Serum Levels of Type II Secretory Phospholipase A2 and the Risk of Future Coronary Artery Disease in Apparently Healthy Men and Women

From the Departments of Cardiology (S.M.B.) and Vascular Medicine (T.T.K., J.J.P.K.), Academic Medical Center, Amsterdam, The Netherlands; Medical Research Council Epidemiology Unit (N.J.W.), Cambridge, the Department of Public Health and Primary Care (R.L., N.E.D., M.S.S., K.T.K.), Institute of Public Health, University of Cambridge, and the Medical Research Council Dunn Nutrition Unit (S.A.B.), Cambridge, United Kingdom; and the Department of Cardiology (J.W.J.), Leiden University Medical Center, Leiden and Sanquin Research at the CLB and Department of Clinical Chemistry (C.E.H.), VU Medical Center, Amsterdam, The Netherlands.

Correspondence to Kay-Tee Khaw, Clinical Gerontology Unit, Box 251 Addenbrooke’s Hospital, University of Cambridge School of Clinical Medicine, Cambridge CB2 2QQ United Kingdom. E-mail kk101@medschl.cam.ac.uk

    Abstract

Objectives— To study the prospective relationship between serum levels of type II secretory phospholipase A2 (sPLA2) and the risk of future coronary artery disease (CAD) in apparently healthy men and women.

Methods and Results— We conducted a prospective nested case-control study among apparently healthy men and women aged 45 to 79 years. Cases (n=1105) were people in whom fatal or nonfatal CAD developed during follow-up. Controls (n=2209) were matched by age, sex, and enrollment time. sPLA2 levels were significantly higher in cases than controls (9.5 ng/mL; interquartile range , 6.4 to 14.8 versus 8.3 ng/mL; IQR, 5.8 to 12.6; P<0.0001). sPLA2 plasma levels significantly correlated with age, body mass index, systolic blood pressure, high-density lipoprotein (HDL) cholesterol levels, and C-reactive protein (CRP) levels. Taking into account matching for sex and age and adjusting for body mass index, smoking, diabetes, systolic blood pressure, low-density lipoprotein cholesterol, HDL cholesterol, and CRP levels, the risk of future CAD was 1.34 (1.02 to 1.71; P=0.02) for people in the highest sPLA2 quartile, compared with those in the lowest (P for linearity=0.03).

Conclusion— Elevated levels of sPLA2 were associated with an increased risk of future CAD in apparently healthy individuals. The magnitude of the association was similar to that observed between CRP and CAD risk, and both associations were independent.

A prospective case-control study was performed to investigate the relationships between levels of secretory phospholipase A2 and the risk of coronary artery disease (CAD). Adjusted for traditional risk factors and C-reactive protein levels, the odds ratio for future CAD was 1.34 for people in the highest quartile (P for linearity=0.03).

Key Words: coronary artery disease ? phospholipase A2 ? oxidation ? inflammation

    Introduction

Inflammation is increasingly considered to play an important role in atherosclerosis.1 Several inflammatory plasma markers, including C-reactive protein (CRP),2 IL-6,3 and IL-8,4 have been shown to predict future coronary artery disease (CAD) in apparently healthy individuals. The retention of low-density lipoprotein (LDL) particles in the arterial wall and their subsequent modification and oxidation could well be the first and key stimulus that elicits this inflammatory process.

Secretory phospholipase A2 (sPLA2) is a member of a family of intracellular and secretory enzymes that can hydrolyze the sn-2 ester bond of phospholipids of cell membranes and lipoproteins.5 sPLA2 has been implicated in atherogenesis in several ways. First, treatment with sPLA2 modifies LDL lipoproteins such that they have a higher affinity for extracellular matrix proteins,67,8 resulting in an increased retention of LDL particles in the arterial wall, which is an early hallmark of atherosclerotic lesion progression.9–11 Second, the sPLA2-mediated hydrolysis of phospholipids yields, among others, lysophospholipids and free fatty acids such as arachidonic acid, known precursors of various proinflammatory mediators such as leukotrienes and prostaglandins.12 Third, sPLA2 has been shown to yield lipoproteins that are more susceptible to lipid peroxidation13 and to generate more bioactive phospholipids.14 Circulating levels of sPLA2 are higher in patients who had documented CAD than in controls.15,16 In addition, a small study has shown that CAD patients with high sPLA2 levels were at an increased risk for recurrent CAD events during follow-up.15 However, thus far, this relationship has not been examined in a large prospective study.

