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
Objectives— We sought to evaluate whether lipoprotein-associated phospholipase A2 (Lp-PLA2), an emerging marker of cardiovascular risk, is associated with prognosis in patients with coronary heart disease (CHD).
Methods and Results— Plasma concentrations and activity of Lp-PLA2 were determined in 1051 patients aged 30 to 70 years with CHD who were followed for &4 years. A Cox proportional hazards model was used to determine the prognostic value of Lp-PLA2 after adjustment for various covariates, including markers of inflammation, renal function, and hemodynamic stress. In multivariable analyses, Lp-PLA2 mass and activity were strongly associated with cardiovascular events after controlling for traditional risk factors, severity of CHD, statin treatment, cystatin C, and N-terminal proBNP. The hazard ratio (HR) for recurrent events was 2.65 (95% confidence interval [CI], 1.47 to 4.76) for the top tertile of Lp-PLA2 mass compared with the bottom tertile and 2.40 (95% CI, 1.35 to 4.29) for Lp-PLA2 activity. After additional adjustment for low-density lipoprotein (LDL), the HRs were only moderately attenuated (mass: 2.09; 95% CI, 1.10 to 3.96; activity: 1.81; 95% CI, 0.94 to 3.49, respectively), but the latter was no longer statistically significant.
Conclusions— Increased concentrations of Lp-PLA2 predict future cardiovascular events in patients with manifest CHD independent of a variety of potential risk factors including markers of inflammation, renal function, and hemodynamic stress.
- cohort study
- coronary heart disease
- lipoprotein-associated phospholipase A2 pathomechanism
Lipoprotein-associated phospholipase A2 (Lp-PLA2) is a calcium-independent member of the phospholipase A2 family.1,2 It is produced mainly by monocytes, macrophages, T-lymphocytes, and mast and liver cells.3–5 Lp-PLA2 activity occurs in association with macrophages and has been found to be upregulated in atherosclerotic lesions, especially in complex plaque,6 as well as in the fibrous cap of coronary lesions prone to rupture.7 In the bloodstream, two-thirds of the Lp-PLA2 plasma isoform circulate primarily bound to low-density lipoprotein (LDL), the other one-third is bounded to between high-density lipoprotein (HDL) and very-low-density lipoprotein.8,9
LDL provides a circulating reservoir, in which Lp-PLA2 remains inactive until LDL undergoes oxidative modification. After LDL oxidation within the arterial wall, a short acyl group at the sn-2 position of phospholipids becomes susceptible to the hydrolytic action of Lp-PLA2 that cleaves an oxidized phosphatidylcholine component of the lipoprotein particle, generating 2 potent pro-inflammatory and pro-atherogenic mediators, namely lysophosphatidylcholine (LysoPC) and oxidized fatty acid (oxFA).10 Of particular note, Lp-PLA2 acts only on oxidatively modified LDLs (oxLDL), and hydrolysis of oxLDL can be performed solely by Lp-PLA2.1,10 Pro-inflammatory actions of LysoPC, as well as those of oxFA, trigger a cascade of events, which might directly promote atherogenesis. LysoPC is a potent chemoattractant for T cells and monocytes, promotes endothelial cell dysfunction, stimulates macrophage proliferation, and induces apoptosis in smooth muscle cells and macrophages.1,11,12
Thus, Lp-PLA2 may represent an important “missing link” between the oxidative modification of LDL in the intimal layer of the arterial wall and local inflammatory processes within the atherosclerotic plaque that may be specific for atherosclerosis. Experimental studies in Watanabe heritable hyperlipidemic rabbits have shown that inhibition of Lp-PLA2 leads to the reduction of atherosclerotic lesion formation.1 The pro-atherogenic role of this enzyme was also implicated by observations from in vitro studies that suggested Lp-PLA2 as a novel therapeutic target. Pyromidones, being noncovalent Lp-PLA2 inhibitors, prevented the production of LysoPC and subsequent monocyte chemotaxis in vitro.13,14 Further in vivo studies revealed a 95% inhibition of Lp-PLA2 in atherosclerotic plaque from Watanabe heritable hyperlipidemic rabbits, observed 2 hours after dosing (30 mg/kg) of SB-480848,15 thereby identifying this compound as a very potent Lp-PLA2 inhibitor with a suitable profile for evaluation in humans. Results from a multi-center trial16 showed a dose-dependent inhibition of Lp-PLA2 plasma activity by 52% and 81% compared with placebo after administration of 40 mg and 80 mg of SB-480848, respectively.
