Lipoprotein-Associated Phospholipase A₂ Activity and Incident Coronary Heart Disease Among Men and Women With Type 2 Diabetes

The Health Professionals Follow-up Study (HPFS) and Nurses' Health Study (NHS) are prospective cohort studies of 51,529 men and 121,700 women, respectively. The present study includes 740 men and 777 women with diabetes who were free of cancer and cardiovascular disease at blood draw, and who had detectable Lp-PLA₂ activity values. These cohorts have been described in detail elsewhere (11,12).

Participants who indicated a diagnosis of diabetes on any biennial follow-up questionnaire were sent a supplementary questionnaire to confirm diabetes. We used the National Diabetes Data Group criteria (13) to define diabetes because our participants were diagnosed before the American Diabetes Association released their criteria in 1997. Diabetes was confirmed in subsamples from the HPFS (97% confirmed) and the NHS (98% confirmed) (14,15).

CHD end points were coronary artery bypass graft surgery or percutaneous transluminal coronary angioplasty (CABG/PTCA), nonfatal myocardial infarction (MI), and fatal CHD. If participants experienced multiple CHD events during follow-up, they were assigned the more severe clinical event, but censored at the date of the first event. Further information about diabetes and CHD diagnoses has been provided previously (11,12) and is provided in the supplementary information (available in an online appendix at http://diabetes.diabetesjournals.org/cgi/content/full/db09-0730/DC1).

Lp-PLA₂ activity was measured by a colorimetric activity test automated assay performed on a clinical chemistry analyzer using a colorimetric substrate for Lp-PLA₂ (7). All samples were run in duplicate with a mean coefficient of variation (CV) of 4.9%. Lp-PLA₂ concentration was measured with an enzyme-linked immunoassay (PLAC test; diaDexus, San Francisco, CA). Other biomarker measurements are described in the supplementary methods. In this study, there was a moderate and statistically significant correlation between concentration and activity (r = 0.30, P < 0.001, among women; r = 0.29, P < 0.001, among men). In preliminary analyses, Lp-PLA₂ concentration was not associated with incident coronary heart disease (data not shown), indicating that Lp-PLA₂ activity may be the more biologically relevant parameterization of Lp-PLA₂. All subsequent analyses therefore focused on Lp-PLA₂ activity.

Follow-up began at the month of blood draw and continued either until June 2004, CHD event, or death. We used Cox proportional hazards analysis stratified by 5-year age categories over each 2-year follow-up period to estimate relative risks (RRs) and 95% CIs for tertiles of Lp-PLA₂ activity. Tests for trend were estimated across median values of Lp-PLA₂ tertile. To assess predictive ability, we calculated the area under the receiver-operating characteristic curves for multivariable-adjusted models with and without Lp-PLA₂ activity included. We assessed potential effect modification by modeling joint effects of Lp-PLA₂ tertile and tertiles of LDL, HDL, C-reactive protein, hormone replacement therapy use, and duration of diabetes. We then compared models with and without a cross-classification term using −2 log likelihood tests. If there was absence of heterogeneity of results between the NHS and HPFS as assessed by the Q-statistic, we pooled results by weighing each estimate by the inverse of its variance (STATA; StataCorp, College Station, TX). All other analyses were performed using SAS statistical software (SAS Institute, Cary, NC).

During 8,010 person-years of follow-up in the men, 178 cases of CHD occurred, including 64 cases of nonfatal MI and fatal CHD and 114 CABG/PTCA. In the women, 146 cases of CHD occurred during 9,643 person-years of follow-up, including 73 cases of nonfatal MI and fatal CHD and 73 cases of CABG/PTCA. Among both men and women, smoking and less aspirin use were related to higher levels of Lp-PLA₂ activity. Additionally, among women only, history of hypertension and family history of heart disease were positively associated, and postmenopausal hormone use was inversely associated with Lp-PLA₂ activity (Table 1). In addition, shorter duration of diabetes was associated with higher Lp-PLA₂ activity. In both men and women, Lp-PLA₂ activity was positively associated with LDL and intercellular adhesion molecule and inversely associated with HDL (supplementary Table 1, available in the online appendix).

