BACKGROUND: Secretory phospholipase A2 (sPLA2) may contribute to atherogenesis. To date, few prospective studies have examined the utility of sPLA2 for risk stratification in coronary artery disease (CAD).
METHODS: We measured plasma sPLA2 activity at baseline in 3708 subjects in the PEACE randomized trial of trandolapril vs placebo in stable CAD. Median follow-up was 4.8 years. We used Cox regression to adjust for demographics, clinical risk factors, apolipoprotein B, apolipoprotein A1, and medications.
RESULTS: After multivariable adjustment, sPLA2 was associated with an increased risk of cardiovascular death, myocardial infarction, or stroke (adjusted hazard ratio Q4:Q1 1.55, 95% CI 1.13–2.14) and cardiovascular death or heart failure (1.91, 1.20–3.03). In further multivariable assessment, increased activity levels of sPLA2 were associated with the risk of cardiovascular death, myocardial infarction, or stroke (adjusted hazard ratio 1.47, 95% CI 1.06–2.04), independent of lipoprotein-associated phospholipase A2 mass and C-reactive protein, and modestly improved the area under the curve (AUC) beyond established clinical risk factors (AUC 0.668–0.675, P = 0.01). sPLA2, N-terminal pro-B-type natriuretic peptide, and high-sensitivity cardiac troponin T all were independently associated with cardiovascular death or heart failure, and each improved risk discrimination (P = 0.02, P < 0.001, P < 0.001, respectively).
CONCLUSIONS: sPLA2 activity provides independent prognostic information beyond established risk markers in patients with stable CAD. These data are encouraging for studies designed to evaluate the role of sPLA2 as a therapeutic target.
The phospholipase A2 enzymes are members of a large family that hydrolyze the sn-2 ester of glycerophospholipids to release free fatty acids and lysophospholipids. The secretory phospholipase A2 (sPLA2)7 family consists of 10 isoenzymes that are involved in a variety of biological processes that include hydrolysis of phospholipids, release of arachidonic acid, and eicosanoid generation (1). sPLA2 enzymes are distinct from lipoprotein-associated phospholipase A2 (Lp-PLA2), another biomarker that has been extensively studied for risk stratification and is now being evaluated as a potential therapeutic target (2).
Growing evidence suggests that sPLA2 may play a causal role in the development of atherosclerosis. sPLA2-X has been shown to promote macrophage foam cell formation in murine models (3), and upregulated sPLA2-IIA or sPLA2-V expression has been shown to increase atherosclerotic lesion size in transgenic mice (4, 5). Additionally, genetic deletion of sPLA2-V or direct inhibition of sPLA2 activity has been shown to reduce atherosclerotic lesion progression in animals (5,–,7).
To date, a small number of studies have evaluated the utility of sPLA2 for risk stratification in primary prevention populations and in patients with acute coronary syndrome (ACS) (1). However, the prognostic utility of sPLA2 activity has not been well established in a large population of patients with stable coronary artery disease (CAD). Determination of this association is particularly relevant given the interest in sPLA2 as a possible therapeutic target for helping to delay the progression of atherosclerosis (8). Furthermore, the relative prognostic utility of sPLA2 compared with other well-established markers of risk, including those measured using new high-sensitivity assays, remains unknown. We hypothesized that sPLA2 activity would provide incremental information for risk stratification beyond established clinical risk factors and biomarkers in a large cohort of subjects with stable CAD.
The design and results of the Prevention of Events with Angiotensin Converting Enzyme Inhibition (PEACE) trial have been reported (9). In brief, the PEACE trial was a double-blind, phase III trial that randomized patients with stable CAD and preserved left ventricular function to trandolapril vs placebo. The median duration of follow-up was 4.8 years.
Clinical endpoints for this analysis included cardiovascular death, nonfatal myocardial infarction (MI), nonfatal stroke, and heart failure (HF). Cardiovascular death, MI, and stroke were adjudicated by an independent clinical events committee who were blinded to assigned treatment arm. HF was classified by centrally trained local investigators and confirmed by outcomes staff through a review of hospital records at the coordinating center.
