Femoral Plaques Confound the Association of Circulating Oxidized Low-Density Lipoprotein With Carotid Atherosclerosis in a General Population Aged 35 to 55 Years
The Asklepios Study
Objective— Reported associations of oxidized low-density lipoprotein (oxLDL) with noninvasive measures of atherosclerosis are inconsistent. In the Asklepios Study cohort of asymptomatic subjects aged 35 to 55 years, we evaluated the relationship of circulating oxLDL with subclinical atherosclerosis in the carotid and femoral arteries.
Methods and Results— Participants (n=2524, 51.5% females) completed a study questionnaire and underwent a clinical examination, blood analysis of oxLDL (mAb-4E6) and other risk markers, and ultrasound examination of intima-media thickness (IMT) and plaques in the left and right carotid and femoral arteries. oxLDL concentrations were highest in subjects with femoral plaques (n=658). In the group of subjects with carotid plaques (n=476), elevated oxLDL concentrations are related to concomitant femoral plaques detected in 54% of these subjects. Multivariate regression analyses (including anthropometric, hemodynamic, biochemical, and lifestyle variables) showed that femoral plaques are independently related to oxLDL whereas femoral IMT, carotid IMT, or carotid plaques were not independently associated with oxLDL.
Conclusion— Circulating oxLDL is independently associated with femoral plaque and not with carotid artery wall damage.
Oxidative stress plays an important role in atherogenesis.1 Oxidative modification of low-density lipoprotein (LDL) in the subendothelial space of the vessel wall is thought to be a key initiating step in atherosclerosis.2 Oxidized LDL (oxLDL) may not only contribute to foam cell generation but also stimulates the synthesis of adhesion molecules by endothelial cells and induces inflammatory processes.1,2⇓ In the last decade, immunoassays were developed using monoclonal antibodies against oxidation-dependent epitopes of LDL which made it possible to directly measure oxLDL in the circulation.3,4⇓
Increased oxLDL concentrations are found in patients with coronary artery disease (CAD) and are related to the severity of CAD.5–7⇓⇓ However, results from clinical studies on LDL oxidation and CAD are not consistent. oxLDL predicts future CAD in healthy men8 but is not an independent predictor of CAD after adjustment of lipid markers in men and women.9
Reports of an association of oxLDL with noninvasive imaging of atherosclerosis have also yielded heterogeneous results. Several investigators found that oxLDL concentrations correlate with carotid intima-media thickness (IMT) in the general population,10,11⇓ in familial hyperlipidemia families,12 and in CAD patients.13 Some studies have reported that oxLDL correlates with plaque occurrence in the carotid and femoral arteries.10,14,15⇓⇓ In general, these ultrasound studies provide an incomplete anatomic picture because most investigators have focused on a single phenotype (IMT or plaque), usually in a single vascular territory (carotid, femoral) or segment, in elderly or “high-risk” populations. Limited data are available from younger populations, where atherosclerosis is expected to be less generalized across multiple arterial sites.
Because atherosclerosis is a slowly progressive process, age is the most important risk determinant for structural changes of the arteries.16,17⇓ The initiation, speed of development, and phenotypic expression of atherosclerotic lesions are mainly artery-related.18 Although lesions of the carotid and femoral arteries generally share common risk factors, several of these risk factors have different impact in different arterial territories.19,20⇓ Some subjects with echographically documented carotid lesions might have concomitant femoral atherosclerosis while others have not,21 and vice versa.
The aim of this study was to investigate the relationship of circulating oxLDL concentration with IMT and plaques measured in both carotid and femoral arterial sites, in the large ASKLEPIOS Study cohort of asymptomatic subjects aged 35 to 55 years.
The ASKLEPIOS study protocol, methodology, and baseline population characteristics have been published elsewhere.22
We recruited a cohort of 2524 apparently healthy, male and female volunteers age 35 to 55 years at study initiation in October 2002. The subjects were randomly sampled from the twinned Belgian communities of Erpe-Mere and Nieuwerkerken, and were free from overt cardiovascular disease. Exclusion criteria were: 1, clinical presence of atherosclerotic or atherothrombotic disease; 2, major concomitant illness; 3, Diabetes mellitus type 1 and 2 if proven clinical macro-vasculopathy or significant renal impairment; 4, pregnancy; 5, inability to provide informed consent. The ethical committee of the Ghent University Hospital approved the study protocol.
