disease • stroke • platelet-activating factor • oxidized phospholipids

Atherosclerosis is characterized by a complex interplay between bloodstream and arterial wall components that leads to a chronic state of vascular oxidative stress and inflammation ( 1 ). Oxidative stress is a key feature of atherogenesis as free radical generation is linked to the forma tion of lipid oxidation products that increase vas-cular infl ammation ( 2 ). Vascular infl ammation plays a key role in the development of early lesions and fatty streaks and is thought to contribute to complications and life-threatening events, such as acute myocardial infarc-tion (MI) and stroke. Thus, infl ammation and oxidative stress are hallmarks of atherosclerosis and are thought to contribute to the initiation, progression, and rupture of lipid-rich vascular lesions.

Several strategies have been used to attenuate oxidation and infl ammation in the vascular wall. Examples of these include antioxidants ( 3 ) and lipoxygenase inhibitors ( 4 ) that limit oxidant stress, and secretory phospholipase A 2 (PLA 2 ) inhibitors that inhibit production of pro-infl ammatory prostaglandins and leukotrienes ( 5, 6 ). Cellular oxidation is controlled through a network of sensors whose function

Abstract Lipoprotein-associated phospholipase A 2 (Lp-PLA 2 ), also known as platelet-activating factor acetylhydrolase (PAF-AH), is a unique member of the phospholipase A 2 superfamily. This enzyme is characterized by its ability to specifi cally hydrolyze PAF as well as glycerophospholipids containing short, truncated, and/or oxidized fatty acyl groups at the sn -2 position of the glycerol backbone. In hu-mans, Lp-PLA 2 circulates in active form as a complex with low- and high-density lipoproteins. Clinical studies have re-ported that plasma Lp-PLA 2 activity and mass are strongly associated with atherogenic lipids and vascular risk. These observations led to the hypothesis that Lp-PLA 2 activity and/or mass levels could be used as biomarkers of cardio-vascular disease and that inhibition of the activity could offer an attractive therapeutic strategy. Darapladib, a com-pound that inhibits Lp-PLA 2 activity, is anti-atherogenic in mice and other animals, and it decreases atherosclerotic plaque expansion in humans. However, disagreement con-tinues to exist regarding the validity of Lp-PLA 2 as an in-dependent marker of atherosclerosis and a scientifi cally justifi ed target for intervention. Circulating Lp-PLA 2 mass and activity are associated with vascular risk, but the strength of the association is reduced after adjustment for basal con-centrations of the lipoprotein carriers with which the enzyme associates. Genetic studies in humans harboring an inacti-vating mutation at this locus indicate that loss of Lp-PLA 2 function is a risk factor for infl ammatory and vascular con-ditions in Japanese cohorts. Consistently, overexpression of Lp-PLA 2 has anti-infl ammatory and anti-atherogenic prop-erties in animal models. This thematic review critically discusses results from laboratory and animal studies, analyzes genetic evidence, reviews clinical work demonstrating asso-ciations between Lp-PLA 2 and vascular disease, and summa-rizes results from animal and human clinical trials in which administration of darapladib was tested as a strategy for the management of atherosclerosis. —Rosenson, R. S., and D. M. Stafforini. Modulation of oxidative stress, infl ammation, and atherosclerosis by lipoprotein-associated phospholipase A 2 . J. Lipid Res. 2012. 53: 1767–1782.

Funding was received from 5P01CA073992 (National Cancer Institute) and Huntsman Cancer Foundation.

Manuscript received 4 January 2012 and in revised form 31 May 2012.

Published, JLR Papers in Press, June 4, 2012 DOI 10.1194/jlr.R024190

Thematic Review Series: New Lipid and Lipoprotein Targets for the Treatment of Cardiometabolic Diseases

Modulation of oxidative stress, infl ammation, and atherosclerosis by lipoprotein-associated phospholipase A 2

Robert S. Rosenson 1, * and Diana M. Stafforini †,§

Mount Sinai School of Medicine ,* New York, NY 10029; and Huntsman Cancer Institute † and Department of Internal Medicine, § University of Utah , Salt Lake City, UT 84112

Abbreviations: AA-PC, arachidonoyl phosphatidylcholine; ACS, acute coronary syndrome; apoB100, apolipoprotein B100; CAD, coro-nary artery disease; CHD, coronary heart disease; CI, confi dence interval; CVD, cardiovascular disease; DM-HC, diabetic and hypercholesterol-emic; Hs-CRP, high-sensitivity C-reactive protein; ICAM-1, intercellular adhesion molecule-1; IL-6, interleukin-6; Lp-PLA 2 , lipoprotein-associated PLA 2 ; MCP-1, monocyte chemotactic protein-1; MI, myocardial infarc-tion; MMP-9, matrix metalloproteinase-9; MPO, myeloperoxidase; NASCET, North American Symptomatic Carotid Endarterectomy Trial; OR, odds ratio; OxFA, oxidized fatty acids; OxPL, oxidized phospho-lipids; OxPL/ApoB, oxidized phospholipid/apolipoprotein B ratio; PAF, platelet-activating factor; PC, phosphatidylcholine; PCI, percuta-neous coronary intervention; PLA 2 , phospholipase A 2 ; RR, relative risk; TNF- � , tumor necrosis factor- � ; VCAM-1, vascular cell adhesion molecule-1; WHHL, Watanabe heritable hyperlipidemic.

1 To whom correspondence should be addressed. e-mail: [email protected]

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1768 Journal of Lipid Research Volume 53, 2012

physiological interactions of the enzyme with lipoproteins gave rise to more descriptive terminology (i.e., Lp-PLA 2 ) that is commonly used, particularly in clinical settings.

This thematic review presents current knowledge of the structural and biochemical properties of Lp-PLA 2 . In addi-tion, we provide a critical discussion of results from genetic, epidemiological, and animal studies and their implication regarding the role of this enzyme in atherosclerosis. Finally, we offer our views on the utility of Lp-PLA 2 as a biomarker and therapeutic target for the treatment of CVD.

LP-PLA 2 : EXPERIMENTAL STUDIES

Biochemical and structural properties Lp-PLA 2 is a Ca 2+ -independent, 45 KDa secreted protein

that circulates in plasma in active form. Sequence analysis of PLA2G7 , the gene encoding Lp-PLA 2 , reveals the pres-ence of a GXSXG motif that is characteristic of neutral lipases and serine esterases ( 24 ). The enzyme harbors a serine/aspartate/histidine catalytic triad whose linear ori-entation and spacing is consistent with the � / � hydrolase conformation of neutral lipases and esterases ( 24 ). This was confi rmed when the solution of the crystal structure of Lp-PLA 2 revealed that the protein indeed has a classic li-pase � / � hydrolase fold ( 25 ). In contrast to most PLA 2 activities that initiate signal transduction and are regulated by the state of cellular activation, Lp-PLA 2 is not acutely regulated ( 19 ). This feature, combined with the indepen-dence of the reaction on Ca 2+ , could potentially threaten the integrity of phospholipid components of cellular mem-branes and lipoproteins ( 26 ). However, the ability of the enzyme to recognize only defi ned types of sn -2 fatty acyl groups ensures that undamaged, structural phospholipids are protected from constitutive hydrolysis ( 27 ). In addi-tion to PAF, Lp-PLA 2 catalyzes hydrolysis of phospholipids generated from structural components in cellular mem-branes and lipoproteins (e.g., LDL) in settings of high oxidant stress ( 28, 29 ). These substrates, which harbor short- and medium-length sn -2 acyl groups, resemble PAF from structural and functional standpoints ( 30 ). The length restriction is relaxed when the sn -2 group contains oxidized functionalities, such as fatty alcohols ( 31 ) and isoprostanes ( 32 ), generated in situ from membrane phospholipids. However, in this case the rate of hydrolysis is much slower. These properties, which were identifi ed using purifi ed substrates ( 28, 32 ), were later confi rmed in elegant studies using human LDL exhaustively oxidized with CuSO 4 in the presence and absence of SB222657, an irreversible inhibitor of Lp-PLA 2 ( 33 ). Lp-PLA 2 lacks a lid region, and its active site has been proposed to be accessible to the solvent ( 34 ). This open active site conformation may account for the ability of the enzyme to accommodate var-ious sn -2 acyl chains in phospholipid substrates.

A key feature of Lp-PLA 2 is its association with lipopro-teins, including HDL, LDL, and to a lesser extent, Lp(a) (reviewed in Ref. 26 ). Differential distribution of Lp-PLA 2 in various plasma lipoproteins may affect its function ( 35–38 ), and thus much effort has been spent elucidating

is to ensure that the redox state of the cell remains within viable parameters. In addition to the multiplicity of com-pensatory responses, individual pathways are biochemically, genetically, and epigenetically regulated by diverse stimuli, environmental cues, receptor ligands, and transcriptional activators whose concentrations are constantly changing to meet homeostatic demands ( 7 ). Similarly, a variety of physiological, often redundant, mechanisms regulate the balance between pro- and anti-infl ammatory responses. These considerations underscore the challenges associ-ated with strategies that rely on targeting a single pathway of infl ammation and/or oxidation ( 5, 8 ). Infl ammation is a physiologic and highly regulated response: although blocking its onset offers therapeutic benefi t in a variety of settings, this mechanism is essential for host defense. Inap-propriate blockade of infl ammation can have detrimental short-term and/or long-term consequences and should be carefully assessed in individual cases.

The development of cardiovascular disease (CVD) is as-sociated with altered expression of multiple infl ammatory gene products ( 1 ). Biomarkers of disease progression sometimes--but not always--offer an opportunity for thera-peutic intervention. When mechanistic studies demonstrate that a biomarker is causally linked to disease pathogenesis, a solid and justifi ed opportunity for intervention arises. Circulating levels of lipoprotein-associated PLA 2 (Lp-PLA 2 ) have been proposed to be markers of CVD on the basis of numerous epidemiological studies ( 9, 10 ). Initial work conducted at least twenty-fi ve years ago showed good correlations between plasma Lp-PLA 2 activity and proven markers of CVD, such as LDL cholesterol and lipoprotein levels ( 11–15 ). Thus, the association between Lp-PLA 2 and CVD has been recognized for many years.

Lp-PLA 2 is a unique member of the PLA 2 super-family that currently contains fi fteen separate, identifi able groups and numerous subgroups ( 16, 17 ). These enzymes are characterized by their ability to hydrolyze the sn -2 ester bond of phospholipid substrates and are assigned to groups based on sequence, molecular weight, disulfi de bonding patterns, the requirement for Ca 2+ , and other features. There are fi ve main categories of PLA 2 activities: secreted small molecular weight sPLA 2 enzymes that include groups I–III, V, IX–XIV, larger cytosolic Ca 2+ -dependent cPLA 2 s (group IV), Ca 2+ -independent iPLA 2 s (group VI), platelet activating factor (PAF) acetylhydrolases (groups VII and VIII), and lysosomal PLA 2 ( 16, 17 ). Lp-PLA 2 has been clas-sifi ed as group VIIA PLA 2 (PLA2G7) owing to its sequence, size, calcium independence, and substrate specifi city ( 16, 17 ). This enzyme was discovered based on its ability to catalyze hydrolysis of the acetyl group at the sn -2 position of PAF to generate lyso-PAF and acetate ( 18 ). To illustrate the ability of Lp-PLA 2 to inactivate PAF, which has been implicated in a variety of infl ammatory diseases ( 19, 20 ), the enzyme was referred to as the secreted/plasma form of PAF acetylhydrolase or PAF-AH. Lp-PLA 2 is primarily pro-duced by macrophages ( 21 ) and circulates in plasma in active form as a complex with LDL and HDL ( 22 ). A small fraction of the enzyme is carried in Lp(a) in sub-jects that express this particle ( 23 ). The biochemical and


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Lp-PLA2 in oxidative stress, infl ammation, and atherosclerosis Q2 1769

and the phenotype of cells of the monocyte/macrophage lineage and that these effects impact key cellular functions.

Independent studies have shown that PAF, which bears structural resemblance to fragmented, short-chain phos-pholipids, upregulates expression of Lp-PLA 2 mRNA in monocyte/macrophages ( 48 ) and HepG2 cells ( 53 ). Con-sistently, a recent study reported elevated plasma levels of both PAF and Lp-PLA 2 mass in a cohort of coronary heart disease (CHD) patients compared with a control group ( 54 ). Increased Lp-PLA 2 mass in this setting may represent in vivo evidence for PAF- and/or OxPL- mediated effects on Lp-PLA 2 expression. De Keyzer and colleagues showed that a high-fat diet, which led to increased levels of oxidized LDL, was accompanied by robust increases in Lp-PLA 2 activity ( 55 ). When this response was modeled in a cellular system, the investigators found that oxidized LDL robustly increased expression of Lp-PLA 2 mRNA in THP1 cells and, to a lesser extent, human monocyte-derived macrophages ( 55 ). These combined results suggest that oxidized LDL, PAF, and perhaps OxPL upregulate expression of Lp-PLA 2 in vivo, possibly by increasing mRNA synthesis or stability, resulting in higher protein expression. This response likely refl ects a homeostatic, negative-feedback mechanism. Additional studies should further investigate changes in PLA2G7 mRNA expression in response to stimulation with various stimuli, such as OxPL, generated during oxidative modifi cation of LDL and in settings of elevated oxidant stress. The impact of the cellular phenotype on these responses should also be investigated.

In addition to transcriptional regulation, Lp-PLA 2 ex-pression is genetically controlled. A recent genome-wide association study that included fi ve population-based re-ports comprising 13,664 subjects identifi ed eight genetic loci associated with variation in Lp-PLA 2 mass and activity ( 56 ). Lp-PLA 2 mass was found to be regulated mainly by PLA2G7 , consistent with another report ( 57 ). Lp-PLA 2 ac-tivity was strongly associated with genetic variants involved in lipid metabolism ( 56 ). The fi ndings of this interesting study suggest that genetic regions known to affect lipopro-tein levels also infl uence Lp-PLA 2 expression.

Functional consequences of Lp-PLA 2 action A number of studies have concluded that the in vivo ac-

tion of Lp-PLA 2 in settings of elevated oxidant stress, al-lergy, and acute infl ammation is benefi cial (see Ref. 26 for a recent review). The mechanism accounting for these ef-fects is thought to involve Lp-PLA 2 -mediated decreases in the bioactivity of phospholipid substrates, such as PAF and OxPL. However, an issue that has eluded clarifi cation de-spite considerable effort over the years is whether the in vivo action of Lp-PLA 2 on endogenous substrates gener-ates products [i.e., lyso-phosphatidylcholine (lyso-PC) plus short and/or oxidized fatty acids] that increase infl am-matory responses in the vascular wall. Specifi cally, it has not been determined whether Lp-PLA 2 is benefi cial or detrimental, or whether the substrates are more, less, or equally atherogenic than the byproducts ( 58 ). This issue has been explored using in vitro approaches to characterize

the molecular basis for these associations. Binding of Lp-PLA 2 to LDL requires W115 and L116, which are lo-cated in a region mostly composed of hydrophobic amino acids ( 39 ). Recent studies have shown that these residues mediate penetration of the enzyme into the lipid mem-brane surface and that they are important for Lp-PLA 2 binding to hydrophobic surfaces ( 34 ). Tyrosine-205, a residue conserved only among Lp-PLA 2 orthologs that associate with endogenous LDL particles, is also required for association with LDL ( 39 ). The enzyme appears to bind to a C-terminal region of apoB100 located between residues 4119-4279 ( 39 ). In recent studies, we found that a carboxyl terminal string of amino acids (H367–K370) unique to human Lp-PLA 2 is required for association with HDL ( 40 ); additional regions may be required for this interaction.