As per study protocol, a sample of venous blood was obtained in EDTA-treated tubes from subjects at the time of enrollment. The plasma component was frozen and shipped to a central laboratory where samples were stored at −70 °C or colder. Plasma sPLA2 activity was measured using a selective fluorescent substrate (Aterovax). Results are expressed in U/mL of sample, with 1 unit defined as the amount of sPLA2 enzyme that catalyzes the release of 1 nmol product in 1 min. The mean intraassay CV for individual human plasma samples was 8.46%. We evaluated the interassay variability by determining the average CV calculated from CV of 8 plasma samples from human subjects run by 2 operators, on 3 different days, on 2 different fluorometers, using 2 different batches of assay substrate and reagents. Total interassay CV for individual human plasma samples ranged from 1.05% to 13.08%, with a mean interassay CV of 5.24%. The mean sPLA2 activity of the 8 plasma samples was 71.2, 73.9, 87.3, 113.6, 148.0, 184.2, 208.7, and 235.9 U/mL. The minimal detectable activity was 10 U/mL. The standard sPLA2 used in the assay was stabilized and presented on liposomes supported by latex beads to optimize activity measurement.
We assessed high-sensitivity cardiac troponin T by use of an autoanalyzer (Cobas e 411, Roche Diagnostics). The lower limit of detection for this high-sensitivity precommercial assay was 0.001 μg/L (10). We performed Lp-PLA2 mass measurements using the PLAC™ test at diaDexus (11) and measured N-terminal pro–brain natriuretic peptide (NT-proBNP) concentrations in plasma with an electrochemiluminescence immunoassay on a modular platform (Roche Diagnostics). High-sensitivity C-reactive protein (hsCRP) measurements were performed with the CRP-Latex (II) immunoturbidimetric assay (Denka Seiken) on a Hitachi 911 immunoanalyzer (Roche Diagnostics) at the Thrombolysis in Myocardial Infarction (TIMI) Clinical Trials laboratory (Boston, MA). This assay has a minimal detectable concentration of 0.03 mg/L. All biomarker testing was performed by personnel who were blinded to treatment arms, outcomes, and results of other biomarker testing.
We compared baseline characteristics using the Wilcoxon rank sum test for continuous measures and the χ2 test for trend for categorical variables. We assessed correlations between sPLA2 activity and other biomarker concentrations using Spearman's correlation coefficient.
Event rates at 5 years were estimated using the Kaplan–Meier method. sPLA2 was modeled by categorization into quartiles of activity. We constructed Cox proportional hazard models to estimate the hazard ratios (HRs) and 95% CIs for clinical events associated with increasing activity levels of sPLA2. The following covariates were included in all multivariable models: age, sex, hypertension, diabetes mellitus, tobacco use (current, prior, never), history of coronary revascularization, use of lipid-lowering therapy, body mass index, systolic blood pressure, estimated glomerular filtration rate (eGFR) by Modification of Diet in Renal Disease equation, apolipoprotein B (apoB), apoA1, and randomized treatment arm (note: there was no evidence of effect modification between sPLA2 activity and randomized treatment arm when an interaction term was included in the model). Additional model permutations included the addition of established biomarkers, such as hsCRP, Lp-PLA2 mass, NT-proBNP, and high-sensitivity cardiac troponin T (by quartiles of biomarker concentration). We also generated covariate-adjusted ROC curves for models with known risk factors with or without the inclusion of sPLA2 and other established biomarkers. Comparisons between ROC curves were determined using the method described by DeLong et al. (12).
Because all analyses were considered to be exploratory, a P value <0.05 was considered to be statistically significant. Analyses were performed using Stata/SE 9.2 (Stata Corp.).
BASELINE DEMOGRAPHICS AND CLINICAL PRESENTATION
sPLA2 activity was available in 3708 subjects who provided a blood sample at randomization. There were no clinically relevant differences in the baseline characteristics of patients who did and did not participate in the biomarker study (10). The baseline characteristics of the study population by quartile of sPLA2 activity are displayed in Table 1. Patients with increasing plasma sPLA2 activity levels were more likely to be female and have a history of hypertension, diabetes mellitus, current tobacco use, and a lower eGFR. Subjects with higher sPLA2 activity were more likely to have higher concentrations of apoB, apoA1, cardiac troponin T, hsCRP, Lp-PLA2 mass, and NT-proBNP.