We hypothesized that among apparently healthy men and women, high serum concentrations of sPLA2 are associated with an increased risk of future CAD. We tested this hypothesis in a large prospective case-control study nested in the European Prospective Investigation into Cancer in Norfolk (EPIC-Norfolk) prospective population study.

    Methods

We performed a nested case-control study among participants of the EPIC-Norfolk (European Prospective Investigation into Cancer and Nutrition) study, a prospective population study of 25 663 men and women aged between 45 and 79 years, resident in Norfolk, UK, who completed a baseline questionnaire survey and attended a clinic visit.17 Participants were recruited from age–sex registers of general practices in Norfolk as part of the 10-country collaborative EPIC study designed to investigate dietary and other determinants of cancer. Additional data were obtained in EPIC-Norfolk to enable the assessment of determinants of other diseases.

The design and methods of the study have been described in detail.17 In short, eligible participants were recruited by mail. At the baseline survey between 1993 and 1997, participants completed a detailed health and lifestyle questionnaire. Blood was taken by venepuncture into plain and citrate bottles. Blood samples were processed for assay at the Department of Clinical Biochemistry, University of Cambridge, or stored at –80°C. All individuals have been flagged for death certification at the UK Office of National Statistics, with vital status ascertained for the entire cohort. In addition, participants admitted to hospital were identified using their unique National Health Service number by data linkage with ENCORE (East Norfolk Health Authority database), which identifies all hospital contacts throughout England and Wales for Norfolk residents. Participants were identified as having CAD during follow-up if they had a hospital admission and/or died with CAD as the underlying cause. CAD was defined as codes 410 to 414 according to the International Classification of Diseases 9th revision. These codes encompass the clinical spectrum of CAD, ie, unstable angina, stable angina, and myocardial infarction. We report results with follow-up up to January 2003, an average of 6 years. The study was approved by the Norwich District Health Authority Ethics Committee and all participants gave signed informed consent.

Participants

We have previously described a similarly designed nested case-control study.4,18 Since then, an extended follow-up has resulted in the identification of additional CAD cases, allowing the present study to be larger. We excluded all individuals who reported a history of heart attack or stroke at the baseline clinic visit. Cases were 1105 individuals in whom a fatal or nonfatal CAD developed during follow-up until November 2003. Controls were study participants who remained free of any cardiovascular disease during follow-up. We attempted to match 2 controls to each case by sex, age (within 5 years), and time of enrollment (within 3 months). A total of 2209 controls could be matched to cases.

Biochemical Analyses

Serum levels of total cholesterol, high-density lipoprotein (HDL) cholesterol, and triglycerides were measured on fresh samples with the RA 1000 (Bayer Diagnostics, Basingstoke, UK), and LDL cholesterol levels were calculated with the Friedewald formula.19 Serum concentrations of sPLA2 were measured with a sandwich-type enzyme-linked immunosorbent assay as previously described.20 The mean intra-assay variation between duplicates was 9.2% and the lower detection limit was 0.4 ng/mL. Plasma concentrations of CRP were measured with a sandwich-type enzyme-linked immunosorbent assay as previously described.21 Results were related to a standard consisting of commercially available CRP (Behringwerke AG, Marburg, Germany). The lower detection limit was 0.1 mg/L. Samples were analyzed in random order to avoid systemic bias. Researchers and laboratory personnel had no access to identifiable information and could identify samples by number only.

