Lipoprotein-Associated Phospholipase A2 and Measures of Extracoronary Atherosclerosis
The Rotterdam Study
Objective— Lipoprotein-associated phospholipase A2 (Lp-PLA2) may be a new and independent predictor of cardiovascular events. The effect of Lp-PLA2 may be exerted through effects of the enzyme on the development of atherosclerosis. Therefore, we investigated the association between Lp-PLA2 activity and measures of extracoronary atherosclerosis.
Methods and Results— Lp-PLA2 activity was determined in a random sample of 1820 participants from the Rotterdam Study, a population-based cohort study in men and women ≥55 years. Common carotid intima-media thickness, carotid plaques, ankle-arm index, and aortic calcification were examined. Atherosclerosis status could be assigned in 1609 participants. The age-adjusted odds ratio of having atherosclerosis at any site for the highest versus the lowest tertile of Lp-PLA2 activity was 1.86 (95% CI, 1.01 to 3.43) in men and 1.60 (95% CI, 1.08 to 2.37) in women. After additional adjustment for cholesterol, these associations attenuated or even disappeared. The odds ratios of having atherosclerosis at specific sites (carotid arteries and aortic-iliac-femoral tract) followed a similar pattern.
Conclusions— Although Lp-PLA2 has been found to be independently associated with cardiovascular events, the association with measures of subclinical extracoronary atherosclerosis found in this study strongly attenuated or even disappeared after adjustment for cholesterol.
Recently, several studies suggested that lipoprotein-associated phospholipase A2 (Lp-PLA2) may be a new and independent risk factor for cardiovascular disease.1–5 Lp-PLA2 hydrolyzes oxidatively modified low-density lipoprotein (LDL) by cleaving oxidized phosphatidylcholines, generating lysophosphatidylcholine and oxidized free fatty acids. Lysophosphatidylcholine and oxidized free fatty acids are both chemoattractants for monocytes and may account for a part of the proinflammatory capacities of oxidized LDL cholesterol.6
Although experimental studies imply that the effect of Lp-PLA2 on cardiovascular disease is exerted through effects of the enzyme on the development of atherosclerosis, little population-based research has been reported on this topic. Several studies have examined the association between Lp-PLA2 and angiographically documented coronary atherosclerosis in case-control settings using high-risk subjects, most of them finding an independent association7–10 and 1 of them finding that the association disappeared after adjusting for clinical and lipid variables.11 Furthermore, in a nested case-control study among young adults participating in the population-based CARDIA study, an independent association was found of Lp-PLA2 mass with calcified coronary plaque assessed by computed tomography.12
In a study among 190 hypercholesterolemic Sicilian individuals, no association was found between Lp-PLA2 activity and carotid intima-media thickness (IMT).13 However, this was a relatively small study, showing only unadjusted values of mean plasma Lp-PLA2 activity for subjects with normal and high carotid IMT. In a study among 247 patients referred for lower extremity arterial evaluation, Lp-PLA2 was a borderline significant predictor of lower ankle-arm index (AAI) after adjustment for conventional cardiovascular risk factors and statin use.14 So far, no population-based studies have investigated whether Lp-PLA2 activity is associated with measures of extracoronary atherosclerosis. We investigated whether Lp-PLA2 activity is associated with atherosclerosis at different sites of the vascular tree in the Rotterdam Study.
The Rotterdam Study is a prospective population-based cohort study comprising 7983 men and women ≥55 years of age. Its overall aim is to investigate the incidence of and risk factors for chronic disabling diseases. From 1990 to 1993, all inhabitants of a suburb of the city of Rotterdam ≥55 years of age were invited to participate in an extensive home interview and 2 visits to the research center. The overall response was 78%. The medical ethics committee of Erasmus MC approved the Rotterdam Study, and written informed consent was obtained from all participants. A more detailed description of the Rotterdam Study and the collection of data have been given previously.15
For the present study, a random sample of 1820 participants was drawn from the source population, and in this subcohort, baseline measurements of Lp-PLA2 activity were conducted. Within this group, measurements of IMT were available for 1430 participants, and assessment of carotid plaques, AAI, and aortic calcification for 1435, 1624, and 1259 participants, respectively. Missing measurements were mainly attributable to logistic reasons. Values for cardiovascular risk factors were missing in <6% of participants.
