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Journal of the American College of Cardiology Vol. 59, No. 8, 2012 2Pub

DownloadeObjectives This study sought to examine the role of lipoprotein-associated phospholipase A2 (Lp-PLA2/PLA2G7) in humaninflammation and coronary atherosclerosis.

Background Lp-PLA2 has emerged as a potential therapeutic target in coronary heart disease. Data supporting Lp-PLA2 areindirect and confounded by species differences; whether Lp-PLA2 is causal in coronary heart disease remains inquestion.

Methods We examined inflammatory regulation of Lp-PLA2 during experimental endotoxemia in humans, probed thesource of Lp-PLA2 in human leukocytes under inflammatory conditions, and assessed the relationship of varia-tion in PLA2G7, the gene encoding Lp-PLA2, with coronary artery calcification.

Results In contrast to circulating tumor necrosis factor-alpha and C-reactive protein, blood and monocyte Lp-PLA2 mes-senger ribonucleic acid decreased transiently, and plasma Lp-PLA2 mass declined modestly during endotoxemia.In vitro, Lp-PLA2 expression increased dramatically during human monocyte to macrophage differentiation andfurther in inflammatory macrophages and foamlike cells. Despite only a marginal association of single nucleo-tide polymorphisms in PLA2G7 with Lp-PLA2 activity or mass, numerous PLA2G7 single nucleotide polymor-phisms were associated with coronary artery calcification. In contrast, several single nucleotide polymorphismsin CRP were significantly associated with plasma C-reactive protein levels but had no relation with coronary ar-tery calcification.

Conclusions Circulating Lp-PLA2 did not increase during acute phase response in humans, whereas inflammatory macro-phages and foam cells, but not circulating monocytes, are major leukocyte sources of Lp-PLA2. Common geneticvariation in PLA2G7 is associated with subclinical coronary atherosclerosis. These data link Lp-PLA2 to athero-sclerosis in humans while highlighting the challenge in using circulating Lp-PLA2 as a biomarker of Lp-PLA2actions in the vasculature. (J Am Coll Cardiol 2012;59:76472) 2012 by the American College of CardiologyFoundation

poprotein-associated phospholipase A2 (Lp-PLA2) haserged as a potential therapeutic target in coronary heartease (CHD) and phase III clinical trials are underway.pporting evidence includes apparent atherogenic bio-emical properties; Lp-PLA2 cleaves oxidized phosphati-lcholine on modified low-density lipoprotein (LDL),

producing inflammatory lysophosphatidylcholine and oxi-dized nonesterified fatty acids (1,2). In addition, enzymaticexpression of Lp-PLA2 is up-regulated in human athero-sclerosis (3), whereas circulating levels are associated withincident CHD (4). Promising proof-of-principle pre-clinical and clinical trials have been carried out (57).

m the *Cardiovascular Institute, Department of Medicine, University of Pennsylvaniaool of Medicine, Philadelphia, Pennsylvania; Institute of Diabetes, Obesity, andtabolism, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania;stitute for Translational Medicine and Therapeutics, University of Pennsylvaniaool of Medicine, Philadelphia, Pennsylvania; Center for Clinical Epidemiology andstatistics, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania;partment of Pathology and Laboratory Medicine, University of Pennsylvania SchoolMedicine, Philadelphia, Pennsylvania; and the Center for Applied Genomics,

and Endocrine Research Center (P20-DK 019525) award (both from the NationalInstitute of Health to the University of Pennsylvania), and P50 HL-083799-SCCORProject award from the National Institute of Health (to Dr. Reilly). GlaxoSmithKlineprovided research grant support for measurement of Lp-PLA2 mass and activity. Drs.Rader and Reilly received research grant support from GlaxoSmithKline. Employeesof GlaxoSmithKline did not contribute to study design, data interpretation, andediting of the report. All other authors have reported that they have no relationshipsrelevant to the contents of this paper to disclose.Translational Studies of LipopPhospholipase A2 in Inflammati

Jane F. Ferguson, PHD,* Christine C. Hinkle, MS,Stephanie L. DerOhannessian, MS,* Rhia Shah, BSJonathan P. Bradfield, BS, Hakon Hakonarson, PStephen R. Master, MD, Daniel J. Rader, MD,*

012 by the American College of Cardiology Foundationlished by Elsevier Inc.ldrens Hospital of Philadelphia, Philadelphia, Pennsylvania. This work was sup-ted by a Clinical and Translational Science Award (UL1RR024134), a Diabetes

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d From: https://content.onlinejacc.org/ on 03/15/2015ein-Associatedand Atherosclerosis

hal N. Mehta, MD,* Roshanak Bagheri, MD,*egan I. Mucksavage, MS,*

Xuexia Wang, PHD,ingyao Li, PHD, Muredach P. Reilly, MB*

ISSN 0735-1097/$36.00doi:10.1016/j.jacc.2011.11.019anuscript received May 13, 2011; revised manuscript received October 31, 2011,pted November 16, 2011.

