lipoprotein associated phospholipase a2: an investigation into one

1

Modulation of oxidative stress, inflammation, and atherosclerosis by lipoprotein-associated phospholipase A2

Robert S. Rosenson, MD Mount Sinai School of Medicine 1425 Madison Ave. Box 1030 New York, NY 10029 Diana M. Stafforini, PhD Huntsman Cancer Institute & Department of Internal Medicine University of Utah 2000 Circle of Hope Salt Lake City, UT 84112

Address for correspondence: Robert S. Rosenson, MD Mount Sinai School of Medicine 1425 Madison Ave. Box 1030 New York, NY 10029 Telephone: 212-659-9174 Facsimile: 212-659-9033 E mail: [email protected]

by guest, on April 7, 2018

ww

w.jlr.org

Dow

nloaded from

mailto:[email protected]

http://www.jlr.org/

2

ABSTRACT

Lipoprotein-associated phospholipase A2 (Lp-PLA2), also known as platelet-activating factor

acetylhydrolase (PAF-AH), is a unique member of the phospholipase A2 superfamily. This enzyme

is characterized by its ability to specifically 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 humans, Lp-PLA2 circulates in active form as a complex with low- and high-density

lipoproteins. Clinical studies have reported that plasma Lp-PLA2 activity and mass are strongly

associated with atherogenic lipids and vascular risk. These observations led to the hypothesis

that Lp-PLA2 activity and/or mass levels could be used as biomarkers of cardiovascular disease

and that inhibition of the activity could offer an attractive therapeutic strategy. Darapladib, a

compound that inhibits Lp-PLA2 activity, is anti-atherogenic in mice and other animals, and

decreases atherosclerotic plaque expansion in humans. However, disagreement continues to

exist regarding the validity of Lp-PLA2 as an independent marker of atherosclerosis and a

scientifically justified target for intervention. Circulating Lp-PLA2 mass and activity are associated

with vascular risk, but the strength of the association is reduced after adjustment for basal

concentrations of the lipoprotein carriers with which the enzyme associates. Genetic studies in

humans harboring an inactivating mutation at this locus indicate that loss of Lp-PLA2 function is

a risk factor for inflammatory and vascular conditions in Japanese cohorts. Consistently, over-

expression of Lp-PLA2 has anti-inflammatory and anti-atherogenic properties in animal models.

This thematic review critically discusses results from laboratory and animal studies, analyzes

genetic evidence, reviews clinical work demonstrating associations between Lp-PLA2 and

vascular disease, and summarizes results from animal and human clinical trials in which

administration of darapladib was tested as a strategy for the management of atherosclerosis.

Keywords: Lipoprotein-associated phospholipase A2, PAF-AH, PLA2G7, darapladib, oxidative stress, inflammation, atherosclerosis, cardiovascular disease, stroke, PAF, platelet-activating factor, oxidized phospholipids.

Abbreviations: AA-PC, arachidonoyl phosphatidylcholine; ACS, acute coronary syndrome; apoB100, apolipoprotein B100; CAD, coronary artery disease; CHD, coronary heart disease; CI, confidence interval; CVD, cardiovascular disease; DM-HC, diabetic and hypercholesterolemic; Hs-CRP, high-sensitivity C-reactive protein; ICAM-1, intracellular adhesion molecule-1; IL-6, interleukin-6; Lp-PLA2, lipoprotein-associated PLA2; MCP-1, monocyte chemotactic protein-1; MI, myocardial infarction; MMP-9, matrix metalloproteinase-9; MPO, myeloperoxidase; NASCET, North American Symptomatic Carotid Endarterectomy Trial; OR, odds ratio; OxFA, oxidized fatty acids; OxPL, oxidized phospholipids; OxPL/ApoB, oxidized phospholipid/apolipoprotein B ratio; PAF, platelet-activating factor; PC, phosphatidylcholine; PCI, percutaneous coronary intervention; PLA2, phospholipase A2; RR, relative risk; TNF-α, tumor necrosis factor-α; VCAM-1, vascular cell adhesion molecule-1; WHHL, Watanabe heritable hyperlipidemic.


ww

w.jlr.org

Dow

nloaded from

http://www.jlr.org/

3

TABLE OF CONTENTS ABSTRACT.........................................................................................................................….. 2 1. INTRODUCTION.............................................................................................................. ….. 4 2. LIPOPROTEIN-ASSOCIATED PHOSPHOLIPASE A2: EXPERIMENTAL STUDIES ........................................ 5

2.1. BIOCHEMICAL AND STRUCTURAL PROPERTIES ................................................................... 5 2.2. CELLULAR SOURCES AND REGULATION OF EXPRESSION ....................................................... 6 2.3. FUNCTIONAL CONSEQUENCES OF LP-PLA2 ACTION ............................................................. 7

3. LIPOPROTEIN-ASSOCIATED PHOSPHOLIPASE A2: EPIDEMIOLOGICAL STUDIES..................................... 8

3.1. LP-PLA2 EXPRESSION IN APPARENTLY HEALTHY SUBJECTS................................................... 8 3.2. LP-PLA2 EXPRESSION IN SUBJECTS WITH STABLE CARDIOVASCULAR DISEASE........................... 9 3.3. LP-PLA2 EXPRESSION IN PATIENTS WITH ACUTE CORONARY SYNDROME................................ 9 3.4. LP-PLA2 EXPRESSION IN STATIN-TREATED PATIENTS.......................................................... 10 3.5. LP-PLA2 EXPRESSION IN ATHEROSCLEROTIC PLAQUES......................................................... 10 3.6. IMPLICATIONS FOR CARDIOVASCULAR RISK ASSESSMENT AND THERAPEUTIC INTERVENTION ....... 11

4. GENETIC STUDIES IN HUMAN POPULATIONS ............................................................................. 12

4.1. GENETIC DEFICIENCY OF LP-PLA2 ............................................................................. ….. 12 4.2. LP-PLA2 DEFICIENCY AND RISK OF CARDIOVASCULAR EVENTS ............................................. 12 4.3. LP-PLA2 POLYMORPHISMS AND CARDIOVASCULAR DISEASE ................................................ 13

5. LIPOPROTEIN-ASSOCIATED PHOSPHOLIPASE A2 INHIBITORS ......................................................... 13

5.1. PHARMACOLOGY, BIOLOGICAL PROPERTIES, AND METABOLIC EFFECTS OF DARAPLADIB............ 13 5.2. IN VIVO STUDIES OF LP-PLA2 OVER-EXPRESSION VERSUS DARAPLADIB ADMINISTRATION………… 15 5.3. BIOMARKER AND IMAGING STUDIES OF LP-PLA2 INHIBITION IN HUMANS AND ANIMAL MODELS 15 5.4. DARAPLADIB AND CARDIOVASCULAR EVENTS: ONGOING CLINICAL TRIALS.............................. 16 5.5. SAFETY ISSUES TO CONSIDER......................................................................................... 16

6. CONCLUSIONS.................................................................................................................... 16 TABLE 1........................................................................................................................... 18 TABLE 2........................................................................................................................... 19 TABLE 3........................................................................................................................... 20 7. REFERENCES...................................................................................................................... 21


ww

w.jlr.org

Dow

nloaded from

http://www.jlr.org/

4

1. INTRODUCTION 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 formation of lipid oxidation products that increase vascular inflammation (2). Vascular inflammation 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 infarction (MI) and stroke. Thus, inflammation 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 inflammation in the vascular wall. Examples of these include antioxidants (3) and lipoxygenase inhibitors (4) that limit oxidant stress, and secretory phospholipase A2 (PLA2) inhibitors that inhibit production of pro-inflammatory prostaglandins and leukotrienes (5, 6). Cellular oxidation is controlled through a network of sensors whose function is to ensure that the redox state of the cell remains within viable parameters. In addition to the multiplicity of compensatory 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-inflammatory responses. These considerations underscore the challenges associated with strategies that rely on targeting a single pathway of inflammation and/or oxidation (5, 8). Inflammation is a physiologic and highly regulated response: while blocking its onset offers therapeutic benefit in a variety of settings, this mechanism is essential for host defense. Inappropriate blockade of inflammation 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 associated with altered expression of multiple inflammatory gene products (1). Biomarkers of disease progression sometimes –but not always— offer an opportunity for therapeutic intervention. When mechanistic studies demonstrate that a biomarker is causally linked to disease pathogenesis, a solid and justified opportunity for intervention arises. Circulating levels of Lipoprotein-associated PLA2 (Lp-PLA2) have been proposed to be markers of CVD on the basis of numerous epidemiological studies (9, 10). Initial work conducted at least twenty-five years ago showed good correlations between plasma Lp-PLA2 activity and proven markers of CVD, such as LDL cholesterol and lipoprotein levels (11-15). Thus, the association between Lp-PLA2 and CVD has been recognized for many years. Lp-PLA2 is a unique member of the PLA2 super-family that currently contains fifteen separate, identifiable 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, disulfide bonding patterns, the requirement for Ca2+, and other features. There are five main categories of PLA2 activities: secreted small molecular weight sPLA2 enzymes that include groups I-III, V, IX-XIV, larger cytosolic Ca2+-dependent cPLA2s (group IV), Ca2+-independent iPLA2s (group VI), platelet activating factor (PAF) acetylhydrolases (groups VII and VIII), and lysosomal PLA2 (16-17). Lp-PLA2 has been classified as group VIIA PLA2 (PLA2G7) owing to its sequence, size, calcium independence and substrate specificity (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-PLA2 to


ww

w.jlr.org

Dow

nloaded from

http://www.jlr.org/

5

inactivate PAF, which has been implicated in a variety of inflammatory diseases (19, 20), the enzyme was referred to as the secreted/plasma form of PAF acetylhydrolase or PAF-AH.Lp-PLA2 is primarily produced 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 subjects that express this particle (23). The biochemical and physiological interactions of the enzyme with lipoproteins gave rise to more descriptive terminology (i.e., Lp-PLA2) which is commonly used, particularly in clinical settings.

This thematic review presents current knowledge of the structural and biochemical properties of Lp-PLA2. In addition, 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-PLA2 as a biomarker and therapeutic target for the treatment of CVD.