Cellular sources and regulation of expression Multiple infl ammatory cells involved in atherogenesis

secrete Lp-PLA 2 , including monocytes, macrophages, neu-trophils, activated bone marrow-derived mast cells, and activated platelets ( 21, 41 ). Monocyte/macrophages are key players in both initiation and progression of athero-sclerosis, and they have been proposed as new potential therapeutic targets for the prevention and treatment of this disease ( 42 ). We previously demonstrated that matu-ration of monocytes into macrophages is accompanied by dramatic increases in Lp-PLA 2 mRNA and protein ( 21, 43 ); these observations were recently confi rmed ( 44 ). Treatment of macrophages with acetylated LDL generates foam cells in vitro and further increases Lp-PLA 2 expres-sion ( 44 ), which is consistent with observations in human atherosclerotic plaques ( 45 ). The properties of macro-phages in atherosclerotic lesions can vary dramatically be-tween two extremes: pro-infl ammatory M1 macrophages and anti-infl ammatory M2 macrophages; these cells can switch from one phenotype to the other depending on mi-croenvironmental cues, such as differentiation factors produced in the lesion and T-cell-derived polarizing cyto-kines ( 46, 47 ). We found that IFN- � , which has been im-plicated as a promoter of foam cell formation and an inducer of M1 pro-infl ammatory phenotypes ( 47 ), de-creased expression of Lp-PLA 2 at the transcriptional level ( 48 ). Conversely, M-CSF, which induces an M2-like, an-ti-infl ammatory phenotype, increased monocyte Lp-PLA 2 expression to a much higher extent compared with the M1-inducer GM-CSF ( 49 ). However, Ferguson and coworkers reported modest increases or no changes in Lp-PLA 2 mRNA levels in M1 and M2 macrophages, respectively, using different induction strategies ( 44 ). In addition to M1 and M2, other macrophage phenotypes have been de-scribed, including subspecies generated in response to stimulation with oxidized phospholipids [OxPL ( 50 )]. Agents such as LPS, IL-1 � , G-CSF, and TNF- � can signifi -cantly increase the synthesis and secretion of functional Lp-PLA 2 by monocytes, RAW264.7, and THP-1 cells ( 51, 52 ). These effects appear to be modulated by the state of cel-lular differentiation ( 26 ). It is clear that signals from the microenvironment dynamically regulate Lp-PLA 2 expression


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mass levels had a strong, positive association with risk of coronary events that was not confounded by classic cardio-vascular risk factors ( 92 ). Since then, plasma and serum Lp-PLA 2 concentration and activity have been deter-mined in a great number of epidemiological studies aimed at determining whether these measurements can be used in patient stratifi cation and/or to predict the like-lihood of future cardiovascular events such as MI and stroke. Several reviews and meta-analyses have recently addressed this issue (e.g., see Refs. 10, 93–95 ). This article will not comprehensively review all the evidence gathered thus far. Instead, we will highlight key features. With few exceptions, we will emphasize studies that assessed the enzymatic activity of Lp-PLA 2 . Inves tigations relying exclusively on Lp-PLA 2 mass determinations should be interpreted within the context of serious methodolog-ical concerns regarding the performance of the leading commercially available assay ( 96–98 ). This includes long-term consistency in the hands of the manufacturer.

Lp-PLA 2 expression in apparently healthy subjects The initial association between Lp-PLA 2 levels and fu-

ture risk of CVD reported by WOSCOPS ( 92 ) was, in gen-eral, confi rmed in smaller studies ( 99–102 ). In contrast, additional trials failed to reproduce these observations ( 95, 103–106 ). The Lp-PLA 2 Studies Collaboration exam-ined associations between circulating Lp-PLA 2 concentra-tion and activity and cardiovascular events from 79,036 subjects who participated in 32 prospective studies ( 94 ). Individuals with no history of vascular disease at baseline (n = 35,945) were included in the analyses, which were adjusted for age, sex, baseline history of vascular disease, and other nonlipid and lipid risk factors. In this group, no association was found between Lp-PLA 2 activity and CHD or ischemic strokes. In summary, Lp-PLA 2 activity levels do not seem to be a useful predictor of future CHD in appar-ently healthy individuals.

Two studies that merit mention include investigations that evaluated plasma Lp-PLA 2 activity, levels of oxidized phospholipids (OxPL) in a subset of apolipoprotein B-100 (apoB) particles (OxPL/apoB), and future vascular events in apparently healthy participants ( 102, 107 ). The fi rst prospective study was relatively small and showed that OxPL/apoB levels were elevated in those who developed coronary, carotid, and femoral artery disease, and acute coronary syndrome (ACS; Ref. 102 ). OxPL/apoB mea-sured at baseline predicted the development of cardiovas-cular events independently of traditional risk factors and high-sensitivity C-reactive protein (hs-CRP). In addition, increased risk was augmented with increasing Lp-PLA 2 ac-tivity. The second study [European Prospective Investiga-tion of Cancer (EPIC)-Norfolk] also included a cohort of 45- to 79-year-old, apparently healthy, men and women ( 107 ). This analysis confi rmed the increased risk of cardiovascular events with increasing levels of oxPL/apoB, but only a weak potentiation of risk was observed with increasing Lp-PLA 2 activity. In sum, the two studies suggest that com-bined assessment of these two mechanistically related bio-markers (Lp-PLA 2 activity and oxPL/apoB ratio) provides

biological properties of relevant substrates and products of the reaction. Both pro- and anti-infl ammatory activities have been ascribed to glycerophospholipids harboring fragmented and/or oxidized sn -2 fatty acyl groups ( 59–69 ). Similarly, lyso-PC ( 66, 70–74 ) and short and/or oxidized fatty acids ( 75–80 ) have been reported to elicit both detri-mental and benefi cial responses. In general, in vitro ap-proaches are strategically useful initially, but they have the usual limitations of studies that do not recapitulate essen-tial in vivo features. This is particularly relevant in studies that involve lipids and lipid metabolites. For example, rel-atively low (nanomolar) concentrations of lyso-PC recruit monocytes and induce pro-infl ammatory cytokine produc-tion in vitro ( 81 ). However, the concentration of lyso-PC in blood plasma of healthy persons usually ranges from 200 to 400 � M ( 82, 83 ), and similar levels have been re-ported in atherosclerotic tissues ( 45 ). Because lyso-PC, un-like PC, is membrane lytic, it is predominantly confi ned to lipoproteins or plasma proteins, such as albumin or immu-noglobulins ( 83, 84 ). As a result, only a fraction of lyso-PC is functionally active, and it is unclear how total concentra-tion relates to bioavailability ( 73, 85 ). Similarly, the bioac-tivity of fatty acids appears to be modulated by the carrier, again underscoring the importance of location on biolog-ical activity ( 86, 87 ). Thus, whether results generated using lyso-PC and oxidized fatty acids in vitro realistically refl ect in vivo responses remains to be rigorously determined.

In view of these limitations, rather than characterizing functional effects of either Lp-PLA 2 substrates or Lp-PLA 2 products, a more informative approach is to assess net changes that result when the action of Lp-PLA 2 alters substrate-to-product ratios. Examples of this strategy include investigations conducted in the presence and absence of Lp-PLA 2 activity, or studies that compare the effect of supplementing active and inactive enzyme. Both pharmacologic and genetic depletion of Lp-PLA 2 in mini-mally modifi ed lipoproteins enhanced monocyte adhesion to aortic endothelial cells compared with minimally modi-fi ed lipoproteins that expressed normal activity levels ( 88 ). Similarly, exogenous Lp-PLA 2 reduced cellular uptake of oxidized LDL and Lp(a), and cholesterol accumula-tion by monocyte-derived macrophages, compared with parallel assays conducted with inactive enzyme ( 89 ). These results suggest that the enzymatic activity of Lp-PLA 2 limits monocyte recruitment and foam cell formation within atherosclerotic lesions ( 89 ). Chen and colleagues found that exogenous Lp-PLA 2 , but not vehicle, inhibited endothelial cell apoptosis induced by minimally modifi ed LDL particles ( 90 ). These approaches, which have also been used in animal models ( 91 ), provide much-needed rigorous, well-controlled comparisons to assess the net effect of altering the balance of metabolites controlled by Lp-PLA 2 .

LP-PLA 2 : EPIDEMIOLOGICAL AND CLINICAL STUDIES

WOSCOPS (West of Scotland Coronary Prevention Study) was the fi rst study to report that circulating Lp-PLA 2


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( 114 ) or cardiac mortality ( 115 ). However, the FRISC II [Fragmin and Fast Revascularization During Instability in Coronary Artery Disease II ( 116 )], GUSTO IV [Global Utilization of Strategies to Open Occluded Arteries IV ( 116 )], and PROVE-IT [Pravastatin or Atorvastatin Evalu-ation and Infection Therapy ( 117 )] studies reported that baseline levels of Lp-PLA 2 mass and/or activity did not predict recurrent events, such as MI, stroke, or death. Analysis of patients diagnosed with an acute ischemic event in the Lp-PLA 2 Studies Collaboration (n = 10,638) revealed no signifi cant associations between Lp-PLA 2 mass or activity and recurrent vascular outcomes. In this meta-analysis, it could be argued that lack of association with recurrence among patients presenting with acute ischemic events could be related to the smaller size of the cohort and the shorter follow-up time (1.1 years vs. 5.8 years). The fact that Lp-PLA 2 measurements were conducted during the acute phase, however, does not appear to ex-plain the observed lack of correlation, as a study from the MIRACL (Myocardial Ischemia Reduction with Acute Cholesterol Lowering) trial in patients presenting with ACS reported no signifi cant changes in Lp-PLA 2 mass or activity after ACS, suggesting that Lp-PLA 2 does not participate to any substantial extent in the acute-phase response of this syndrome ( 118 ). In addition, the authors found no discernible relationship between Lp-PLA 2 levels and risk of future ischemic events or death in place-bo-treated patients ( 118 ). These combined studies strongly suggest that measurement of Lp-PLA 2 levels is unlikely to improve stratifi cation or risk prediction in patients pre-senting with ACS .

Lp-PLA 2 expression in statin-treated patients The Heart Protection Collaboration Group evaluated

whether baseline Lp-PLA 2 activity was associated with CVD in a randomized, placebo-controlled clinical trial of simvastatin that included individuals at high risk of de-veloping vascular disease ( 119 ). Although a modest as-sociation was initially observed in analyses that adjusted for nonlipid risk factors, after adjustment for apoB, the association with activity became nonsignifi cant ( 120 ). How-ever, the study population was diverse, and confounding factors from cardiovascular medications and prior vascu-lar disease may have affected the results. The JUPITER (Justifi cation for the Use of statins in Prevention: an Inter-vention Trial Evaluating Rosuvastatin) trial compared ro-suvastatin to placebo in 17,802 men and women without CVD or diabetes at study entry ( 98 ). This study found only modest associations with risk in subjects allocated to the placebo arm and whose cholesterol levels were, therefore, not managed ( 98 ). Importantly, the JUPITER trial showed that Lp-PLA 2 did not predict risk or clinical outcomes in rosuvastatin-treated patients. The MIRACL trial, which included placebo-treated and atorvastatin-treated individ-uals whose Lp-PLA 2 mass and activity were signifi cantly re-duced, found no discernible relationship between Lp-PLA 2 levels and risk of future ischemic events or death in either atorvastatin-treated or placebo-treated patients ( 118 ). The studies show that Lp-PLA 2 levels are unlikely to predict

modest incremental information to predict risk of future cardiovascular events in apparently healthy individuals.

A nested case-control analysis evaluated whether in-creased Lp-PLA 2 activity correlates with MI in 1,221 ap-parently disease-free women matched for age, smoking status, and date of blood draw. During a 14-year follow-up period, there were 421 cases of incident MI ( 108 ). Lp-PLA 2 activity was associated with incident MI [RR = 1.75; 95% CI (1.09–2.84)] even after multivariable adjustment for clinical, lipid, and infl ammatory risk factors. The differ-ences in the risk of CHD events associated with increased Lp-PLA 2 activity in this observational study of participants without known vascular disease may relate to sex-specifi c disease associations and longer average duration of fol-low-up compared with results included in the Lp-PLA 2 Studies Collaboration ( 94 ).

Lp-PLA 2 expression in subjects with stable cardiovascular disease

Several studies reported that Lp-PLA 2 mass predicts future cardiovascular events in patients with stable CVD ( 95, 109–111 ). Since Lp-PLA 2 is carried in the circulation as a complex with lipoproteins, such as apoB-containing LDL, there is a strong relationship between Lp-PLA 2 and LDL concentrations ( 96 ). Thus, the strength of the asso-ciation of Lp-PLA 2 with CVD depends substantially on apoB or its associated lipid moieties ( 96 ). It is therefore essential to evaluate whether the predictive power of Lp-PLA 2 is independent of common risk factors, such as high apoB/LDL levels. In one study, adjustment for mul-tiple risk factors [e.g., hypertension, smoking, family his-tory of coronary artery disease (CAD), and prior MI] and medication use did not affect the ability of Lp-PLA 2 to predict CAD risk, but it abolished the potential to pre-dict all-cause mortality, non-CAD CV death, new MI, nonfatal MI, or a composite endpoint of all-cause mor-tality, MI, cerebrovascular accident, and revascularization at follow-up ( 112 ). In another study, however, the associa-tion remained signifi cant ( 111 ). Lp-PLA 2 activity levels also appear to predict future cardiovascular events and cardiac mortality in patients with stable CVD ( 109, 113 ). The Lp-PLA 2 Studies Collaboration analyzed 32 pro-spective analyses in 35,494 patients with a history of stable vascular disease ( 94 ). Risk ratios for CHD and ischemic strokes increased progressively for every 1-standard devi-ation higher Lp-PLA 2 activity or concentration in models adjusted for age, sex, baseline history of vascular disease, and other nonlipid and lipid risk factors. In summary, the evidence available thus far indicates that patients with stable CVD have increased risk for MI, ischemic stroke, and cardiac death with progressively increased Lp-PLA 2 activity or mass.

Lp-PLA 2 expression in patients with ACS Initial epidemiological analyses in patients with ACS

led to promising results that failed to be confi rmed in subsequent work ( 114 ). Two small studies suggested that elevated Lp-PLA 2 levels may help in the identifi cation of patients who will develop cardiovascular complications


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dismutase-2 and glutathione peroxidase ( 127 ). The ex-pression of these proteins may be upregulated to coun-teract overproduction of reactive oxygen species in atherosclerotic lesions ( 128 ). Thus, although multiple studies report high Lp-PLA 2 expression in advanced atherosclerotic lesions, this feature cannot be used as surrogate evidence for active contribution of this enzyme to the disease phenotype.