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Downloadewever, whether Lp-PLA2 is causal and whether itsibition will prevent CHD events remain undetermined.Data for Lp-PLA2 in human atherosclerosis remainirect and confounded by species differences in physiologyd actions. Lp-PLA2 circulates in blood bound to lipopro-ns that modulate its actions. In rodents, Lp-PLA2 isried mostly on high-density lipoprotein particles,ereas in humans, the enzyme is bound to LDL particles.us, confounding may be particularly marked for plasma-PLA2 relative to other inflammatory markers, as regu-ion of atherogenic lipoproteins is a major influence onculating Lp-PLA2 levels and activity (8). Indeed, whetherulating Lp-PLA2 is associated with CHD beyond a completeessment of atherogenic lipoproteins remains uncertain (9).Arterial Lp-PLA2 biosynthesis by macrophages and foamls, rather than circulating levels or activity, may deter-ne its atherogenicity (10). Lp-PLA2 expression withinnecrotic core and surrounding macrophages of vulner-

le and ruptured plaques, compared with less-advancedions, is increased (11), suggesting a potential role inmoting plaque instability. The extent to which human-PLA2 is regulated in circulation by systemic inflamma-n, however, versus locally controlled in arterialcrophage-foam cells is uncertain. Further, lesion biosyn-sis is difficult to measure in humans, limiting our abilitymonitor Lp-PLA2 activity in disease-relevant tissue andassess vascular efficacy of pharmacological inhibition.In this report, we examined inflammatory regulation ofculating Lp-PLA2 during experimental endotoxemia inmans, probed the source of Lp-PLA2 in human leuko-es under inflammatory conditions, and determined theationship of genetic variation in phospholipase A2, groupI (PLA2G7), the gene encoding Lp-PLA2, to coronaryery calcification (CAC) as well as plasma levels of-PLA2 mass and activity. We found that, unlike bloodor necrosis factor (TNF)-alpha and C-reactive protein

RP), circulating Lp-PLA2 did not increase during thete phase response in humans; that inflammatory macro-ages and foam cells, but not circulating or ex vivonocytes, are primary leukocyte sources of Lp-PLA2; andt common genetic variation in PLA2G7 is associatedth subclinical coronary atherosclerosis. These data link-PLA2 to atherosclerosis in humans while providing aman physiological context for the difficulty in usingculating Lp-PLA2 as a biomarker of disease or pharma-logical efficacy in atherosclerosis.

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inical studies. HUMAN ENDOTOXEMIA. Healthy volun-rs on no medications and no significant medical history 32, 50% women; mean age 25.7 3.90 years) weredied as described previously (12,13) and in the Online

ruary 21, 2012:76472pendix. Serial blood samples were collected before ander intravenous bolus infusion of 3 ng/kg U.S. standard

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d From: https://content.onlinejacc.org/ on 03/15/2015erence endotoxin and wereepared for plasma, whole-od ribonucleic acid (RNA)d monocyte RNA (12).

NETIC ASSOCIATION STUDIES.

e PennCAC (Penn Coronarytery Calcification) resource in-ded European-ancestry subjectsruited to 3 separate studies atUniversity of Pennsylvania: theCA (Study of Inherited Risk

Coronary Atherosclerosis) (n9), the PDHS (Penn Diabetesart Study) (n 782), and theMSyN (Philadelphia Area Met-lic Syndrome Network) (n 0). These studies are described inail previously (14,15) and in theline Appendix. In each study,jects with clinical atheroscleroticdiovascular disease were ex-ded. PLA2G7 single nucleotidelymorphisms (SNPs) were geno-ed in all 3 studies. Plasma Lp-A2 mass and activity data wereilable in SIRCA and PDHS. Global CAC scores weretermined by electron beam tomography (Imatron, San Fran-co, California) according to the method of Agatston et al.). For all human studies described, the University ofnnsylvania Institutional Review Board approved each study,d written informed consent was provided by all participants.boratory methods. HUMAN MONOCYTE, MACROPHAGE,D FOAM CELL STUDIES. Humanmonocyte isolation, macro-age (M1 and M2 phenotype) differentiation (17), and foaml preparation were performed as described elsewhere (12) andthe Online Appendix. Experiments were performed in batchesng freshly isolated monocytes, macrophages, and foam cellsived from the same human volunteer.

ASMA LP-PLA2, INFLAMMATORY, AND METABOLIC MARKERS.

sma and cell-media levels of Lp-PLA2 mass and activity,F-alpha, and CRP, as well as lipid and biochemicalrkers were measured as described elsewhere (13,14,18)d in the Online Appendix.

