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Prospective studies reporting a positive association of lipoprotein-associated phospholipase A2 (Lp-PLA2) mass and activity with incident cardiovascular disease (CVD) have included primarily white individuals. We evaluated associations of Lp-PLA2 and first-time cardiovascular events in a healthy multi-ethnic cohort characterized by presence or absence of baseline subclinical atherosclerosis.
Lp-PLA2 mass and activity were measured at baseline in 5456 participants in the Multi-Ethnic Study of Atherosclerosis. Individuals were characterized for presence of baseline subclinical disease (coronary artery calcium score>0 or carotid intima-media thickness value>80th percentile) and followed prospectively for development of CVD events (coronary heart disease, ischemic stroke, and cardiovascular death).
516 incident CVD events occurred over median follow-up of 10.2 years. In adjusted Cox proportional hazards models, each higher standard deviation of both Lp-PLA2 activity and mass was associated with an increased risk of cardiovascular events; hazard ratios (HR; 95% confidence intervals (CI)) 1.12 (1.01–1.26) for Lp-PLA2 activity and 1.10 (1.01–1.21) for mass. Associations did not differ by subclinical disease status (p-value for interaction 0.99 for Lp-PLA2 activity and 0.32 for Lp-PLA2 mass) and there was no confounding by subclinical atherosclerosis measures. Associations of Lp-PLA2 activity but not mass were weaker in Chinese participants but there were relatively few events among Chinese in race-stratified analysis.
In this multi-ethnic cohort, Lp-PLA2 was positively associated with CVD risk, regardless of the presence of coronary artery calcium or a thickened carotid-intimal media.
Lipoprotein-associated phospholipase A2 (Lp-PLA2) is a 50-kd calcium-independent enzyme highly expressed by macrophages in atherosclerotic lesions.1,2 Lp-PLA2 is responsible for the hydrolysis of oxidized phospholipids on LDL particles.3,4 The presence and activity of Lp-PLA2 within a plaque appear to be associated with vulnerable, rupture-prone plaques.5 Thus, Lp-PLA2 may be a marker specific to vascular inflammation.6
Prior studies in individuals free of prevalent cardiovascular disease (CVD) have documented an association between higher Lp-PLA2 mass and elevated Lp-PLA2 activity with incident coronary heart disease and ischemic stroke.7–12 These studies included primarily white individuals, with data in non-whites largely limited to Asian populations. Additionally, prior studies did not evaluate whether the risk of incident cardiovascular events associated with Lp-PLA2 differed based on presence of subclinical atherosclerosis. Individuals with subclinical atherosclerosis are at higher risk for developing incident CVD compared to those without subclinical atherosclerosis.13 If associations of Lp-PLA2 with incident CVD are larger for those with subclinical disease compared to those without subclinical disease, this might identify a group more likely to experience a reduction in primary cardiovascular disease with Lp-PLA2 inhibition. In patients with stable coronary heart disease (CHD), however, oral Lp-PLA2 inhibition did not significantly reduce the composite outcome cardiovascular death, myocardial infarction (MI), or stroke although there was a reduced risk of coronary events.14
We evaluated associations of both Lp-PLA2 mass and activity with incident cardiovascular events in a healthy multi-ethnic cohort characterized at baseline for subclinical atherosclerosis. We hypothesized larger associations of Lp-PLA2 with cardiovascular events in those with subclinical atherosclerosis.
MESA recruited 6814 adults aged 45 to 84 years from 6 field centers (Baltimore, Maryland; Chicago, Illinois; Forsyth County, North Carolina; Los Angeles, California; New York, New York; and St Paul, Minnesota) to a baseline examination between July 2000 and September 2002.15 The study participants were white (38%), African American (28%), Hispanic (22%), and Chinese American (12%) and without known clinical CVD. MESA conducted 3 subsequent examinations of the cohort between 2002 and 2007. Institutional review boards at each site approved the study, and all participants gave written informed consent.