We calculated the relative risk associated with tertiles of Lp-PLA₂ activity for specific CHD end points and for total CHD (Table 2). With no differences in risk estimates between sexes, we pooled the results from the two studies (Q = 0.12, P = 0.73 for total CHD; Q = 0.15, P = 0.70 for nonfatal MI and fatal CHD; and Q = 0.85, P = 0.36 for CABG/PTCA); sex-specific results are presented in supplementary Tables 2 and 3. The age-adjusted RR between the extreme tertiles of Lp-PLA₂ activity was 1.93 (95% CI 1.25–2.97; P for trend = 0.001) for nonfatal MI and fatal CHD, 1.61 (1.12–2.30; P for trend = 0.009) for CABG/PTCA, and 1.76 (1.34–2.32; P = 0.001) for total CHD. After adjusting for lifestyle and clinical risk factors other than LDL, these relative risks were only modestly attenuated. However, after additional adjustment for LDL, the relative risk was strongly attenuated for CABG/PTCA (RR 1.12; 95% CI 0.74–1.70), but not so for nonfatal MI and fatal CHD (RR 1.75; 1.05–2.92). These results were slightly stronger for fatal CHD (RR 2.01; 0.90–4.50; P for trend <0.001 for fully adjusted extreme tertiles) than nonfatal MI (RR 1.62; 0.81–3.22; P for trend = 0.002 for fully adjusted extreme tertiles), but there was no statistically significant difference between these two end points (Q = 0.16; P = 0.69). Lp-PLA₂ activity added significantly to the area under the receiver-operating characteristic curve for MI in men (P = 0.02), but did not significantly add to predictive ability for any other end point in men or women. Additional adjustment for hypertension medications did not alter these results.

We next examined whether Lp-PLA₂ activity is more strongly related to cardiovascular events occurring closer to the date of Lp-PLA₂ measurement. Over the first 6 years of follow-up, there were 138 total cases of CHD among the men and women, and over the next 6 years in the HPFS and 8 years in the NHS, there were 186 total cases of CHD. Lp-PLA₂ activity was more strongly related to incident CHD during the first 6 years of follow-up (RR 2.00, 95% CI 1.16–3.44 for extreme tertiles) but less so after 6 years of follow-up (1.23, 0.81–1.89) (Table 3), irrespective of type of CHD (data not shown).

Because the risk of CHD associated with Lp-PLA₂ may vary by levels of other lifestyle and biological markers, we tested for interactions in the combined datasets and found no significant or meaningful interactions with LDL, HDL, C-reactive protein, hormone replacement therapy use, or duration of diabetes (data not shown).

In the present study, higher levels of Lp-PLA₂ activity were associated with increased risk of incident coronary heart disease among diabetic men and women. This is the first prospective study specifically to examine Lp-PLA₂ and incident CHD among diabetic subjects, and the relative risks found in this study are similar to those from studies that have examined general community and clinical populations.

We found that Lp-PLA₂ activity was more strongly associated with nonfatal MI and fatal CHD than with CABG/PTCA. It is possible that CABG/PTCA is a weaker clinical end point, since reasons to clinically intervene may be more subjective and less likely to be conducted in the sickest patients. In the only other study explicitly to investigate several different CHD outcomes, Oei et al. (9) found virtually no difference in relative risk by outcome. We also found that Lp-PLA₂ activity was more strongly related to cardiovascular events that occurred within the first 6 years of follow-up. Our study is the first to examine the relation between Lp-PLA₂ and incident CHD stratified by time to event in a nonclinical population. Although Lp-PLA₂ concentration has been associated with coronary artery calcification (16,17), Lp-PLA₂ activity has consistently demonstrated lack of association with coronary artery calcification (17,18). This, in combination with our observations that both later occurrence of CHD and CABG/PTCA were more weakly associated with Lp-PLA₂, could indicate that Lp-PLA₂ activity is related to unstable or rupture-prone lesions rather than to atherosclerosis alone. This is supported by recent evidence from a randomized trial that demonstrated that use of an Lp-PLA₂ activity inhibitor decreased plaque severity over 12 weeks (19).