AL-TIME QUANTITATIVE PCR AND EXPRESSION

ANTITATIVE TRAIT LOCUS ANALYSIS. Whole-blood, iso-ed circulating monocyte, and human cultured monocyte,crophage, and foam cell messenger ribonucleic acidRNA) were subjected to quantitative polymerase chainction (PCR) using primers and probes (Applied Biosys-s 7300 Real-Time PCR System, Foster City, Califor-) as previously described (12) for measurement of Lp-A , TNF-alpha, and beta-actin mRNA (Online

Abbreviationsand Acronyms

CAC coronary arterycalcification

CHD coronary heartdisease

CRP C-reactive protein

CT cycle threshold

eQTL expressionquantitative trait locus

LDL low-densitylipoprotein

Lp-PLA2 lipoprotein-associated phospholipase A2

LPS lipopolysaccharide

mRNA messengerribonucleic acid

PCR polymerase chainreaction

RNA ribonucleic acid

SNP single nucleotidepolymorphism

TNF tumor necrosisfactor

Human Translation of Lp-PLA22

pendix). The relative quantitationd to determine fold-change from b2-(Ct) method wasaseline (19). Explor-

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Downloadery expression quantitative trait locus (eQTL) analysis isscribed in the Online Appendix.

NOTYPING. As described previously (20) and in the Onlinependix, PennCAC participants were genotyped using theMAT Broad Care CARDIOVASCULAR DISEASEdidate gene array, which surveys50,000 SNPs in2,000didate genes (21). SNP data for PLA2G7 (n 19) andP (n 16) were selected for the present analysis.atistical analysis. The effect of endotoxemia on plasma-PLA2 mass and activity, TNF-alpha, and CRP, as wellwhole blood and monocyte mRNA was tested byeated-measures analysis of variance. Analysis of variances applied also to in vitro cell data. Post hoc t tests wered to compare specific time points and treatments. Weserved heterogeneity of variance in several variables fol-ing lipopolysaccharide (LPS) challenge, which was to beected given the known variation in responses to endo-in. We tested for homogeneity of variance using Levenet, and in cases where the assumption of homogeneity ofiance was violated, we confirmed whether the groupferences were significant using Tamhane post-hoc test.In PennCAC, CAC scores were transformed by the naturalafter adding 1, [Ln(CAC 1)], to correct for skewed

tribution. This variable was used as the outcome in a linearression model, with PLA2G7 and CRP SNPs, adjusting for, sex, and agesex interaction. For linear regression analysisSNP associations with plasma proteins, Lp-PLA2 mass and

igure 1 Human Endotoxemia Does Not Induce Circulating Lp-PLA

ndotoxemia (3 ng/kg lipopolysaccharide [LPS] intravenously) markedly increased pla 0.001) but not (B) lipoprotein-associated phospholipase A2 (Lp-PLA2) mass or a

Human Translation of Lp-PLA2NF-alpha messenger ribonucleic acid (mRNA) was markedly induced but (D) Lp-PLA2 mRNANF-alpha but not of (F) Lp-PLA2.

d From: https://content.onlinejacc.org/ on 03/15/2015ivity were normally distributed and, therefore, used astcomes, whereas CRP was log-transformed. The linearression model included adjustments for age, sex, and smok-. Analysis used PLINK (version 1.06, Shaun Purcell,ston, Massachusetts). Analyses of CAC and plasma pro-ns were performed separately in each sample and thenjected to meta-analysis. Meta-analysis applied a weightedscore method using METAL (Center for Statistical Genet-, University of Michigan, Ann Arbor, Michigan) (22,23) asapplied it in (24) as described in the Online Appendix. Inalysis of SNP data, we corrected for the number of inde-ndent tests within each gene (10 tests for 19 PLA2G7 SNPs,adjusted p value threshold of 0.005, and 15 tests for 15 CRPPs, unadjusted p value threshold of 0.0033) using thethod of Nyholt (25).

sults

-PLA2 is not induced in a human model of acutease response. As we described previously (13,26), endo-emia produced an acute, febrile illness associated with arked, transient induction of plasma TNF-alpha (p 01), followed by a delayed 100-fold induction ofsma CRP at 24 h (p 0.001) (Fig. 1A). In contrast,sma Lp-PLA2 mass and activity did not increase follow-LPS (Fig. 1B). Indeed, levels of Lp-PLA2 mass tended to

cline (by 18% at 6 h, p 0.01). The mRNA response toS in whole blood for TNF-alpha (Fig. 1C) and Lp-PLA2

tein or Leukocyte Lp-PLA2 mRNA In Vivo

vels of (A) tumor necrosis factor (TNF)-alpha and C-reactive protein (CRP)which declined transiently (p 0.01). Following LPS, whole blood (C)