To date, 4 large prospective studies17–20 in initially healthy subjects support the notion that Lp-PLA2 may be considered a new and independent cardiovascular biomarker. However, to our knowledge, only 1 study in patients with manifest coronary heart disease (CHD) has investigated the association between Lp-PLA2 and future adverse cardiovascular events,21 and no study so far has determined whether Lp-PLA2 mass or activity are more important.
Thus, because it is presently unclear to what extent Lp-PLA2 mass and activity carry the same prognostic information, we sought to investigate simultaneously the value of both parameters for the prediction of future cardiovascular events in a large cohort of patients with manifest CHD. Furthermore, we wanted to compare its prognostic value with that of other emerging risk markers, like C-reactive protein (CRP), cystatin C as an indicator of renal function, and N-terminal-pro brain natriuretic peptide (NT-proBNP) as a measure of hemodynamic stress.
Materials and Methods
All patients with CHD (International Classification of Diseases, 9th Revision codes 410 to 414) aged 30 to 70 years and participating in an in-hospital rehabilitation program between January 1999 and May 2000 in 2 cooperating clinics (Schwabenland-Klinik, Isny and Klinik im Südpark, Bad Nauheim, Germany) were enrolled in the study (initial response 58%). In Germany all patients, after acute coronary syndrome or coronary artery revascularization, are offered a comprehensive in-hospital rehabilitation program after discharge from the acute care hospital. The aim of this 3-week program is the reduction of cardiovascular risk factors, improvement of health related quality of life, and the preservation of the ability to work (the latter only if a subject was still at work at the onset of disease, otherwise the prevention of nursing care). This in-hospital rehabilitation program usually starts &3 weeks after the acute event or coronary artery revascularization. In the current study, only patients who were admitted within 3 months after the acute event or coronary artery revascularization have been included.
All subjects gave written informed consent. The study was approved by the Ethics Boards of the Universities of Ulm and Heidelberg and of the Physicians’ chamber of the States of Baden-Wuerttemberg and Hessen (Germany).
At the beginning of the in-hospital rehabilitation program all subjects filled out a standardized questionnaire containing sociodemographic information and medical history. In addition, information was taken from the patients’ hospital charts. In all patients active follow-up was conducted 1, 3, and 4.5 years after discharge from the rehabilitation center. Information was obtained from the patients using a mailed standardized questionnaire. Information regarding secondary cardiovascular events and treatment since discharge from the in-hospital rehabilitation clinic was obtained from the primary care physicians also by means of a standardized questionnaire. If a subject had died during follow-up, the death certificate was obtained from the local Public Health Department and the main cause of death was coded according to the International Classification of Diseases (9th Revision). Secondary cardiovascular events were defined either as cardiovascular disease (CVD) as the main cause of death (as stated in the death certificate), nonfatal myocardial infarction (MI), or ischemic cerebrovascular event (stroke). All nonfatal secondary events were reported by the primary care physicians.