Implications for cardiovascular risk assessment and therapeutic intervention

The strength of the association between Lp-PLA 2 mass and activity with vascular risk is markedly reduced after ad-justment for basal concentrations of lipids and apolipo-proteins ( 96, 129 ). Cohort stratifi cation ( 94 ) combined with individual analyses of the most rigorously designed studies ( 96, 98 ) indicate that Lp-PLA 2 activity and risk of future cardiovascular events or death are not signifi cantly associated in individuals without a history of vascular disease, in patients treated with effective statin doses ( 96 ), or in patients presenting with ACS. In view of the fi ndings outlined above, it is unlikely that measurement of plasma or serum Lp-PLA 2 activity would yield additional power to predict risk of CHD or help in risk stratifi cation, beyond that already provided by assessment of traditional risk factors ( 130 ). A second issue to consider is whether these results affect the suitability of Lp-PLA 2 as a target for inter-vention. Aside from mechanistic considerations that are discussed elsewhere in this article, the epidemiological evidence summarized above does not support the hypoth-esis that targeting Lp-PLA 2 would improve outcomes above and beyond what is already achieved by current strategies to manage CVD.

GENETIC STUDIES IN HUMAN POPULATIONS

Genetic defi ciency of Lp-PLA 2 Much information can be gleaned from genetic analyses

in human populations combined with molecular studies in biochemical, cellular, and animal models. In addition, merging results from genetic and biomarker studies may help establish disease causality ( 131 ). If the relationship between biomarker and outcome is causal, then genotype should associate with outcome ( 131 ). This approach has been used to elucidate the real function of Lp-PLA 2 in vivo and its impact in human physiology and disease. Fourteen years ago, one of us identified the molecular basis of Lp-PLA 2 defi ciency as a missense mutation at position 994 (G>T) of the PLA2G7 gene, which results in a valine to phenylalanine transversion near the active site of the en-zyme [V279F ( 132 )]. Homozygous carriers of this variant express virtually no enzymatic activity in plasma and heterozygous subjects have a 50% decrease in activity com-pared with those expressing two copies of the wild-type al-lele ( 132, 133 ). The V279F allele is common in Japanese populations: 25–30% of healthy subjects express one copy of this allele and 3–4% carry two copies ( 134–136 ). The mutation has been reported in Korean ( 137 ), Taiwanese

future risk when cholesterol levels are adequately man-aged by statin therapy. These observations have important clinical implications that will be discussed later in this article. An additional important observation from the MIRACL ( 118 ) and JUPITER ( 98 ) trials is that the data are consistent with conclusions described in an earlier section that, in placebo-treated, apparently healthy subjects, Lp-PLA 2 levels did not predict future CHD.

Lp-PLA 2 expression in atherosclerotic plaques A number of studies have reported increased Lp-PLA 2

expression in macrophages from human and rabbit ath-erosclerotic lesions ( 121 ). A prospective study of 167 ca-rotid endarterectomy patients reported increased local expression of Lp-PLA 2 and lyso-PC in the vulnerable plaques of symptomatic compared with asymptomatic pa-tients ( 45 ). Lp-PLA 2 expression was associated with plaque vulnerability in experimental animals and in humans ( 121, 122 ). Early lesions stained weakly for Lp-PLA 2 , but necrotic cores and surrounding macrophages of vulnerable and ruptured plaques displayed robust Lp-PLA 2 expression ( 122 ). Interestingly, Lp-PLA 2 was detected in apoptotic macrophages. This observation may represent events me-diated by accumulated OxPL, which are known to be po-tent inducers of apoptosis ( 65 ). Elevated plaque Lp-PLA 2 expression is a feature that appears to be refl ected in the circulation. Among 42 patients with high-grade carotid stenoses ( � 70% by the NASCET criteria), circulating Lp-PLA 2 levels were higher in patients with unstable plaque compared with those who had stable lesions ( 123 ). A larger study that included 467 patients with a fi rst ischemic stroke reported that increased Lp-PLA 2 concentration was pre-dictive of recurrent ischemic neurological events ( 124 ). Finally, in a study of 40 ACS patients with successful percu-taneous coronary intervention (PCI), changes in the vol-ume of nonculprit lesions after 6 months were signifi cantly associated with changes in both LDL cholesterol levels ( r = 0.444) and Lp-PLA 2 concentration [ r = 0.496 ( 125 )]. Al-though Lp-PLA 2 expression is elevated in advanced ath-erosclerotic plaques and in the circulation, it is not evident from these results that Lp-PLA 2 levels would be a more useful clinical marker of plaque instability than simple de-termination of LDL cholesterol levels.

It is important to emphasize that protein expression pat-terns in vascular lesions refl ect cellular populations pre-sent at the time of lesion collection, along with differential regulation in response to the cellular and physical envi-ronment ( 126 ). Thus, differential gene/protein expression in atherosclerotic plaques refl ects both damage occurring in the tissue and mechanisms engaged to limit additional adverse responses ( 126 ). Although molecular character-izations of diseased tissues, such as atherosclerotic plaques, have a proven record of success for biomarker purposes, this approach cannot be used to determine causal versus consequential links. The tissue-protective nature of some of the genes upregulated in atherosclerotic plaques sug-gests that their regulation is a result, rather than a cause, of atherosclerosis ( 126 ). Examples include upregulated expression of antioxidant proteins such as superoxide


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These observations point at potentially important dif-ferences in Japanese compared with Korean subjects, assuming these fi ndings are recapitulated in indepen-dent Korean cohorts. Thus, with the exception of these two studies by a single Korean group, the evidence avail-able to date strongly suggests that defi ciency of Lp-PLA 2 is associated with increased risk of CAD.

Lp-PLA 2 polymorphisms and CAD In contrast to the V279F allele associated primarily with

Asian populations, three relatively well-studied nonsynon-ymous Lp-PLA 2 polymorphisms (R92H, I198T, and A379V) have been observed in all populations studied so far ( 142, 156 ). The variants have been functionally characterized using molecular approaches ( 157 ). The purifi ed A379V variant displays decreased substrate affi nity and increased rate of hydrolysis ( 157 ), but these features are not always recapitulated in whole-plasma activity determinations ( 142 ). In a case-control study of Chinese Han, the risk of MI was higher among CAD patients harboring two VV al-leles compared with AA (wild-type) homozygotes ( 153 ). In a Taiwanese cohort, A379V was associated with increased risk of MI and increased severity of CAD ( 138 ). In con-trast, a study of European Caucasians revealed reduced risk of MI in homozygotes for A379V ( 158 ), but meta-analyses of European Caucasian populations reported no association with CHD risk ( 56, 156 ). In South Koreans, a similar lack of association between A379V and CVD disease was reported ( 137 ). These observations suggest that Lp-PLA 2 polymorphisms that modestly impact enzymatic activity are unlikely to be associated with cardiovascular risk.

LP-PLA 2 INHIBITORS

Epidemiological and correlative studies showing that Lp-PLA 2 expression is elevated in CVD, combined with in vitro analyses, have led to the proposition that the en-zyme has a predominantly pro-infl ammatory role in ath-erogenesis ( 75, 159 ). Mechanistically, proponents of this model argue that Lp-PLA 2 -mediated lyso-PC and oxidized fatty acid generation contribute to several aspects of plaque formation and actively contribute to the patho-genesis of vascular disease. In turn, this led to the propo-sition that inhibiting the enzymatic activity of Lp-PLA 2 could be potentially benefi cial for the prevention and/or treatment of atherosclerosis and related disorders ( 160 ). A number of azetidinone inhibitors that target the active-site serine residue of the enzyme have been developed ( 161 ). One agent, darapladib (SB480848) is being tested in two large phase III clinical trials to assess clinical ben-efi t in patients diagnosed with CHD ( 9, 162 ) and fol-lowing ACS ( 163 ).

Pharmacology, biological properties, and metabolic effects of darapladib

Darapladib reversibly and noncovalently binds to human recombinant Lp-PLA 2 with a Ki of 110 pM and an off-rate of 27 min ( 161 ). It is lipophilic and has good membrane

( 138 ), and Chinese ( 139, 140 ) populations, and in sub-jects from Turkey, Azerbaijan, and Kyrgyzstan ( 141 ). Allele frequency varies widely in these populations: the highest prevalence was reported in Japan and in Taiwan, fol-lowed by Korea and China (reviewed in Ref. 142 ). In contrast, this variant has been observed rarely in Euro-peans ( 142, 143 ); thus far, only one individual of Swiss ancestry has been shown to be a carrier of the V279F allele ( 143 ). Other functionally validated null alleles [i.e., Q281R, I317N, and Ins 191] have been observed in Caucasian and Japanese populations with extremely low frequencies ( 143–146 ).

Lp-PLA 2 defi ciency and CAD Diverse studies have reported that the V279F null allele

is associated with increased risk of developing atheroscle-rosis, CAD, stroke, and MI in Japanese subjects ( 134, 147–150 ). A study using strict criteria for statistical signifi cance ( P < 0.005) showed that the V279F allele is signifi cantly associated with CAD in Japanese men with nonfamilial hypercholesterolemia ( 151 ). Based on the same crite-ria, the presence of the mutated allele did not increase (or decrease) CAD risk in females or normolipidemic men ( 151 ). V279F was associated with increased plasma oxLDL/LDL ratio in a recessive manner, but it had no effect on intima media thickness ( 152 ). A recent meta-analysis involving predominantly Asian individuals ana-lyzed seven case-control studies involving 3,614 patients with CHD and 4,334 controls ( 139 ). Although the crude ORs were not signifi cant, analyses stratifi ed by study size, ethnicity, case defi nition, and source of controls revealed strong associations with Japanese ethnicity and other parameters, and suggested that V279F contributes to CHD. Similarly, in a study of Chinese Han men and women, the frequency of the V279F allele was more common in CAD patients compared with healthy controls (13.5% vs. 9.3% in controls, P = 0.024). Importantly, the severity of atherosclerosis was greater in V279F carriers; these associations remained signifi cant after multivariate ad-justment [OR = 1.922; 95% CI (1.146–3.224); P = 0.013 ( 153 )].

Interestingly, two studies in Korean men concluded that incidence of the V279F allele is lower in CVD patients compared with apparently healthy controls [OR = 0.646; 95% CI (0.490–0.850), P = 0.002], even after adjustments for age, body mass index, waist circumference, waist to hip ratio, cigarette smoking, and alcohol consumption ( 137 ). However, these Korean cohorts had unique clin-ical characteristics. In the initial study, LDL and total cholesterol levels were lower in patients, including those not treated with lipid-lowering drugs, compared with healthy subjects ( 137 ). The second study included mostly patients whose LDL cholesterol levels were pharmacologi-cally decreased by statin therapy ( 154 ). In healthy Kore-ans, homozygosity for the V279F allele is associated with decreased LDL-cholesterol levels [V/V: 120.9 ± 0.69; F/F: 109.2 ± 4.84 mg/dl, P = 0.025 ( 155 )]. In contrast, no such relationship has been observed in Japanese cohorts [V/V: 126.0 ± 1.56; F/F: 128.8 ± 0.77 mg/dl, P = 0.77 ( 136, 152 )].


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a diabetic-hypercholesterolemic (DM-HC) phenotype decreased the content of sn -2 arachidonoyl-containing PCs ( 165 ), possibly owing to increased oxidation and/or activation of PLA 2 activities other than Lp-PLA 2 (dis-cussed earlier in this article). Surprisingly, darapladib treatment signifi cantly reduced this response ( Table 1 ), which points at anti-oxidant properties or inhibitory ef-fects of darapladib on long-chain PLA 2 activities with specifi city for arachidonoyl-containing substrates. Although darapladib treatment attenuated the elevated arterial lyso-PC abundance in a manner similar to its in vitro effects on LDL lipids, the compound had no effect on the levels of truncated oxidized PC species known to be Lp-PLA 2 substrates ( 33 ). Notably, darapladib failed to alter serum PAF levels in two murine models of athero-sclerosis ( 167, 168 ). The possibility that other circu-lating PLA 2 activities are targets of darapladib was evaluated in in vitro experiments showing modest (8.7%) inhibition of sPLA2GX and no effect on sPLA2GIIa or sPLA2GV (reviewed in Ref. 164 ). However, it is unknown whether darapladib inhibits other PLA 2 activities, such as sPLA2GIII (e.g., Ref. 73 ).

Aside from effects on lipid metabolism, darapladib treatment decreased caspase-3 and caspase-8 activity in vivo ( 52, 169 ), and a compound related to darapladib (SB222657) inhibited macrophage apoptosis induced by oxidized LDL in vitro ( 170 ). These observations may ac-count for the ability of darapladib to limit necrotic core expansion in humans ( 169 ), but it is not clear whether these effects occur as a consequence of Lp-PLA 2 inhibi-tion. Inhibition of Lp-PLA 2 activity (and substrate accumu-lation) is predicted to increase apoptosis through caspase activation, based on multiple rigorous laboratory investi-gations (see Ref. 65 for a recent comprehensive review), which is opposite to the observed effect of darapladib on caspase-3 and caspase-8 inhibition ( 52, 169 ).

In conclusion, although darapladib and related com-pounds inhibit Lp-PLA 2 activity as determined using ex-ogenous substrates in vitro, it is not clear whether enzyme inhibition accounts for observed in vivo effects. An ideal

permeability ( 164 ). Darapladib has minimal interaction with cytochrome P450 isozymes and has an oral bioavail-ability of 11 ± 2% and ≈ 21% in fed and fasted rats, respec-tively, and 28 ± 4% in dogs ( 161 ). Darapladib has been repeatedly shown to inhibit hydrolysis of Lp-PLA 2 sub-strates [i.e., PAF, 1-myristoyl-2-(4-nitrophenylsuccinyl) PC, and others]. Darapladib inhibits Lp-PLA 2 -mediated ex-ogenous substrate hydrolysis in plasma and LDL in vitro ( 161 ). In addition, plasma and atherosclerotic plaque lysates from darapladib-treated Watanabe heritable hyper-lipidemic (WHHL) rabbits ( 161 ) and diabetic and hyper-cholesterolemic swine ( 165 ) display highly impaired (up to 95% in plaques) exogenous substrate hydrolysis ( Table 1 ). However, it is essential to assess effects on endogenous phospholipids ( Table 1 ). A number of studies evaluated the impact of darapladib-related compounds on endoge-nous substrate levels using ex vivo and in vivo approaches. Exhaustive in vitro oxidation of LDL with CuSO 4 in the absence and presence of SB222657 confi rmed previous work ( 31, 32 ) showing that truncated PC oxidation prod-ucts are the predominant endogenous substrates for Lp-PLA 2 ( 33 ). PCs that undergo oxidative modifi cation without chain fragmentation are clearly much less effi cient substrates for Lp-PLA 2 , but because they are quantitatively the most abundant PC products of LDL oxidation ( 33 ), the enzyme is likely to affect their levels to a certain extent. In fact, lipopolysaccharide-stimulated whole-blood speci-mens from metabolic syndrome patients treated with various dosages of the Lp-PLA 2 inhibitor SB677116, a com-pound structurally related to darapladib, attenuated the production of C-18 (unfragmented) oxidized fatty acids from endogenous precursors during a 6 h incubation pe-riod ( 166 ) ( Table 1 ).