At baseline, standardized questionnaires were used to obtain demographic information, level of education, annual household income, smoking history, and medication usage for high blood pressure, high cholesterol, or diabetes. Cigarette smoking was calculated in pack-years and also defined as current, former, or never. Body mass index (BMI) was calculated as weight in kilograms divided by height in meters squared. Systolic and diastolic resting blood pressures were measured in seated participants.16
Total and high-density lipoprotein (HDL) cholesterol, triglycerides, and glucose levels were measured from blood samples obtained after a 12-h fast. Low-density lipoprotein cholesterol was calculated by the Friedewald equation. Diabetes was defined as fasting glucose >125 mg/dl or use of hypoglycemic medication. C-reactive protein (CRP) was quantified by a high-sensitivity assay (N-High-Sensitivity CRP; Dade Behring, Deerfield, IL; inter-assay coefficient of variation: 2.1–5.7%).
Both Lp-PLA2 mass and activity were measured in plasma samples from the baseline examination. Measurements were performed by diaDexus Inc. (South San Francisco, CA).17 Lp-PLA2 mass was measured with a sandwich enzyme immunoassay (PLAC™ Test; diaDexus). Lp-PLA2 activity was measured by an enzymatic assay using a tritium-labeled platelet activating factor (PAF) analog as the substrate. The interassay coefficients of variation were 6.0% for Lp-PLA2 mass and 5.0% for Lp-PLA2 activity. LpPLA2 values were not available in 1328 participants, mostly due to lack of consent for research involving a commercial entity.
Scanning centers assessed coronary artery calcium (CAC) by CT using either a cardiac-gated electron-beam CT scanner or a multidetector CT system.18 Participants were scanned twice consecutively over phantoms of known physical calcium concentration. The phantom contained 4 bars of known calcium density and was used to calibrate the x-ray attenuation level between measurements conducted on different machines. A radiologist or cardiologist read all CT scans at a central reading center (Harbor-UCLA Medical Center/Los Angeles Biomedical Research Institute, Torrance, California). An abnormal CAC was defined as a value greater than zero. The mean phantom-adjusted Agatston score was used in all analyses.19
Carotid intimal medial thickness (CIMT) ultrasound measurements were interpreted at Tufts-New England Medical Center, Boston, Massachusetts and have been previously described.20 Maximal IMT was defined as the mean maximum IMT value taken from the near and far walls of the right and left sides for both the internal carotid artery (ICA) and common carotid artery (CCA). An abnormal CIMT was defined as either an ICA or CCA value in the highest overall quintile.
At 9–12 month intervals, participants or family members were contacted regarding interim hospital admissions, outpatient diagnoses of CVD, and deaths. Follow-up for this analysis extended through 2011. To verify self-reported diagnoses, trained personnel abstracted data from hospital records. Next of kin and physicians were contacted for participants with out-of-hospital cardiovascular deaths. Two physician members of the MESA mortality and morbidity review committee independently classified events. The full committee made final classifications if there were disagreements.
Events were classified as due to CVD or CHD. CVD events included nonfatal MI, resuscitated cardiac arrest, CVD death, definite angina and probable angina associated with revascularization, and ischemic stroke. CHD events included nonfatal MI, resuscitated cardiac arrest, CHD death, definite angina and probable angina associated with revascularization. A subset of CHD events was defined as ‘hard’ CHD events and included CHD death or nonfatal MI. Revascularizations not preceded by a diagnosis of angina were not included in the CVD endpoint. The diagnosis of MI was based on symptoms, electrocardiographic findings, and levels of circulating cardiac biomarkers. A death was considered related to CHD if it occurred within 28 days after a myocardial infarction, if the participant had had chest pain within 72 hours before death, or if the participant had a history of CHD and there was no known nonatherosclerotic, non-cardiac cause of death. Reviewers classified resuscitated cardiac arrest when a patient successfully recovered from full cardiac arrest through cardiopulmonary resuscitation (including cardioversion). Adjudicators graded angina using their clinical judgment. A classification of definite or probable angina required clear and definite documentation of symptoms distinct from the diagnosis of MI. Classification of definite angina also required objective evidence of reversible myocardial ischemia or obstructive coronary artery disease. A more detailed description of the MESA follow-up methods is available at http://www.mesa-nhlbi.org/followup.aspx.
Baseline characteristics were compared between CVD event cases and non-cases using t-tests for continuous variables and chi-square tests for categorical variables.