Blood at baseline was drawn at discharge from the rehabilitation center (on the average 43 days after the acute event; first quartile, 36 days; third quartile, 51 days) in a fasting state under standardized conditions. Plasma concentrations of Lp-PLA2 were determined by a commercially available Lp-PLA2 enzyme-linked immunosorbent assay (ELISA) kit (second generation PLACTM Test; diaDexus Inc, South San Francisco, Calif).22 The lower detection limit of Lp-PLA2 in this assay is &2 ng/mL. The interassay coefficient of variation (CV) was between 6% and 7%. Lp-PLA2 activity was measured in a 96-well microplate with a colorimetric substrate that is converted on hydrolysis by the phospholipase enzyme. Briefly, 25 μL of sample, or standard, or control are added per well, followed by addition of assay buffer plus substrate. The change in absorbance is immediately measured at 405 nm. The level of Lp-PLA2 activity in nmol/min per mL was calculated from the slope, based on a standard conversion factor from a p-Nitrophenol calibration curve. Mass and activity were moderately correlated (r=0.573, P<0.001).
CRP concentrations in plasma were measured by immunonephelometry on a Behring Nephelometer II (N Latex CRP mono; Dade-Behring, Marburg). Cystatin C was determined on the same device (Dade Behring, Marburg). NT-proBNP was measured by electrochemiluminescence on an Elecsys 170 (Roche Diagnostics, Mannheim, Germany). Interassay CV for CRP was 4.1%, for cystatin C it was 3.8%, and for NT-proBNP it was between 3% and 7%. All markers were measured in a blinded fashion. Blood lipids and leukocyte count were done by routine methods in both participating clinics.
First, the study population was described with respect to various sociodemographic and medical characteristics. The associations of sociodemographic characteristics, various cardiovascular risk factors, and medication with Lp-PLA2 mass and activity (distribution in top tertile versus first and second) were quantified by means of a χ2 test. Partial Spearman correlation coefficients, adjusted for age and gender, were calculated for Lp-PLA2 concentrations and activity and blood lipids, CRP, creatinine clearance, cystatin C, and NT-proBNP.
The relation of Lp-PLA2 mass and activity with CVD events during follow-up was assessed by the Kaplan-Meier and life table method and quantified by means of the log-rank test. Then the Cox proportional hazards model was used to assess the independent association of Lp-PLA2 mass and activity distribution with the risk of secondary CVD events. A basic model was adjusted for age (years) and gender. In addition (and to avoid over-adjustment), besides the main factor Lp-PLA2 and the variables age, gender and hospital site, from a set of covariates (body mass index [BMI] kg/m2), smoking status (never, current, ex-smoker), duration of school education (<10 years, ≥10 years), family status (married, other), history of MI (yes, no), history of diabetes mellitus (yes, no), severity of CHD (number of affected vessels at baseline), HDL cholesterol (mg/dL), CRP (mg/L), cystatin C (mg/L), NT-proBNP (ng/mL), intake of β-blockers, intake of ACE inhibitors, intake of diuretics, and hospital site (Isny, Bad Nauheim), only those were added to the model that were significant predictors of a secondary event at an α level of 0.1 or that changed the parameter estimates for the main variables (Lp-PLA2) by >10%. In further analyses LDL cholesterol was included also.
A receiver-operating curve was constructed after adjustment for covariates and the area under the curve (AUC) with its 95% confidence interval (CI) was calculated. In addition, Somer’s D, a measure of association between ordinal variables that provides a rank correlation between predicted and observed probabilities, was calculated for the various models. Somer’s D ranges between −1 and +1; 0 reflects no association at all. All statistical procedures were performed with the SAS statistical software package (release 8.2; SAS Institute Inc, Cary, NC) using the SAS-macro-package for prognostic modeling.23
Overall, 1206 patients with a diagnosis of CHD within the past 3 months were included in the study at baseline during the in-hospital rehabilitation program; 4.5-year follow-up information was complete for 1051 patients (87.2%). A total of 95 (9.0%) fatal and nonfatal CVD events occurred (30 cardiovascular deaths, 35 nonfatal MIs, and 30 strokes) during a mean follow-up time of 48.7 months (SD 15.9).