Statistical Analysis

Baseline characteristics were compared between cases and controls taking into account the matching between them. A mixed effect model was used for continuous variables and conditional logistic regression was used for categorical variables. Because triglycerides, CRP, and sPLA2 levels had a skewed distribution, values were log-transformed before being used as continuous variables in statistical analyses; however, in the Tables, we show untransformed medians and corresponding interquartile ranges (IQR). To determine relationships between sPLA2 and traditional cardiovascular risk factors, we calculated mean risk factor levels per sPLA2 quartile. Quartiles were based on the distribution in the controls. For sex-specific analyses, sex-specific quartiles were used, and for pooled analyses, we used quartiles based on the sexes combined. In addition, Pearson correlation coefficients and corresponding probability values were calculated to assess the relationship between sPLA2 as a continuous variable and other continuous risk factors. Odds ratios (ORs) and corresponding 95% confidence intervals (95% CIs) as an estimate of the relative risk of incident CAD were calculated using conditional logistic regression analysis, which takes into account the matching for sex and age. The lowest sPLA2 quartile was used as the reference category. ORs were adjusted for the following cardiovascular risk factors: body mass index, diabetes, systolic blood pressure, LDL cholesterol, HDL cholesterol, and smoking (never, previous, current). ORs were also calculated after additional adjustment for CRP levels. The statistical interaction between sex and sPLA2 was calculated to assess the validity of pooling sexes. Statistical analyses were performed using SPSS software (version 12.0.1; Chicago, Ill). P<0.05 was considered to indicate statistical significance.

    Results

Baseline characteristics of cases and controls are presented in Table 1. Matching ensured that age and sex were comparable between cases and controls. As expected, individuals in whom CAD developed during follow-up were more likely than controls to smoke and have diabetes. Levels of total cholesterol, LDL cholesterol, triglycerides, systolic and diastolic blood pressure, body mass index, and CRP were significantly higher in cases than controls, and HDL cholesterol levels were significantly lower.

   TABLE 1. Baseline Characteristics of CAD Cases and Matched Controls in EPIC-Norfolk 1993–2003

sPLA2 levels were higher in cases than controls among men (8.3 ng/mL; IQR, 5.8 to 12.5 versus 7.3 ng/mL; IQR, 5.3 to 10.6; P<0.0001) and women (12.3 ng/mL; IQR, 7.9 to 18.3 versus 10.4 ng/mL; IQR, 7.3 to 16.3; P<0.0001). Notably, sPLA2 levels were substantially higher in women than men (11.0 ng/mL; IQR, 7.5 to 16.9 versus 7.7 ng/mL; IQR 5.5 to 11.3; P<0.0001). Among men and women, sPLA2 plasma levels significantly correlated with age, body mass index, systolic blood pressure, HDL cholesterol, and CRP levels (Table 2).

   TABLE 2. Distribution of Cardiovascular Risk Factors According to sPLA2 Quartile in EPIC-Norfolk 1993–2003

We conducted pooled analyses for men and women because no significant interaction was observed between sPLA2 levels and sex for CAD risk (P=0.7). Using sPLA2 quartiles based on the distribution among controls, the risk of future CAD increased continuously (P for linearity=0.001). Taking into account matching for sex and age and adjusting for body mass index, smoking, diabetes, systolic blood pressure, LDL cholesterol, HDL cholesterol, and CRP levels, the risk of future CAD was 1.34 (1.02 to 1.71; P=0.02) for people in the highest sPLA2 quartile, compared with those in the lowest. The sex-specific analyses and the pooled analyses based on sex-specific quartiles showed similar patterns.

To assess the independence of the associations of CRP and sPLA2 with CAD risk, we calculated the increase in CAD risk that was associated with 1 SD increase of each. Taking into account the matching for sex and age and adjusting for body mass index, systolic blood pressure, LDL cholesterol, HDL cholesterol, diabetes, and smoking, 1 SD increase in CRP level was associated with an OR of 1.18 (95% CI, 1.08 to 1.29); the equivalent value for sPLA2 was 1.19 (95% CI, 1.09 to 1.30). When CRP quartiles and sPLA2 quartiles were both entered into the same conditional logistic regression model, both retained an independent and statistically significant association with CAD risk (Table 3).

   TABLE 3. Risk of CAD in Apparently Healthy Individuals by Quartiles of C-Reactive Protein and sPLA2 in EPIC-Norfolk 1993–2003

    Discussion

In this large prospective study among apparently healthy men and women, we observed that serum levels of sPLA2 are associated with an increased risk of future coronary artery disease. After adjustment for classical cardiovascular risk factors and CRP levels, people in the highest sPLA2 quartile had a 34% increased risk compared with those in the lowest quartile. This observation suggests that sPLA2 levels may reflect a pathophysiological process relevant in the development of atherosclerosis, which is different from that mirrored by CRP levels.