Measurement of Lp-PLA2 Activity
Plasma aliquots prepared from nonfasting blood samples were collected at baseline and stored at −80°C, and Lp-PLA2 activity was measured with a high throughput radiometric activity assay. Briefly, plasma samples were aliquoted into 96-well microtiter plates and mixed with a substrate solution consisting of 0.4 μmol/L [3H]-platelet-activating factor (Specific Activity 21.5 Ci/mmol; Perkin Elmer Life Sciences) and 99.6 μmol/L C16-platelet-activating factor (Avanti Polar Lipids Inc) in assay buffer (100 mmol/L HEPES, 150 mmol/L NaCl, and 5 mmol/L EDTA, pH 7.4). The reactions were allowed to proceed at room temperature for 5 minutes before sequestering of the phospholipid substrates by an ice-cold fatty acid–free BSA solution at a final concentration of 16.1 mg/mL. The BSA–lipid complexes were then precipitated with ice-cold trichloroacetic acid at a final concentration of 7.8% and pelleted by centrifugation at &6000g for 15 minutes at 4°C. Aliquots of the supernatant containing the reaction products were transferred to another microplate (Perkin Elmer) and the radioactivity counted in a Topcount liquid scintillation counter (Perkin Elmer Life Sciences) on addition of Microscint-20 scintillation cocktail (Perkin Elmer Life Sciences). Lp-PLA2 activity was expressed as nanomoles of platelet-activating factor hydrolyzed per minute per milliliter of plasma samples.
Before analysis of plasma samples from the Rotterdam Study, a prestudy validation was conducted to determine the reliability of the Lp-PLA2 activity assay. Six plasma samples were tested in triplicate, and the coefficient of variation (CV) for intra-assay precision ranged from 3.51% to 8.96%. To assess interassay precision, 6 plasma samples were tested on 3 occasions, and CV ranged from 8.48% to 15.08%. Three cycles of freeze-thaw of frozen plasma did not result in appreciable loss of activity. The assay was therefore considered suitable for analysis of the Rotterdam Study samples, which were tested in duplicate. Samples were retested if the replicate CV was >25%. The range of detection was 8 to 150 nmol/min per mL.
Measures of Atherosclerosis
Ultrasonography of both carotid arteries was performed with a 7.5-MHz linear-array transducer and a duplex scanner (ATL UltraMark IV; Advanced Technology Laboratories). Measurements of the common carotid IMT involved regions of the common carotid arteries proximal to the carotid bulb, starting at a distance of 1 cm from the bulb. IMT was determined as the average of mean near- and far-wall measurements, computing the average of left and right common carotid IMT.16 We considered carotid IMT <1 mm as absence of atherosclerosis according to this measurement.
The internal carotid artery, carotid bifurcation, and common carotid artery were examined both left and right for the presence of plaques. Plaques were defined as a focal widening relative to adjacent segments, with the protrusion into the lumen composed either of only calcified deposits or a combination of calcification and noncalcified material.16 The anterior and posterior wall were evaluated for the presence of a plaque. Carotid plaques were dichotomized into presence or absence of carotid plaques.
Using a random-zero sphygmomanometer, sitting blood pressure was measured at the right upper arm. The average of 2 measurements obtained at 1 occasion was used. Systolic blood pressure at the ankles (posterior tibial artery) was measured in supine position with a random-zero sphygmomanometer and an 8-MHz continuous wave Doppler probe (Huntleigh 500D; Huntleigh Technology). The ratio of the systolic blood pressure at the ankle to the systolic blood pressure at the arm was computed to obtain the AAI. For the analyses, we used the lowest value of 2 legs. Values of the AAI larger than 1.50 were considered invalid.17 We considered AAI >0.90 as absence of atherosclerosis according to this measurement.