Each examination component was performed with a fixed order: after 10 to 15 minutes of rest in a temperature-controlled environment, (1) informed consent and the study questionnaire were reviewed, (2) basic clinical data were measured, and (3) blood samples were taken by one study nurse, followed by (4) the vascular ultrasound measurements by one cardiologist. The questionnaire included information on smoking history (including pack-years), weekly alcohol consumption, fruit and vegetable intake, physical activity, and educational level.22 Subjects smoking at least 1 cigarette (or any nicotine) per week were categorized as “active smokers,” subjects who had stopped smoking (at least 1 week) and who had a cumulative exposure of at least 100 cigarettes were categorized as ex-smokers. Fruit and vegetable intake was assessed by a semiquantitative food frequency questionnaire; a daily fruit or vegetable intake of ≥400 g was classified as “Sufficient”. Physical activity was categorized based on intensity (metabolic equivalents; MET) and on frequency averaged throughout the year; subjects were defined as “Active” when performing activities ≥5 MET at least twice weekly. Body mass index (BMI) was calculated as weight (kg)/height (m).2 Blood pressure (BP) was recorded using cuff-patient matched bilateral triplicate measurements on a rested sitting subject using a validated oscillometric device.
Subjects underwent ultrasonographic examination of the left and right carotid and femoral arteries (VIVID 7, GE Vingmed Ultrasound).22–25⇓⇓⇓ Analysis of the ECG-gated RAW DICOM cineloops was performed offline by a single, blinded, measurement-dedicated reader. Carotid and femoral arteries were scanned bilaterally for the presence of plaque (focal protrusion >50% compared to adjacent sites, IMT >1.5 mm). IMT was defined as the distance from the leading edge of the lumen-intima interface to the leading edge of the media-adventitia interface, measured in end diastole, at the far wall, 1 to 2 cm before the bifurcations.22,25⇓ Intraobserver variability of IMT was 5.24%. Failure to measure IMT was associated with obesity or plaque with 8, 168, and 198 missing values for the right carotid, left femoral, and right femoral arteries, respectively. The reported IMT is the mean and maximum of the right and left segments. When the measure was missing on one side, the IMT estimate was based on the measurement of the site for which a value was available. In 41 cases both right and left femoral IMT were missing (bilateral plaque occurrence), so that no IMT value for the femoral artery was available. Because segments with an IMT >1.5 mm were classified as plaque (and because of the bilateral screening) it was possible to classify all subjects. Carotid or femoral “vascular target organ damage” (TOD) was defined as unilateral or bilateral IMT ≥0.9 mm or presence of plaque.
All subjects were fasting and had refrained from smoking for at least 6 hours. Subjects with recent or active infection/inflammation returned for blood sampling after their symptoms had subsided for at least 10 days. Serum glucose, creatinine, uric acid, triglycerides, total cholesterol, and HDL-cholesterol concentrations were measured on a Modular P system (Roche Diagnostics). LDL-cholesterol was calculated using the Friedewald formula.26 In 42 subjects (1.7%), triglycerides exceeded 350 mg/dL and LDL-cholesterol was not calculated. Serum C-reactive protein (hs-CRP) concentrations were measured by high-sensitive immunoturbidimetry (Roche). Plasma fibrinogen was assayed on an automated coagulation analyser (STAR, Roche). White blood cells (WBC) were counted in EDTA blood on a XE-2100 hematology analyser (Sysmex). Total homocysteine (tHcy) concentration in plasma was measured using a fluorescence polarization immunoassay on AxSYM (Abbott). Serum oxLDL concentration was measured by a sandwich enzyme-linked immunosorbent assay (Mercodia) as described previously,27 based on the murine monoclonal antibody, mAb-4E6, specific for a neoepitope in the aldehyde-substituted lysine residues of the apolipoprotein B-100 moiety of oxLDL. Coefficient of variation of all tests was <3.0% except for tHcy (<5.1%) and oxLDL (<7.4%).
Study variables are given as mean±SD or as median (interquartile range) where appropriate. Because of significant skew toward higher values, triglycerides, hs-CRP, and pack-years were ln-transformed. Comparison between 2 subgroups was performed using the t test for normally distributed variables and the Mann–Whitney test when not normally distributed. Correlations between variables were examined by Spearman rank analysis. Stepwise multivariate regression analyses were performed with oxLDL as dependent variable. Collinearity was checked for all variables included in the regression analyses. Logistic regression analyses was performed to evaluate whether oxLDL is associated with atherosclerosis independently of LDL-cholesterol. Statistical analysis was performed using SPSS for Windows 14.0 and Medcalc version 9.3. Statistical significance was considered at the level of P<0.05.