2. LIPOPROTEIN-ASSOCIATED PHOSPHOLIPASE A2: EXPERIMENTAL STUDIES 2.1. BIOCHEMICAL AND STRUCTURAL PROPERTIES Lp-PLA2 is a Ca2+-independent, 45 KDa secreted protein that circulates in plasma in active

form. Sequence analysis of PLA2G7, the gene encoding Lp-PLA2, reveals the presence 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 orientation and spacing is consistent with the α/β hydrolase conformation of neutral lipases and esterases (24). This was confirmed when the solution of the crystal structure of Lp-PLA2 revealed that the protein indeed has a classic lipase α/β hydrolase fold (25). In contrast to most PLA2 activities that initiate signal transduction and are regulated by the state of cellular activation, Lp-PLA2 is not acutely regulated (19). This feature, combined with the independence of the reaction on Ca2+, could potentially threaten the integrity of phospholipid components of cellular membranes and lipoproteins (26). However, the ability of the enzyme to recognize only defined types of sn-2 fatty acyl groups insures that undamaged, structural phospholipids are protected from constitutive hydrolysis (27). In addition to PAF, Lp-PLA2 catalyzes hydrolysis of phospholipids generated from structural components in cellular membranes 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 identified using purified substrates (28, 32), were later confirmed in elegant studies using human LDL exhaustively oxidized with CuSO4 in the presence and absence of SB222657, an irreversible inhibitor of Lp-PLA2 (33). Lp-PLA2 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 various sn-2 acyl chains in phospholipid substrates.

A key feature of Lp-PLA2 is its association with lipoproteins, including HDL, LDL and, to a lesser extent, Lp(a) [reviewed in (26)]. Differential distribution of Lp-PLA2 in various plasma lipoproteins may impact its function (35-38) and thus much effort has been spent elucidating the molecular basis for these associations. Binding of Lp-PLA2 to LDL requires W115 and L116, which are located 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 membrane surface, and that they are important for Lp-PLA2 binding to hydrophobic surfaces (34). Tyrosine-


ww

w.jlr.org

Dow

nloaded from

http://www.jlr.org/

6

205, a residue conserved only among Lp-PLA2 orthologs that associate with endogenous LDL particles, is also required for association with LDL (39). The enzyme appears to bind to apolipoprotein B100 (apoB100) through domains located in a 160 amino stretch comprised between apoB100 residues 4119 and 4279 (39). In recent studies, we found that a carboxyl terminal string of amino acids (H367-K370) unique to human Lp-PLA2 is required for association with HDL (40); additional regions may be required for this interaction.

2.2. CELLULAR SOURCES AND REGULATION OF EXPRESSION Multiple inflammatory cells involved in atherogenesis secrete Lp-PLA2, including monocytes,

macrophages, neutrophils, activated bone marrow-derived mast cells, and activated platelets (21, 41). Monocyte/macrophages are key players in both initiation and progression of atherosclerosis, and have been proposed as new potential therapeutic targets for the prevention and treatment of this disease (42). We previously demonstrated that maturation of monocytes into macrophages is accompanied by dramatic increases in Lp-PLA2 mRNA and protein (21, 43); these observations were recently confirmed (44). Treatment of macrophages with acetylated LDL generates foam cells in vitro and further increases Lp-PLA2 expression (44), which is consistent with observations in human atherosclerotic plaques (45). The properties of macrophages in atherosclerotic lesions can vary dramatically between two extremes: pro-inflammatory M1 macrophages and anti-inflammatory M2 macrophages; these cells can switch from one phenotype to the other depending on micro-environmental cues such as differentiation factors produced in the lesion and T-cell-derived polarizing cytokines (46, 47). We found that IFN-γ, which has been implicated as a promoter of foam cell formation and an inducer of M1 pro-inflammatory phenotypes (47), decreased expression of Lp-PLA2 at the transcriptional level (48). Conversely, M-CSF, which induces an M2-like, anti-inflammatory phenotype, increased monocyte Lp-PLA2 expression to a much higher extent compared with the M1-inducer GM-CSF (49). However, Ferguson and co-workers reported modest increases or no changes in Lp-PLA2 mRNA levels in M1 and M2 macrophages, respectively, using different induction strategies (44). In addition to M1 and M2, other macrophage phenotypes have been described, including sub-species generated in response to stimulation with oxidized phospholipids [OxPL, (50)]. Agents such as LPS, IL-1β, G-CSF, and TNF-α can significantly increase the synthesis and secretion of functional Lp-PLA2 by monocytes, RAW264.7, and THP-1 cells (51, 52). These effects appear to be modulated by the state of cellular differentiation (26). It is clear that signals from the microenvironment dynamically regulate Lp-PLA2 expression 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 phospholipids, up-regulates expression of Lp-PLA2 mRNA in monocyte/macrophages (48) and HepG2 cells (53). Consistently, a recent study reported elevated plasma levels of both PAF and Lp-PLA2 mass in a cohort of coronary heart disease (CHD) patients compared with a control group (54). Increased Lp-PLA2 mass in this setting may represent in vivo evidence for PAF- and/or OxPL mediated effects on Lp-PLA2 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-PLA2 activity (55). When this response was modeled in a cellular system, the investigators found that oxidized LDL robustly increased expression of Lp-PLA2 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, up-regulate expression of Lp-PLA2 in vivo, possibly by increasing mRNA synthesis or stability, resulting in


ww

w.jlr.org

Dow

nloaded from

http://www.jlr.org/

7

higher protein expression. This response likely reflects 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 modification 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-PLA2 expression is genetically controlled. A recent a genome-wide association study that included five population-based reports comprising 13,664 subjects identified eight genetic loci associated with variation in Lp-PLA2 mass and activity (56). Lp-PLA2 mass was found to be regulated mainly by PLA2G7, consistent with another report (57). Lp-PLA2 activity was strongly associated with genetic variants involved in lipid metabolism (56). The findings of this interesting study suggest that genetic regions known to affect lipoprotein levels also influence Lp-PLA2 expression.

2.3. FUNCTIONAL CONSEQUENCES OF LP-PLA2 ACTION A number of studies have concluded that the in vivo action of Lp-PLA2 in settings of elevated

oxidant stress, allergy and acute inflammation is beneficial [see (26) for a recent review]. The mechanism accounting for these effects is thought to involve Lp-PLA2-mediated decreases in the bioactivity of phospholipid substrates such as PAF and OxPL. However, an issue that has eluded clarification despite considerable effort over the years is whether the in vivo action of Lp-PLA2 on endogenous substrates, generates products [i.e., lyso-phosphatidylcholine (lyso-PC) plus short and/or oxidized fatty acids] that increase inflammatory responses in the vascular wall. Specifically, it has not been determined whether Lp-PLA2 is beneficial or detrimental, and whether the substrates are more, less, or equally atherogenic than the byproducts (58). This issue has been explored using in vitro approaches to characterize biological properties of relevant substrates and products of the reaction. Both pro- and anti-inflammatory 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 detrimental and beneficial responses. In general, in vitro approaches are strategically useful initially, but they have the usual limitations of studies that do not recapitulate essential in vivo features. This is particularly relevant in studies that involve lipids and lipid metabolites. For example, relatively low (nanomolar) concentrations of lyso-PC recruit monocytes and induce pro-inflammatory cytokine production in vitro (81). However, the concentration of lyso-PC in blood plasma of healthy persons usually ranges from 200 to 400 μM (82, 83) and similar levels have been reported in atherosclerotic tissues (45). Since lyso-PC, unlike PC, is membrane lytic, it is predominantly confined to lipoproteins or plasma proteins, such as albumin or immunoglobulins (83, 84). As a result, only a fraction of lyso-PC is functionally active and it is unclear how total concentration relates to bioavailability (73, 85). Similarly, the bioactivity of fatty acids appears to be modulated by the carrier, again underscoring the importance of location on biological activity (86, 87). Thus, whether results generated using lyso-PC and oxidized fatty acids in vitro realistically reflect in vivo responses remains to be rigorously determined.

In view of these limitations, rather than characterizing functional effects of either Lp-PLA2 substrates or Lp-PLA2 products, a more informative approach is to assess net changes that result when the action of Lp-PLA2 alters substrate-to-product ratios. Examples of this strategy include investigations conducted in the presence and absence of Lp-PLA2 activity, or studies that compare the effect of supplementing active and inactive enzyme. Both pharmacologic and genetic depletion of Lp-PLA2 in minimally modified lipoproteins enhanced monocyte adhesion to


ww

w.jlr.org

Dow

nloaded from

http://www.jlr.org/

8

aortic endothelial cells compared with minimally modified lipoproteins that expressed normal activity levels (88). Similarly, exogenous Lp-PLA2 reduced cellular uptake of oxidized LDL and Lp(a), and cholesterol accumulation by monocyte-derived macrophages, compared with parallel assays conducted with inactive enzyme (89). These results suggest that the enzymatic activity of Lp-PLA2 limits monocyte recruitment and foam cell formation within atherosclerotic lesions (89). Chen and colleagues found that exogenous Lp-PLA2, but not vehicle, inhibited endothelial cell apoptosis induced by minimally modified 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-PLA2.

3. LIPOPROTEIN-ASSOCIATED PHOSPHOLIPASE A2: EPIDEMIOLOGICAL AND CLINICAL STUDIES WOSCOPS (West of Scotland Coronary Prevention Study) was the first study to report that

circulating Lp-PLA2 mass levels had a strong, positive association with risk of coronary events that was not confounded by classic cardiovascular risk factors (92). Since then, plasma and serum Lp-PLA2 concentration and activity have been determined in a great number of epidemiological studies aimed at determining whether these measurements can be used in patient stratification and/or to predict the likelihood of future cardiovascular events such as MI and stroke. Several reviews and meta-analyses have recently addressed this issue [see, for example, 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-PLA2. Investigations relying exclusively on Lp-PLA2 mass determinations should be interpreted within the context of serious methodological concerns regarding the performance of the leading commercially available assay (96-98). This includes long-term consistency in the hands of the manufacturer.