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Downloadeg. 1D), as well as in circulating monocytes for TNF-alphag. 1E) and Lp-PLA2 (Fig. 1F), was similar to that ofsma proteins. The mRNA levels of Lp-PLA2 in circulatingnocytes were low but detectable (baseline cycle thresholdTs] 30, varying from CTs of 28 to 32 post-LPS).-PLA2 expression is induced in inflammatory humancrophages and foam cells. Lp-PLA2 mRNA levels were(CTs 30) in freshly isolated human monocytes but

reased markedly (CTs 20) following 6 days of differenti-on to mature macrophages (p 0.0001) (Fig. 2A) andreased modestly during further polarization to M1 (p 001) but not M2 macrophages (Fig. 2B). Lp-PLA2 proteinss also was induced during differentiation to macrophages,th increases in both the cell-associated protein (p 0.0001)d the secreted protein (p 0.0004) (Fig. 2C). Followingding of human monocyte-derived macrophages with acety-ed LDL-cholesterol for 48 h, cholesterol ester (128 vs. 0.6cholesterol ester /mg protein) and total cholesterol (422 vs.6 g cholesterol/mg protein) were significantly higher inded versus unloaded cells, which is consistent with findingsin vivo foam cells (27). Lp-PLA2 mRNA levels were

nificantly greater in foam cells than in mature macrophages 0.01) (Fig. 3A). Similarly, cell-associated (p 0.05) andreted (p 0.008) Lp-PLA2 protein levels were higher inm cells than in macrophages (Fig. 3B). There was no-PLA2 protein detectable in the media or acetylated LDLd to treat cells. Overall, these data are consistent with lackin vivo increase in plasma or monocyte levels of Lp-PLA2ring the acute phase and suggest that, in human atheroscle-is, Lp-PLA2 may be generated by macrophages and foamls rather than by circulating leukocytes.Exploratory interrogation of PLA2G7 SNP eQTLs for-PLA2 mRNA expression in publicly available dataealed nominal associations of several SNPs in theA2G7 region with exon probe levels in peripheral bloodnonuclear cells (best p 0.0059, rs12181971) and brain (best0.008, rs12195701) (28), as well as skin (best p 0.021,

6874962), fat (best p 0.019, rs16874962), and lymphoblas-d cells (best p 0.037, rs7745519) (MuTHER [Multiplesue Human Expression Resource] twin2 study (29)). Thesedest associations, however, were not significant after correctionmultiple testing (see the Online Appendix). Macrophage andm cell expression datasets were not available for testing a moreerosclerosis-relevant cell type.mmon polymorphisms in PLA2G7 are associated withronary calcification but only weakly with plasma Lp-A2 mass or activity. Individually in SIRCA or PDHSples, there were no significant associations betweenA2G7 SNPs and Lp-PLA2 mass or activity. In thembined meta-analysis, only 1 SNP (rs1805017) hadminal association with Lp-PLA2 mass (p 0.02; p 0.2er Bonferroni correction) (Table 1A). As a positive control,performed similar analysis of plasma CRP using com-

ruary 21, 2012:76472n CRP SNPs. In contrast to findings for PLA2G7--PLA2, there were significant associations between nu-rous CRP SNPs and plasma CRP in the SIRCA and

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d From: https://content.onlinejacc.org/ on 03/15/2015HS samples and in the overall meta-analysis (Table 1B).

igure 2Lp-PLA2 mRNA and ProteinIncrease During Differentiation ofHuman Monocytes to Macrophages In Vitro

p-PLA2 mRNA levels increased markedly during differentiation from monocytesmature macrophages (p 0.0001) (A) and increased modestly during fur-

er polarization to M1 macrophages (p 0.001) but fell during M2 polariza-on (p 0.001) (B). Lp-PLA2 protein mass also increased significantly duringifferentiation to macrophages, with increases in both the cell-associated pro-in (p 0.0001) and the secreted protein (p 0.0004) (C). Note: As mono-

ytes were grown in suspension, protein levels were measured in monocyteell lysates but could not be measured in media. (Analysis of variance andonferroni post hoc tests). Abbreviations as in Figure 1.

Human Translation of Lp-PLA216 CRP SNPs, 9 had nominal (p 0.05) associationsth CRP levels and 8 of these SNPs had significantociations after Bonferroni correction.

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DownloadeAssociation of PLA2G7 SNPs with CAC was assessedtially in SIRCA and PAMSyN together with follow-upPDHS. Multiple PLA2G7 SNPs had nominal associa-ns with CAC in SIRCA and PAMSyN (11 with p 5; lowest p 0.0001 for rs1421378). Replication signalsPDHS were modest (strongest rs10948300, p 0.02)ely due to limited power; however, in PDHS, 16 of 19 SNPsd effects in the same direction as in SIRCA or PAMSyNi squared: 8.9, p 0.003). Meta-analysis of the combinedple found several SNP associations with CAC (rs9349373,0.002; rs2216465, p 0.002; rs12195701, p 0.004) that

re significant after Bonferroni correction (Table 2A). In-ding plasma Lp-PLA2 mass or activity in the model did notenuate the association between PLA2G7 SNPs and CAC.ese findings support recent associations of variation inA2G7 with CHD (3032).

igure 3 Lp-PLA2 mRNA and Protein Are Up-Regulatedin Human Foamlike Cells In Vitro

p-PLA2 mRNA was significantly greater (p 0.01) in foam cells than in matureacrophages (A). Similarly, cell-associated (p 0.05) and secreted (p .008) Lp-PLA2 protein levels were higher in foam cells than in macrophages). (Note: As monocytes were grown in suspension, protein levels were mea-

ured in monocyte cell lysates but could not be measured in media. (Analysisf variance and Bonferroni post hoc tests). Abbreviations as in Figure 1.