The associations of standard deviation increments of baseline Lp-PLA2 mass and activity with incident cardiovascular events were evaluated using Cox proportional-hazard models, adjusting for age, gender, race/ethnicity, body-mass index (BMI), diabetes mellitus, smoking status, systolic blood pressure, total and HDL cholesterol, statin use, anti-hypertensive use, education, CRP, and continuous measures of subclinical atherosclerosis (maximal CCA IMT, maximal ICA IMT, and CAC). Models were run with and without adjustment for subclinical disease measures because they could be in the pathway between Lp-PLA2 and CVD. We used the transformation ln (CAC+1) for analysis. These analyses were performed for each of the following cardiovascular endpoints: (1) CVD, (2) CHD, and (3) CHD (hard). We used generalized additive models to evaluate whether the assumption of linearity was significantly violated. All relationships seemed well represented by the linear model. We tested for multiplicative interactions between the Lp-PLA2 variables and age, gender, race/ethnicity and subclinical CVD. A p-value for interaction of less than 0.05 was considered statistically significant and stratified results shown by race/ethnicity and presence of absence of subclinical atherosclerosis by design.
5,486 (80.5%) MESA participants had Lp-PLA2 mass and activity measured at baseline, of which 30 were excluded because they did not have follow-up data. Chinese individuals were less likely to have missing Lp-PLA2 measurements while Black individuals were more likely to have missing Lp-PLA2 measurements. Otherwise, there were no meaningful differences in baseline characteristics between participants with versus without Lp-PLA2 measurements.
Of 516 validated cardiovascular events occurring during follow-up, 358 were due to CHD and 223 of these were ‘hard’ events. Baseline characteristics of the study population are shown in Table 1. Compared with noncases, individuals who developed CVD were older and more likely male. They had significantly higher systolic and diastolic blood pressures, and lower HDL-C levels. Diabetes, statin use, and anti-hypertensive use were more prevalent in CVD cases. Individuals who developed CVD had a higher proportion of current and former smokers compared with individuals who did not. Mean levels of both Lp-PLA2 mass and Lp-PLA2 activity were higher in CVD cases, CHD cases, and hard CHD cases when compared to noncases (Table 2).
In a Cox proportional hazards model adjusted for age, sex, race/ethnicity, diabetes, smoking, total and HDL cholesterol, systolic blood pressure, lipid and anti-hypertensive medication use, BMI, education, and CRP, higher Lp-PLA2 mass and activity were both associated with an increased risk of incident CVD, CHD, and ‘hard’ CHD with the largest associations for hard CHD (Table 3). After additional adjustment for continuous measures of subclinical atherosclerosis (maximal CCA IMT, maximal ICA IMT, and CAC), the hazard ratios for incident CVD, CHD and hard CHD were nearly identical for both Lp-PLA2 mass and activity.
In the subset of patients on baseline statin therapy (n=879), higher Lp-PLA2 mass was not associated with an increased risk of incident CVD (HR 1.06, 95% CI 0.87– 1.29), CHD (HR 1.14, 95% CI 0.91–1.41), or ‘hard’ CHD (HR 1.24, 95% CI 0.93–1.64) after adjustment for age, gender, and race/ethnicity. Similarly, higher Lp-PLA2 activity was also not associated with an increased risk of incident CVD (HR 1.12, 95% CI 0.89–1.41), CHD (HR 1.24, 95% CI 0.96–1.60), or ‘hard’ CHD (HR 1.39, 95% CI 0.99–1.97).
When performing tests for interaction between Lp-PLA2 and sex a significant interaction was seen for Lp-PLA2 mass and the endpoints of CHD (p interaction=0.01) and ‘hard’ CHD (p interaction=0.02). Lp-PLA2 mass was more strongly associated with incident CHD in women than men. The interaction terms for Lp-PLA2 activity and sex for CHD and ‘hard’ CHD were 0.19 and 0.12 respectively. No significant interactions were seen between Lp-PLA2 mass and activity and either CRP or sex for the CVD endpoint.