3.1. LP-PLA2 EXPRESSION IN APPARENTLY HEALTHY SUBJECTS The initial association between Lp-PLA2 levels and future risk of CVD reported by WOSCOPS

(92) was, in general, confirmed in smaller studies (99-102). In contrast, additional trials failed to reproduce these observations (95, 103-106). The Lp-PLA2 Studies Collaboration examined associations between circulating Lp-PLA2 concentration 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 non-lipid and lipid risk factors. In this group, no association was found between Lp-PLA2 activity and CHD or ischemic strokes. In summary, Lp-PLA2 activity levels do not seem to be a useful predictor of future CHD in apparently healthy individuals.

Two studies that merit mention include investigations that evaluated plasma Lp-PLA2 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 first 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, (102)]. OxPL/apoB measured at baseline predicted the development of cardiovascular events independently of traditional risk factors and high-sensitivity C-reactive protein (hs-CRP). In addition, increased risk was augmented with increasing Lp-PLA2 activity. The second study [European Prospective Investigation of Cancer (EPIC)-Norfolk] also included a cohort of 45-79 year-old, apparently healthy, men and women (107). This analysis confirmed the


ww

w.jlr.org

Dow

nloaded from

http://www.jlr.org/

9

increased risk of cardiovascular events with increasing levels of oxPL/apoB but only a weak potentiation of risk was observed with increasing Lp-PLA2 activity. In sum, the two studies suggest that combined assessment of these two mechanistically related biomarkers (Lp-PLA2 activity and oxPL/apoB ratio) provides modest incremental information to predict risk of future cardiovascular events in apparently healthy individuals.

A nested case-control analysis evaluated whether increased Lp-PLA2 activity correlates with MI in 1,221 apparently 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-PLA2 activity was associated with incident MI [RR=1.75; 95% CI (1.09-2.84)] even after multivariable adjustment for clinical, lipid and inflammatory risk factors. The differences in the risk of CHD events associated with increased Lp-PLA2 activity in this observational study of participants without known vascular disease may relate to sex-specific disease associations and longer average duration of follow-up compared with results included in the Lp-PLA2 Studies Collaboration (94).

3.2. LP-PLA2 EXPRESSION IN SUBJECTS WITH STABLE CARDIOVASCULAR DISEASE Several studies reported that Lp-PLA2 mass predicts future cardiovascular events in patients

with stable CVD (95, 109-111). Since Lp-PLA2 is carried in the circulation as a complex with lipoproteins such as apoB-containing LDL, there is a strong relationship between Lp-PLA2 and LDL concentrations (96). Thus, the strength of the association of Lp-PLA2 with CVD depends substantially on apoB or its associated lipid moieties (96). It is therefore essential to evaluate whether the predictive power of Lp-PLA2 is independent of common risk factors such as high apoB/LDL levels. In one study, adjustment for multiple risk factors [e.g., hypertension, smoking, family history of coronary artery disease (CAD), and prior MI] and medication use did not affect the ability of Lp-PLA2 to predict CAD risk, but abolished the potential to predict all-cause mortality, non-CAD CV death, new MI, nonfatal MI, or a composite end point of all-cause mortality, MI, cerebrovascular accident, and revascularization at follow-up (112). In another study, however, the association remained significant (111). Lp-PLA2 activity levels also appear to predict future cardiovascular events and cardiac mortality in patients with stable CVD (109, 113). The Lp-PLA2 Studies Collaboration analyzed 32 prospective 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 deviation higher Lp-PLA2 activity or concentration in models adjusted for age, sex, baseline history of vascular disease and other non-lipid 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-PLA2 activity or mass.

3.3. LP-PLA2 EXPRESSION IN PATIENTS WITH ACUTE CORONARY SYNDROME Initial epidemiological analyses in patients with ACS led to promising results that failed to be

confirmed in subsequent work (114). Two small studies suggested that elevated Lp-PLA2 levels may help in the identification of patients who will develop cardiovascular complications (114) or cardiac mortality (115). However, the Fragmin and Fast Revascularization During Instability in Coronary Artery Disease II [FRISC II (116)], the Global Utilization of Strategies to Open Occluded Arteries IV [GUSTO IV (116)] and Pravastatin or Atorvastatin Evaluation and Infection Therapy [PROVE-IT (117)] studies reported that baseline levels of Lp-PLA2 mass and/or activity did not predict recurrent events such as MI, stroke, or death. Analysis of patients diagnosed with an


ww

w.jlr.org

Dow

nloaded from

http://www.jlr.org/

10

acute ischemic event in the Lp-PLA2 Studies Collaboration (n=10,638) revealed no significant associations between Lp-PLA2 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-PLA2 measurements were conducted during the acute phase, however, does not appear to explain the observed lack of correlation as a study from the Myocardial Ischemia Reduction With Acute Cholesterol Lowering (MIRACL) trial in patients presenting with ACS reported no significant changes in Lp-PLA2 mass or activity after ACS, suggesting that Lp-PLA2 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-PLA2 levels and risk of future ischemic events or death in placebo-treated patients (118). These combined studies strongly suggest that measurement of Lp-PLA2 levels is unlikely to improve stratification or risk prediction in patients presenting with ACS.

3.4. LP-PLA2 EXPRESSION IN STATIN-TREATED PATIENTS

The Heart Protection Collaboration Group evaluated whether baseline Lp-PLA2 activity was associated with CVD in a randomized, placebo-controlled clinical trial of simvastatin that included individuals at high risk of developing vascular disease (119). While a modest association was initially observed in analyses that adjusted for non-lipid risk factors, after adjustment for apoB, the association with activity became non-significant (120). However, the study population was diverse and confounding factors from cardiovascular medications and prior vascular disease may have affected the results. The JUPITER (Justification for the Use of Statins in Prevention: an Intervention Trial Evaluating Rosuvastatin) trial compared rosuvastatin 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-PLA2 did not predict risk or clinical outcomes in rosuvastatin-treated patients. The MIRACL trial, which included placebo-treated and atorvastatin-treated individuals whose Lp-PLA2 mass and activity were significantly reduced, found no discernible relationship between Lp-PLA2 levels and risk of future ischemic events or death in either atorvastatin-treated or placebo-treated patients (118). The studies show that Lp-PLA2 levels are unlikely to predict future risk when cholesterol levels are adequately managed by statin therapy. These observations have important clinical implications that will be discussed in 3.6. An additional important observation from the MIRACL (118) and JUPITER (98) trials is that the data are consistent with conclusions reached in 3.1 above, as in placebo-treated, apparently healthy subjects, Lp-PLA2 levels did not predict future CHD.

3.5. LP-PLA2 EXPRESSION IN ATHEROSCLEROTIC PLAQUES A number of studies have reported increased Lp-PLA2 expression in macrophages from

human and rabbit atherosclerotic lesions (121). A prospective study of 167 carotid endarterectomy patients reported increased local expression of Lp-PLA2 and lyso-PC in the vulnerable plaques of symptomatic compared with asymptomatic patients (45). Lp-PLA2 expression was associated with plaque vulnerability in experimental animals and in humans (121, 122). Early lesions stained weakly for Lp-PLA2 but necrotic cores and surrounding macrophages of vulnerable and ruptured plaques displayed robust Lp-PLA2 expression (122). Interestingly, Lp-PLA2 was detected in apoptotic macrophages. This observation may represent


ww

w.jlr.org

Dow

nloaded from

http://www.jlr.org/

11

events mediated by accumulated OxPL which are known to be potent inducers of apoptosis (65). Elevated plaque Lp-PLA2 expression is a feature that appears to be reflected in the circulation. Among 42 patients with high-grade carotid stenoses (≥70 percent by the NASCET criteria), circulating Lp-PLA2 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 first ischemic stroke reported that increased Lp-PLA2 concentration was predictive of recurrent ischemic neurological events (124). Finally, in a study of 40 ACS patients with successful percutaneous coronary intervention (PCI), changes in the volume of non-culprit lesions after 6 months were significantly associated with changes in both LDL cholesterol levels (r=0.444) and Lp-PLA2 concentration [r=0.496, (125)]. While Lp-PLA2 expression is elevated in advanced atherosclerotic plaques and in the circulation, it is not evident from these results that Lp-PLA2 levels would be a more useful clinical marker of plaque instability than simple determination of LDL cholesterol levels.

It is important to emphasize that protein expression patterns in vascular lesions reflect cellular populations present at the time of lesion collection, along with differential regulation in response to the cellular and physical environment (126). Thus, differential gene/protein expression in atherosclerotic plaques reflects both damage occurring in the tissue and mechanisms engaged to limit additional adverse responses (126). While molecular characterizations 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 up-regulated in atherosclerotic plaques suggests that their regulation is a result, rather than a cause, of atherosclerosis (126). Examples include up-regulated expression of antioxidant proteins such as superoxide dismutase-2 and glutathione peroxidase (127). The expression of these proteins may be up-regulated to counteract overproduction of reactive oxygen species in atherosclerotic lesions (128). Thus, while multiple studies report high Lp-PLA2 expression in advanced atherosclerotic lesions, this feature cannot be used as surrogate evidence for active contribution of this enzyme to the disease phenotype.

3.6. IMPLICATIONS FOR CARDIOVASCULAR RISK ASSESSMENT AND THERAPEUTIC INTERVENTION The strength of the association between Lp-PLA2 mass and activity with vascular risk is

markedly reduced after adjustment for basal concentrations of lipids and apolipoproteins (96, 129). Cohort stratification (94) combined with individual analyses of the most rigorously designed studies (96, 98) indicate that Lp-PLA2 activity and risk of future cardiovascular events or death are not significantly 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 findings outlined above, it is unlikely that measurement of plasma or serum Lp-PLA2 activity would yield additional power to predict risk of CHD, or help in risk stratification, beyond that already provided by assessment of traditional risk factors (130). A second issue to consider is whether these results impact the suitability of Lp-PLA2 as a target for intervention. Aside from mechanistic considerations that are discussed elsewhere in this article, the epidemiological evidence summarized above does not support the hypothesis that targeting Lp-PLA2 would improve outcomes above and beyond what is already achieved by current strategies to manage CVD.


ww

w.jlr.org

Dow

nloaded from

http://www.jlr.org/

12

4. GENETIC STUDIES IN HUMAN POPULATIONS

4.1. GENETIC DEFICIENCY OF LP-PLA2 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-PLA2 in vivo and its impact in human physiology and disease. Fourteen years ago, one of us identified the molecular basis of Lp-PLA2 deficiency 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 enzyme [V279F, (132)]. Homozygous carriers of this variant express virtually no enzymatic activity in plasma and heterozygous subjects have a 50% decrease in activity compared with those expressing two copies of the wild-type allele (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 (138), and Chinese (139, 140) populations, and in subjects from Turkey, Azerbaijan, and Kyrgyzstan (141). Allele frequency varies widely in these populations: the highest prevalence was reported in Japan and in Taiwan, followed by Korea and China [reviewed in (142)]. In contrast, this variant has been observed rarely in Europeans (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).