sPLA2 and CRP as CAD Risk Markers

Our observations are consistent with a small prospective study that showed that among patients with symptomatic CAD, those with high sPLA2 levels were at an increased risk for recurrent CAD events during follow-up.15 They are also consistent with 2 large prospective studies that investigated the association between a family member of sPLA2, lipoprotein-associated phospholipase A2 (Lp-PLA2), and CAD risk. Plasma levels of Lp-PLA2 were shown to predict future CAD in men with elevated LDL-c levels22 and in apparently healthy individuals.23

Both sPLA2 and CRP are acute-phase proteins and as such might be regarded as surrogate markers of vascular inflammation reflecting endothelial dysfunction. CRP has been established as plasma marker of CAD risk and, only recently, other evidence is starting to accumulate that it may play an active role in atherogenesis as well.242526 Conversely, substantial evidence already exists to support the causality of sPLA2 in a number of pathophysiological mechanisms potentially relevant in atherosclerosis but sound epidemiological evidence for a relationship with CAD risk is lacking.

We observed that sPLA2 levels and CRP levels were strongly correlated in men and in women. Adjustment for CRP did attenuate the relationship between sPLA2 and CAD risk, but both remained significant independent predictors of CAD. Second, we observed an inverse correlation between sPLA2 levels and HDL cholesterol levels in men and women. In recent years, evidence is accumulating that HDL cholesterol and inflammation are inversely related.27 For instance, hydrolysis of acute-phase HDL particles by sPLA2 was 2-fold to 3-fold more rapid and intensive than of normal HDL.28 In addition, sPLA2 is known to cause increased catabolism of HDL particles under noninflammatory circumstances as well,29 which may explain our observation. Finally, we observed that, on average, women had substantially higher sPLA2 levels than men. This observation is consistent with the observation that women have higher CRP levels than men.30 The high levels of sPLA2 observed among women could not be explained by the use of hormone replacement therapy, which may increase CRP levels. When we investigated men and women separately, the association after adjustment for CRP became borderline and nonsignificant in men and women, respectively. As apparent in Table 4, this reduction in statistical significance was comparable in men and women, and was also observed in men and women combined. Thus, the fact that the pooled analysis retained statistical significance, whereas the sex-specific analyses did not, cannot be explained by sex differences of sPLA2 levels, but should be ascribed to the lower number of observations in the sex-stratified analyses.

   TABLE 4. Risk of Future CAD Per sPLA2 Quartile for Apparently Healthy Men, Women, and Sexes Combined

Several lines of biochemical evidence support the involvement of sPLA2 in atherogenesis. Transgenic mice expressing human sPLA2 have more atherosclerosis.31 It is well-established that sPLA2 detrimentally affects lipid metabolism,29,31–33 but this could not explain its effect on atherogenesis because it also occurred in transgenic mice kept on a low-fat diet.31 Subsequently, it was demonstrated that mice that did not express sPLA2 systemically but received bone marrow-derived cells expressing human sPLA2 also had significantly more atherosclerosis.34 Thus, macrophage-expressed sPLA2 may also play an important role in atherogenesis.

In humans, sPLA2 is highly expressed in atherosclerotic tissue and colocalizes with monocyte-derived macrophages.35–38 sPLA2 is expressed in response to a variety of inflammatory cytokines, including IL-1?, IL-6, and tumor necrosis factor (TNF)-.5,39,40 Vice versa, sPLA2 itself directly induces the expression of chemokines and adhesion molecules in microvascular endothelium.41 Thus, sPLA2 may play a role in the signaling pathways during inflammation but it also has direct atherogenic effects, possibly via the modification of the structure of lipoproteins. First, treatment of LDL lipoproteins with sPLA2 causes a substantial reduction of phosphatidylcholine in the surface monolayer of LDL particles, resulting in smaller and denser LDL particles and altering the configuration of the apolipoprotein B molecule on the lipoprotein.7 This altered configuration may expose more arginine-rich and lysine-rich segments, which can form strong interactions with glycosaminoglycans in the extracellular matrix,6 explaining the higher affinity for extracellular matrix components of sPLA2-modified lipoproteins compared with control LDL.7,8 This increased affinity for extracellular matrix components results in increased retention of LDL particles in the arterial wall, an early marker of atherosclerotic lesion progression,9–11 possibly because matrix-bound lipoproteins are more susceptible to form aggregated lipid droplets and vesicles in the vessel wall.42 Second, the sPLA2-mediated hydrolysis of the sn-2 ester bond in phospholipids liberates a number of biologically active agents, including nonesterified fatty acids and lysophospholipids, which are precursors of various proinflammatory mediators, including leukotrienes and prostaglandines.12 Third, sPLA2 may increase the susceptibility of lipoproteins to undergo lipid peroxidation,13 yielding oxidized lipoproteins that may in turn enhance macrophage growth.43 In addition, sPLA2 may also generate more bioactive phospholipids, which stimulate endothelial cells to bind monocytes.14