Table 1 shows the main characteristics of the study population. Of the 1051 patients with a diagnosis of CHD, 58.2% had reported a history of MI, and 42.7% of patients (with coronary angiography) had 3-vessel disease. The mean age of CHD patients was 59 years; most of them (56.5%) were between 60 to 70 years old, and 84.9% were male. The initial invasive management of CHD in the acute care hospital was percutaneous coronary intervention (PCI) in 361 (21.9%) and coronary artery bypass grafting (CABG) in 499 (28.1%) patients.
Table 2 shows the relationship of various cardiovascular risk factors, clinical severity of CHD, and medication with Lp-PLA2 mass and activity. For Lp-PLA2 mass, females were more likely to be in the top tertile of the Lp-PLA2 distribution than men. Also, higher age, history of MI, more advanced coronary artery disease (as determined by angiography), not taking an ACE inhibitor and, as expected, particularly the lack of statin treatment, were all strongly and positively related to Lp-PLA2 distribution; there was no association with BMI, smoking status, history of diabetes, and β-blocker or diuretic intake. For Lp-PLA2 activity, similar relationships were seen with gender, clinical score, and statin treatment, but not with age, history of MI, and ACE inhibitor intake. High Lp-PLA2 activity was seen more frequently in those on a diuretic compared with those without.
Table 3 shows correlations between lipid variables, other emerging risk factors and Lp-PLA2 mass and activity (adjusted for age and gender). Total cholesterol (r=0.345 and r=0.490) and LDL cholesterol (r=0.423 and r=0.564) were strongly and positively correlated with Lp-PLA2 mass and activity; HDL cholesterol showed a moderately negative correlation, and comparable small positive correlations for both parameters were seen with CRP, cystatin C, and with NT-proBNP. All correlation coefficients were statistically significant (P<0.0001).
Figurea and b shows Kaplan-Meier curves presenting the proportion of patients with secondary CVD events according to tertiles of Lp-PLA2 mass (and activity) at baseline. Of patients in the bottom tertile of Lp-PLA2, 5.8% (5.2%) experienced an event compared with 10.7% (10.0%) and 10.6% (11.9%) in the middle and upper tertile, respectively (P<0.03 and P<0.01, respectively).
Table 4 shows the results of multivariable analysis to estimate the independent association of Lp-PLA2 concentrations and activity at baseline with risk of fatal and nonfatal cardiovascular events during follow-up. In age- and gender-adjusted analysis, patients in the top tertile compared with those in the bottom tertile of the Lp-PLA2 mass distribution at baseline had a hazard ratio (HR) of 1.79 (95% confidence interval [CI], 1.04 to 3.08) for a CVD event (P for trend <0.05). The HR increased after adjustment for classical risk factors, severity of CHD, and all factors from Table 2 contributing significantly (P<0.1) to the model or which changed the main effect of Lp-PLA2 by >10% (which were BMI, HDL cholesterol, history of MI, history of diabetes mellitus, treatment with ACE inhibitors, cystatin C, and NT-proBNP), resulting in a HR for the top tertile of 2.65 (95% CI, 1.47 to 4.76). If CRP, which did not qualify for an inclusion into the model, was included, the HR even increased slightly further (HR in the top tertile 2.70) (95% CI, 1.49 to 4.90). Final adjustment for LDL cholesterol led to an expected decrease of the association (HR for top tertile 2.09; 95% CI, 1.10 to 3.96), which, however, was still statistically significant. For Lp-PLA2 activity, in the multivariate model, the HR was slightly lower (HR in the top tertile 2.40; 95% CI, 1.35 to 4.29) and also decreased after adjustment for LDL cholesterol (HR in the top tertile 1.81; 95% CI, 0.94 to 3.49), but in contrast to Lp-PLA2 mass the 95% CI included the null value.
In Table 5 we quantified the incremental contribution of Lp-PLA2 mass to risk prediction in the presence of classical risk factors, renal function, and hemodynamic stress. As can be seen from receiver operating characteristics (ROC) curve analyses, the addition of cystatin C and NT-proBNP to a basic model improved the predictive accuracy of the model (AUC from 0.67 to 0.71). After additional inclusion of Lp-PLA2 mass, there was still a further, however smaller, increase (AUC from 0.71 to 0.73).