4.2. LP-PLA2 DEFICIENCY AND CORONARY ARTERY DISEASE

Diverse studies have reported that the V279F null allele is associated with increased risk of developing atherosclerosis, CAD, stroke, and MI in Japanese subjects (134, 147-150). A study using strict criteria for statistical significance (p<0.005) showed that the V279F allele is significantly associated with CAD in Japanese men with nonfamilial hypercholesterolemia (151). (151). Based on the same criteria, 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 had no effect on intima media thickness (152). A recent meta-analysis involving predominantly Asian individuals analyzed seven case-control studies involving 3,614 patients with CHD and 4334 controls (139). While the crude ORs were not significant, analyses stratified by study size, ethnicity, case definition, 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 significant after multivariate adjustment [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 clinical characteristics. In the initial study, LDL and total cholesterol levels were


ww

w.jlr.org

Dow

nloaded from

http://www.jlr.org/

13

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 pharmacologically decreased by statin therapy (154). In healthy Koreans, 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)]. These observations point at potentially important differences in Japanese compared with Korean subjects, assuming these findings are re-capitulated in independent Korean cohorts. Thus, with the exception of these two studies by a single Korean group, the evidence available to date strongly suggests that deficiency of Lp-PLA2 is associated with increased risk of CAD.

4.3. LP-PLA2 POLYMORPHISMS AND CORONARY ARTERY DISEASE In contrast to the V279F allele associated primarily with Asian populations, three relatively

well-studied non-synonymous Lp-PLA2 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 purified A379V variant displays decreased substrate affinity 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 alleles 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 contrast, 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-PLA2 polymorphisms that modestly impact enzymatic activity are unlikely to be associated with cardiovascular risk.

5. LP-PLA2 INHIBITORS Epidemiological and correlative studies showing that Lp-PLA2 expression is elevated in CVD,

combined with in vitro analyses have led to the proposition that the enzyme has a predominantly pro-inflammatory role in atherogenesis (75, 159). Mechanistically, proponents of this model argue that Lp-PLA2-mediated lyso-PC and oxidized fatty acid generation contribute to several aspects of plaque formation, and actively contribute to the pathogenesis of vascular disease. In turn, this led to the proposition that inhibiting the enzymatic activity of Lp-PLA2 could be potentially beneficial 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 benefit in patients diagnosed with CHD (9, 162) and following ACS (163).

5.1. PHARMACOLOGY, BIOLOGICAL PROPERTIES, AND METABOLIC EFFECTS OF DARAPLADIB Darapladib reversibly and non-covalently binds to human recombinant Lp-PLA2 with a Ki of

110 pM and an off-rate of 27 min (161). It is lipophilic and has good membrane permeability (164). Darapladib has minimal interaction with cytochrome P450 isozymes and has an oral bioavailability of 11 ± 2% and ≈21% in fed and fasted rats, respectively, and 28 ± 4% in dogs


ww

w.jlr.org

Dow

nloaded from

http://www.jlr.org/

14

(161). Darapladib has been repeatedly shown to inhibit hydrolysis of Lp-PLA2 substrates [i.e., PAF, 1-myristoyl-2-(4-nitrophenylsuccinyl) PC, and others]. Darapladib inhibits Lp-PLA2-mediated exogenous substrate hydrolysis in plasma and LDL in vitro (161). In addition, plasma and atherosclerotic plaque lysates from darapladib-treated Watanabe heritable hyperlipidemic (WHHL) rabbits (161) and diabetic and hypercholesterolemic 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 endogenous substrate levels using ex vivo and in vivo approaches. Exhaustive in vitro oxidation of LDL with CuSO4 in the absence and presence of SB222657, confirmed previous work (31, 32) showing that truncated PC oxidation products are the predominant endogenous substrates for Lp-PLA2 (33). PCs that undergo oxidative modification without chain fragmentation are clearly much less efficient substrates for Lp-PLA2, but since they are quantitatively the most abundant PC products of LDL oxidation (33), the enzyme is likely to impact their levels to a certain extent. In fact, lipopolysaccharide-stimulated whole blood specimens from metabolic syndrome patients treated with various dosages of the Lp-PLA2 inhibitor SB677116, a compound structurally related to darapladib, attenuated the production of C-18 (unfragmented) oxidized fatty acids from endogenous precursors during a 6-h incubation period (166) (Table 1).

In vivo studies showed that darapladib treatment reduced the content of lyso-PC in pig atherosclerotic lesions, owing to inhibition of hydrolysis of endogenous glycerophospholipids (165). The nature of possible endogenous substrates was evaluated in studies that compared lipid profiles in darapladib-treated versus placebo-treated animals. In Yorkshire domestic farm pigs, the induction of 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 PLA2 activities other than Lp-PLA2 (see Section 2). Surprisingly, darapladib treatment significantly reduced this response (Table 1), which points at anti-oxidant properties or inhibitory effects of darapladib on long chain PLA2 activities with specificity for arachidonoyl-containing substrates. While 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-PLA2 substrates (33). Notably, darapladib failed to alter serum PAF levels in two murine models of atherosclerosis (167, 168). The possibility that other circulating PLA2 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 (164)]. However, it is unknown whether darapladib inhibits other PLA2 activities such as sPLA2GIII, for example (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 account 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-PLA2 inhibition. Inhibition of Lp-PLA2 activity (and substrate accumulation) is predicted to increase apoptosis through caspase activation, based on multiple rigorous laboratory investigations [see (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, while darapladib and related compounds inhibit Lp-PLA2 activity as determined using exogenous substrates in vitro, it is not clear whether enzyme inhibition accounts for observed in vivo effects. An ideal way to rigorously investigate the mechanism of darapladib action would be to test the effect of this agent in patients genetically deficient in Lp-


ww

w.jlr.org

Dow

nloaded from

http://www.jlr.org/

15

PLA2. Efficacy in this patient population would strongly suggest Lp-PLA2-independent effects. Clinical studies of this nature are difficult to implement, although safety/tolerability trials of darapladib in healthy Japanese subjects have been completed (NCT00551317 & NCT00622830). An excellent alternative would be to test this issue in mice. Since darapladib limited atherosclerosis in ApoE―/― and Ldlr―/― mice (167, 168), these models appear adequate for future investigation. Specifically, comparing the effect of darapladib in control and Lp-PLA2-deficient mice in a model of atherosclerosis (e.g., ApoE―/― or Ldlr―/―) could provide definitive answers regarding the real mechanism of darapladib action.

5.2. IN VIVO STUDIES OF LP-PLA2 OVER-EXPRESSION VERSUS DARAPLADIB ADMINISTRATION Similarly to what is observed in humans, enhanced levels of sPLA2s and Lp-PLA2 correlate

with increased atherosclerosis in animal models (95), with the exception of a study by Forte an co-workers (171). Secretory PLA2s, unlike Lp-PLA2, have been demonstrated to actively contribute to atherogenesis, and so correlate with disease status (85). An excellent review by Öörni and colleagues highlights multiple studies showing that development of atherosclerosis is decreased in mice lacking sPLA2 expression, and increased with over-expression (85). Parallel studies addressing the role of Lp-PLA2 have clearly shown that over-expression of Lp-PLA2 diminishes atherosclerosis in mice (172-175) and rabbits (172, 173, 176) (Table 2). This series of studies suggested that Lp-PLA2 may play a protective role in vascular and inflammatory diseases. Lp-PLA2-deficient mice displayed decreased intestinal inflammation in vivo (177) but the impact of Lp-PLA2 deletion on vascular inflammation in mice has not yet been reported.

Studies in which Lp-PLA2 was over-expressed in mice have recently been complemented by preclinical trials of darapladib, also in mice (167, 168). The effects of darapladib administration were similar to those observed when Lp-PLA2 was over-expressed (Table 2). Darapladib had multiple anti-inflammatory 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-inflammatory pathways relevant to atherogenesis. However, as mentioned previously, it is not clear whether the mechanism that mediates the action of darapladib in vivo occurs via Lp-PLA2. The comparisons provided in this section strongly argue against this possibility.

5.3. BIOMARKER AND IMAGING STUDIES OF LP-PLA2 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-PLA2 activity (43, 55, and 66 % inhibition at doses of 40 mg, 80 mg, and 160 mg, respectively [Table 3, (178)]. Interleukin-6 (IL-6) levels and hs-CRP were reduced by 12.3 % and 13 %, respectively, suggesting a reduction in inflammatory burden. The Integrated Biomarker and Imaging Study (IBIS-2) found no effect of darapladib on coronary atheroma deformability, plasma hs-CRP levels and other endpoints in patients with angiographically documented CAD. [Table 3, (169)]. However, assessment of lipid necrotic core by virtual histology revealed

increased size in the placebo group (4.5 7.9 mm3; p=0.009) but not in darapladib-treated

patients (0.5 13.9 mm3; p=0.71), resulting in a significant treatment difference of -5.2 mm3 [Table 3, (169)]. These findings are consistent with results from the swine model [Table 3, (165)] and suggest that darapladib stabilizes rupture-prone plaques.


ww

w.jlr.org

Dow

nloaded from

http://www.jlr.org/

16

5.4. DARAPLADIB AND CARDIOVASCULAR EVENTS: ONGOING CLINICAL TRIALS The clinical efficacy of darapladib is currently being examined in two large-scale trials that

investigate its effects on nonfatal MI, nonfatal stroke, and cardiovascular death in CHD patients. The STABILITY trial (Stabilization of Atherosclerotic Plaque by Initiation of Darapladib Therapy) is a multicenter, randomized, double blind, placebo-controlled 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 (Stabilization of plaques using darapladib thrombolysis in myocardial infarction) 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 relationships between cardiovascular events and baseline/on-trial Lp-PLA2 activity, lipid profiles, and changes in the extent of pro- and anti-inflammatory responses (6, 8). These measurements will provide key information to establish the therapeutic utility of darapladib and help determine whether its effects are mediated through Lp-PLA2 or other mechanisms.