Aortic calcification was diagnosed by radiographic detection of calcified deposits in the abdominal aorta on a lateral abdominal film.18 Aortic calcification was dichotomized into presence or absence of aortic calcification.
Based on the above 4 measurements, we assigned atherosclerosis status to the participants. Atherosclerosis status could be assigned to 1609 participants, which were subsequently used for the analysis. Participants without atherosclerosis were defined as those with either 3 or 4 measures of atherosclerosis available, which all showed absence of atherosclerosis. Presence of atherosclerosis was defined as presence of atherosclerosis at any site measured.
Furthermore, presence of atherosclerosis was classified according to the 2 vessel beds involved, being the carotid arteries and the aortic-iliac-femoral tract. Atherosclerosis in the carotid arteries was defined as IMT >1 mm or presence of carotid plaques, and atherosclerosis in the aortic-iliac-femoral tract was defined as AAI <0.9 or presence of aortic calcification. Atherosclerosis in both the carotid arteries and the aortic-iliac-femoral tract was defined as IMT >1 mm or presence of carotid plaques concomitant with AAI <0.9 or presence of aortic calcification.
Assessment of Covariates
At baseline, covariates were ascertained using standard procedures as described previously.4 C-reactive protein was measured using a nephelometric method (Immage; Beckman Coulter) in serum which was kept frozen at −20°C. This system has a within-run precision <5.0%, a total precision <7.5%, and a reliability coefficient of 0.995. To compute the correlation between total cholesterol and LDL cholesterol, we determined LDL cholesterol in a random sample of 42 subjects using an enzymatic method (Roche). The correlation coefficient between total cholesterol and LDL cholesterol was high (r=0.91; P<0.001).
First, we tested for differences between the subcohort used for analysis and the remainder of the Rotterdam Study by using a t test for continuous and a χ2 test for dichotomous variables. Because the distribution of C-reactive protein was skewed, the Mann–Whitney test was used for this variable. Second, age-adjusted (except for age) and sex-adjusted (except for sex) correlation coefficients were computed for the association of age, sex, and cardiovascular risk factors with Lp-PLA2.
Lp-PLA2 activity was divided into tertiles using cutoff values of 39 and 48 nmol/min per mL. Because gender differences may exist for Lp-PLA2 activity, we also did sex-specific analyses using sex-specific tertiles (cutoffs 37 and 47 nmol/min per mL for women and 41 and 51 nmol/min per mL for men). We used binary logistic regression to examine the association between tertiles of Lp-PLA2 activity and presence of any sign of atherosclerosis in men and women. In model 1, we adjusted for age. In model 2, total cholesterol and high-density lipoprotein (HDL) cholesterol were added. In model 3, body mass index, systolic blood pressure, diabetes, smoking, cholesterol-lowering medication, and C-reactive protein were entered additionally for men, and for women, hormone replacement therapy was also added. We used multinomial logistic regression to examine the association between tertiles of Lp-PLA2 activity and presence of atherosclerosis in the carotid arteries, the aortic-iliac-femoral tract, or both, in men and women. Again, 3 models were constructed as described above.
Missing values for cardiovascular risk factors were handled by imputing the mean for normally distributed variables, the median for skewed variables, and the value with the highest prevalence for nominal variables. A sensitivity analysis was performed to investigate whether the results changed when only subjects with complete information on all covariates were used.
The characteristics of the subcohort used for the analysis were similar to the baseline characteristics of the remaining population of the Rotterdam Study with a few minor exceptions. Subjects in the subcohort were slightly younger (68.8 versus 71.0 years of age), had a slightly lower mean systolic blood pressure (138 versus 140 mm Hg) and diastolic blood pressure (73 versus 74 mm Hg), a slightly higher total cholesterol level (6.7 versus 6.6 mmol/L), and a somewhat lower prevalence of myocardial infarction (11% versus 14%). Table 1 shows the baseline characteristics of the participants according to sex and atherosclerosis status.