Finally, in addition to its potential role in the initiation and progression of atherosclerosis, sPLA2 may also have detrimental effects in the setting of ischemic events. Depositions of sPLA2 have been demonstrated in the necrotic center of infarcted human myocardium.44 Interestingly, sPLA2 was also found to be localized in myocardium adjacent to the border zone where cardiomyocytes do not show morphological signs of cell death.44 This suggests that sPLA2 may play a role in the enlargement of the necrotic myocardium during ischemia by enhancing damage in cells that are presumably only reversibly damaged by the ischemic challenge. In vitro evidence suggests that sPLA2 can transform flip-flopped but viable cardiomyocytes into apoptotic/necrotic cells.45 This observation suggests that sPLA2 may enhance myocardial cell damage either by a direct cytotoxic effect or by enhancing the inflammatory response.

Considerations

Certain aspects of this study merit further consideration. First, levels of sPLA2 were determined in a serum sample that was stored at –80°C, so we cannot exclude some degree of protein degradation in these samples. However, this would introduce an increased random measurement error, which is likely to lead to an underestimation of any relationship and therefore does not negate our findings.

Second, CAD events were ascertained through death certification and hospital admission data, which are likely to lead to underascertainment and to misclassification of cases. Previous validation studies in our cohort indicate high specificity of such case ascertainment.46 Again, case underascertainment and misclassification are likely to attenuate any relationships. Third, the current data do not allow us to study the causality of the relationship between sPLA2 and CAD. We cannot exclude the possibility that in this population study, sPLA2 concentration was a marker of subclinical atherosclerosis rather than an effector, although this hypothesis would not be consistent with a number of in vitro observations supporting a causal role of sPLA2 in lipoprotein modification.

The independent relationship of sPLA2 with CAD may be of clinical interest in light of the development of pharmacological inhibitors of PLA2 activity. Recently, an inhibitor of Lp-PLA2 has been tested in phase II trials. This enzyme is distinct from its family member sPLA2 in terms of catalytic activity, properties, and chromosome localization, but their hypothesized proatherosclerotic properties in the atherosclerotic plaques (local production of lyso-phosphatidylcholine and free fatty acids) are similar. The results of the present analysis raise several issues concerning the potential effects of currently tested inhibitors of sPLA2 activity.5 First, we have studied apparently healthy individuals, although sPLA2 inhibitors will be used, if ever, in people at high cardiovascular risk. Second, we have studied sPLA2 under physiological conditions in which sPLA2 activity and sPLA2 concentration are assumed to be strongly correlated. This contrasts with the reduced sPLA2 activity on pharmacological inhibition. Taking these considerations into account, care is warranted when extrapolating our finding that high sPLA2 concentrations are associated with a moderately reduced elevated risk of CAD in healthy individuals to the potential effects of sPLA2 inhibition on CAD risk in patients.

Conclusion

Elevated levels of sPLA2 are associated with an increased risk of future CAD in apparently healthy individuals. The observed relationship was continuous and was independent of classical cardiovascular risk factors. The magnitude of the relationship was similar to that observed between CRP levels and CAD risk, and these relationships were independent. These prospective data support the hypothesis that sPLA2 plays a role in the pathogenesis of atherosclerosis or its major clinical manifestation, CAD. Inhibitors of sPLA2 activity may hold promise for the therapeutic future.

    Acknowledgments

We thank the participants, general practitioners, and staff in EPIC-Norfolk. EPIC-Norfolk is supported by program grants from the Medical Research Council UK and Cancer Research UK, and with additional support from the European Union, Stroke Association, British Heart Foundation, Department of Health, Food Standards Agency, and the Wellcome Trust. J.J.P. Kastelein is an established investigator (2000 D039) and J.W. Jukema is an established clinical investigator (2001 D032) of the Netherlands Heart Foundation, The Hague, The Netherlands.

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