5.5. 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 treatment (169, 178). Regardless of the mechanism whereby darapladib affects events relevant to atherosclerosis, there is no doubt that this compound inhibits Lp-PLA2 activity. Thus, an immediate issue that merits consideration is the potential impact of widespread use of this drug in populations with a large spectrum of conditions. A research team in Korea has reported that decreased Lp-PLA2 activity is beneficial in certain settings (137, 154) and it is clear that partial/complete deficiency of Lp-PLA2 does not by itself cause disease (26, 27). However, several independent studies have shown that decreased Lp-PLA2 expression in humans increases the severity of inflammatory, vascular and other conditions [reviewed in (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 underlying acute and/or chronic inflammatory conditions (e.g., asthma, anaphylaxis, sepsis) in which decreased or absent Lp-PLA2 may increase disease severity (135, 180). An example of this paradigm was reported in a clinical trial focused on type II diabetes subjects undergoing hemodialysis (181). This study serendipitously found that patients whose Lp-PLA2 activity decreased by 25 % or more over a 6-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-PLA2 in mortality. However, it provides an example of a patient population in which inhibiting Lp-PLA2 may not be advisable. In cardiovascular medicine, therapies directed at surrogate end points can be associated with excess deaths (182). The safety and efficacy of darapladib should be very carefully evaluated before potential harmful effects become widespread.

6. CONCLUSIONS It has been proposed that one of the strongest arguments in favor of a pro-atherogenic role

of Lp-PLA2 is the substantial number of epidemiological studies (66). While it is clear that there is a positive association between Lp-PLA2 activity and CAD, the strength of this association appears


ww

w.jlr.org

Dow

nloaded from

http://www.jlr.org/

17

to be almost exclusively accounted for by the fact that most of the plasma Lp-PLA2 activity associates with LDL. These observations raise questions regarding the utility of Lp-PLA2 as a bio-marker, particularly when one considers that current guidelines direct clinicians to treat at-risk patients with cholesterol-lowering drugs and that for these patients Lp-PLA2 levels no longer predict vascular risk (96, 98, 120).

It is unlikely that strategies based on inhibiting the enzymatic activity of Lp-PLA2 will be efficacious for the prevention or treatment of CVD in humans. The evidence currently available, which includes the majority of genetic analyses in humans and all over-expression studies in mice and rabbits, do not mechanistically support this rationale. Darapladib has been shown to reduce development of experimental atherosclerosis and prevent the expansion of necrotic core lesions in humans, but these effects may not be mediated by Lp-PLA2. Our ability to establish meaningful links between Lp-PLA2 and CVD depends on our willingness to go well beyond observational studies that show associations between expression and outcome.


ww

w.jlr.org

Dow

nloaded from

http://www.jlr.org/

18

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-PLA2 Agent Study Substrate source

Effect Ref.

Recombinant Lp-PLA2 (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

(165)

Endogenous

Increased AA-PC; no

change in short chain OxPL

LDL exposed to CuSO4 (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


(168)

Endogenous No change in PAF levels

Serum (Ldlr ―/―mice)

Exogenous


(167)

Endogenous No change in PAF levels

Plasma (human) Exogenous


(169)


ww

w.jlr.org

Dow

nloaded from

http://www.jlr.org/

19

TABLE 2. OVEREXPRESSION OF LP-PLA2 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-PLA2

New Zealand white rabbits

• ↓ Necrotic area

• ↓ Systolic shortening and wall thickness

• ↓ Neutrophil infiltration

(172)

Aortic injury induced by balloon

denudation

Adenovirus-mediated Lp-PLA2

gene transfer

Cholesterol-fed New Zealand white rabbits

• ↓ Intima/media ratio

• ↓ Apoptosis

(173)

Atherosclerosis

ApoE ―/― mice

• ↓ Modified LDL autoantibodies

• ↓ Neointimal area

• ↓ Atherosclerosis

(174)

• ↓ Macrophage homing to endothelium

(175)

• ↓ Aortic wall thickness (183)

Balloon-mediated

injury of carotid arteries

Normolipidemic

New Zealand white rabbits

•↓ Accumulation of oxidized lipoproteins

• ↓ Intima/media ratio • Anti-inflammatory effects • Anti-thrombotic effects • Anti-proliferative effects

(176)

Atherosclerosis

Darapladib

Ldlr ―/― mice •↓ Inflammatory burden

•↓ Plaque area

(167)

ApoE ―/― mice • ↓ Inflammatory burden

• ↓ Plaque area

(168)

DM-HC swine

• General anti-inflammatory action

• ↓ Plaque and necrotic core area

• ↓ Medial destruction

(165)

Coronary disease patients

• Halted necrotic core volume expansion

• ↔ coronary atheroma deformability

• ↔ Plaque volume

(169)


ww

w.jlr.org

Dow

nloaded from

http://www.jlr.org/

20

TABLE 3. EFFECT OF DARAPLADIB TREATMENT ON MARKERS OF INFLAMMATION IN ANIMAL MODELS OF

ATHEROSCLEROSIS AND IN HUMAN PATIENTS

In vivo model Biomarker effects Ref.

Ldlr ―/― mice

• ↓Serum Lp-PLA2 activity • ↓ Hs-CRP, IL-6, MCP-1, VCAM-1 • ↔ Serum PAF

(167)

ApoE ―/― mice

• ↓Serum Lp-PLA2 activity • ↓ Hs-CRP, IL-6, MCP-1, VCAM-1 and TNF- α • ↔ Serum PAF • ↓ Macrophage content in atherosclerotic lesions • ↑ Collagen content in atherosclerotic lesions

(168)

DM-HC swine

• ↓Plasma Lp-PLA2 activity • ↓Lesion Lp-PLA2 activity and lyso-PC content • ↓24 genes associated with macrophage and T

lymphocyte functions • ↓ Macrophage content in atherosclerotic lesions

(165)

Healthy men

• ↔ ADP-induced platelet aggregation • ↔ Collagen-induced platelet aggregation

(184)

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

• ↓Plasma Lp-PLA2

• ↓ Hs-CRP, IL-6 • ↔ MPO, MMP-9 activity, P-selectin, CD40L • ↔ HDL & LDL cholesterol

(178)

Patients with angiographically documented CHD (IBIS-2)

• ↓Plasma Lp-PLA2 • ↔ Hs-CRP, MPO, ICAM-1, MMP-9 activity, OxPL/apoB • ↔ HDL & LDL cholesterol

(169)


ww

w.jlr.org

Dow

nloaded from

http://www.jlr.org/

21

7. REFERENCES 1. Libby, P., Ridker, P.M., and Maseri, A. 2002. Inflammation and atherosclerosis.

Circulation 105:1135-1143. 2. Witztum, J.L., and Berliner, J.A. 1998. Oxidized phospholipids and isoprostanes in

atherosclerosis. Curr Opin Lipidol 9:441-448. 3. Tardif, J.C., McMurray, J.J., Klug, E., Small, R., Schumi, J., Choi, J., Cooper, J., Scott, R.,

Lewis, E.F., L'Allier, P.L., et al. 2008. Effects of succinobucol (AGI-1067) after an acute coronary syndrome: a randomised, double-blind, placebo-controlled trial. Lancet 371:1761-1768.

4. Tardif, J.C., L'Allier P, L., Ibrahim, R., Gregoire, J.C., Nozza, A., Cossette, M., Kouz, S., Lavoie, M.A., Paquin, J., Brotz, T.M., 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., Hislop C., McConnell D., Elliott M., Stasiv Y., Wang N., Waters D.D.; PLASMA Investigators. 2009. Effects of 1-H-indole-3-glyoxamide (A-002) on concentration of secretory phospholipase A2 (PLASMA study): a phase II double-blind, randomised, placebo-controlled trial. Lancet 373:649-658.

6. Rosenson, R.S. 2010. Phospholipase A2 inhibition and atherosclerotic vascular disease: prospects for targeting secretory and lipoprotein-associated phospholipase A2 enzymes. Curr Opin Lipidol 21:473-480.

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

8. Rosenson R.S. 2009. Future role for selective phospholipase A2 inhibitors in the prevention of atherosclerotic cardiovascular disease. Cardiovasc Drugs Ther 23:93-101.

9. White, H. 2010. Why inhibition of lipoprotein-associated phospholipase A2 has the potential to improve patient outcomes. Curr Opin Cardiol 25:299-301.

10. Garza, C.A., Montori, V.M., McConnell, J.P., Somers, V.K., Kullo, I.J., and Lopez-Jimenez, F. 2007. Association between lipoprotein-associated phospholipase A2 and cardiovascular disease: a systematic review. Mayo Clin Proc 82:159-165.

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

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

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

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

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

16. Burke, J.E., and Dennis, E.A. 2009. Phospholipase A2 biochemistry. Cardiovasc Drugs Ther 23:49-59.


ww

w.jlr.org

Dow

nloaded from

http://www.jlr.org/

22

17. Schaloske, R.H., and Dennis, E.A. 2006. The phospholipase A2 superfamily and its group numbering system. Biochim Biophys Acta 1761:1246-1259.

18. Farr, R.S., Cox, C.P., Wardlow, M.L., and Jorgensen, R. 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., Zimmerman, G.A., Stafforini, D.M., and McIntyre, T.M. 2000. Platelet-activating factor and related lipid mediators. Annu Rev Biochem 69:419-445.

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

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

22. Stafforini, D.M., McIntyre, T.M., Carter, M.E., and Prescott, S.M. 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., Hermetter, A., Kostner, G.M., and Deigner, H.P. 1995. Enhanced association of platelet-activating factor acetylhydrolase with lipoprotein (a) in comparison with low density lipoprotein. J Biol Chem 270:31151-31157.

24. Tjoelker, L.W., Eberhardt, C., Unger, J., Trong, H.L., Zimmerman, G.A., McIntyre, T.M., Stafforini, D.M., Prescott, S.M., and Gray, P.W. 1995. Plasma platelet-activating factor acetylhydrolase is a secreted phospholipase A2 with a catalytic triad. J Biol Chem 270:25481-25487.