In vivo studies showed that darapladib treatment re-duced the content of lyso-PC in pig atherosclerotic lesions, owing to inhibition of hydrolysis of endogenous glycero-phospholipids ( 165 ). The nature of possible endogenous substrates was evaluated in studies that compared lipid profi les in darapladib-treated versus placebo-treated ani-mals. In Yorkshire domestic farm pigs, the induction of

TABLE 1. Darapladib and related compounds inhibit hydrolysis of exogenous phospholipid substrates but have varied effects on the metabolism of endogenous phospholipids

Source of Lp-PLA 2 Agent Study Substrate Source Effect Ref.

Recombinant Lp-PLA 2 (human)LDL particles (human)

Darapladib (SB480848)

In vitro Exogenous Inhibition of hydrolysis

( 161 )

Plasma and extracts from atherosclerotic plaques (WHHL rabbits)

( 161 )

Plasma and iliac artery tissue (DM-HC male Yorkshire pigs)

In vivo Endogenous Increased AA-PC; no change in short chain OxPL

( 165 )

LDL exposed to CuSO 4 (human) SB222657 In vitro Increased truncated OxPL

( 33 )

LPS-treated blood (human) SB677116 Decreased C18-OxFA content

( 166 )

Serum ( ApoE �/� mice) Darapladib (SB480848)

In vivo Exogenous Inhibition of hydrolysis

( 168 )

Endogenous No change in PAF levelsSerum ( Ldlr �/� mice) Exogenous Inhibition of hydrolysis ( 167 )

Endogenous No change in PAF levelsPlasma (human) Exogenous Inhibition of hydrolysis ( 169 )


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mice ( 172–175 ) and rabbits ( 172, 173, 176 ) ( Table 2 ). T his series of studies suggested that Lp-PLA 2 may play a protective role in vascular and infl ammatory diseases. Lp-PLA 2 -defi cient mice displayed decreased intestinal in-fl ammation in vivo ( 177 ), but the effect of Lp-PLA 2 deletion on vascular infl ammation in mice has not been reported.

Studies in which Lp-PLA 2 was overexpressed in mice have recently been complemented by preclinical trials of darapladib, also in mice ( 167, 168 ). The effects of darap-ladib administration were similar to those observed when Lp-PLA 2 was overexpressed ( Table 2 ). Darapladib had multiple anti-infl ammatory and anti-atherogenic effects in mice fed atherogenic diets ( 167, 168 ), in diabetic and hypercholesterolemic swine ( 165 ), and in humans ( 169 ) ( Table 2 ). These data suggest that darapladib may have important effects mitigating key pro-infl ammatory path-ways relevant to atherogenesis. However, as mentioned previously, it is not clear whether the mechanism that me-diates the action of darapladib in vivo occurs via Lp-PLA 2 . The comparisons provided in this section strongly argue against this possibility.

Biomarker and imaging studies of Lp-PLA 2 inhibition in humans and in animal models

Two phase II clinical trials of darapladib, including a biomarker study and an atherosclerosis imaging trial, have been completed. Among stable CHD patients with achieved LDL cholesterol concentrations lower than 115 mg/dl on atorvastatin, treatment with darapladib reduced plasma Lp-PLA 2 activity (43, 55, and 66% inhibition at doses of 40, 80, and 160 mg, respectively ( Table 3 , Ref. 178 ). Interleukin-6

way to rigorously investigate the mechanism of darapladib action would be to test the effect of this agent in patients genetically defi cient in Lp-PLA 2 . Effi cacy in this patient population would strongly suggest Lp-PLA 2 -independent effects. Clinical studies of this nature are difficult to implement, although safety/tolerability trials of darap-ladib in healthy Japanese subjects have been completed (NCT00551317 and NCT00622830). An excellent alter-native would be to test this issue in mice. Because darap-ladib limited atherosclerosis in ApoE �/� and Ldlr �/� mice ( 167, 168 ), these models appear adequate for future in-vestigation. Specifi cally, comparing the effect of darap-ladib in control and Lp-PLA 2 -defi cient mice in a model of atherosclerosis (e.g., ApoE �/� or Ldlr �/� ) could provide defi nitive answers regarding the real mechanism of dara-pladib action.

In vivo studies of Lp-PLA 2 overexpression versus darapladib administration

Similar to what is observed in humans, enhanced levels of sPLA 2 s and Lp-PLA 2 correlate with increased athero-sclerosis in animal models ( 95 ), with the exception of a study by Forte and coworkers ( 171 ). Secretory PLA 2 s, un-like Lp-PLA 2 , have been demonstrated to actively contrib-ute to atherogenesis, and so correlate with disease status ( 85 ). An excellent review by Oörni and colleagues high-lights multiple studies showing that development of ath-erosclerosis is decreased in mice lacking sPLA 2 expression and is increased with overexpression ( 85 ). Parallel studies addressing the role of Lp-PLA 2 have clearly shown that overexpression of Lp-PLA 2 diminishes atherosclerosis in

TABLE 2. Overexpression of Lp-PLA 2 and darapladib treatment do not have opposite effects in vivo

Experimental Model Intervention In Vivo Model Effects Ref.

Ischemia reperfusion injury induced by coronary ligation

Administration of recombinant Lp-PLA 2

New Zealand white rabbits

↓ Necrotic area ↓ Systolic shortening and wall

thickness

( 172 )

↓ Neutrophil infi ltrationAortic injury induced by

balloon denudationAdenovirus-mediated

Lp-PLA 2 gene transfer

Cholesterol-fed New Zealand white rabbits

↓ Intima/media ratio ↓ Apoptosis

( 173 )

Atherosclerosis ApoE �/� mice ↓ Modifi ed LDL autoantibodies ( 174 ) ↓ Neointimal area ↓ Atherosclerosis ↓ Macrophage homing to

endothelium( 175 )

↓ Aortic wall thickness ( 183 )Balloon-mediated injury

of carotid arteriesNormolipidemic

New Zealand white rabbits

↓ Accumulation of oxidized lipoproteins

↓ Intima/media ratio

( 176 )

Anti-infl ammatory effectsAnti-thrombotic effectsAnti-proliferative effects

Atherosclerosis Darapladib Ldlr �/� mice ↓ Infl ammatory burden ( 167 ) ↓ Plaque area

ApoE �/� mice ↓ Infl ammatory burden ( 168 ) ↓ Plaque area

DM-HC swine General anti-infl ammatory action ( 165 ) ↓ Plaque and necrotic core area ↓ Medial destruction

Coronary disease patients

Halted necrotic core volume expansion

( 169 )

↔ Coronary atheroma deformability

↔ Plaque volume


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determine whether its effects are mediated through Lp-PLA 2 or other mechanisms.

Safety issues to consider In phase II clinical studies, darapladib was well tolerated

at dosages of 40, 80 and 160 mg daily ( 169, 178 ). The most common adverse reactions, reported in 16–36% of study subjects, included diarrhea and malodor of the urine and feces. In addition, CD40L levels and mean systolic blood pressure were higher in the darapladib group during treat-ment ( 169, 178 ). Regardless of the mechanism whereby darapladib affects events relevant to atherosclerosis, there is no doubt that this compound inhibits Lp-PLA 2 activity. Thus, an immediate issue that merits consideration is the potential impact of widespread use of this drug in popula-tions with a large spectrum of conditions. A research team in Korea has reported that decreased Lp-PLA 2 activity is benefi cial in certain settings ( 137, 154 ), and it is clear that partial/complete defi ciency of Lp-PLA 2 does not by itself cause disease ( 26, 27 ). However, several independent studies have shown that decreased Lp-PLA 2 expression in humans increases the severity of infl ammatory, vascular, and other conditions (reviewed in Refs. 26, 142, 179 ). It has been argued that these concerns are “theoretical” ( 9 ), but it is important to point out that studies in relatively small populations are unlikely to include patients with un-derlying acute and/or chronic infl ammatory conditions (e.g., asthma, anaphylaxis, sepsis) in which decreased or absent Lp-PLA 2 may increase disease severity ( 135, 180 ). An example of this paradigm was reported in a clinical trial focused on type II diabetes subjects undergoing he-modialysis ( 181 ). This study serendipitously found that patients whose Lp-PLA 2 activity decreased by 25% or more over a six-month period suffered from a dramatic increase in mortality [HR = 2.48; CI (1.56–3.95), P < 0·001 ( 181 )]. This observation is only correlative and, therefore, does not demonstrate a causal role for decreased Lp-PLA 2 in

(IL-6) levels and hs-CRP were reduced by 12.3% and 13%, respectively, suggesting a reduction in infl ammatory bur-den. The IBIS-2 (Integrated Biomarker and Imaging Study) found no effect of darapladib on coronary ath-eroma deformability, plasma hs-CRP levels, or other endpoints in patients with angiographically documented CAD ( Table 3 , Ref. 169 ). However, assessment of lipid necrotic core by virtual histology revealed increased size in the placebo group (4.5 ± 7.9 mm 3 , P = 0.009) but not in darapladib-treated patients (0.5 ± 13.9 mm 3 , P = 0.71), resulting in a signifi cant treatment difference of � 5.2 mm 3 ( Table 3 , Ref. 169 ). These fi ndings are con-sistent with results from the swine model ( Table 3 , Ref. 165 ) and suggest that darapladib stabilizes rupture-prone plaques.

Darapladib and cardiovascular events: ongoing clinical trials

The clinical effi cacy of darapladib is currently being ex-amined in two large-scale trials that investigate its effects on nonfatal MI, nonfatal stroke, and cardiovascular death in CHD patients. The STABILITY (Stabilization of Athero-sclerotic Plaque by Initiation of Darapladib Therapy) trial is a multicenter, randomized, double-blind, placebo-con-trolled trial that compares the effect of darapladib and placebo on cardiovascular events in 15,500 patients with stable CHD ( 162 ). In addition, SOLID-TIMI 52 (Stabiliza-tion Of pLaques usIng Darapladib-Thrombolysis In Myo-cardial Infarction 52) is designed to investigate the effects of darapladib on major recurrent cardiovascular events in 11,500 MI/stroke patients ( 163 ). In these two secondary prevention trials, it will be important to investigate rela-tionships between cardiovascular events and baseline/on-trial Lp-PLA 2 activity, lipid profi les, and changes in the extent of pro- and anti-infl ammatory responses ( 6, 8 ). These measurements will provide key information to es-tablish the therapeutic utility of darapladib and help

TABLE 3. Effect of darapladib treatment on markers of infl ammation in animal models of atherosclerosis and in human patients

In Vivo Model Biomarker Effects Ref.

Ldlr �/� mice ↓ Serum Lp-PLA 2 activity ( 167 ) ↓ Hs-CRP, IL-6, MCP-1, VCAM-1 ↔ Serum PAF

ApoE �/� mice ↓ Serum Lp-PLA 2 activity ( 168 ) ↓ Hs-CRP, IL-6, MCP-1, VCAM-1 and TNF- � ↔ Serum PAF ↓ Macrophage content in atherosclerotic lesions ↑ Collagen content in atherosclerotic lesions

DM-HC swine ↓ Plasma Lp-PLA 2 activity ( 165 ) ↓ Lesion Lp-PLA 2 activity and lyso-PC content ↓ Expression of 24 genes associated with macrophage

and T lymphocyte functions ↓ Macrophage content in atherosclerotic lesions

Healthy men ↔ ADP-induced platelet aggregation ( 184 ) ↔ Collagen-induced platelet aggregation

CHD patients (LDL cholesterol< 115 mg/dl on atorvastatin)

↓ Plasma Lp-PLA 2 ↓ Hs-CRP, IL-6

( 178 )

↔ MPO, MMP-9 activity, P-selectin, CD40L ↔ HDL and LDL cholesterol

Patients with angiographically documented CHD (IBIS-2)

↓ Plasma Lp-PLA 2 ↔ Hs-CRP, MPO, ICAM-1, MMP-9 activity, OxPL/apoB

( 169 )

↔ HDL and LDL cholesterol


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mortality. However, it provides an example of a patient population in which inhibiting Lp-PLA 2 may not be advis-able. In cardiovascular medicine, therapies directed at sur-rogate endpoints can be associated with excess deaths ( 182 ). The safety and effi cacy of darapladib should be evaluated very carefully before potential harmful effects become widespread.

CONCLUSIONS

It has been proposed that one of the strongest argu-ments in favor of a pro-atherogenic role of Lp-PLA 2 is the substantial number of epidemiological studies ( 66 ). Al-though it is clear that there is a positive association between Lp-PLA 2 activity and CAD, the strength of this as-sociation appears to be almost exclusively accounted for by the fact that most of the plasma Lp-PLA 2 activity asso-ciates with LDL. These observations raise questions re-garding the utility of Lp-PLA 2 as a biomarker, particularly when one considers that current guidelines direct clini-cians to treat at-risk patients with cholesterol-lowering drugs and that for these patients Lp-PLA 2 levels no longer predict vascular risk ( 96, 98, 120 ).

It is unlikely that strategies based on inhibiting the en-zymatic activity of Lp-PLA 2 will be effi cacious for the pre-vention or treatment of CVD in humans. The evidence currently available, which includes the majority of genetic analyses in humans and all overexpression studies in mice and rabbits, does not mechanistically support this ratio-nale. Darapladib has been shown to reduce development of experimental atherosclerosis and prevent the expan-sion of necrotic core lesions in humans, but these effects may not be mediated by Lp-PLA 2 . Our ability to establish meaningful links between Lp-PLA 2 and CVD depends on our willingness to go well beyond observational studies that show associations between expression and outcome.

REFERENCES

1 . Libby , P. , P. M. Ridker , and A. Maseri . 2002 . Infl ammation and atherosclerosis. Circulation . 105 : 1135 – 1143 .

2 . Witztum , J. L. , and J. A. Berliner . 1998 . Oxidized phospho-lipids and isoprostanes in atherosclerosis. Curr. Opin. Lipidol. 9 : 441 – 448 .

3 . Tardif , J. C. , J. J. McMurray , E. Klug , R. Small , J. Schumi , J. Choi , J. Cooper , R. Scott , E. F. Lewis , P. L. L’Allier , et al . 2008 . Effects of succi-nobucol (AGI-1067) after an acute coronary syndrome: a randomised, double-blind, placebo-controlled trial. Lancet . 371 : 1761 – 1768 .

4 . Tardif , J. C. , P. L. L’allier , R. Ibrahim, J. C. Gregoire, A. Nozza, M. Cossette, S. Kouz, M. A. Lavoie, J. Paquin, T. M. Brotz, et al. 2010 . Treatment with 5-lipoxygenase inhibitor VIA-2291 (Atreleuton) in patients with recent acute coronary syndrome. Circ. Cardiovasc. Imaging . 3 : 298 – 307 .