Human Translation of Lp-PLA2As an expected negative control (14,33,34), we examinedP variant associations with CAC in the same sample and

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d From: https://content.onlinejacc.org/ on 03/15/2015nd minimal signal, with 1 SNP having nominal associ-on in SIRCA and PAMSyN (rs3093068, p 0.04);wever, there were no associations in PDHS nor inmbined meta-analysis.

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e provide novel insight into the pathophysiology of Lp-A2 in humans. First, we show that unlike TNF-alpha andP, circulating Lp-PLA2 does not increase during experi-ntal endotoxemia and, therefore, does not contribute toman acute phase response. Second, we found that inflam-tory macrophages and foam cells, but not circulating mono-es or cultured primary monocytes, generate significant-PLA2. This is consistent with the concept that the majorityLp-PLA2 in atherosclerotic plaque is derived from localsynthesis by inflammatory macrophage and foam cellsher than from circulating leukocytes. Third, we found thatmon variants in PLA2G7 are associated with CAC but hadited relation to circulating Lp-PLA2 mass or activity. Thisports an atherogenic role for PLA2G7-Lp-PLA2 in hu-ns that may be independent of circulating Lp-PLA2 massactivity.-PLA2 does not contribute to human acute phaseponse. We demonstrate that Lp-PLA2 is not an acutease protein in humans. This is in contrast to rodentdels in which LPS challenge was shown to induce a rapidrease in plasma and tissue levels of Lp-PLA2 (35). Thisvides further evidence of fundamental differences be-een humans and rodents in the physiology and action of-PLA2 (36). Lack of induction in blood and circulatingnocytes by endotoxemia in vivo also suggests limited, ify, role for circulating leukocyte production of Lp-PLA2atherosclerosis. In contrast, marked in vitro up-regulationmacrophages and foam cells is consistent with a specifice for local vascular production of Lp-PLA2 in atheroscle-is. Although it is possible that local macrophage Lp-A2 production in plaque may contribute to a portion ofculating Lp-PLA2, it is unlikely to render circulatingels useful as independent biomarkers of Lp-PLA2 actionsatherosclerosis because published data show that circu-ing Lp-PLA2 mass and activity do not correlate withque Lp-PLA2 in patients undergoing elective carotiddarterectomy (37) and because there is substantial con-nding of plasma Lp-PLA2 by circulating lipoproteinsardless of tissue source. Overall, these data suggest thatels of Lp-PLA2 mRNA and protein in blood may beor surrogates of PLA2G7 actions in arterial plaque.oinflammatory macrophages and foam cells, but notnocytes, generate significant Lp-PLA2. We foundt Lp-PLA2 expression was markedly increased duringdifferentiation of monocytes to macrophages, and fur-r induced in vitro in foam celllike macrophages. This is

February 21, 2012:76472nsistent with constitutive expression and activity in in-mmatory macrophages (38) and foam cells in atheroscle-is. Indeed, Lp-PLA2 expression is increased in athero-

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Downloadeerotic lesions in humans (10). In this environment,reted Lp-PLA2 can hydrolyze oxidized phospholipidsd fatty acids on atherogenic lipoproteins, generatingctive lipid mediators thought to promote plaque in-bility. Inhibition of Lp-PLA2 suppressed oxidized-Linduced macrophage apoptosis (39), a feature ofammatory plaque. Further, in a porcine model of com-x atherosclerosis, suppression of Lp-PLA retarded ath-

k of Association Between SNPs in PLA2G7 and Circulating Lp-PLAt Significant Relation of Multiple SNPs in CRP With Circulating CRable 1 Lack of Association Between SNPs in PLA2G7 and CircuBut Significant Relation of Multiple SNPs in CRP With Ci

PLA2G7 SNPllele: Minor/Major) Type of SNP MAF

1805017 (A/G) Nonsynonymous coding 0.26

2216465 (C/G) Intronic 0.34

1421378 (G/A) Upstream 0.41

17288905 (G/A) Intronic 0.08

10948300 (T/A) Intronic 0.21

12195701 (A/G) Intronic 0.21

1051931 (A/G) Nonsynonymous coding 0.21

7756935 (C/A) Intronic 0.21

12528807 (C/A) Intronic 0.08

1362931 (A/C) Intronic 0.20

1421372 (A/G) Intronic 0.20

3799861 (A/G) Intronic 0.20

9472830 (A/G) Intronic 0.20

16874962 (A/G) Intronic 0.07

3799863 (A/T) Intronic 0.05

1421368 (G/A) Intronic 0.10

9349373 (A/G) Upstream 0.20

16874967 (A/G) Intronic 0.06

1805018 (G/A) Nonsynonymous coding 0.06

CRP SNPllele: Minor/Major)