There was some evidence of effect modification by race/ethnicity for Lp-PLA2 activity and the endpoints of CVD (p interaction=0.16), CHD (p interaction=0.06), and ‘hard’ CHD (p interaction=0.07), but it did not meet our stringent significance level. In age and gender adjusted analysis, higher Lp-PLA2 activity was not associated with an increased risk of incident CVD (HR 0.95, 95% CI 0.71–1.28), CHD (HR 0.90, 95% CI 0.64–1.26), or ‘hard’ CHD (HR 0.79, 95% CI 0.50–1.26) in Chinese participants. The number of events for incident CVD, CHD, and ‘hard’ CHD among Chinese participants were 41, 32, and 17 respectively. The association of higher Lp-PLA2 activity and incident CVD in Blacks reached borderline significance (p=0.10) but all other associations of higher Lp-PLA2 activity and all three endpoints were significant and similar in Blacks, Hispanics, and Whites (Table 4).
3,228 participants had evidence of baseline subclinical disease and 450 of these individuals experienced a cardiovascular event. After adjusting for age, sex, and total cholesterol, Lp-PLA2 mass and activity had no significant correlation with either carotid IMT or CAC. Both Lp-PLA2 mass and Lp-PLA2 activity were associated with increased risk of incident CVD in the subgroup of individuals with baseline subclinical disease (Table 5). Among the 2,228 participants without evidence of baseline subclinical disease, only 66 experienced a cardiovascular event. Lp-PLA2 activity but not mass was associated with increased risk for a CVD event. Despite the apparent difference in the association of Lp-PLA2 mass with CVD by subclinical disease status, the interaction terms for Lp-PLA2 mass with subclinical disease was not statistically significant (p= 0.32 in a model adjusted for age, sex and race/ethnicity).
Higher Lp-PLA2 mass and activity were both associated with increased incidence of CVD and CHD in a multiethnic cohort without clinical CVD at baseline. Higher Lp-PLA2 activity was associated with a similar increased CVD risk in individuals with or without baseline subclinical disease, defined by the presence of calcified coronary artery disease or a thickened carotid intima-media. Although higher Lp-PLA2 mass was associated with increased CVD risk in individuals with subclinical disease but not in those without subclinical disease, the difference by subclinical disease status failed to achieve statistical significance.
The association between higher Lp-PLA2 and CVD risk has been previously reported in both white and Asian populations and our hazard ratios are consistent with prior studies.8–10,12,21–24 In a recent meta-analysis of 32 prospective studies, associations with incident coronary heart disease and ischemic stroke were 1.11 and 1.14 respectively per standard deviation increment of Lp-PLA2 mass and 1.10 and 1.08 respectively per standard deviation increment of Lp-PLA2 activity.7 In this meta-analysis, however, Lp-PLA2 activity was not associated with incident coronary heart disease in the subset of patients without a history of vascular disease, which is in contrast to what we found.7
Our results extend these previous findings to a multi-ethnic population, which has been underrepresented in research to date. The increased risk was similar across different ethnicities with the exception of no association of Lp-PLA2 activity in Chinese individuals. A higher prevalence of certain gene polymorphisms in Chinese individuals may be responsible for these findings. Mutations in the V279F allele of the PLA2G7 gene are associated with lower Lp-PLA2 activity.25, 26 The carrier frequency of these polymorphisms is high in Asian populations, 25% in Japanese individuals and greater than 10% in Korean and Chinese individuals.27–29 In a cross-sectional study of Korean males, presence of this polymorphism was associated with a 20% less CAD prevalence. 30 The polymorphism, however, was not associated with risk of ischemic stroke in a study of Chinese individuals.31 In two large meta-analyses that included individuals of Europoid ancestery, a reduction in incident CVD risk associated with these polymorphisms was not demonstrated.29,32 Our null findings in Chinese subjects may also simply be due to small event numbers or chance.
Lp-PLA2 can be measured by quantification of either its mass or activity. Previous studies that measured both mass and activity reported only moderate correlations between the two measurement methods.22 Activity may be more reflective of the inflammatory state induced by Lp-PLA2.22 With respect to cardiovascular outcomes the two measurements perform similarly, with a recent meta-analysis reporting that the risk ratios for Lp-PLA2 were similar whether mass or activity of the Lp-PLA2 enzyme was measured, in agreement with our results.7 Although our results suggest there is effect modification by sex for Lp-PLA2 mass and CHD, these findings are likely due to chance since the same meta-analyses showed no difference by sex in the association between Lp-PLA2 mass and CHD.7
There were a significantly higher proportion of patients on baseline statin therapy who experienced a cardiovascular event compared to those not on statin therapy. Hazard ratios of Lp-PLA2 mass and activity with incident cardiovascular disease in the subset of participants on baseline statin therapy, although not statistically significant, were similar to the associations in the entire cohort. The lack of significance is likely attributed to limited power and Lp-PLA2 levels are likely a risk factor regardless of statin use at baseline.