5 . Rosenson , R. S. , C. Hislop , D. McConnell , M. Elliott , Y. Stasiv , N. Wang , D. D. Waters , and PLASMA Investigators. 2009 . Effects of 1-H-indole-3-glyoxamide (A-002) on concentration of secretory phospholipase A 2 (PLASMA study): a phase II double-blind, ran-domised, placebo-controlled trial. Lancet . 373 : 649 – 658 .

6 . Rosenson , R. S. 2010 . Phospholipase A 2 inhibition and atheroscle-rotic vascular disease: prospects for targeting secretory and lipo-protein-associated phospholipase A 2 enzymes. Curr. Opin. Lipidol. 21 : 473 – 480 .

7 . Wierda , R. J. , S. B. Geutskens , J. W. Jukema , P. H. Quax , and P. J. van den Elsen . 2010 . Epigenetics in atherosclerosis and infl amma-tion. J. Cell. Mol. Med. 14 : 1225 – 1240 .

8 . Rosenson , R. S. 2009 . Future role for selective phospholipase A 2 inhibitors in the prevention of atherosclerotic cardiovascular disease. Cardiovasc. Drugs Ther. 23 : 93 – 101 .

9 . White , H. 2010 . Why inhibition of lipoprotein-associated phos-pholipase A 2 has the potential to improve patient outcomes. Curr. Opin. Cardiol. 25 : 299 – 301 .

10 . Garza , C. A. , V. M. Montori , J. P. McConnell , V. K. Somers , I. J. Kullo , and F. Lopez-Jimenez . 2007 . Association between lipopro-tein-associated phospholipase A 2 and cardiovascular disease: a sys-tematic review. Mayo Clin. Proc. 82 : 159 – 165 .

11 . Stephens , C. J. , R. M. Graham , O. P. Yadava , L. L. Leong , M. J. Sturm , and R. R. Taylor . 1992 . Plasma platelet activating factor degradation and serum lipids after coronary bypass surgery. Cardiovasc. Res. 26 : 25 – 31 .

12 . Guerra , R. , B. Zhao , V. Mooser , D. Stafforini , J. M. Johnston , and J. C. Cohen . 1997 . Determinants of plasma platelet-activating factor acetylhydrolase: heritability and relationship to plasma li-poproteins. J. Lipid Res. 38 : 2281 – 2288 .

13 . Graham , R. M. , C. J. Stephens , M. J. Sturm , and R. R. Taylor . 1992 . Plasma platelet-activating factor degradation in patients with severe coronary artery disease. Clin. Sci. (Lond.) . 82 : 535 – 541 .

14 . Ostermann , G. , A. Lang , H. Holtz , K. Ruhling , L. Winkler , and U. Till . 1988 . The degradation of platelet-activating factor in serum and its discriminative value in atherosclerotic patients. Thromb. Res. 52 : 529 – 540 .

15 . Ostermann , G. , K. Ruhling , R. Zabel-Langhennig , L. Winkler , B. Schlag , and U. Till . 1987 . Plasma from atherosclerotic patients ex-erts an increased degradation of platelet-activating factor. Thromb. Res. 47 : 279 – 285 .

16 . Burke , J. E. , and E. A. Dennis . 2009 . Phospholipase A 2 biochemis-try. Cardiovasc. Drugs Ther. 23 : 49 – 59 .

17 . Schaloske , R. H. , and E. A. Dennis . 2006 . The phospholipase A 2 superfamily and its group numbering system. Biochim. Biophys. Acta . 1761 : 1246 – 1259 .

18 . Farr , R. S. , C. P. Cox , M. L. Wardlow , and R. Jorgensen . 1980 . Preliminary studies of an acid-labile factor (ALF) in human sera that inactivates platelet-activating factor (PAF). Clin. Immunol. Immunopathol. 15 : 318 – 330 .

19 . Prescott , S. M. , G. A. Zimmerman , D. M. Stafforini , and T. M. McIntyre . 2000 . Platelet-activating factor and related lipid media-tors. Annu. Rev. Biochem. 69 : 419 – 445 .

20 . Prescott , S. M. , G. A. Zimmerman , and T. M. McIntyre . 1990 . Platelet-activating factor. J. Biol. Chem. 265 : 17381 – 17384 .

21 . Stafforini , D. M. , M. R. Elstad , T. M. McIntyre , G. A. Zimmerman , and S. M. Prescott . 1990 . Human macrophages secret platelet-activating factor acetylhydrolase. J. Biol. Chem. 265 : 9682 – 9687 .

22 . Stafforini , D. M. , T. M. McIntyre , M. E. Carter , and S. M. Prescott . 1987 . Human plasma platelet-activating factor acetylhydrolase. Association with lipoprotein particles and role in the degradation of platelet-activating factor. J. Biol. Chem. 262 : 4215 – 4222 .

23 . Blencowe , C. , A. Hermetter , G. M. Kostner , and H. P. Deigner . 1995 . Enhanced association of platelet-activating factor acetylhy-drolase with lipoprotein (a) in comparison with low density lipo-protein. J. Biol. Chem. 270 : 31151 – 31157 .

24 . Tjoelker , L. W. , C. Eberhardt , J. Unger , H. L. Trong , G. A. Zimmerman , T. M. McIntyre , D. M. Stafforini , S. M. Prescott , and P. W. Gray . 1995 . Plasma platelet-activating factor acetylhydrolase is a secreted phospholipase A 2 with a catalytic triad. J. Biol. Chem. 270 : 25481 – 25487 .

25 . Samanta , U. , and B. J. Bahnson . 2008 . Crystal structure of human plasma platelet-activating factor acetylhydrolase: structural im-plication to lipoprotein binding and catalysis. J. Biol. Chem. 283 : 31617 – 31624 .

26 . Stafforini , D. M. 2009 . Biology of platelet-activating factor acetyl-hydrolase (PAF-AH, lipoprotein associated phospholipase A 2 ). Cardiovasc. Drugs Ther. 23 : 73 – 83 .

27 . McIntyre , T. M. , S. M. Prescott , and D. M. Stafforini . 2009 . The emerging roles of PAF acetylhydrolase. J. Lipid Res. 50 ( Suppl. ): S255 – S259 .

28 . Stremler , K. E. , D. M. Stafforini , S. M. Prescott , and T. M. McIntyre . 1991 . Human plasma platelet-activating factor acetylhydrolase. Oxidatively fragmented phospholipids as substrates. J. Biol. Chem. 266 : 11095 – 11103 .

29 . Steinbrecher , U. P. , and P. H. Pritchard . 1989 . Hydrolysis of phos-phatidylcholine during LDL oxidation is mediated by platelet-activating factor acetylhydrolase. J. Lipid Res. 30 : 305 – 315 .


ww

w.jlr.org

Dow

nloaded from

http://www.jlr.org/


47 . Wolfs , I. M. , M. M. Donners , and M. P. de Winther . 2011 . Differentiation factors and cytokines in the atherosclerotic plaque micro-environment as a trigger for macrophage polarisation. Thromb. Haemost. 106 : 763 – 771 .

48 . Cao , Y. , D. M. Stafforini , G. A. Zimmerman , T. M. McIntyre , and S. M. Prescott . 1998 . Expression of plasma platelet-activating factor acetylhydrolase is transcriptionally regulated by mediators of infl ammation. J. Biol. Chem. 273 : 4012 – 4020 .

49 . Al-Darmaki , S. , H. A. Schenkein , J. G. Tew , and S. E. Barbour . 2003 . Differential expression of platelet-activating factor acetylhy-drolase in macrophages and monocyte-derived dendritic cells. J. Immunol. 170 : 167 – 173 .

50 . Kadl , A. , A. K. Meher , P. R. Sharma , M. Y. Lee , A. C. Doran , S. R. Johnstone , M. R. Elliott , F. Gruber , J. Han , W. Chen , et al . 2010 . Identifi cation of a novel macrophage phenotype that develops in response to atherogenic phospholipids via Nrf2. Circ. Res. 107 : 737 – 746 .

51 . Wu , X. , G. A. Zimmerman , S. M. Prescott , and D. M. Stafforini . 2004 . The p38 MAPK pathway mediates transcriptional activation of the plasma platelet-activating factor acetylhydrolase gene in macrophages stimulated with lipopolysaccharide. J. Biol. Chem. 279 : 36158 – 36165 .

52 . Shi , Y. , A. Zalewski , C. H. Macphee , and M. Dawson . 2007 . Selective inhibition of lipoprotein-associated phospholipase A 2 attenuates markers of plaque vulnerability in humans. (Abstract 594). Circulation . 116 : II-108 .

53 . Satoh , K. , T. Imaizumi , Y. Kawamura , H. Yoshida , M. Hiramoto , S. Takamatsu , and M. Takamatsu . 1991 . Platelet-activating factor (PAF) stimulates the production of PAF acetylhydrolase by the human hepatoma cell line, HepG2. J. Clin. Invest. 87 : 476 – 481 .

54 . Zheng , G. H. , S. Q. Xiong , L. J. Mei , H. Y. Chen , T. Wang , and J. F. Chu . Elevated plasma platelet activating factor, platelet activating factor acetylhydrolase levels and risk of coronary heart disease or blood stasis syndrome of coronary heart disease in Chinese: a case control study. Infl ammation . Epub ahead of print. March 20, 2012; doi:10.1007/s10753-012-9455-4.

55 . De Keyzer , D. , S. A. Karabina , W. Wei , B. Geeraert , D. Stengel , J. Marsillach , J. Camps , P. Holvoet , and E. Ninio . 2009 . Increased PAFAH and oxidized lipids are associated with infl ammation and atherosclerosis in hypercholesterolemic pigs. Arterioscler. Thromb. Vasc. Biol. 29 : 2041 – 2046 .

56 . Grallert , H. , J. Dupuis , J. C. Bis , A. Dehghan , M. Barbalic , J. Baumert , C. Lu , N. L. Smith , A. G. Uitterlinden , R. Roberts , et al . 2012 . Eight genetic loci associated with variation in lipoprotein-associated phospholipase A 2 mass and activity and coronary heart disease: meta-analysis of genome-wide association studies from fi ve community-based studies. Eur. Heart J. 33 : 238 – 251 .

57 . Suchindran , S. , D. Rivedal , J. R. Guyton , T. Milledge , X. Gao , A. Benjamin , J. Rowell , G. S. Ginsburg , and J. J. McCarthy . 2010 . Genome-wide association study of Lp-PLA 2 activity and mass in the Framingham Heart Study. PLoS Genet. 6 : e1000928 .

58 . McConnell , J. P. , and D. M. Hoefner . 2006 . Lipoprotein-associated phospholipase A 2 . Clin. Lab. Med. 26 : 679 – 697 .

59 . Birukov , K. G. , V. N. Bochkov , A. A. Birukova , K. Kawkitinarong , A. Rios , A. Leitner , A. D. Verin , G. M. Bokoch , N. Leitinger , and J. G. Garcia . 2004 . Epoxycyclopentenone-containing oxidized phos-pholipids restore endothelial barrier function via Cdc42 and Rac. Circ. Res. 95 : 892 – 901 .

60 . Watson , A. D. , G. Subbanagounder , D. S. Welsbie , K. F. Faull , M. Navab , M. E. Jung , A. M. Fogelman , and J. A. Berliner . 1999 . Structural identifi cation of a novel pro-infl ammatory epoxyiso-prostane phospholipid in mildly oxidized low density lipoprotein. J. Biol. Chem. 274 : 24787 – 24798 .

61 . Lusis , A. J. 2000 . Atherosclerosis. Nature . 407 : 233 – 241 . 62 . Subbanagounder , G. , J. W. Wong , H. Lee , K. F. Faull , E. Miller , J.

L. Witztum , and J. A. Berliner . 2002 . Epoxyisoprostane and epoxy-cyclopentenone phospholipids regulate monocyte chemotactic protein-1 and interleukin-8 synthesis. Formation of these oxidized phospholipids in response to interleukin-1beta. J. Biol. Chem. 277 : 7271 – 7281 .

63 . Podrez , E. A. , E. Poliakov , Z. Shen , R. Zhang , Y. Deng , M. Sun , P. J. Finton , L. Shan , B. Gugiu , P. L. Fox , et al . 2002 . Identifi cation of a novel family of oxidized phospholipids that serve as ligands for the macrophage scavenger receptor CD36. J. Biol. Chem. 277 : 38503 – 38516 .

64 . Gao , S. , R. Zhang , M. E. Greenberg , M. Sun , X. Chen , B. S. Levison , R. G. Salomon , and S. L. Hazen . 2006 . Phospholipid hydroxyalkenals,

30 . Marathe , G. K. , K. A. Harrison , R. C. Murphy , S. M. Prescott , G. A. Zimmerman , and T. M. McIntyre . 2000 . Bioactive phospholipid oxidation products. Free Radic. Biol. Med. 28 : 1762 – 1770 .

31 . Kriska , T. , G. K. Marathe , J. C. Schmidt , T. M. McIntyre , and A. W. Girotti . 2007 . Phospholipase action of platelet-activating factor acetylhydrolase, but not paraoxonase-1, on long fatty acyl chain phospholipid hydroperoxides. J. Biol. Chem. 282 : 100 – 108 .

32 . Stafforini , D. M. , J. R. Sheller , T. S. Blackwell , A. Sapirstein , F. E. Yull , T. M. McIntyre , J. V. Bonventre , S. M. Prescott , and L. J. Roberts 2nd . 2006 . Release of free F 2 -isoprostanes from es-terifi ed phospholipids is catalyzed by intracellular and plasma platelet-activating factor acetylhydrolases. J. Biol. Chem. 281 : 4616 – 4623 .

33 . Davis , B. , G. Koster , L. J. Douet , M. Scigelova , G. Woffendin , J. M. Ward , A. Smith , J. Humphries , K. G. Burnand , C. H. Macphee , et al . 2008 . Electrospray ionization mass spectrometry identifi es substrates and products of lipoprotein-associated phospholipase A 2 in oxidized human low density lipoprotein. J. Biol. Chem. 283 : 6428 – 6437 .

34 . Cao , J. , Y. H. Hsu , S. Li , V. L. Woods , and E. A. Dennis . 2011 . Lipoprotein-associated phospholipase A 2 interacts with phos-pholipid vesicles via a surface-disposed hydrophobic alpha-helix. Biochemistry . 50 : 5314 – 5321 .

35 . Karabina , S. A. , M. Elisaf , E. Bairaktari , C. Tzallas , K. C. Siamopoulos , and A. D. Tselepis . 1997 . Increased activity of plate-let-activating factor acetylhydrolase in low-density lipoprotein sub-fractions induces enhanced lysophosphatidylcholine production during oxidation in patients with heterozygous familial hypercho-lesterolaemia. Eur. J. Clin. Invest. 27 : 595 – 602 .

36 . Tsimihodimos , V. , S. A. Karabina , A. P. Tambaki , E. Bairaktari , G. Miltiadous , J. A. Goudevenos , M. A. Cariolou , M. J. Chapman , A. D. Tselepis , and M. Elisaf . 2002 . Altered distribution of platelet-activating factor-acetylhydrolase activity between LDL and HDL as a function of the severity of hypercholesterolemia. J. Lipid Res. 43 : 256 – 263 .