Lp-PLA2 activity was positively associated with male sex (Spearman correlation coefficient r=0.16), body mass index (r=0.074), systolic blood pressure (r=0.070), and total cholesterol (r=0.42). An inverse association was present with HDL cholesterol (r=−0.28). Lp-PLA2 activity was not significantly associated with age, diabetes, smoking, and C-reactive protein.
In Table 2, the association between Lp-PLA2 activity and presence of atherosclerosis in all participants and in men and women separately is displayed. A total of 303 participants were classified as not having atherosclerosis, and 1306 participants were classified as having atherosclerosis. After adjusting for age and sex, the overall odds ratio of having atherosclerosis was 1.77 (95% CI, 1.26 to 2.50) for the highest compared with the lowest tertile of Lp-PLA2 activity. In men, the age-adjusted odds ratio was 1.86 (95% CI, 1.01 to 3.43), and in women, it was 1.60 (95% CI, 1.08 to 2.37). After adjustment for total and HDL cholesterol, the effect disappeared; what was left was a nonsignificant, attenuated odds ratio of 1.40 (95% CI, 0.70 to 2.77) in men. Additional adjustment for body mass index, systolic blood pressure, diabetes, smoking, cholesterol-lowering medication, C-reactive protein, and, for women, hormone replacement therapy, did not materially alter the risk estimates. The middle versus the lowest tertile of Lp-PLA2 did not show any association with atherosclerosis.
Table 3 shows odds ratios of presence of atherosclerosis in the carotid arteries, the aortic-iliac-femoral tract, or both, according to tertiles of Lp-PLA2 activity. The age- and sex-adjusted odds ratio for having aortic-iliac-femoral atherosclerosis was 1.97 (95% CI, 1.34 to 2.90) for the highest versus the lowest tertile of Lp-PLA2 activity in all participants. In men, the corresponding age-adjusted odds ratio was 1.95 (95% CI, 0.98 to 3.87), and in women, it was 1.70 (95% CI, 1.08 to 1.13). These associations disappeared after adjustment for total and HDL cholesterol. The odds ratios for carotid atherosclerosis were somewhat lower and did not reach statistical significance. Again, these were attenuated by adjustment for cholesterol. A strong association was found with having both carotid and aortic-iliac-femoral atherosclerosis; the overall age- and sex-adjusted odds ratio for the highest versus the lowest tertile of Lp-PLA2 activity was 1.97 (95% CI, 1.34 to 2.90). For men and women, the age-adjusted odds ratios were 2.04 (95% CI, 1.06 to 3.95) and 1.82 (95% CI, 1.15 to 2.88), respectively. Again, the associations disappeared after adjustment for total and HDL cholesterol. Performing the analysis using only subjects with complete information on all covariates did not materially change the results.
Lp-PLA2 activity has been found previously to be independently associated with cardiovascular events in the Rotterdam Study.4 In the present study, Lp-PLA2 activity was associated with extracoronary atherosclerosis at different sites of the arterial tree after adjustment for age and sex. However, after adjustment for total cholesterol and HDL cholesterol, the associations between Lp-PLA2 activity and measures of atherosclerosis strongly attenuated or even disappeared.
The inconsistency between the association of Lp-PLA2 with clinical and subclinical atherosclerosis merits attention. First, the atherosclerosis measurements need to be evaluated. This study was performed within the Rotterdam Study, a large population-based study in subjects ≥55 years of age. We used several techniques to measure atherosclerosis. Ultrasound was used to measure the IMT of the common carotid artery and to detect plaques in the common carotid artery, bifurcation, and internal carotid artery. We took x-ray films to assess the amount of aortic calcification, which has been shown to be a highly specific technique for the measurement of aortic intimal atherosclerosis,19 and we used AAI as a measure of lower extremity atherosclerosis. The measures of carotid, aortic, and lower extremity atherosclerosis have all shown to be associated with cardiovascular risk factors and risk of cardiovascular events and are considered to be measures of generalized atherosclerosis.16,20–23 In a previous study, we found that C-reactive protein is independently associated with all these measures of atherosclerosis.24 This suggests that measures of atherosclerosis were determined appropriately in our study.