These data from a large cohort of patients with manifest CHD strongly suggest a role for Lp-PLA2 as a novel predictor of cardiovascular risk in a population at high risk for future CVD events. In multivariable analyses after adjusting for a wide range of established risk factors including markers of inflammation, renal function, and hemodynamic stress, there was still an &2-fold increased risk for future CVD events in patients in the upper 2 tertiles of Lp-PLA2 mass compared with the bottom tertile; for Lp-PLA2 activity, risk estimates were only slightly smaller and failed to reach statistical significance after full adjustment for covariates. Therefore, especially Lp-PLA2 mass may be a promising biomarker for risk prediction in secondary prevention of CVD.
Associations of Lp-PLA2 With Other Risk Markers
Lp-PLA2 mass and activity were strongly positively correlated with LDL and total cholesterol and moderately negatively with HDL cholesterol, as described in previous studies17–21 reflecting mainly the close association with LDL cholesterol. Markers of inflammation, renal function, and hemodynamic stress were also positively correlated in our study. Small associations with age have also been seen in these studies.17–21 A relationship with gender was no longer present in the study by Brilakis21 after adjustment for HDL cholesterol. Initial concentrations in men were higher than in women, which was the opposite way in our study. In univariate analysis, we found an association with the severity of CHD (P<0.001), which was also reported by Brilakis et al.21 However, in the latter study, the association was no longer present after adjusting for clinical and lipid variables.
Furthermore, an expected association was seen with statin intake; statins lower Lp-PLA2 mass and activity,24,25 thus it is conceivable that those not on a statin had higher concentrations and activity. However, the inverse association for Lp-PLA2 mass with ACE inhibitor intake as seen in univariate analysis may represent confounding by indication as patients at high risk may more likely receive these drugs. Yet there are no observations that ACE inhibitors may lower plasma concentrations of Lp-PLA2.
Lp-PLA2 and Prediction of Cardiovascular Events in Patients With Manifest CHD
In the only other study in patients with mainly stable CHD in which the prognostic value of Lp-PLA2 has been studied,21 466 consecutive patients scheduled for coronary angiography were followed for a median of 4 years. During this time period, 72 events occurred in 61 patients. Baseline concentrations of Lp-PLA2 mass were higher in CHD patients who later on developed an adverse event compared with those who did not. The relative risk for a future event for a one standard deviation increase in Lp-PLA2 mass was 1.30 after multivariable adjustments and thus comparable to data from primary risk studies such as WOSCOPS17 and MONICA.19
Our study participants were about the same age as those in the study by Brilakis et al,21 Lp-PLA2 concentrations were in the same range, and the follow-up period was similar. When looking at Kaplan-Meier estimates, striking similarities between the 2 studies can be seen. Using comparable cut-points, the second and third tertiles were clearly different from the bottom tertile in both studies, suggesting a cut-point for increased risk rather than a linear association. Similar results were seen for Lp-PLA2 activity, which is also in accordance with the Rotterdam study in initially healthy subjects.20
However, our study has several advantages and extends the results of the previous study. First, the patient population was more than twice as large as the one by Brilakis et al and had more atherosclerosis specific end points as we did not include the need for revascularization or all-cause mortality. Second, the study population was more homogeneous because only patients weeks after an acute event were included. Finally, we were able to measure simultaneously Lp-PLA2 mass and activity and considered also markers of inflammation like CRP, cystatin C, a strong indicator of renal function, and NT-proBNP, an established marker of hemodynamic stress. The latter 2 markers have been reported to be independently linked to future cardiovascular outcome in patients with CHD.26–30 Even after taking into account these emerging predictors, Lp-PLA2 mass in our cohort added incremental prognostic information. Thus, it seems hat the risk prediction associated with elevated concentrations of Lp-PLA2 is rather stable and yields clinically relevant information to the already known established and laboratory risk factors in a population at already high cardiovascular risk.