37 . Okamura , K. , S. Miura , B. Zhang , Y. Uehara , K. Matsuo , K. Kumagai , and K. Saku . 2007 . Ratio of LDL- to HDL-associated platelet-activating factor acetylhydrolase may be a marker of in-fl ammation in patients with paroxysmal atrial fi brillation. Circ. J. 71 : 214 – 219 .

38 . Papavasiliou , E. C. , C. Gouva , K. C. Siamopoulos , and A. D. Tselepis . 2006 . PAF-acetylhydrolase activity in plasma of patients with chronic kidney disease. Effect of long-term therapy with erythropoietin. Nephrol. Dial. Transplant. 21 : 1270 – 1277 .

39 . Stafforini , D. M. , L. W. Tjoelker , S. P. McCormick , D. Vaitkus , T. M. McIntyre , P. W. Gray , S. G. Young , and S. M. Prescott . 1999 . Molecular basis of the interaction between plasma platelet-activating factor acetylhydrolase and low density lipoprotein. J. Biol. Chem. 274 : 7018 – 7024 .

40 . Gardner , A. A. , E. C. Reichert , M. K. Topham , and D. M. Stafforini . 2008 . Identifi cation of a domain that mediates association of platelet-activating factor acetylhydrolase with high density lipo-protein. J. Biol. Chem. 283 : 17099 – 17106 .

41 . Tselepis , A. D. , and M. John Chapman . 2002 . Inflammation, bioactive lipids and atherosclerosis: potential roles of a lipoprotein-associated phospholipase A 2 , platelet activating factor-acetylhydrolase. Atheroscler. Suppl. 3 : 57 – 68 .

42 . Linton , M. F. , and S. Fazio . 2003 . Macrophages, infl ammation, and atherosclerosis. Int. J. Obes. Relat. Metab. Disord. 27 ( Suppl. 3 ): S35 – S40 .

43 . Tjoelker , L. W. , C. Wilder , C. Eberhardt , D. M. Stafforini , G. Dietsch , B. Schimpf , S. Hooper , H. Le Trong , L. S. Cousens , G. A. Zimmerman , et al . 1995 . Anti-infl ammatory properties of a platelet-activating factor acetylhydrolase. Nature . 374 : 549 – 553 .

44 . Ferguson , J. F. , C. C. Hinkle , N. N. Mehta , R. Bagheri , S. L. Derohannessian , R. Shah , M. I. Mucksavage , J. P. Bradfi eld , H. Hakonarson , X. Wang , et al . 2012 . Translational studies of lipoprotein- associated phospholipase A 2 in infl ammation and atherosclerosis. J. Am. Coll. Cardiol. 59 : 764 – 772 .

45 . Mannheim , D. , J. Herrmann , D. Versari , M. Gossl , F. B. Meyer , J. P. McConnell , L. O. Lerman , and A. Lerman . 2008 . Enhanced ex-pression of Lp-PLA 2 and lysophosphatidylcholine in symptomatic carotid atherosclerotic plaques. Stroke . 39 : 1448 – 1455 .

46 . Lee , S. , S. Huen , H. Nishio , S. Nishio , H. K. Lee , B. S. Choi , C. Ruhrberg , and L. G. Cantley . 2011 . Distinct macrophage pheno-types contribute to kidney injury and repair. J. Am. Soc. Nephrol. 22 : 317 – 326 .


ww

w.jlr.org

Dow

nloaded from

http://www.jlr.org/


83 . Taylor , L. A. , J. Arends , A. K. Hodina , C. Unger , and U. Massing . 2007 . Plasma lyso-phosphatidylcholine concentration is decreased in cancer patients with weight loss and activated infl ammatory status. Lipids Health Dis. 6 : 17 – 24 .

84 . Cunningham , T. J. , L. Yao , and A. Lucena . 2008 . Product inhibi-tion of secreted phospholipase A 2 may explain lysophosphatidyl-cholines’ unexpected therapeutic properties. J. Infl amm. (Lond.) . 5 : 17 – 26 .

85 . Oörni , K. , and P. T. Kovanen . 2009 . Lipoprotein modifi cation by se-cretory phospholipase A 2 enzymes contributes to the initiation and progression of atherosclerosis. Curr. Opin. Lipidol. 20 : 421 – 427 .

86 . Edwards , I. J. , I. M. Berquin , H. Sun , J. T. O’Flaherty, L. W. Daniel, M. J. Thomas, L. L. Rudel, R. L. Wykle, and Y. Q. Chen. 2004 . Differential effects of delivery of omega-3 fatty acids to human cancer cells by low-density lipoproteins versus albumin. Clin. Cancer Res. 10 : 8275 – 8283 .

87 . Morton , R. E. , and D. J. Greene . 2000 . The capacity of various non-esterifi ed fatty acids to suppress lipid transfer inhibitor protein activity is related to their perturbation of the lipoprotein surface. Biochim. Biophys. Acta . 1486 : 275 – 284 .

88 . Lee , C. , F. Sigari , T. Segrado , S. Horkko , S. Hama , P. V. Subbaiah , M. Miwa , M. Navab , J. L. Witztum , and P. D. Reaven . 1999 . All ApoB-containing lipoproteins induce monocyte chemotaxis and adhesion when minimally modifi ed. Modulation of lipoprotein bioactivity by platelet-activating factor acetylhydrolase. Arterioscler. Thromb. Vasc. Biol. 19 : 1437 – 1446 .

89 . Yang , M. , E. M. Chu , M. J. Caslake , C. Edelstein , A. M. Scanu , and J. S. Hill . 2010 . Lipoprotein-associated phospholipase A 2 de-creases oxidized lipoprotein cellular association by human macro-phages and hepatocytes. Biochim. Biophys. Acta . 1801 : 176 – 182 .

90 . Chen , C. H. , T. Jiang , J. H. Yang , W. Jiang , J. Lu , G. K. Marathe , H. J. Pownall , C. M. Ballantyne , T. M. McIntyre , P. D. Henry , et al . 2003 . Low-density lipoprotein in hypercholesterolemic human plasma induces vascular endothelial cell apoptosis by in-hibiting fi broblast growth factor 2 transcription. Circulation . 107 : 2102 – 2108 .

91 . Angeli , V. , J. Llodra , J. X. Rong , K. Satoh , S. Ishii , T. Shimizu , E. A. Fisher , and G. J. Randolph . 2004 . Dyslipidemia associated with atherosclerotic disease systemically alters dendritic cell mobiliza-tion. Immunity . 21 : 561 – 574 .

92 . Packard , C. J. , D. S. O’Reilly , M. J. Caslake , A. D. McMahon , I. Ford , J. Cooney , C. H. Macphee , K. E. Suckling , M. Krishna , F. E. Wilkinson , et al . 2000 . Lipoprotein-associated phospholipase A 2 as an independent predictor of coronary heart disease. West of Scotland Coronary Prevention Study Group. N. Engl. J. Med. 343 : 1148 – 1155 .

93 . Liu , J. , Y. Hong, Y. Qi, F. Zhao, and D. Zhao. 2011 . Systematic review of the association between lipoprotein-associated phospho-lipase A 2 and atherosclerosis. N. Am. J. Med. Sci. 4 : 201 – 211 .

94 . Thompson , A. , P. Gao , L. Orfei , S. Watson , E. Di Angelantonio , S. Kaptoge , C. Ballantyne , C. P. Cannon , M. Criqui , M. Cushman , et al . 2010 . Lipoprotein-associated phospholipase A 2 and risk of coronary disease, stroke, and mortality: collaborative analysis of 32 prospective studies. Lancet . 375 : 1536 – 1544 .

95 . Mallat , Z. , G. Lambeau , and A. Tedgui . 2010 . Lipoprotein-associated and secreted phospholipases A in cardiovascular disease: roles as biological effectors and biomarkers. Circulation . 122 : 2183 – 2200 .

96 . Stein , E. A. 2012 . Lipoprotein-associated phospholipase A 2 measure-ments: mass, activity, but little productivity. Clin. Chem. 58 : 814 – 817 .

97 . McConnell , J. P. , and A. S. Jaffe . 2008 . Variability of lipopro-tein-associated phospholipase A 2 measurements. Clin. Chem. 54 : 932 – 933 .

98 . Ridker , P. M. , J. G. Macfadyen , R. L. Wolfert , and W. Koenig . 2012 . Relationship of lipoprotein-associated phospholipase A 2 mass and activity with incident vascular events among primary prevention patients allocated to placebo or to statin therapy: an analysis from the JUPITER trial. Clin. Chem. 58 : 877 – 886 .

99 . Koenig , W. , N. Khuseyinova , H. Lowel , G. Trischler , and C. Meisinger . 2004 . Lipoprotein-associated phospholipase A 2 adds to risk prediction of incident coronary events by C-reactive protein in apparently healthy middle-aged men from the general popu-lation: results from the 14-year follow-up of a large cohort from southern Germany. Circulation . 110 : 1903 – 1908 .

100 . Oei , H. H. , I. M. van der Meer , A. Hofman , P. J. Koudstaal , T. Stijnen , M. M. Breteler , and J. C. Witteman . 2005 . Lipoprotein-associated phospholipase A 2 activity is associated with risk of

a subset of recently discovered endogenous CD36 ligands, spon-taneously generate novel furan-containing phospholipids lacking CD36 binding activity in vivo. J. Biol. Chem. 281 : 31298 – 31308 .

65 . McIntyre , T. M. Bioactive oxidatively truncated phospholipids in infl ammation and apoptosis: Formation, targets, and inactiva-tion. Biochim. Biophys. Acta . Epub ahead of print. March 16, 2012; doi:10.1016/j.bbamem.2012.03.004.

66 . Suckling , K. 2010 . Phospholipase A 2 s: developing drug targets for atherosclerosis. Atherosclerosis . 212 : 357 – 366 .

67 . Oskolkova , O. V. , T. Afonyushkin , B. Preinerstorfer , W. Bicker , E. von Schlieffen , E. Hainzl , S. Demyanets , G. Schabbauer , W. Lindner , A. D. Tselepis , et al . 2010 . Oxidized phospholipids are more potent antagonists of lipopolysaccharide than inducers of infl ammation. J. Immunol. 185 : 7706 – 7712 .

68 . Bochkov , V. N. , A. Kadl , J. Huber , F. Gruber , B. R. Binder , and N. Leitinger . 2002 . Protective role of phospholipid oxidation prod-ucts in endotoxin-induced tissue damage. Nature . 419 : 77 – 81 .

69 . Berliner , J. A. , N. Leitinger , and S. Tsimikas . 2009 . The role of oxidized phospholipids in atherosclerosis. J. Lipid Res. 50 ( Suppl. ): S207 – S212 .

70 . Ozaki , H. , K. Ishii , H. Arai , N. Kume , and T. Kita . 1999 . Lysophosphatidylcholine activates mitogen-activated protein ki-nases by a tyrosine kinase-dependent pathway in bovine aortic endothelial cells. Atherosclerosis . 143 : 261 – 266 .

71 . Ueno , Y. , N. Kume , S. Miyamoto , M. Morimoto , H. Kataoka , H. Ochi , E. Nishi , H. Moriwaki , M. Minami , N. Hashimoto , et al . 1999 . Lysophosphatidylcholine phosphorylates CREB and acti-vates the jun2TRE site of c-jun promoter in vascular endothelial cells. FEBS Lett. 457 : 241 – 245 .

72 . Kohno , M. , K. Yokokawa , K. Yasunari , M. Minami , H. Kano , T. Hanehira , and J. Yoshikawa . 1998 . Induction by lysophosphatidyl-choline, a major phospholipid component of atherogenic lipo-proteins, of human coronary artery smooth muscle cell migration. Circulation . 98 : 353 – 359 .

73 . Matsumoto , T. , T. Kobayashi , and K. Kamata . 2007 . Role of lyso-phosphatidylcholine (LPC) in atherosclerosis. Curr. Med. Chem. 14 : 3209 – 3220 .

74 . Chen , G. , J. Li , X. Qiang , C. J. Czura , M. Ochani , K. Ochani , L. Ulloa , H. Yang , K. J. Tracey , P. Wang , et al . 2005 . Suppression of HMGB1 release by stearoyl lysophosphatidylcholine:an additional mechanism for its therapeutic effects in experimental sepsis. J. Lipid Res. 46 : 623 – 627 .

75 . MacPhee , C. H. , K. E. Moores , H. F. Boyd , D. Dhanak , R. J. Ife , C. A. Leach , D. S. Leake , K. J. Milliner , R. A. Patterson , K. E. Suckling , et al . 1999 . Lipoprotein-associated phospholipase A 2 , platelet-activating factor acetylhydrolase, generates two bioactive products during the oxidation of low-density lipoprotein: use of a novel inhibitor. Biochem. J. 338 : 479 – 487 .

76 . Obinata , H. , T. Hattori , S. Nakane , K. Tatei , and T. Izumi . 2005 . Identifi cation of 9-hydroxyoctadecadienoic acid and other oxi-dized free fatty acids as ligands of the G protein-coupled receptor G2A. J. Biol. Chem. 280 : 40676 – 40683 .

77 . Mas , S. , R. Martinez-Pinna , J. L. Martin-Ventura , R. Perez , D. Gomez-Garre , A. Ortiz , A. Fernandez-Cruz , F. Vivanco , and J. Egido . 2010 . Local non-esterifi ed fatty acids correlate with in-fl ammation in atheroma plaques of patients with type 2 diabetes. Diabetes . 59 : 1292 – 1301 .

78 . Marantos , C. , V. Mukaro , J. Ferrante , C. Hii , and A. Ferrante . 2008 . Inhibition of the lipopolysaccharide-induced stimulation of the members of the MAPK family in human monocytes/macro-phages by 4-hydroxynonenal, a product of oxidized omega-6 fatty acids. Am. J. Pathol. 173 : 1057 – 1066 .

79 . Luckey , S. W. , M. Taylor , B. P. Sampey , R. I. Scheinman , and D. R. Petersen . 2002 . 4-hydroxynonenal decreases interleukin-6 expression and protein production in primary rat Kupffer cells by inhibiting nu-clear factor-kappaB activation. J. Pharmacol. Exp. Ther. 302 : 296 – 303 .

80 . McGrath , C. E. , K. A. Tallman , N. A. Porter , and L. J. Marnett . 2011 . Structure-activity analysis of diffusible lipid electrophiles as-sociated with phospholipid peroxidation: 4-hydroxynonenal and 4-oxononenal analogues. Chem. Res. Toxicol. 24 : 357 – 370 .

81 . Olofsson , K. E. , L. Andersson , J. Nilsson , and H. Bjorkbacka . 2008 . Nanomolar concentrations of lysophosphatidylcholine recruit monocytes and induce pro-infl ammatory cytokine production in macrophages. Biochem. Biophys. Res. Commun. 370 : 348 – 352 .

82 . Croset , M. , N. Brossard , A. Polette , and M. Lagarde . 2000 . Characterization of plasma unsaturated lysophosphatidylcholines in human and rat. Biochem. J. 345 : 61 – 67 .


ww

w.jlr.org

Dow

nloaded from

http://www.jlr.org/


A 2 and prognosis after myocardial infarction in the community. Arterioscler. Thromb. Vasc. Biol. 26 : 2517 – 2522 .