ASSOCIATION OF sPLA2 ACTIVITY WITH CLINICAL OUTCOMES
There was a stepwise increase in the risk of cardiovascular death, MI, or stroke by quartile of sPLA2 activity (P for trend <0.001) (Table 2). The cumulative incidence of cardiovascular events by quartile of sPLA2 activity is displayed in Fig. 1. Directional consistency was observed across all elements of the composite endpoint (Table 2). In particular, subjects with sPLA2 activity levels in the highest quartile had a 2-fold higher risk of cardiovascular death (HR 2.00, 95% CI 1.18–3.37, P = 0.01) compared with patients with sPLA2 in the lowest quartile. A strong association was also observed between increasing sPLA2 and the risk of heart failure, such that subjects with sPLA2 activity levels in the highest quartile had nearly a 3-fold higher incidence of heart failure during follow-up (HR Q4:Q1 2.73, 95% CI 1.38–5.38, P = 0.004).
After adjusting for known clinical risk factors and potential confounders, sPLA2 activity remained independently associated with an increased risk of cardiovascular death, MI, or stroke during long-term follow-up (adjusted HR 1.55, 95% CI 1.13–2.14, P = 0.007) (Table 2) compared with patients with the lowest activity levels of sPLA2. Directional consistency was also seen for sPLA2 and the risk of individual endpoints, including cardiovascular death (adjusted HR Q4:Q1, 1.66, 95% CI 0.95–2.88), MI (1.49, 0.96–2.32), and stroke (1.59, 0.74–3.41). Likewise, increased activity levels of sPLA2 were independently associated with an increased risk of CVD or HF (adjusted HR Q4:Q1 1.91, 95% CI 1.20–3.03) and HF alone (2.63, 1.19–5.80).
We subsequently evaluated the independent prognostic utility of sPLA2 activity compared with other established biomarkers of cardiovascular risk and clinical risk factors. These included biomarkers of inflammation (hsCRP and Lp-PLA2 mass), and biomarkers of hemodynamic stress (NT-proBNP) and myonecrosis (high-sensitivity cardiac troponin T).
Biomarkers of inflammation.
There was a modest correlation between sPLA2 activity and concentrations of hsCRP (r = 0.26, P < 0.001). Although statistically significant, only a weak correlation was found between sPLA2 activity and Lp-PLA2 mass (r = 0.05, P < 0.001). After inclusion of hsCRP and Lp-PLA2 in a multivariable model that included established clinical risk factors, sPLA2 activities in the highest quartile remained significantly associated with an increased risk of cardiovascular death, MI, or stroke (adjusted HR Q4:Q1 1.47, 1.06–2.04, P = 0.019) (Fig. 2). In contrast, the association with hsCRP and Lp-PLA2 mass and the risk of cardiovascular death, MI, or stroke was attenuated (Fig. 2).
In ROC analyses, sPLA2 activity was the only marker of inflammation to significantly improve the area under the curve (AUC) for identifying patients at increased risk of cardiovascular death, MI, or stroke (0.668 to 0.675, P = 0.01) compared with clinical risk factors alone.
Biomarkers of hemodynamic stress and myonecrosis.
We observed a weak but statistically significant correlation between sPLA2 activity and concentrations of NT-proBNP (r = 0.06, P < 0.001) and high-sensitivity cardiac troponin T (r = 0.05, P = 0.002).
Because NT-proBNP and high-sensitivity cardiac troponin T have been shown to be associated with the risk of cardiovascular death or heart failure (rather than MI or stroke), we evaluated NT-proBNP, high-sensitivity cardiac troponin T, and sPLA2 in the context of this endpoint. In a model that included both NT-proBNP and cardiac troponin T, sPLA2 activity levels in the highest quartile remained significantly associated with an increased risk of cardiovascular death or HF (adjusted HR 1.79, 95% CI 1.12–2.86, P = 0.015) compared with patients with the lowest activity levels of sPLA2. There also existed a strong and independent association between NT-proBNP and cardiac troponin T concentrations in the highest quartile and the risk of cardiovascular death or HF (Fig. 2), thereby suggesting that all 3 markers provided complementary information for risk stratification.