1205 (A/G) 3 UTR 0.34

1800947 (G/C) Synonymous coding 0.07

3091244 (A/G) 5 upstream 0.37

12084589 (A/C) 5 upstream 0.07

12068753 (A/T) 5 upstream 0.07

3093059 (G/A) 5 upstream 0.07

3093068 (G/C) 3 downstream 0.06

2794521 (G/A) 5 upstream 0.25

3093066 (A/C) 3 UTR 0.001

2808634 (A/G) 5 upstream 0.27

1130864 (A/G) 3 UTR 0.30

2808631 (G/A) 3 downstream 0.002

3093070 (C/A) 3 downstream 0.02

3093069 (C/A) 3 downstream 0.001

3093071 (A/C) 3 downstream 0.01

19 SNPs in PLA2G7 fall into 10 LD blocks (r2 0.8); therefore, we used Bonferroni multiple testRP fall into 15 LD blocks (r2 0.8); therefore, we used Bonferroni multiple testing correction for 1A (Study of Inherited Risk of Coronary Atherosclerosis) and the PDHS (Penn Diabetes Heart Stud linkage disequilibrium; Lp-PLA2 lipoprotein-associated phospholipase A2; MAF minor all

ruary 21, 2012:764722

sclerosis progression and decreased plaque inflammation,crosis, and fibrous cap erosion (7). Compared with

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d From: https://content.onlinejacc.org/ on 03/15/2015cebo, short-term Lp-PLA2 inhibition in humans alsouced several markers of plaque inflammation in carotidions examined ex vivo (5,6). Overall, these data provideirect evidence for atherogenic actions of Lp-PLA2 incular lesions. Indeed, Lp-PLA2 inhibition is currentlying tested in large phase III clinical trials of CHD inh-risk patients (NCT01000727).netic variation inPLA2G7may relate toCHDindependent

tein Mass or Activityels*Lp-PLA2 Protein Mass or Activityting CRP Levels*

Lp-PLA2 Mass Lp-PLA2 Activity

Meta-Analysis(n 1,723)


Z-Score p Value Z-Score p Value

2.34 0.02 0.23 0.82

1.54 0.12 0.66 0.51

1.37 0.17 0.37 0.71

1.74 0.08 0.79 0.43

1.77 0.08 0.31 0.76

1.46 0.14 0.77 0.44

1.53 0.13 0.07 0.95

1.53 0.13 0.07 0.95

0.36 0.72 0.44 0.66

1.15 0.25 0.06 0.96

1.15 0.25 0.06 0.96

1.15 0.25 0.06 0.96

1.07 0.29 0.08 0.94

1.12 0.26 0.46 0.64

0.71 0.48 1.01 0.31

0.42 0.68 0.02 0.99

0.23 0.82 0.49 0.63

0.34 0.74 0.68 0.49

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Z-Score p Value

5.63 1.8 108

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3.23 1.0 103

3.18 1.0 103

3.14 2.0 103

2.94 3.0 103

0.29 0.77

1.99 0.05

0.39 0.69

2.94 0.003

1.49 0.14

0.82 0.41

1.37 0.17

1.23 0.22

ection for 10 effective tests, giving a p value threshold of significance of 0.005. The 15 SNPsive tests, giving a p value threshold of significance of 0.0033. *Meta-analysis of data from ther all analyses, the effect allele corresponds to the minor allele.uency; SNP single nucleotide polymorphism; UTR untranslated region.

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Downloadeivity levels with risk of CHD (9,4042). Meta-analysesport a modest CHD relationship independent of tradi-nal risk factors and plasma CRP (40,43,44). Publisheddies, however, may underestimate the degree of con-nding because of incomplete measurement and controlall atherogenic lipoproteins (9). In circulation, Lp-PLA2ociates with apolipoprotein B and high-density lipopro-ns, with the majority found on LDL particles. Because-PLA2 protein and activity are closely linked to circulat-apolipoprotein B lipoproteins (36,45), it is not surpris-that genetic factors (e.g., APOC1, PSRC1, ZNF259)t regulate plasma apolipoprotein B lipoproteins are also

sociation of SNPs in PLA2G7 But Not in CRP With CACable 2 Association of SNPs in PLA2G7 But Not in CRP With CAC