116 . Oldgren , J. , S. K. James , A. Siegbahn , and L. Wallentin . 2007 . Lipoprotein-associated phospholipase A 2 does not predict mor-tality or new ischaemic events in acute coronary syndrome pa-tients. Eur. Heart J. 28 : 699 – 704 .

117 . O’Donoghue , M. , D. A. Morrow , M. S. Sabatine , S. A. Murphy , C. H. McCabe , C. P. Cannon , and E. Braunwald . 2006 . Lipoprotein-associated phospholipase A 2 and its association with cardiovascu-lar outcomes in patients with acute coronary syndromes in the PROVE IT-TIMI 22 (PRavastatin Or atorVastatin Evaluation and Infection Therapy-Thrombolysis In Myocardial Infarction) trial. Circulation . 113 : 1745 – 1752 .

118 . Ryu , S. K. , Z. Mallat , J. Benessiano , A. Tedgui , A. G. Olsson , W. Bao , G. G. Schwartz , and S. Tsimikas . 2012 . Phospholipase A 2 en-zymes, high-dose atorvastatin, and prediction of ischemic events after acute coronary syndromes. Circulation . 125 : 757 – 766 .

119 . Heart Protection Study Collaborative Group . 2010 . Lipoprotein-associated phospholipase A 2 activity and mass in relation to vascular disease and nonvascular mortality. J. Intern. Med. 268 : 348 – 358 .

120 . Rosenson , R. S. 2010 . Physicochemically modifi ed apolipoprotein B-containing lipoproteins and the risk of cardiovascular disease. J. Intern. Med. 268 : 316 – 319 .

121 . Häkkinen , T. , J. S. Luoma , M. O. Hiltunen , C. H. Macphee , K. J. Milliner , L. Patel , S. Q. Rice , D. G. Tew , K. Karkola , and S. Yla-Herttuala . 1999 . Lipoprotein-associated phospholipase A 2 , plate-let-activating factor acetylhydrolase, is expressed by macrophages in human and rabbit atherosclerotic lesions. Arterioscler. Thromb. Vasc. Biol. 19 : 2909 – 2917 .

122 . Kolodgie , F. D. , A. P. Burke , K. S. Skorija , E. Ladich , R. Kutys , A. T. Makuria , and R. Virmani . 2006 . Lipoprotein-associated phospholipase A 2 protein expression in the natural progression of human coronary atherosclerosis. Arterioscler. Thromb. Vasc. Biol. 26 : 2523 – 2529 .

123 . Sarlon-Bartoli , G. , A. Boudes , C. Buffat , M. A. Bartoli , M. D. Piercecchi-Marti , E. Sarlon , L. Arnaud , Y. Bennis , B. Thevenin , C. Squarcioni , et al . 2012 . Circulating lipoprotein-associated phospholipase A 2 in high-grade carotid stenosis: a new biomarker for predicting unstable plaque. Eur. J. Vasc. Endovasc. Surg. 43 : 154 – 159 .

124 . Elkind , M. S. , W. Tai , K. Coates , M. C. Paik , and R. L. Sacco . 2009 . Lipoprotein-associated phospholipase A 2 activity and risk of re-current stroke. Cerebrovasc. Dis. 27 : 42 – 50 .

125 . Dohi , T. , K. Miyauchi , S. Okazaki , T. Yokoyama , R. Ohkawa , K. Nakamura , N. Yanagisawa , S. Tsuboi , M. Ogita , K. Yokoyama , et al . 2011 . Decreased circulating lipoprotein-associated phospholipase A 2 levels are associated with coronary plaque regression in patients with acute coronary syndrome. Atherosclerosis . 219 : 907 – 912 .

126 . Eyster , K. M. , S. E. Appt , C. J. Mark-Kappeler , A. Chalpe , T. C. Register , and T. B. Clarkson . 2011 . Gene expression signatures differ with extent of atherosclerosis in monkey iliac artery. Menopause . 18 : 1087 – 1095 .

127 . Zelko , I. N. , T. J. Mariani , and R. J. Folz . 2002 . Superoxide dis-mutase multigene family: a comparison of the CuZn-SOD (SOD1), Mn-SOD (SOD2), and EC-SOD (SOD3) gene structures, evolution, and expression. Free Radic. Biol. Med. 33 : 337 – 349 .

128 . Bonomini , F. , S. Tengattini , A. Fabiano , R. Bianchi , and R. Rezzani . 2008 . Atherosclerosis and oxidative stress. Histol. Histopathol. 23 : 381 – 390 .

129 . Rosenson , R. S. 2010 . Lp-PLA 2 and risk of atherosclerotic vascular disease. Lancet . 375 : 1498 – 1500 .

130 . Steinberg , D. I. , and A. Mayer . 2007 . Lipoprotein-associated phos-pholipase A 2 and risk stratifi cation for cardiovascular disease: not ready for “prime time”. Mayo Clin. Proc. 82 : 155 – 157 .

131 . Shah , S. H. , and J. A. de Lemos . 2009 . Biomarkers and cardiovas-cular disease: determining causality and quantifying contribution to risk assessment. JAMA . 302 : 92 – 93 .

132 . Stafforini , D. M. , K. Satoh , D. L. Atkinson , L. W. Tjoelker , C. Eberhardt , H. Yoshida , T. Imaizumi , S. Takamatsu , G. A. Zimmerman , T. M. McIntyre , et al . 1996 . Platelet-activating factor acetylhydrolase defi -ciency. A missense mutation near the active site of an anti-infl amma-tory phospholipase. J. Clin. Invest. 97 : 2784 – 2791 .

133 . Miwa , M. , T. Miyake , T. Yamanaka , J. Sugatani , Y. Suzuki , S. Sakata , Y. Araki , and M. Matsumoto . 1988 . Characterization of se-rum platelet-activating factor (PAF) acetylhydrolase. Correlation between defi ciency of serum PAF acetylhydrolase and respiratory symptoms in asthmatic children. J. Clin. Invest. 82 : 1983 – 1991 .

coronary heart disease and ischemic stroke: the Rotterdam Study. Circulation . 111 : 570 – 575 .

101 . Daniels , L. B. , G. A. Laughlin , M. J. Sarno , R. Bettencourt , R. L. Wolfert , and E. Barrett-Connor . 2008 . Lipoprotein-associated phospholipase A 2 is an independent predictor of incident coro-nary heart disease in an apparently healthy older population: the Rancho Bernardo Study. J. Am. Coll. Cardiol. 51 : 913 – 919 .

102 . Kiechl , S. , J. Willeit , M. Mayr , B. Viehweider , M. Oberhollenzer , F. Kronenberg , C. J. Wiedermann , S. Oberthaler , Q. Xu , J. L. Witztum , et al . 2007 . Oxidized phospholipids, lipoprotein(a), lipoprotein-associated phospholipase A 2 activity, and 10-year car-diovascular outcomes: prospective results from the Bruneck study. Arterioscler. Thromb. Vasc. Biol. 27 : 1788 – 1795 .

103 . Blake , G. J. , N. Dada , J. C. Fox , J. E. Manson , and P. M. Ridker . 2001 . A prospective evaluation of lipoprotein-associated phos-pholipase A 2 levels and the risk of future cardiovascular events in women. J. Am. Coll. Cardiol. 38 : 1302 – 1306 .

104 . Ballantyne , C. M. , R. C. Hoogeveen , H. Bang , J. Coresh , A. R. Folsom , G. Heiss , and A. R. Sharrett . 2004 . Lipoprotein-associated phospholipase A 2 , high-sensitivity C-reactive protein, and risk for incident coronary heart disease in middle-aged men and women in the Atherosclerosis Risk in Communities (ARIC) study. Circulation . 109 : 837 – 842 .

105 . Rana , J. S. , B. J. Arsenault , J. P. Despres , M. Cote , P. J. Talmud , E. Ninio , J. Wouter Jukema , N. J. Wareham , J. J. Kastelein , K. T. Khaw , et al . 2011 . Infl ammatory biomarkers, physical activity, waist circumference, and risk of future coronary heart disease in healthy men and women. Eur. Heart J. 32 : 336 – 344 .

106 . Melander , O. , C. Newton-Cheh , P. Almgren , B. Hedblad , G. Berglund , G. Engstrom , M. Persson , J. G. Smith , M. Magnusson , A. Christensson , et al . 2009 . Novel and conventional biomarkers for prediction of incident cardiovascular events in the commu-nity. JAMA . 302 : 49 – 57 .

107 . Tsimikas , S. , Z. Mallat , P. J. Talmud , J. J. Kastelein , N. J. Wareham , M. S. Sandhu , E. R. Miller , J. Benessiano , A. Tedgui , J. L. Witztum , et al . 2010 . Oxidation-specifi c biomarkers, lipoprotein(a), and risk of fatal and nonfatal coronary events. J. Am. Coll. Cardiol. 56 : 946 – 955 .

108 . Hatoum , I. J. , N. R. Cook , J. J. Nelson , K. M. Rexrode , and E. B. Rimm . 2011 . Lipoprotein-associated phospholipase A 2 activity improves risk discrimination of incident coronary heart disease among women. Am. Heart J. 161 : 516 – 522 .

109 . Koenig , W. , D. Twardella , H. Brenner , and D. Rothenbacher . 2006 . Lipoprotein-associated phospholipase A 2 predicts future cardiovascular events in patients with coronary heart disease in-dependently of traditional risk factors, markers of infl ammation, renal function, and hemodynamic stress. Arterioscler. Thromb. Vasc. Biol. 26 : 1586 – 1593 .

110 . Brilakis , E. S. , J. P. McConnell , R. J. Lennon , A. A. Elesber , J. G. Meyer , and P. B. Berger . 2005 . Association of lipoprotein-associated phospholipase A 2 levels with coronary artery disease risk factors, angiographic coronary artery disease, and major adverse events at follow-up. Eur. Heart J. 26 : 137 – 144 .

111 . Sabatine , M. S. , D. A. Morrow , M. O’Donoghue , K. A. Jablonksi , M. M. Rice , S. Solomon , Y. Rosenberg , M. J. Domanski , and J. Hsia . 2007 . Prognostic utility of lipoprotein-associated phospholi-pase A 2 for cardiovascular outcomes in patients with stable coro-nary artery disease. Arterioscler. Thromb. Vasc. Biol. 27 : 2463 – 2469 .

112 . May , H. T. , B. D. Horne , J. L. Anderson , R. L. Wolfert , J. B. Muhlestein , D. G. Renlund , J. L. Clarke , M. J. Kolek , T. L. Bair , R. R. Pearson , et al . 2006 . Lipoprotein-associated phospholipase A 2 independently predicts the angiographic diagnosis of coronary artery disease and coronary death. Am. Heart J. 152 : 997 – 1003 .

113 . Winkler , K. , M. M. Hoffmann , B. R. Winkelmann , I. Friedrich , G. Schafer , U. Seelhorst , B. Wellnitz , H. Wieland , B. O. Boehm , and W. Marz . 2007 . Lipoprotein-associated phospholipase A 2 pre-dicts 5-year cardiac mortality independently of established risk factors and adds prognostic information in patients with low and medium high-sensitivity C-reactive protein (the Ludwigshafen risk and cardiovascular health study). Clin. Chem. 53 : 1440 – 1447 .

114 . Möckel , M. , R. Muller , J. O. Vollert , C. Muller , O. Danne , R. Gareis , T. Stork , R. Dietz , and W. Koenig . 2007 . Lipoprotein-associated phospholipase A 2 for early risk stratifi cation in patients with sus-pected acute coronary syndrome: a multi-marker approach: the North Wuerttemberg and Berlin Infarction Study-II (NOBIS-II). Clin. Res. Cardiol. 96 : 604 – 612 .

115 . Gerber , Y. , J. P. McConnell , A. S. Jaffe , S. A. Weston , J. M. Killian , and V. L. Roger . 2006 . Lipoprotein-associated phospholipase


ww

w.jlr.org

Dow

nloaded from

http://www.jlr.org/


of gene polymorphisms with coronary artery disease in individuals with or without nonfamilial hypercholesterolemia. Atherosclerosis . 172 : 167 – 173 .

152 . Wang , T. , K. Karino , M. Yamasaki , Y. Zhang , J. Masuda , S. Yamaguchi , K. Shiwaku , and T. Nabika . 2009 . Effects of G994T in the Lp-PLA 2 gene on the plasma oxidized LDL level and ca-rotid intima-media thickness in Japanese: the Shimane study. Am. J. Hypertens. 22 : 742 – 747 .

153 . Li , L. , L. Qi , N. Lv , Q. Gao , Y. Cheng , Y. Wei , J. Ye , X. Yan , and A. Dang . 2011 . Association between lipoprotein-associated phospho-lipase A 2 gene polymorphism and coronary artery disease in the Chinese Han population. Ann. Hum. Genet. 75 : 605 – 611 .

154 . Jang , Y. , D. Waterworth , J. E. Lee , K. Song , S. Kim , H. S. Kim , K. W. Park , H. J. Cho , I. Y. Oh , J. E. Park , et al . 2011 . Carriage of the V279F null allele within the gene encoding Lp-PLA 2 is protective from coronary artery disease in South Korean males. PLoS ONE . 6 : e18208 .

155 . Paik , J. K. , J. S. Chae , Y. Jang , J. Y. Kim , O. Y. Kim , T. S. Jeong , S. H. Lee , and J. H. Lee . 2010 . Effects of V279F in the Lp-PLA 2 gene on markers of oxidative stress and infl ammation in Koreans. Clin. Chim. Acta . 411 : 486 – 493 .

156 . Casas , J. P. , E. Ninio , A. Panayiotou , J. Palmen , J. A. Cooper , S. L. Ricketts , R. Sofat , A. N. Nicolaides , J. P. Corsetti , F. G. Fowkes , et al . 2010 . PLA2G7 genotype, lipoprotein-associated phos-pholipase A 2 activity, and coronary heart disease risk in 10 494 cases and 15 624 controls of European Ancestry. Circulation . 121 : 2284 – 2293 .

157 . Kruse , S. , X. Q. Mao , A. Heinzmann , S. Blattmann , M. H. Roberts , S. Braun , P. S. Gao , J. Forster , J. Kuehr , J. M. Hopkin , et al . 2000 . The Ile198Thr and Ala379Val variants of plasmatic PAF-acetylhydrolase impair catalytical activities and are associated with atopy and asthma. Am. J. Hum. Genet. 66 : 1522 – 1530 .

158 . Abuzeid , A. M. , E. Hawe , S. E. Humphries , and P. J. Talmud . 2003 . Association between the Ala379Val variant of the lipo-protein associated phospholipase A 2 and risk of myocardial infarction in the north and south of Europe. Atherosclerosis . 168 : 283 – 288 .

159 . Macphee , C. H. , J. Nelson , and A. Zalewski . 2006 . Role of lipopro-tein-associated phospholipase A 2 in atherosclerosis and its poten-tial as a therapeutic target. Curr. Opin. Pharmacol. 6 : 154 – 161 .