Compared with clinical risk factors alone, the addition of sPLA2 significantly improved model discrimination (AUC 0.734 to 0.742, P = 0.02). When considered individually, both NT-proBNP (AUC 0.734 to 0.765, P < 0.001) and high-sensitivity cardiac troponin (AUC 0.734 to 0.757, P < 0.001) also increased the AUC compared with clinical risk factors alone. Moreover, the inclusion of sPLA2 activity into a model that already included both NT-proBNP and cardiac troponin T further improved model discrimination (AUC 0.778 to 0.782, P = 0.03).
The current findings demonstrate that higher levels of plasma sPLA2 activity are associated with an increased risk of cardiovascular events in a large population of patients with stable CAD. In particular, higher levels of sPLA2 activity were associated with an increased risk of cardiovascular death, MI, or stroke and cardiovascular death or heart failure, independent of traditional risk factors. Moreover, sPLA2 provided modest additive information for risk stratification beyond several established markers of risk, including hsCRP, Lp-PLA2 mass, NT-proBNP, and high-sensitivity cardiac troponin T.
Prior studies have evaluated the prognostic utility of sPLA2 in primary and secondary prevention populations. In 2 nested case-control studies in the European Prospective Investigation of Cancer (EPIC)-Norfolk Study, increasing sPLA2 mass and activity were associated with the first occurrence of a coronary event during 6 years of follow-up in otherwise healthy individuals (13, 14). As well, higher sPLA2 activity levels, but not mass, were shown to be associated with a higher risk of recurrent cardiovascular events in patients hospitalized with ACS (15).
Three prior studies have examined the prognostic role of sPLA2 in patients with stable CAD. In a retrospective case-control study of 142 patients with angiographically proven CAD, sPLA2 mass was significantly higher in cases than controls and independently associated with a higher risk of major adverse cardiovascular events (16). In a second study of 247 patients undergoing percutaneous coronary intervention (PCI), an increased sPLA2 concentration post-PCI was associated with an increased risk of coronary events during 2 years of follow-up compared with controls (17). In the largest prior study to date of patients with stable CAD, sPLA2 was measured in a cohort of 1024 subjects between the ages of 30 and 70 who were engaged in cardiac rehabilitation after ACS. Higher sPLA2 activity levels or mass were associated with an increased risk of cardiovascular events after adjustment for traditional risk factors and markers including hsCRP, cystatin C, Lp-PLA2 mass, and NT-proBNP (18). The study population proved to be at relatively low risk, however, and few fatal events occurred during study follow-up (2.8% incidence of cardiovascular death after median follow-up of 4.6 years). Additionally, the study design did not allow for adjudication of nonfatal events, and the incidence of HF was not reported.
The current analysis is, to our knowledge, the largest study to date to evaluate the prognostic utility of sPLA2 in patients with stable CAD. We found that individuals with sPLA2 activity levels in the highest quartile had a 58% higher risk of cardiovascular death, MI, or stroke independent of established risk factors. The association between sPLA2 and vascular events is consistent with sPLA2 and its hypothesized role in inflammation and atherogenesis. In addition, the current study was able to compare the prognostic utility of sPLA2 activity relative to other established biomarkers using modern high-sensitivity assays. Among biomarkers of inflammation, sPLA2 but not hsCRP or Lp-PLA2 mass modestly improved risk discrimination for identifying patients at increased risk of major adverse cardiovascular events. In contrast, prior analyses in the same study population have demonstrated that Lp-PLA2 mass appears to have a stronger association with nonfatal cardiac outcomes, including coronary revascularization and unstable angina (11). Higher sPLA2 activity levels also identified patients at increased risk of cardiovascular death or HF in the setting of a preserved ejection fraction and provided additive information to high-sensitivity cardiac troponin T and NT-proBNP. These latter findings were unanticipated because sPLA2 is not recognized as a marker of myonecrosis or hemodynamic stress. Additional studies will be needed to determine the basis for the observed association.