Since Lp-PLA2 is considered a marker of vascular inflammation, we hypothesized that levels of Lp-PLA2 in individuals with known subclinical atherosclerosis may be more predictive of incident CVD compared to levels in those without subclinical atherosclerosis; however, this was not observed in our study. By contrast, in a report from the Cardiovascular Health Study, higher CRP was associated with CVD risk only in those participants with detectable atherosclerosis on carotid ultrasound.33
Both Lp-PLA2 mass and activity were weakly correlated with carotid IMT and CAC in our study. Studies on associations between Lp-PLA2 and measures of subclinical atherosclerosis in individuals have reported mixed results. The Cardiovascular Health Study and the Malmo Diet and Cancer Study have both reported associations with higher Lp-PLA2 activity and higher carotid IMT.34,35 Conversely, other studies reported no associations between Lp-PLA2 and carotid artery atherosclerosis.36,37 Regarding CAC, in the Coronary Artery RIsk Development in Young Adults study higher Lp-PLA2 mass, but not activity, was associated with presence and severity of calcified coronary plaque after adjustment for cardiovascular risk factors including both LDL and HDL cholesterol.38 In other studies, there were no associations between Lp-PLA2 and either coronary artery calcification or carotid artery atherosclerosis when adjusted for total and HDL cholesterol.39,40
Our findings suggest that the association of Lp-PLA2 with incident CVD appears to be through mechanisms independent of those associated with the presence of measurable subclinical disease as assessed here. In a study of patients with a zero coronary artery calcium score, 11% had evidence of noncalcified plaque on CT coronary angiogram.41 A prior study demonstrated that reduction in Lp-PLA2 reduces progression of necrotic core volume but not total atheroma volume in human coronary atherosclerotic plaque.42 Our findings suggest that Lp-PLA2 identifies characteristics of vulnerable plaque not associated with traditional measures of subclinical disease. Lp-PLA2 may, therefore, potentially help to identify individuals at higher risk for cardiovascular events without regard to presence of measurable subclinical atherosclerosis.
Our study has limitations. The population included individuals with no known baseline clinical CVD and findings cannot be generalized to dissimilar populations. The number of CVD events was low for some strata in our stratified analyses. The lack of an association between Lp-PLA2 and incident cardiovascular disease when stratified by subclinical disease or across certain ethnicities may be due to limited power. Other studies or longer term follow-up in MESA is required to further investigate these questions. Non-ischemic cardiac causes cannot be excluded as an etiology for resuscitated cardiac arrest; however, only 7 of 516 CVD events were exclusively due to resuscitated cardiac arrest and it is unlikely to affect the results. Lastly, our detection of atherosclerosis is based on surrogate measures and does not capture all participants with evidence of subclinical disease.
In conclusion, Lp-PLA2 mass and activity were both associated with CVD and CHD risk in a multiethnic cohort characterized for the presence of subclinical disease at baseline using presence of calcified coronary disease or a thickened carotid intima-media. The increased CVD risk was similar in individuals with or without baseline subclinical disease. Our findings suggest that the association of Lp-PLA2 with incident CVD appears to be through mechanisms independent of those associated with the measures of subclinical disease as assessed in this cohort.
We thank the other investigators, the staff, and the participants of the MESA study for their valuable contributions. A full list of participating MESA investigators and institutions can be found at http://www.mesa-nhlbi.org
Sources of funding:
This research was supported by contracts N01-HC-95159, N01-HC-95160, N01-HC-95161, N01-HC-95162, N01-HC-95163, N01-HC-95164, N01-HC-95165, N01-HC-95166, N01-HC-95167, N01-HC-95168, N01-HC-95169 and grant K12-HL083790 from the National Heart, Lung, and Blood Institute and by grants UL1-TR-000040 and UL1-TR-001079 from the National Center for Research Resources. Additional support was from an investigator initiated grant from GlaxoSmithKline.
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