Interestingly, our data are supported by those published by O’Donoghue et al31 from the PROVE IT-TIMI 22 trial suggesting that Lp-PLA2 activity or mass may not be predictive for recurrent events when measured at the time of admission to hospital or early after an acute coronary syndrome, but indeed may be able to modify risk prediction when measured some time apart from the acute events eg, at day 30. In that study, in 3648 patients with acute coronary syndrome, Lp-PLA2 activity and mass were measured at baseline and 30 days later (n=3625). Patients with an elevated Lp-PLA2 activity but not mass in the top quintile at day 30 had a 33% increased risk for recurrent events (RR, 1.33; 95% CI, 1.01 to 1.74) over 24 months of follow-up.
In contrast to prospective cohorts with clinical end points, data from a large cross-sectional study using intima media thickness of the carotid arteries as a measure of subclinical atherosclerosis32 showed no independent association with Lp-PLA2 activity after adjustment for cholesterol. Because Lp-PLA2 travels with LDL cholesterol in the peripheral circulation, a consistent correlation between these 2 variables has been reported in all studies. Thus, adjusting for (LDL) cholesterol clearly is associated with an attenuation of the association between Lp-PLA2 and the end point under study. In the majority of studies on (inflammatory) biomarkers, a moderate or even strong association has been reported between elevated concentrations in blood and clinical end points but the association with subclinical atherosclerosis or atherosclerotic burden/extent has been negative in almost all studies.
Measurement of Lp-PLA2 Mass Versus Activity
Results from WOSCOPS17 and MONICA,19 which measured mass, and from the Rotterdam study,20 which measured activity, suggest reasonable agreement between both methods. However, there is a lack of data comparing the predictive value of Lp-PLA2 mass and activity in the same population.
Most recently, in the PROVE-IT TIMI 22 trial31 only a modest correlation of r=0.36 between activity and mass was found. Irribarren et al33 measured both parameters in the CARDIA study and found a correlation between mass and activity of r=0.61, which is comparable to our study (r=0.57). These authors also reported a stronger correlation between activity compared with mass with LDL cholesterol (r=0.52 and r=0.39, respectively), which is similar to correlations we found (activity r=0.56 and mass r=0.42, respectively). Thus, as suggested by these authors, the loss of statistical significance for activity versus mass in multivariable prediction models, may be, at least in part, attributable to its stronger correlation with LDL cholesterol. Certainly, more studies are needed to resolve these issues.
The following limitations of our study should be considered. Although we had a large sample of patients with CHD (>50% with a history of MI), fatal CVD events were limited in this study population. This is explained by the fact that mortality of MI is highest during the prehospital and early in-hospital phase. Because the acute events leading to diagnosis of CHD or MI had occurred at least 3 weeks before inclusion in this study, selection of patients with a better prognosis compared with a patient population within the early phase of a newly diagnosed CHD must be assumed. Furthermore, not all patients were willing or able to participate in an in-hospital rehabilitation program. This may be a further reason for the under-representation of severely ill patients in our study sample. However, this does not explain the positive findings between Lp-PLA2 and CVD events, but suggests that the true prognostic value of Lp-PLA2 may even be stronger than shown in our study.
These data are in support of an important prognostic value of Lp-PLA2 among patients with known CHD. These data strongly suggest that especially Lp-PLA2 mass is a useful clinical biomarker, which is independent of traditional risk factors and other emerging risk factors like CRP, cystatin C, and NT-proBNP, and may be superior to measurements of activity. If causal involvement in the pathogenesis of atherosclerosis can be proven, Lp-PLA2 may become a promising target for intervention in the future.
We highly appreciate the technical assistance of Gerlinde Trischler.
Sources of Funding
This research was supported by an unrestricted grant from diaDexus, Inc, South San Francisco, California.
W.K. has received honoraria for lectures from diaDexus and GSU. The other authors have no disclosures.
Original received February 22, 2005; final version accepted April 6, 2006.
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