160 . Zalewski , A. , C. Macphee , and J. J. Nelson . 2005 . Lipoprotein-associated phospholipase A 2 : a potential therapeutic target for atherosclerosis. Curr. Drug Targets Cardiovasc. Haematol. Disord. 5 : 527 – 532 .

161 . Blackie , J. A. , J. C. Bloomer , M. J. Brown , H. Y. Cheng , B. Hammond , D. M. Hickey , R. J. Ife , C. A. Leach , V. A. Lewis , C. H. Macphee , et al . 2003 . The identifi cation of clinical candidate SB-480848: a potent inhibitor of lipoprotein-associated phospho-lipase A 2 . Bioorg. Med. Chem. Lett. 13 : 1067 – 1070 .

162 . White , H. , C. Held , R. Stewart , D. Watson , R. Harrington , A. Budaj , P. G. Steg , C. P. Cannon , S. Krug-Gourley , J. Wittes , et al . 2010 . Study design and rationale for the clinical outcomes of the STABILITY Trial (STabilization of Atherosclerotic plaque By Initiation of dara-pLadIb TherapY) comparing darapladib versus placebo in patients with coronary heart disease. Am. Heart J. 160 : 655 – 661 .

163 . O’Donoghue , M. L. , E. Braunwald , H. D. White , P. Serruys , P. G. Steg , J. Hochman , A. P. Maggioni , C. Bode , D. Weaver , J. L. Johnson , et al . 2011 . Study design and rationale for the Stabilization Of pLaques usIng Darapladib-Thrombolysis In Myocardial Infarction (SOLID-TIMI 52) trial in patients after an acute coronary syndrome. Am. Heart J. 162: 613–619.e1.

164 . Corson , M. A. 2010 . Darapladib: an emerging therapy for athero-sclerosis. Ther. Adv. Cardiovasc. Dis. 4 : 241 – 248 .

165 . Wilensky , R. L. , Y. Shi , E. R. Mohler 3rd , D. Hamamdzic , M. E. Burgert , J. Li , A. Postle , R. S. Fenning , J. G. Bollinger , B. E. Hoffman , et al . 2008 . Inhibition of lipoprotein-associated phos-pholipase A 2 reduces complex coronary atherosclerotic plaque development. Nat. Med. 14 : 1059 – 1066 .

166 . Rosenson , R. S. , M. Vracar-Grabar , and I. Helenowski . 2008 . Lipoprotein associated phospholipase A 2 inhibition reduces gen-eration of oxidized fatty acids: Lp-PLA 2 reduces oxidized fatty acids. Cardiovasc. Drugs Ther. 22 : 55 – 58 .

167 . Hu , M. M. , J. Zhang , W. Y. Wang , W. Y. Wu , Y. L. Ma , W. H. Chen , and Y. P. Wang . 2011 . The inhibition of lipoprotein-associated phospholipase A 2 exerts benefi cial effects against atherosclerosis in LDLR-defi cient mice. Acta Pharmacol. Sin. 32 : 1253 – 1258 .

134 . Yamada , Y. , H. Yoshida , S. Ichihara , T. Imaizumi , K. Satoh , and M. Yokota . 2000 . Correlations between plasma platelet-activating factor acetylhydrolase (PAF-AH) activity and PAF-AH genotype, age, and atherosclerosis in a Japanese population. Atherosclerosis . 150 : 209 – 216 .

135 . Stafforini , D. M. , T. Numao , A. Tsodikov , D. Vaitkus , T. Fukuda , N. Watanabe , N. Fueki , T. M. McIntyre , G. A. Zimmerman , S. Makino , et al . 1999 . Defi ciency of platelet-activating factor acetylhydrolase is a severity factor for asthma. J. Clin. Invest. 103 : 989 – 997 .

136 . Zhang , S. Y. , H. Shibata , K. Karino , B. Y. Wang , S. Kobayashi , J. Masuda , and T. Nabika . 2007 . Comprehensive evaluation of ge-netic and environmental factors infl uencing the plasma lipopro-tein-associated phospholipase A 2 activity in a Japanese population. Hypertens. Res. 30 : 403 – 409 .

137 . Jang , Y. , O. Y. Kim , S. J. Koh , J. S. Chae , Y. G. Ko , J. Y. Kim , H. Cho , T. S. Jeong , W. S. Lee , J. M. Ordovas , et al . 2006 . The Val279Phe variant of the lipoprotein-associated phospholipase A 2 gene is associated with catalytic activities and cardiovascular disease in Korean men. J. Clin. Endocrinol. Metab. 91 : 3521 – 3527 .

138 . Liu , P. Y. , Y. H. Li , H. L. Wu , T. H. Chao , L. M. Tsai , L. J. Lin , G. Y. Shi , and J. H. Chen . 2006 . Platelet-activating factor-acetylhy-drolase A379V (exon 11) gene polymorphism is an independent and functional risk factor for premature myocardial infarction. J. Thromb. Haemost. 4 : 1023 – 1028 .

139 . Zheng , G. H. , H. Y. Chen , S. Q. Xiong , and J. F. Chu . 2011 . Lipoprotein-associated phospholipase A 2 gene V279F polymor-phisms and coronary heart disease: a meta-analysis. Mol. Biol. Rep. 38 : 4089 – 4099 .

140 . Hou , L. , S. Chen , H. Yu , X. Lu , J. Chen , L. Wang , J. Huang , Z. Fan , and D. Gu . 2009 . Associations of PLA2G7 gene polymorphisms with plasma lipoprotein-associated phospholipase A 2 activity and coronary heart disease in a Chinese Han population: the Beijing atherosclerosis study. Hum. Genet. 125 : 11 – 20 .

141 . Balta , G. , A. Gurgey , D. K. Kudayarov , B. Tunc , and C. Altay . 2001 . Evidence for the existence of the PAF acetylhydrolase mutation (Val279Phe) in non-Japanese populations: a preliminary study in Turkey, Azerbaijan, and Kyrgyzstan. Thromb. Res. 101 : 231 – 234 .

142 . Stafforini , D. M. 2009 . Functional consequences of mutations and polymorphisms in the coding region of the PAF acetylhydrolase (PAF-AH) gene. Pharmaceuticals . 2 : 94 – 117 .

143 . Song , K. , M. R. Nelson , J. Aponte , E. S. Manas , S. A. Bacanu , X. Yuan , X. Kong , L. Cardon , V. E. Mooser , J. C. Whittaker , et al . Sequencing of Lp-PLA 2 -encoding PLA2G7 gene in 2000 Europeans reveals several rare loss-of-function mutations. Pharmacogenomics J. Epub ahead of print. May 24, 2011; doi:10.1038/tpj.2011.20.

144 . Yamada , Y. , and M. Yokota . 1997 . Loss of activity of plasma plate-let-activating factor acetylhydrolase due to a novel Gln281 → Arg mutation. Biochem. Biophys. Res. Commun. 236 : 772 – 775 .

145 . Ishihara , M. , T. Iwasaki , M. Nagano , J. Ishii , M. Takano , T. Kujiraoka , M. Tsuji , H. Hattori , and M. Emi . 2004 . Functional impairment of two novel mutations detected in lipoprotein-asso-ciated phospholipase A 2 (Lp-PLA 2 ) defi ciency patients. J. Hum. Genet. 49 : 302 – 307 .

146 . Kujiraoka , T. , T. Iwasaki , M. Ishihara , M. Ito , M. Nagano , A. Kawaguchi , S. Takahashi , J. Ishi , M. Tsuji , T. Egashira , et al . 2003 . Altered distribution of plasma PAF-AH between HDLs and other lipoproteins in hyperlipidemia and diabetes mellitus. J. Lipid Res. 44 : 2006 – 2014 .

147 . Yamada , Y. , S. Ichihara , T. Fujimura , and M. Yokota . 1998 . Identifi cation of the G994 → T missense in exon 9 of the plasma platelet-activating factor acetylhydrolase gene as an independent risk factor for coronary artery disease in Japanese men. Metabolism . 47 : 177 – 181 .

148 . Yamada , Y. , H. Izawa , S. Ichihara , F. Takatsu , H. Ishihara , H. Hirayama , T. Sone , M. Tanaka , and M. Yokota . 2002 . Prediction of the risk of myocardial infarction from polymorphisms in candi-date genes. N. Engl. J. Med. 347 : 1916 – 1923 .

149 . Hiramoto , M. , H. Yoshida , T. Imaizumi , N. Yoshimizu , and K. Satoh . 1997 . A mutation in plasma platelet-activating factor acetyl-hydrolase (Val279 → Phe) is a genetic risk factor for stroke. Stroke . 28 : 2417 – 2420 .

150 . Unno , N. , T. Nakamura , H. Kaneko , T. Uchiyama , N. Yamamoto , J. Sugatani , M. Miwa , and S. Nakamura . 2000 . Plasma platelet-activating factor acetylhydrolase defi ciency is associated with ath-erosclerotic occlusive disease in Japan. J. Vasc. Surg. 32 : 263 – 267 .

151 . Shimokata , K. , Y. Yamada , T. Kondo , S. Ichihara , H. Izawa , K. Nagata , T. Murohara , M. Ohno , and M. Yokota . 2004 . Association


ww

w.jlr.org

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nloaded from

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168 . Wang , W. Y. , J. Zhang , W. Y. Wu , J. Li , Y. L. Ma , W. H. Chen , H. Yan , K. Wang , W. W. Xu , J. H. Shen , et al . 2011 . Inhibition of lipoprotein-associated phospholipase A 2 ameliorates infl ammation and decreases atherosclerotic plaque formation in ApoE-defi cient mice. PLoS ONE . 6 : e23425 .

169 . Serruys , P. W. , H. M. Garcia-Garcia , P. Buszman , P. Erne , S. Verheye , M. Aschermann , H. Duckers , O. Bleie , D. Dudek , H. E. Botker , et al . 2008 . Effects of the direct lipoprotein-associated phospholipase A 2 inhibitor darapladib on human coronary ath-erosclerotic plaque. Circulation . 118 : 1172 – 1182 .

170 . Carpenter , K. L. , I. F. Dennis , I. R. Challis , D. P. Osborn , C. H. Macphee , D. S. Leake , M. J. Arends , and M. J. Mitchinson . 2001 . Inhibition of lipoprotein-associated phospholipase A 2 diminishes the death-inducing effects of oxidised LDL on human monocyte-macrophages. FEBS Lett. 505 : 357 – 363 .

171 . Forte , T. M. , G. Subbanagounder , J. A. Berliner , P. J. Blanche , A. O. Clermont , Z. Jia , M. N. Oda , R. M. Krauss , and J. K. Bielicki . 2002 . Altered activities of anti-atherogenic enzymes LCAT, paraoxonase, and platelet-activating factor acetylhydrolase in atherosclerosis-susceptible mice. J. Lipid Res. 43 : 477 – 485 .

172 . Morgan , E. N. , E. M. Boyle , Jr ., W. Yun , J. C. Kovacich , T. G. Canty , Jr ., E. Chi , T. H. Pohlman , and E. D. Verrier . 1999 . Platelet-activating factor acetylhydrolase prevents myocardial ischemia-reperfusion injury. Circulation . 100 : II365 – II368 .

173 . Turunen , P. , H. Puhakka , J. Rutanen , M. O. Hiltunen , T. Heikura , M. Gruchala , and S. Yla-Herttuala . 2005 . Intravascular adenovirus-mediated lipoprotein-associated phospholipase A 2 gene transfer reduces neointima formation in balloon-denuded rabbit aorta. Atherosclerosis . 179 : 27 – 33 .

174 . Quarck , R. , B. De Geest , D. Stengel , A. Mertens , M. Lox , G. Theilmeier , C. Michiels , M. Raes , H. Bult , D. Collen , et al . 2001 . Adenovirus-mediated gene transfer of human platelet-activating factor-acetylhydrolase prevents injury-induced neointima forma-tion and reduces spontaneous atherosclerosis in apolipoprotein E-defi cient mice. Circulation . 103 : 2495 – 2500 .

175 . Theilmeier , G. , B. De Geest , P. P. Van Veldhoven , D. Stengel , C. Michiels , M. Lox , M. Landeloos , M. J. Chapman , E. Ninio , D. Collen , et al . 2000 . HDL-associated PAF-AH reduces endothelial adhesiveness in apoE � / � mice. FASEB J. 14 : 2032 – 2039 .

176 . Arakawa , H. , J. Y. Qian , D. Baatar , K. Karasawa , Y. Asada , Y. Sasaguri , E. R. Miller , J. L. Witztum , and H. Ueno . 2005 . Local expression of platelet-activating factor-acetylhydrolase reduces accumulation of oxidized lipoproteins and inhibits infl amma-tion, shear stress-induced thrombosis, and neointima formation in balloon-injured carotid arteries in nonhyperlipidemic rabbits. Circulation . 111 : 3302 – 3309 .

177 . Lu , J. , M. Pierce , A. Franklin , T. Jilling , D. M. Stafforini , and M. Caplan . 2010 . Dual roles of endogenous platelet-activating factor acetylhydrolase in a murine model of necrotizing enterocolitis. Pediatr. Res. 68 : 225 – 230 .

178 . Mohler 3rd , E. R. , C. M. Ballantyne , M. H. Davidson , M. Hanefeld , L. M. Ruilope , J. L. Johnson , and A. Zalewski . 2008 . The effect of darapladib on plasma lipoprotein-associated phospholipase A 2 activity and cardiovascular biomarkers in patients with stable coronary heart disease or coronary heart disease risk equivalent: the results of a multicenter, randomized, double-blind, placebo-controlled study. J. Am. Coll. Cardiol. 51 : 1632 – 1641 .

179 . Karasawa , K. 2006 . Clinical aspects of plasma platelet-activating factor-acetylhydrolase. Biochim. Biophys. Acta . 1761 : 1359 – 1372 .

180 . Vadas , P. , M. Gold , B. Perelman , G. M. Liss , G. Lack , T. Blyth , F. E. Simons , K. J. Simons , D. Cass , and J. Yeung . 2008 . Platelet-activating factor, PAF acetylhydrolase, and severe anaphylaxis. N. Engl. J. Med. 358 : 28 – 35 .

181 . Winkler , K. , M. M. Hoffmann , V. Krane , C. Drechsler , and C. Wanner . 2012 . Lipoprotein-associated phospholipase A 2 and out-come in patients with type 2 diabetes on haemodialysis. Eur. J. Clin. Invest. 42: 693–701.

182 . Topol , E. J. 2005 . Nesiritide - not verifi ed. N. Engl. J. Med. 353 : 113 – 116 .

183 . Hase , M. , M. Tanaka , M. Yokota , and Y. Yamada . 2002 . Reduction in the extent of atherosclerosis in apolipoprotein E-defi cient mice induced by electroporation-mediated transfer of the human plasma platelet-activating factor acetylhydrolase gene into skeletal muscle. Prostaglandins Other Lipid Mediat. 70 : 107 – 118 .

184 . Shaddinger , B. C. , L. Gao , G. Cai , L. M. Wentzell , and A. G. Johnson . 2005 . A study to evaluate the effect of repeat oral doses of SB-480848 on platelet function as compared to placebo in healthy subjects. Clin. Pharmacol. Ther. 79 : 36 .


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