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

26. Stafforini, D.M. 2009. Biology of platelet-activating factor acetylhydrolase (PAF-AH, lipoprotein associated phospholipase A2). Cardiovasc Drugs Ther 23:73-83.

27. McIntyre, T.M., Prescott, S.M., and Stafforini, D.M. 2009. The emerging roles of PAF acetylhydrolase. J Lipid Res 50 Suppl:S255-259.

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

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

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

31. Kriska, T., Marathe, G.K., Schmidt, J.C., McIntyre, T.M., and Girotti, A.W. 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., Sheller, J.R., Blackwell, T.S., Sapirstein, A., Yull, F.E., McIntyre, T.M., Bonventre, J.V., Prescott, S.M., and Roberts, L.J., 2nd. 2006. Release of free F2-isoprostanes from esterified phospholipids is catalyzed by intracellular and plasma platelet-activating factor acetylhydrolases. J Biol Chem 281:4616-4623.

33. Davis, B., Koster, G., Douet, L.J., Scigelova, M., Woffendin, G., Ward, J.M., Smith, A., Humphries, J., Burnand, K.G., Macphee, C.H., et al. 2008. Electrospray ionization mass


ww

w.jlr.org

Dow

nloaded from

http://www.jlr.org/

23

spectrometry identifies substrates and products of lipoprotein-associated phospholipase A2 in oxidized human low density lipoprotein. J Biol Chem 283:6428-6437.

34. Cao, J., Hsu, Y.H., Li, S., Woods, V.L., and Dennis, E.A. 2011. Lipoprotein-associated phospholipase A2 interacts with phospholipid vesicles via a surface-disposed hydrophobic alpha-helix. Biochemistry 50:5314-5321.

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

36. Tsimihodimos, V., Karabina, S.A., Tambaki, A.P., Bairaktari, E., Miltiadous, G., Goudevenos, J.A., Cariolou, M.A., Chapman, M.J., Tselepis, A.D., and Elisaf, M. 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., Miura, S., Zhang, B., Uehara, Y., Matsuo, K., Kumagai, K., and Saku, K. 2007. Ratio of LDL- to HDL-associated platelet-activating factor acetylhydrolase may be a marker of inflammation in patients with paroxysmal atrial fibrillation. Circ J 71:214-219.

38. Papavasiliou, E.C., Gouva, C., Siamopoulos, K.C., and Tselepis, A.D. 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., Tjoelker, L.W., McCormick, S.P., Vaitkus, D., McIntyre, T.M., Gray, P.W., Young, S.G., and Prescott, S.M. 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., Reichert, E.C., Topham, M.K., and Stafforini, D.M. 2008. Identification of a domain that mediates association of platelet-activating factor acetylhydrolase with high density lipoprotein. J Biol Chem 283:17099-17106.

41. Tselepis, A.D., and John Chapman, M. 2002. Inflammation, bioactive lipids and atherosclerosis: potential roles of a lipoprotein-associated phospholipase A2, platelet activating factor-acetylhydrolase. Atheroscler Suppl 3:57-68.

42. Linton, M.F., and Fazio, S. 2003. Macrophages, inflammation, and atherosclerosis. Int J Obes Relat Metab Disord 27 Suppl 3:S35-40.

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

44. Ferguson, J.F., Hinkle, C.C., Mehta, N.N., Bagheri, R., Derohannessian, S.L., Shah, R., Mucksavage, M.I., Bradfield, J.P., Hakonarson, H., Wang, X., et al. 2012. Translational studies of lipoprotein-associated phospholipase A2 in inflammation and atherosclerosis. J Am Coll Cardiol 59:764-772.

45. Mannheim, D., Herrmann, J., Versari, D., Gossl, M., Meyer, F.B., McConnell, J.P., Lerman, L.O., and Lerman, A. 2008. Enhanced expression of Lp-PLA2 and lysophosphatidylcholine in symptomatic carotid atherosclerotic plaques. Stroke 39:1448-1455.

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

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


ww

w.jlr.org

Dow

nloaded from

http://www.jlr.org/

24

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

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

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

51. Wu, X., Zimmerman, G.A., Prescott, S.M., and Stafforini, D.M. 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., Zalewski, A., Macphee, C.H., and Dawson, M. 2007. Selective Inhibition of Lipoprotein-associated phospholipase A2 attenuates markers of plaque vulnerability in humans. Circulation 116:II-108.

53. Satoh, K., Imaizumi, T., Kawamura, Y., Yoshida, H., Hiramoto, M., Takamatsu, S., and Takamatsu, M. 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., Xiong, S.Q., Mei, L.J., Chen, H.Y., Wang, T., and Chu, J.F. 2012. 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. Inflammation March 20 [Epub ahead of print].

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

56. Grallert, H., Dupuis, J., Bis, J.C., Dehghan, A., Barbalic, M., Baumert, J., Lu, C., Smith, N.L., Uitterlinden, A.G., Roberts, R., et al. 2012. Eight genetic loci associated with variation in lipoprotein-associated phospholipase A2 mass and activity and coronary heart disease: meta-analysis of genome-wide association studies from five community-based studies. Eur Heart J 33:238-251.

57. Suchindran, S., Rivedal, D., Guyton, J.R., Milledge, T., Gao, X., Benjamin, A., Rowell, J., Ginsburg, G.S., and McCarthy, J.J. 2010. Genome-wide association study of Lp-PLA2 activity and mass in the Framingham Heart Study. PLoS Genet 6:e1000928.

58. McConnell, J.P., and Hoefner, D.M. 2006. Lipoprotein-associated phospholipase A2. Clin Lab Med 26:679-697.

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

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

61. Lusis, A.J. 2000. Atherosclerosis. Nature 407:233-241.


ww

w.jlr.org

Dow

nloaded from

http://www.jlr.org/

25

62. Subbanagounder, G., Wong, J.W., Lee, H., Faull, K.F., Miller, E., Witztum, J.L., and Berliner, J.A. 2002. Epoxyisoprostane and epoxycyclopentenone 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., Poliakov, E., Shen, Z., Zhang, R., Deng, Y., Sun, M., Finton, P.J., Shan, L., Gugiu, B., Fox, P.L., et al. 2002. Identification 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., Zhang, R., Greenberg, M.E., Sun, M., Chen, X., Levison, B.S., Salomon, R.G., and Hazen, S.L. 2006. Phospholipid hydroxyalkenals, a subset of recently discovered endogenous CD36 ligands, spontaneously generate novel furan-containing phospholipids lacking CD36 binding activity in vivo. J Biol Chem 281:31298-31308.

65. McIntyre, T.M. 2012. Bioactive oxidatively truncated phospholipids in inflammation and apoptosis: Formation, targets, and inactivation. Biochim Biophys Acta Mar 16 [Epub ahead of print].

66. Suckling, K. 2010. Phospholipase A2s: developing drug targets for atherosclerosis. Atherosclerosis 212:357-366.

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

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

69. Berliner, J.A., Leitinger, N., and Tsimikas, S. 2009. The role of oxidized phospholipids in atherosclerosis. J Lipid Res 50 Suppl:S207-212.

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

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

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

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

74. Chen, G., Li, J., Qiang, X., Czura, C.J., Ochani, M., Ochani, K., Ulloa, L., Yang, H., Tracey, K.J., Wang, P., 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., Moores, K.E., Boyd, H.F., Dhanak, D., Ife, R.J., Leach, C.A., Leake, D.S., Milliner, K.J., Patterson, R.A., Suckling, K.E., et al. 1999. Lipoprotein-associated phospholipase A2, 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.


ww

w.jlr.org

Dow

nloaded from

http://www.jlr.org/

26

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

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

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

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

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

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

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

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

84. Cunningham, T.J., Yao, L., and Lucena, A. 2008. Product inhibition of secreted phospholipase A2 may explain lysophosphatidylcholines' unexpected therapeutic properties. J Inflamm (Lond) 5:17-26.

85. Öörni, K., and Kovanen, P.T. 2009. Lipoprotein modification by secretory phospholipase A2 enzymes contributes to the initiation and progression of atherosclerosis. Curr Opin Lipidol 20:421-427.

86. Edwards, I.J., Berquin, I.M., Sun, H., O'Flaherty J, T., Daniel, L.W., Thomas, M.J., Rudel, L.L., Wykle, R.L., and Chen, Y.Q. 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 Greene, D.J. 2000. The capacity of various non-esterified 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., Sigari, F., Segrado, T., Horkko, S., Hama, S., Subbaiah, P.V., Miwa, M., Navab, M., Witztum, J.L., and Reaven, P.D. 1999. All ApoB-containing lipoproteins induce monocyte chemotaxis and adhesion when minimally modified. Modulation of lipoprotein bioactivity by platelet-activating factor acetylhydrolase. Arterioscler Thromb Vasc Biol 19:1437-1446.

89. Yang, M., Chu, E.M., Caslake, M.J., Edelstein, C., Scanu, A.M., and Hill, J.S. 2010. Lipoprotein-associated phospholipase A2 decreases oxidized lipoprotein cellular


ww

w.jlr.org

Dow

nloaded from

http://www.jlr.org/

27

association by human macrophages and hepatocytes. Biochim Biophys Acta 1801:176-182.

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

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

92. Packard, C.J., O'Reilly, D.S., Caslake, M.J., McMahon, A.D., Ford, I., Cooney, J., Macphee, C.H., Suckling, K.E., Krishna, M., Wilkinson, F.E., et al. 2000. Lipoprotein-associated phospholipase A2 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., H., Qi, Y., Zhao, F., and Zhao, D. 2011. Systematic review of the association between lipoprotein – associated phospholipase A2 and atherosclerosis. N A J Med Sci. 4:201-211.

94. Thompson, A., Gao, P., Orfei, L., Watson, S., Di Angelantonio, E., Kaptoge, S., Ballantyne, C., Cannon, C.P., Criqui, M., Cushman, M., et al. 2010. Lipoprotein-associated phospholipase A2 and risk of coronary disease, stroke, and mortality: collaborative analysis of 32 prospective studies. Lancet 375:1536-1544.

95. Mallat, Z., Lambeau, G., and Tedgui, A. 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 A2 measurements: mass, activity, but little productivity. Clin Chem 58:814-817.