There exist limitations to the current analysis that warrant consideration. Because we did not quantify sPLA2 mass, we are unable to evaluate the relative prognostic utility of sPLA2 activity vs mass for risk assessment in this study population. Furthermore, the current analysis is exploratory, and future studies will be required to validate our findings or to consider potential cut points for clinical use.
In summary, we have demonstrated that sPLA2 activity is associated with an increased risk of cardiovascular events in patients with stable CAD and provides incremental and complementary information for risk stratification beyond several established risk markers, including hsCRP, Lp-PLA2 mass, NT-proBNP, and high-sensitivity cardiac troponin T. These data not only strengthen the association between sPLA2 activity and the risk of CV outcomes, but also are encouraging for clinical trials of an sPLA2 inhibitor (8). If such trials show a clinical benefit, then measuring sPLA2 might be useful for targeting therapy.
Manuscript presented as an abstract at the American Heart Association Scientific Sessions 2009; 2009 Nov 17; Orlando, FL.
↵7 Nonstandard abbreviations:
- secretory phospholipase A2;
- lipoprotein-associated PLA2;
- acute coronary syndrome;
- coronary artery disease;
- Prevention of Events with Angiotensin Converting Enzyme Inhibition;
- myocardial infarction; HF, heart failure;
- N-terminal pro–brain natriuretic peptide;
- high-sensitivity C-reactive protein;
- Thrombolysis in Myocardial Infarction;
- hazard ratio;
- estimated glomerular filtration rate;
- area under the curve;
- European Prospective Investigation of Cancer;
- percutaneous coronary intervention
Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article.
Authors' Disclosures or Potential Conflicts of Interest: Upon manuscript submission, all authors completed the Disclosures of Potential Conflict of Interest form. Potential conflicts of interest:
Employment or Leadership: Z. Mallat, Aterovax.
Consultant or Advisory Role: Z. Mallat, Aterovax; D.A. Morrow, AstraZeneca, Beckman-Coulter, Boehringher Ingelheim, Gilead, Instrumentation Laboratory, Merck, OrthoClinical Diagnostics, and Roche; J. Benessiano, Aterovax; A. Tedgui, Aterovax.
Stock Ownership: Z. Mallat, Aterovax; A. Tedgui, Aterovax.
Honoraria: M.L. O'Donoghue, Eli Lilly and Daiichi Sankyo; Z. Mallat, Aterovax; T. Omland, Roche Diagnostics, Abbott Laboratories, and Otsuka; A. Tedgui, Aterovax.
Research Funding: The PEACE trial was supported by a contract from the National Heart, Lung, and Blood Institute and by Knoll Pharmaceuticals and Abbott Laboratories. The assay for sPLA2 activity was conducted at Aterovax. M.L. O'Donoghue, AstraZeneca (grant), GlaxoSmithKline (grant), and Eisai (grant); D.A. Morrow, AstraZeneca, Merck, GlaxoSmithKline, Beckman-Coulter, Siemens, Buhlmann, Singulex, Brahms, and BGMedicine; T. Omland, Abbott Laboratories and Roche Diagnostics; E. Braunwald, GlaxoSmithKline, grant support for the SOLID-TIMI 52 Trial; M.S. Sabatine, diaDexus, OrthoClinical Diagnostics, Singulex, and NIH (R01 HL094390).
Expert Testimony: None declared.
Other Remuneration: Z. Mallat, coinventor on 2 biomarker patents filed by INSERM; D.A. Morrow, consulting fees from Siemens; J. Benessiano, coinventor on 2 biomarker patents filed by INSERM; A. Tedgui, coinventor on 2 biomarker patents filed by INSERM.
Role of Sponsor: The funding organizations played no role in the design of study, choice of enrolled patients, review and interpretation of data, or preparation or approval of manuscript.
- Received for publication March 31, 2011.
- Accepted for publication June 29, 2011.
- © 2011 The American Association for Clinical Chemistry