In a recent investigation in the population-based Atherosclerosis Risk in Communities (ARIC) study, the authors found that of 19 novel biomarkers, Lp-PLA₂ concentration was the only marker to add significantly to the area under the receiver operating curves, although this magnitude was relatively small (20). This is of considerable importance for diabetic populations because of their increased risk of CHD. In the present study, Lp-PLA₂ activity added significantly to risk prediction for MI among men, but not for other end points in men or women. It is possible that our study is underpowered. Future studies with larger populations should examine the predictive ability of Lp-PLA₂ because markers such as Lp-PLA₂ that can add to risk prediction can help uncover important etiologic pathways for cardiovascular disease.

In this study, although Lp-PLA₂ activity was associated with incident CHD, Lp-PLA₂ concentration was not. It is possible that the diabetes-associated disruption of metabolic parameters also affects Lp-PLA₂ concentrations and renders these values less informative. This will need to be confirmed in other populations. It is unclear which parameterization of Lp-PLA₂ will ultimately be most relevant for prediction and etiology, and future studies with both measurements should tease out these distinct pathways.

Lp-PLA₂ is hypothesized to contribute to the development of atherosclerosis through the propagation of inflammatory processes in the arterial intima (21). Several characteristics of Lp-PLA₂ suggest that this enzyme could be specifically relevant in people with diabetes. First, diabetic subjects have increased circulating levels of small dense LDL (2), potentially providing an efficient vehicle for delivery of Lp-PLA₂ to the intima, since Lp-PLA₂ has apparent binding preference for small dense LDL (22). Second, free radicals from oxidation of excess glucose may increase the oxidation of lipoproteins (23) and thereby increase levels of the substrate for Lp-PLA₂. Third, diabetes is characterized by increased macrophage infiltration of the arterial wall (2). Lp-PLA₂ is secreted by macrophages, although the actual level of Lp-PLA₂ production within the artery wall is unclear (21). Finally, in addition to diabetic and insulin-resistant conditions providing a milieu that would foster increased Lp-PLA₂ concentration and activity, Lp-PLA₂ could potentially perpetuate insulin resistance through its generation of free fatty acids and increased chemotaxis, both of which could lead to persistent chronic inflammation (21,24,25).

This study has several limitations. First, although we have a relatively large number of men and women with diabetes in this study, we have less power to detect relationships when stratifying by type of CHD. Second, Lp-PLA₂ was only measured at baseline, which may lead to modest misclassification over time. However, in a pilot study from the Health Professionals Follow-Up Study, men provided two samples 1 year apart, and Lp-PLA₂ activity was shown to be highly reproducible (intraclass r = 0.87).

In conclusion, among male and female participants with type 2 diabetes, Lp-PLA₂ activity was an independent risk factor for coronary heart disease, particularly for nonfatal MI and fatal CHD. This is the first prospective study to document this relationship among diabetic subjects who were free of CVD at baseline. Although the risk for CHD potentially conferred by Lp-PLA₂ among diabetic subjects appears to be consistent with those in the general population, further research is needed to explore whether reduction in Lp-PLA₂ reduces risk of CHD in diabetic subjects.

This study was supported by research Grant DK58845 from the National Institute of Diabetes and Digestive and Kidney Diseases. Additional support was provided by GlaxoSmithKline. GlaxoSmithKline had no access to the data, and the academic institution had full and final right to publish. I.J.H. and E.B.R. reported research support from GlaxoSmithKline. J.J.N. reported employment by GlaxoSmithKline. No other potential conflicts of interest relevant to this article were reported.