97. McConnell, J.P., and Jaffe, A.S. 2008. Variability of lipoprotein-associated phospholipase A2 measurements. Clin Chem 54:932-933.

98. Ridker, P.M., Macfadyen, J.G., Wolfert, R.L., and Koenig, W. 2012. Relationship of Lipoprotein-associated phospholipase A2 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., Khuseyinova,N., Lowel, H., Trischler, G., and Meisinger, C. 2004. Lipoprotein-associated phospholipase A2 adds to risk prediction of incident coronary events by C-reactive protein in apparently healthy middle-aged men from the general population: results from the 14-year follow-up of a large cohort from southern Germany. Circulation 110:1903-1908.

100. Oei, H.H., van der Meer, I.M., Hofman, A., Koudstaal, P.J., Stijnen, T., Breteler, M.M., and Witteman, J.C. 2005. Lipoprotein-associated phospholipase A2 activity is associated with risk of coronary heart disease and ischemic stroke: the Rotterdam Study. Circulation 111: 570-575.

101. Daniels, L.B., Laughlin, G.A., Sarno, M.J., Bettencourt, R., Wolfert, R.L., and Barrett-Connor, E. 2008. Lipoprotein-associated phospholipase A2 is an independent predictor of incident coronary heart disease in an apparently healthy older population: the Rancho Bernardo Study. J Am Coll Cardiol 51:913-919.

102. Kiechl, S., Willeit, J., Mayr, M., Viehweider, B., Oberhollenzer, M., Kronenberg, F., Wiedermann, C.J., Oberthaler, S., Xu, Q., Witztum, J.L., et al. 2007. Oxidized phospholipids, lipoprotein(a), lipoprotein-associated phospholipase A2 activity, and 10-


ww

w.jlr.org

Dow

nloaded from

http://www.jlr.org/

28

year cardiovascular outcomes: prospective results from the Bruneck study. Arterioscler Thromb Vasc Biol 27:1788-1795.

103. Blake, G.J., Dada, N., Fox, J.C., Manson, J.E., and Ridker, P.M. 2001. A prospective evaluation of lipoprotein-associated phospholipase A2 levels and the risk of future cardiovascular events in women. J Am Coll Cardiol 38:1302-1306.

104. Ballantyne, C.M., Hoogeveen, R.C., Bang, H., Coresh, J., Folsom, A.R., Heiss, G., and Sharrett, A.R. 2004. Lipoprotein-associated phospholipase A2, 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., Arsenault, B.J., Despres, J.P., Cote, M., Talmud, P.J., Ninio, E., Wouter Jukema, J., Wareham, N.J., Kastelein, J.J., Khaw, K.T., et al. 2011. Inflammatory 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., Newton-Cheh, C., Almgren, P., Hedblad, B., Berglund, G., Engstrom, G., Persson, M., Smith, J.G., Magnusson, M., Christensson, A., et al. 2009. Novel and conventional biomarkers for prediction of incident cardiovascular events in the community. JAMA 302:49-57.

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

108. Hatoum, I.J., Cook, N.R., Nelson, J.J., Rexrode, K.M., and Rimm, E.B. 2011. Lipoprotein-associated phospholipase A2 activity improves risk discrimination of incident coronary heart disease among women. Am Heart J 161:516-522.

109. Koenig, W., Twardella, D., Brenner, H., and Rothenbacher, D. 2006. Lipoprotein-associated phospholipase A2 predicts future cardiovascular events in patients with coronary heart disease independently of traditional risk factors, markers of inflammation, renal function, and hemodynamic stress. Arterioscler Thromb Vasc Biol 26:1586-1593.

110. Brilakis, E.S., McConnell, J.P., Lennon, R.J., Elesber, A.A., Meyer, J.G., and Berger, P.B. 2005. Association of lipoprotein-associated phospholipase A2 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., Morrow, D.A., O'Donoghue, M., Jablonksi, K.A., Rice, M.M., Solomon, S., Rosenberg, Y., Domanski, M.J., and Hsia, J. 2007. Prognostic utility of lipoprotein-associated phospholipase A2 for cardiovascular outcomes in patients with stable coronary artery disease. Arterioscler Thromb Vasc Biol 27:2463-2469.

112. May, H.T., Horne, B.D., Anderson, J.L., Wolfert, R.L., Muhlestein, J.B., Renlund, D.G., Clarke, J.L., Kolek, M.J., Bair, T.L., Pearson, R.R., et al. 2006. Lipoprotein-associated phospholipase A2 independently predicts the angiographic diagnosis of coronary artery disease and coronary death. Am Heart J 152:997-1003.

113. Winkler, K., Hoffmann, M.M., Winkelmann, B.R., Friedrich, I., Schafer, G., Seelhorst, U., Wellnitz, B., Wieland, H., Boehm, B.O., and Marz, W. 2007. Lipoprotein-associated phospholipase A2 predicts 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.


ww

w.jlr.org

Dow

nloaded from

http://www.jlr.org/

29

114. Mockel, M., Muller, R., Vollert, J.O., Muller, C., Danne, O., Gareis, R., Stork, T., Dietz, R., and Koenig, W. 2007. Lipoprotein-associated phospholipase A2 for early risk stratification in patients with suspected 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., McConnell, J.P., Jaffe, A.S., Weston, S.A., Killian, J.M., and Roger, V.L. 2006. Lipoprotein-associated phospholipase A2 and prognosis after myocardial infarction in the community. Arterioscler Thromb Vasc Biol 26:2517-2522.

116. Oldgren, J., James, S.K., Siegbahn, A., and Wallentin, L. 2007. Lipoprotein-associated phospholipase A2 does not predict mortality or new ischaemic events in acute coronary syndrome patients. Eur Heart J 28:699-704.

117. O'Donoghue, M., Morrow, D.A., Sabatine, M.S., Murphy, S.A., McCabe, C.H., Cannon, C.P., and Braunwald, E. 2006. Lipoprotein-associated phospholipase A2 and its association with cardiovascular 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., Mallat, Z., Benessiano, J., Tedgui, A., Olsson, A.G., Bao, W., Schwartz, G.G., and Tsimikas, S. 2012. Phospholipase A2 Enzymes, 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 A2 activity and mass in relation to vascular disease and nonvascular mortality. J Intern Med 268:348-358.

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

121. Hakkinen, T., Luoma, J.S., Hiltunen, M.O., Macphee, C.H., Milliner, K.J., Patel, L., Rice, S.Q., Tew, D.G., Karkola, K., and Yla-Herttuala, S. 1999. Lipoprotein-associated phospholipase A2, platelet-activating factor acetylhydrolase, is expressed by macrophages in human and rabbit atherosclerotic lesions. Arterioscler Thromb Vasc Biol 19:2909-2917.

122. Kolodgie, F.D., Burke, A.P., Skorija, K.S., Ladich, E., Kutys, R., Makuria, A.T., and Virmani, R. 2006. Lipoprotein-associated phospholipase A2 protein expression in the natural progression of human coronary atherosclerosis. Arterioscler Thromb Vasc Biol 26:2523-2529.

123. Sarlon-Bartoli, G., Boudes, A., Buffat, C., Bartoli, M.A., Piercecchi-Marti, M.D., Sarlon, E., Arnaud, L., Bennis, Y., Thevenin, B., Squarcioni, C., et al. 2011. Circulating lipoprotein-associated phospholipase A2 in high-grade carotid stenosis: a new biomarker for predicting unstable plaque. Eur J Vasc Endovasc Surg 43:154-159.

124. Elkind, M.S., Tai, W., Coates, K., Paik, M.C., and Sacco, R.L. 2009. Lipoprotein-associated phospholipase A2 activity and risk of recurrent stroke. Cerebrovasc Dis 27:42-50.

125. Dohi, T., Miyauchi, K., Okazaki, S., Yokoyama, T., Ohkawa, R., Nakamura, K., Yanagisawa, N., Tsuboi, S., Ogita, M., Yokoyama, K., et al. 2011. Decreased circulating lipoprotein-associated phospholipase A2 levels are associated with coronary plaque regression in patients with acute coronary syndrome. Atherosclerosis 219:907-912.

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


ww

w.jlr.org

Dow

nloaded from

http://www.jlr.org/

30

127. Zelko, I.N., Mariani, T.J., and Folz, R.J. 2002. Superoxide dismutase 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., Tengattini, S., Fabiano, A., Bianchi, R., and Rezzani, R. 2008. Atherosclerosis and oxidative stress. Histol Histopathol 23:381-390.

129. Rosenson, R.S. 2010. Lp-PLA2 and risk of atherosclerotic vascular disease. Lancet 375:1498-1500.

130. Steinberg, D.I., and Mayer, A. 2007. Lipoprotein-associated phospholipase A2 and risk stratification for cardiovascular disease: not ready for "prime time". Mayo Clin Proc 82:155-157.

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

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

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

134. Yamada, Y., Yoshida, H., Ichihara, S., Imaizumi, T., Satoh, K., and Yokota, M. 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., Numao, T., Tsodikov, A., Vaitkus, D., Fukuda, T., Watanabe, N., Fueki, N., McIntyre, T.M., Zimmerman, G.A., Makino, S., et al. 1999. Deficiency of platelet-activating factor acetylhydrolase is a severity factor for asthma. J Clin Invest 103:989-997.

136. Zhang, S.Y., Shibata, H., Karino, K., Wang, B.Y., Kobayashi, S., Masuda, J., and Nabika, T. 2007. Comprehensive evaluation of genetic and environmental factors influencing the plasma lipoprotein-associated phospholipase A2 activity in a Japanese population. Hypertens Res 30:403-409.

137. Jang, Y., Kim, O.Y., Koh, S.J., Chae, J.S., Ko, Y.G., Kim, J.Y., Cho, H., Jeong, T.S., Lee, W.S., Ordovas, J.M., et al. 2006. The Val279Phe variant of the lipoprotein-associated phospholipase A2 gene is associated with catalytic activities and cardiovascular disease in Korean men. J Clin Endocrinol Metab 91:3521-3527.

138. Liu, P.Y., Li, Y.H., Wu, H.L., Chao, T.H., Tsai, L.M., Lin, L.J., Shi, G.Y., and Chen, J.H. 2006. Platelet-activating factor-acetylhydrolase 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., Chen, H.Y., Xiong, S.Q., and Chu, J.F. 2011. Lipoprotein-associated phospholipase A2 gene V279F polymorphisms and coronary heart disease: a meta-analysis. Mol Biol Rep 38:4089-4099.