Second, it needs to be considered that molecules that regulate inflammation will not necessarily correlate with plaque burden measures, as illustrated by inflammatory markers such as C-reactive protein. Although C-reactive protein is associated with coronary events, it has not been a good predictor of the extent of atherosclerotic disease and may measure other characteristics than atherosclerotic mass, such as the activity of lymphocyte and macrophage populations within plaque or the degree of plaque destabilization and ongoing ulceration or thrombosis.25 The same may apply to Lp-PLA2. In a study enrolling 504 patients undergoing clinically indicated coronary angiography, Lp-PLA2 mass was found to be associated with severity of angiographically determined coronary artery disease.11 However, after adjustment for clinical and lipid variables, this association disappeared. In the same study, LpPLA2 was independently associated with coronary events. Furthermore, Lp-PLA2 has been found to be independently associated with cardiovascular events in the West of Scotland Coronary Prevention Study (WOSCOPS), Atherosclerosis Risk in Communities (ARIC), Monitoring of Trends and Determinants in Cardiovascular Disease (MONICA), and Rotterdam studies,1–5 and univariately associated in the Women’s Health Study.26 Also, Lp-PLA2 was found to be independently associated with a positive history of coronary artery disease in a small randomized controlled trial.27
Lp-PLA2 is bound to LDL cholesterol and therefore highly correlated with LDL cholesterol levels. In the present study, no LDL cholesterol levels were available, and therefore, we adjusted for total cholesterol levels. Because of the high correlation between LDL and total cholesterol in a random sample in the present study and because the correlation between Lp-PLA2 and total cholesterol in our study was even higher than that of Lp-PLA2 with LDL cholesterol in the WOSCOPS1 and the ARIC study,2 we believe that we adjusted sufficiently for LDL cholesterol. Furthermore, whereas residual confounding would lead to an overestimation of the effect, we no longer observed an association between Lp-PLA2 activity and measures of atherosclerosis after adjustment for total and HDL cholesterol, showing that adjustment for total cholesterol exerts its effect on the risk estimates.
Few studies have been reported on the association between Lp-PLA2 and extracoronary atherosclerosis. In a study among 190 hypercholesterolemic Sicilian individuals, no association was found between Lp-PLA2 activity and a carotid IMT >1 mm.13 However, this was a small study in high-risk subjects, and only unadjusted values of mean plasma Lp-PLA2 activity for patients with normal and high carotid IMT were presented. Sex-specific values were not given. Furthermore, none of the established cardiovascular risk factors was found to be associated with IMT in this study, most likely because of small sample size. In a study among 247 patients referred for lower extremity arterial evaluation, Lp-PLA2 was a borderline significant predictor of lower AAI after adjustment for conventional cardiovascular risk factors and statin use.14
Several studies have examined the association between Lp-PLA2 and angiographically documented coronary atherosclerosis in case-control settings using high-risk subjects, most of them finding an independent association7–10 and 1 of them finding that the association disappeared after adjusting for clinical and lipid variables.11 Furthermore, in a nested case-control study among young adults participating in the population-based CARDIA study, an independent association was found of Lp-PLA2 mass with calcified coronary plaque assessed by computed tomography.12 These studies have been conducted in various populations and were different in design and are therefore not strictly comparable to our study. Discrepancies between these studies and our study may be attributable to different sites of the atherosclerosis measurements (coronary versus extracoronary), use of high-risk subjects in the case-control studies, and differences in the age range of the participants.
In conclusion, although Lp-PLA2 activity has been found to be independently associated with cardiovascular events in the Rotterdam Study, the association with measures of subclinical extracoronary atherosclerosis found in this study strongly attenuated or even disappeared after adjustment for cholesterol. Future studies are needed to further elucidate the role of Lp-PLA2 in the stages of the atherosclerotic process and the development of cardiovascular disease.
This study was supported by an unrestricted grant from GlaxoSmithKline. The authors thank Yun-Fu Hu and Rena Vora at GlaxoSmithKline for development of the high throughput radiometric activity assay for Lp-PLA2.
- Received February 3, 2005.
- Accepted December 12, 2005.
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