Type of SNP MAF

LA2G7 SNP (allele: minor/major)*

rs1421378 (G/A) Upstream 0.41

rs2216465 (C/G) Intronic 0.34

rs12528807 (C/A) Intronic 0.08

rs9349373 (A/G) Upstream 0.20

rs10948300 (T/A) Intronic 0.21

rs1805017 (A/G) Nonsynonymous coding 0.26

rs3799863 (A/T) Intronic 0.05

rs16874967 (A/G) Intronic 0.06

rs1805018 (G/A) Nonsynonymous coding 0.06


rs1421368 (G/A) Intronic 0.10


rs1362931 (A/C) Intronic 0.20



rs7756935 (C/A) Intronic 0.21

rs1051931 (A/G) Nonsynonymous coding 0.21


rs17288905 (G/A) Intronic 0.084

RP SNP (allele: minor/major)*

rs3093068 (G/C) 3 downstream 0.06

rs3093059 (G/A) 5 upstream 0.07

rs12068753 (A/T) 5 upstream 0.07

rs12084589 (A/C) 5 upstream 0.07

rs2808631 (G/A) 3 downstream 0.002

rs1800947 (G/C) Synonymous coding 0.07

rs2794521 (G/A) 5 upstream 0.25

rs1130864 (A/G) 3 UTR 0.30

rs2808634 (A/G) 5 upstream 0.27

rs3093071 (A/C) 3 downstream 0.01

rs3093066 (A/C) 3 UTR 0.001

rs3093069 (C/A) 3 UTR 0.001

rs1205 (A/G) 3 downstream 0.34

rs3093070 (C/A) 5 upstream 0.02

rs3091244 (A/G) 3 downstream 0.37

r all analyses, the effect allele corresponds to the minor allele.AC coronary artery calcification; PAMSyN Philadelphia Area Metabolic Syndrome Network; PDreviations as in Table 1.

Human Translation of Lp-PLA2ociated with plasma Lp-PLA2 (46). Parenthetically, wend modest association of lipid-related genes (e.g., LRP2,

onmR

d From: https://content.onlinejacc.org/ on 03/15/2015L, APOA2) with plasma Lp-PLA2 likely reflecting thisirect post-translational influence (Online Tables 1A and). Interpretation of studies of plasma Lp-PLA2 in CHDchallenging partly because circulating lipoproteins mayssly confound the association of plasma Lp-PLA2 withD (8) and furthermore because lesion macrophageduction may be more relevant to the disease thanculating protein.While we failed to detect significant association betweensma Lp-PLA2 and common SNPs in PLA2G7, the sameA2G7 variants were associated with CAC within ourdy samples. Our preliminary exploration also revealed

A and PAMSyN 1,279)

PDHS(n 782)


p Value Beta p Value Z Score p Value

9.8 105 0.05 0.65 3.35 8.1 104

3.9 104 0.05 0.67 3.06 0.002

0.004 0.40 0.07 3.36 7.7 104

0.008 0.22 0.13 3.04 0.002

0.013 0.32 0.02 3.36 7.8 104

0.014 0.09 0.49 2.37 0.018

0.016 0.23 0.39 1.37 0.17

0.02 0.14 0.56 1.41 0.16

0.02 0.14 0.56 1.41 0.16

0.03 0.26 0.07 2.85 0.004

0.04 0.17 0.37 2.16 0.03

0.05 0.16 0.23 2.26 0.02

0.05 0.16 0.23 2.25 0.02

0.05 0.16 0.23 2.25 0.02

0.05 0.16 0.23 2.25 0.02

0.11 0.15 0.25 1.98 0.05

0.11 0.16 0.23 2.01 0.04

0.44 0.15 0.46 1.06 0.29

0.76 0.02 0.94 0.29 0.77

0.04 0.09 0.70 1.82 0.07

0.06 0.11 0.65 1.78 0.07

0.06 0.08 0.75 1.70 0.09

0.06 0.05 0.83 1.63 0.10

0.07 0.52 0.64 1.16 0.25

0.23 0.29 0.18 1.77 0.08

0.23 0.01 0.93 0.90 0.37

0.27 0.15 0.21 0.10 0.92

0.30 0.00 0.98 0.84 0.40

0.54 0.21 0.67 0.75 0.45

0.57 1.52 0.33 1.04 0.30

0.57 1.54 0.49 0.01 0.99

0.65 0.16 0.17 1.20 0.23

0.69 0.67 0.56 0.22 0.50

0.99 0.00 0.15 0.17 0.37

enn Diabetes Heart Study; SIRCA Study of Inherited Risk of Coronary Atherosclerosis; other

February 21, 2012:76472LPind1BisgroCHprocir

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0.21

0.27

0.19

0.19

0.19

0.19

0.16

0.16

0.12

0.04

0.32

0.31

0.31

0.31

1.52

0.19

0.11

0.10

0.09

0.23

0.57

0.57

0.04

0.13

0.89

HS Ply nominal associations of PLA2G7 SNPs with Lp-PLA2NA levels in multiple cells and tissues. These eQTL

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Downloadedings should be interpreted cautiously because of limitedwer, relatively low levels of Lp-PLA2 expression in testedls, and (unlike CRP) well-characterized cis-acting SNPsPLA2G7 are lacking. Further, appropriately powereddies are needed to determine whether PLA2G7 SNPs areated to expression of Lp-PLA2 in inflammatory macro-ages and foam cells, sources that may be most relevant toerosclerosis. However, our data suggest caution in usingculating leukocyte Lp-PLA2 mRNA levels as surrogateseffects of PLA2G7 variation on arterial pathology.