140. Hou, L., Chen, S., Yu, H., Lu, X., Chen, J., Wang, L., Huang, J., Fan, Z., and Gu, D. 2009. Associations of PLA2G7 gene polymorphisms with plasma lipoprotein-associated phospholipase A2 activity and coronary heart disease in a Chinese Han population: the Beijing atherosclerosis study. Hum Genet 125:11-20.


ww

w.jlr.org

Dow

nloaded from

http://www.jlr.org/

31

141. Balta, G., Gurgey, A., Kudayarov, D.K., Tunc, B., and Altay, C. 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., Nelson, M.R., Aponte, J., Manas, E.S., Bacanu, S.A., Yuan, X., Kong, X., Cardon, L., Mooser, V.E., Whittaker, J.C., et al. 2011. Sequencing of Lp-PLA2-encoding PLA2G7 gene in 2000 Europeans reveals several rare loss-of-function mutations. Pharmacogenomics J. May 24 [Epub ahead of print].

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

145. Ishihara, M., Iwasaki, T., Nagano, M., Ishii, J., Takano, M., Kujiraoka, T., Tsuji, M., Hattori, H., and Emi, M. 2004. Functional impairment of two novel mutations detected in lipoprotein-associated phospholipase A2 (Lp-PLA2) deficiency patients. J Hum Genet 49:302-307.

146. Kujiraoka, T., Iwasaki, T., Ishihara, M., Ito, M., Nagano, M., Kawaguchi, A., Takahashi, S., Ishi, J., Tsuji, M., Egashira, T., 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., Ichihara, S., Fujimura, T., and Yokota, M. 1998. Identification 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., Izawa, H., Ichihara, S., Takatsu, F., Ishihara, H., Hirayama, H., Sone, T., Tanaka, M., and Yokota, M. 2002. Prediction of the risk of myocardial infarction from polymorphisms in candidate genes. N Engl J Med 347:1916-1923.

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

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

151. Shimokata, K., Yamada, Y., Kondo, T., Ichihara, S., Izawa, H., Nagata, K., Murohara, T., Ohno, M., and Yokota, M. 2004. Association of gene polymorphisms with coronary artery disease in individuals with or without nonfamilial hypercholesterolemia. Atherosclerosis 172:167-173.

152. Wang, T., Karino, K., Yamasaki, M., Zhang, Y., Masuda, J., Yamaguchi, S., Shiwaku, K., and Nabika, T. 2009. Effects of G994T in the Lp-PLA2 gene on the plasma oxidized LDL level and carotid intima-media thickness in Japanese: the Shimane study. Am J Hypertens 22:742-747.

153. Li, L., Qi, L., Lv, N., Gao, Q., Cheng, Y., Wei, Y., Ye, J., Yan, X., and Dang, A. 2011. Association between lipoprotein-associated phospholipase A2 gene polymorphism and coronary artery disease in the Chinese Han population. Ann Hum Genet 75:605-611.

154. Jang, Y., Waterworth, D., Lee, J.E., Song, K., Kim, S., Kim, H.S., Park, K.W., Cho, H.J., Oh, I.Y., Park, J.E., et al. 2011. Carriage of the V279F null allele within the gene encoding Lp-


ww

w.jlr.org

Dow

nloaded from

http://www.jlr.org/

32

PLA2 is protective from coronary artery disease in South Korean males. PLoS One 6:e18208.

155. Paik, J.K., Chae, J.S., Jang, Y., Kim, J.Y., Kim, O.Y., Jeong, T.S., Lee, S.H., and Lee, J.H. 2010. Effects of V279F in the Lp-PLA2 gene on markers of oxidative stress and inflammation in Koreans. Clin Chim Acta 411:486-493.

156. Casas, J.P., Ninio, E., Panayiotou, A., Palmen, J., Cooper, J.A., Ricketts, S.L., Sofat, R., Nicolaides, A.N., Corsetti, J.P., Fowkes, F.G., et al. 2010. PLA2G7 genotype, lipoprotein-associated phospholipase A2 activity, and coronary heart disease risk in 10 494 cases and 15 624 controls of European Ancestry. Circulation 121:2284-2293.

157. Kruse, S., Mao, X.Q., Heinzmann, A., Blattmann, S., Roberts, M.H., Braun, S., Gao, P.S., Forster, J., Kuehr, J., Hopkin, J.M., 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., Hawe, E., Humphries, S.E., and Talmud, P.J. 2003. Association between the Ala379Val variant of the lipoprotein associated phospholipase A2 and risk of myocardial infarction in the north and south of Europe. Atherosclerosis 168:283-288.

159. Macphee, C.H., Nelson, J., and Zalewski, A. 2006. Role of lipoprotein-associated phospholipase A2 in atherosclerosis and its potential as a therapeutic target. Curr Opin Pharmacol 6:154-161.

160. Zalewski, A., Macphee, C., and Nelson, J.J. 2005. Lipoprotein-associated phospholipase A2: a potential therapeutic target for atherosclerosis. Curr Drug Targets Cardiovasc Haematol Disord 5:527-532.

161. Blackie, J.A., Bloomer, J.C., Brown, M.J., Cheng, H.Y., Hammond, B., Hickey, D.M., Ife, R.J., Leach, C.A., Lewis, V.A., Macphee, C.H., et al. 2003. The identification of clinical candidate SB-480848: a potent inhibitor of lipoprotein-associated phospholipase A2. Bioorg Med Chem Lett 13:1067-1070.

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

163. O'Donoghue, M.L., Braunwald, E., White, H.D., Serruys, P., Steg, P.G., Hochman, J., Maggioni, A.P., Bode, C., Weaver, D., Johnson, J.L., 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 e611.

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

165. Wilensky, R.L., Shi, Y., Mohler, E.R., 3rd, Hamamdzic, D., Burgert, M.E., Li, J., Postle, A., Fenning, R.S., Bollinger, J.G., Hoffman, B.E., et al. 2008. Inhibition of lipoprotein-associated phospholipase A2 reduces complex coronary atherosclerotic plaque development. Nat Med 14:1059-1066.

166. Rosenson, R.S., Vracar-Grabar, M., and Helenowski, I. 2008. Lipoprotein associated phospholipase A2 inhibition reduces generation of oxidized fatty acids: Lp-PLA2 reduces oxidized fatty acids. Cardiovasc Drugs Ther 22:55-58.

167. Hu, M.M., Zhang, J., Wang, W.Y., Wu, W.Y., Ma, Y.L., Chen, W.H., and Wang, Y.P. 2011. The inhibition of lipoprotein-associated phospholipase A2 exerts beneficial effects against atherosclerosis in LDLR-deficient mice. Acta Pharmacol Sin 32:1253-1258.


ww

w.jlr.org

Dow

nloaded from

http://www.jlr.org/

33

168. Wang, W.Y., Zhang, J., Wu, W.Y., Li, J., Ma, Y.L., Chen, W.H., Yan, H., Wang, K., Xu, W.W., Shen, J.H., et al. 2011. Inhibition of lipoprotein-associated phospholipase A2 ameliorates inflammation and decreases atherosclerotic plaque formation in ApoE-deficient mice. PLoS One 6:e23425.

169. Serruys, P.W., Garcia-Garcia, H.M., Buszman, P., Erne, P., Verheye, S., Aschermann, M., Duckers, H., Bleie, O., Dudek, D., Botker, H.E., et al. 2008. Effects of the direct lipoprotein-associated phospholipase A2 inhibitor darapladib on human coronary atherosclerotic plaque. Circulation 118:1172-1182.

170. Carpenter, K.L., Dennis, I.F., Challis, I.R., Osborn, D.P., Macphee, C.H., Leake, D.S., Arends, M.J., and Mitchinson, M.J. 2001. Inhibition of lipoprotein-associated phospholipase A2 diminishes the death-inducing effects of oxidised LDL on human monocyte-macrophages. FEBS Lett 505:357-363.

171. Forte, T.M., Subbanagounder, G., Berliner, J.A., Blanche, P.J., Clermont, A.O., Jia, Z., Oda, M.N., Krauss, R.M., and Bielicki, J.K. 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., Boyle, E.M., Jr., Yun, W., Kovacich, J.C., Canty, T.G., Jr., Chi, E., Pohlman, T.H., and Verrier, E.D. 1999. Platelet-activating factor acetylhydrolase prevents myocardial ischemia-reperfusion injury. Circulation 100:II365-368.

173. Turunen, P., Puhakka, H., Rutanen, J., Hiltunen, M.O., Heikura, T., Gruchala, M., and Yla-Herttuala, S. 2005. Intravascular adenovirus-mediated lipoprotein-associated phospholipase A2 gene transfer reduces neointima formation in balloon-denuded rabbit aorta. Atherosclerosis 179:27-33.

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

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

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

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

178. Mohler, E.R., 3rd, Ballantyne, C.M., Davidson, M.H., Hanefeld, M., Ruilope, L.M., Johnson, J.L., and Zalewski, A. 2008. The effect of darapladib on plasma lipoprotein-associated phospholipase A2 activity and cardiovascular biomarkers in patients with stable coronary heart disease or coronary heart disease risk equivalent: the results of a multicenter, randomized, 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.


ww

w.jlr.org

Dow

nloaded from

http://www.jlr.org/

34

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

181. Winkler, K., Hoffmann, M.M., Krane, V., Drechsler, C., and Wanner, C. 2011. Lipoprotein-associated phospholipase A2 and outcome in patients with type 2 diabetes on haemodialysis. Eur J Clin Invest Dec 13 [Epub ahead of print].

182. Topol, E.J. 2005. Nesiritide - not verified. N Engl J Med 353:113-116. 183. Hase, M., Tanaka, M., Yokota, M., and Yamada, Y. 2002. Reduction in the extent of

atherosclerosis in apolipoprotein E-deficient 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., Gao, L., Cai , G., Wentzell, L.M., and Johnson, A.G. 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.


ww

w.jlr.org

Dow

nloaded from

http://www.jlr.org/

lipoprotein associated phospholipase a2: an investigation into one

Documents