erall, our findings support the concept that PLA2G7 mayate to atherosclerosis independent of circulating Lp-A2 mRNA and protein.Published studies of PLA2G7 in CHD are conflicting. Inmeta-analysis of individuals of European ancestry,A2G7 SNPs did not associate with risk of CHD (n,000) (42), although there were relationships between-PLA2 activity and CHD and between PLA2G7 SNPsd Lp-PLA2 activity. However in a meta-analysis of over,000 Asians, a common nonsynonymous PLA2G7 SNPwed evidence of association with CHD (31). Additionalnsynonymous SNPs have been associated with carotidque in Japanese (32) and recently a loss-of-functioniant in PLA2G7 was shown to protect against CHD inreans (30). Due to the absence in Caucasian samples offunctional PLA2G7 SNP found in Asians (rs76863441V279F), we were not able to evaluate the effect of thisctional variant in our samples. However, common vari-on in PLA2G7 is well covered on the ITMAT Broadre array platform (tag SNP-coverage r2 0.8 for allelesth minor allele frequency 2% in the gene 5 kilobases)). Therefore, we are confident that we achieved excellenterage of common variation in this gene region in Cauca-ns. Whereas ethnic difference in the presence of alleliciation may exist, most published data suggest a relationshipPLA2G7 with clinical CHD supporting our CAC findings.Findings for CRP in our samples are consistent withblished data and contrast with that observed forA2G7-Lp-PLA2. Thus, even though a number of SNPsCRP had strong associations with circulating CRP levels,re was no relationship between these same SNPs andC. These data are in line with hallmark Mendeliandomization studies of clinical CHD outcomes (33,34)d support a model of confounding or reverse causation forP associations with CAC and CHD.

udy limitations. First, our studies are correlative and dot define causality. We have not studied loss-of-functiongain-of-function variants in PLA2G7 for their relation toC or CHD and, therefore, cannot infer Lp-PLA2ectional actions in atherosclerosis. However, expressionta in inflammatory macrophages and foam cells coupledpreliminary studies of Lp-PLA2 inhibition in humanerosclerosis support an atherogenic role for human

ruary 21, 2012:76472A2G7. Second, recent studies have shown stronger asso-tions of PLA2G7 with circulating Lp-PLA2 measuresn were found in our sample. This may relate to our

d From: https://content.onlinejacc.org/ on 03/15/2015aller sample size, heterogeneity in the SIRCA andHS study samples, or differences in Lp-PLA2 assaysd across studies. The PLA2G7-Lp-PLA2 system, how-r, may be a poor target for Mendelian randomizationdies for several reasons including: heterogeneous envi-mental and genetic influences on circulating levels;A2G7 actions in atherosclerosis are likely to be indepen-nt of circulating Lp-PLA2; and well-characterized cis-ing SNPs to use as instrumental variables for PLA2G7lacking. Finally, although not a direct measure of

ronary atherosclerosis, studies show that CAC provides aantitative estimate of coronary atherosclerosis (47) and isseful predictor of CHD events (48).

nclusions

e have demonstrated that Lp-PLA2, in contrast to CRP,not an acute phase protein in humans. Lp-PLA2 hasited expression in circulating leukocytes or unstimulatednocytes ex vivo, but it is induced during differentiation tocrophages and in foam cells. Thus, robust biomarkers of-PLA2 action in atherosclerosis and of its pharmacolog-l modulation in vascular tissues are lacking. Commoniation in PLA2G7, but not in CRP, is related to therden of CAC, suggesting that PLA2G7 may indeeddulate human atherosclerosis. Our data provide supportthe atherogenicity of Lp-PLA2 in humans while high-hting the challenges in using plasma Lp-PLA2 as amarker of CHD and in determining drug-dosing andrapeutic efficacy in atherosclerosis.

print requests and correspondence: Dr. Jane F. Ferguson,rdiovascular Institute, University of Pennsylvania, DepartmentMedicine, TRC 11th Floor, 3400 Civic Center Boulevard,iladelphia, Pennsylvania 19104-6160. E-mail: [email protected].

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y Words: coronary artery calcification y lipoprotein-associatedspholipase A2 y PLA2G7.

APPENDIXsupplementary methods, please see the online version of this paper.

Translational Studies of Lipoprotein-Associated Phospholipase A2 in Inflammation and Atheroscler ...MethodsClinical studiesHuman EndotoxemiaGenetic Association Studies

Laboratory methodsHuman Monocyte, Macrophage, and Foam Cell StudiesPlasma Lp-PLA2, Inflammatory, and Metabolic MarkersReal-Time Quantitative PCR and Expression Quantitative Trait Locus AnalysisGenotyping

Statistical analysis

ResultsLp-PLA2 is not induced in a human model of acute phase responseLp-PLA2 expression is induced in inflammatory human macrophages and foam cellsCommon polymorphisms in PLA2G7 are associated with coronary calcification but only weakly with p ...

DiscussionLp-PLA2 does not contribute to human acute phase responseProinflammatory macrophages and foam cells, but not monocytes, generate significant Lp-PLA2Genetic variation in PLA2G7 may relate to CHD independent of circulating Lp-PLA2Study limitations

ConclusionsReferencesAppendix

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