Ultrasonographic TOD phenotypes (IMT-thickening, plaque) were more pronounced among men compared to women (P<0.001 for all ultrasound variables; Table 1⇑). Occurrence of intimal thickening and plaque were higher in the femoral arteries than in the carotid arteries in both men and women. Plaque burden (total number of plaques, left and right) was also higher in femoral arteries (P<0.001, data not shown). On the other hand, mean and maximal carotid IMT were larger than mean and maximal femoral IMT (in both genders). These seemingly inconsistent data have an anatomic explanation: femoral IMT measurement sites were more often thickened (>1.5 mm) and part of a large plaque extending in the bifurcation and the posterior wall, and thus high femoral IMT values were more often classified as plaque.
oxLDL concentrations were relatively higher in subjects with carotid TOD (P<0.05), but much more elevated in those with femoral TOD (P<0.001), when compared to subjects with undetectable lesions in both arterial territories (Table 2). No significant differences were found when comparing oxLDL between subjects with unilateral (left or right) versus bilateral (left and right) lesions in the carotid and femoral arteries (data not shown).
Associations of oxLDL
Univariate correlations between oxLDL and other continuous variables (including mean IMT of the carotid and femoral arteries) are summarized in Table 3. Associations of oxLDL with plaque occurrence as dichotomous variable are shown in the Figure. Among subjects with carotid plaques, highest oxLDL concentrations were associated with concomitant femoral plaques detected in 54% (n=255) of these subjects.
High oxLDL concentrations were also associated with obesity (BMI ≥30 kg/m2) (P<0.0001), abdominal obesity (P<0.0001), the metabolic syndrome (P<0.0001), impaired fasting glucose (IFG; ≥100 mg/dL including diabetes; P=0.0008), active smoking (P=0.009), physical inactivity (P=0.041), insufficient fruit and vegetable intake (P=0.016), and low educational level (≤secondary; P=0.017).
Multivariate Regression Analyses
Stepwise multivariate regression analyses were then performed for oxLDL as dependent variable. The independent variables included were age, gender, BMI, abdominal obesity, systolic BP, heart rate, HDL-cholesterol, non-HDL-cholesterol, ln(triglycerides), ln(CRP), fibrinogen, WBC, IFG (incl. diabetes), creatinine, uric acid, tHcy, active smoking, ln(packyears +1), alcohol consumption, fruit and vegetable intake, physical activity, educational level, drug-treated hypertension, drug-treated hyperlipidemia, and 1 of the following ultrasound indices: (1) mean carotid IMT, (2) carotid plaque occurrence, (3) mean femoral IMT, or (4) femoral plaque occurrence.
Femoral plaques were independently associated with oxLDL (Table 4). In contrast, none of the other ultrasonographic indices entered into the multivariate regression models (1), (2), and (3) for oxLDL (data not shown).
Logistic Regression Analyses
In a logistic regression model with femoral plaque occurrence as dependent variable, both LDL-cholesterol and oxLDL were independently of each other related to plaque occurrence (Table 5). In contrast, oxLDL was not significantly associated in the models for carotid plaque occurrence, carotid IMT thickening, and femoral IMT thickening wherein LDL-cholesterol was an independent predictor (data not shown).
Atherosclerotic disease has a long subclinical and asymptomatic period before its sudden and often fatal first manifestation. Therefore, early detection and recognition of subclinical atherosclerosis has received increased attention in the last decades. A plethora of noninvasive phenotypes are currently being used in research and some have found their way into clinical practice. Several of these vascular phenotypes have been reported to correlate with circulating oxLDL concentration.10–15⇓⇓⇓⇓⇓
Although the literature evidence linking oxLDL to subclinical atherosclerosis seems robust, it is also heterogeneous because of lack of uniformity in definitions, measurement protocols, choice of arterial site (carotid versus femoral) or phenotype (IMT, plaque, …), and “health” or “risk” of the study populations. These studies have been performed mostly in elderly populations, whereas less data are available in younger “low-risk” populations using multiple phenotypes across multiple arterial sites, such as we report here.
In the ASKLEPIOS cohort, we found that males and females with “vascular TOD” (defined as IMT-thickening or plaques), detected either unilaterally or bilaterally in the carotid and femoral arteries, show elevated oxLDL concentrations compared to subjects with undetectable TOD. However, highest oxLDL concentrations were observed in subjects with femoral TOD. Femoral plaque was the sole echographic variable that was independently associated with oxLDL. Serum LDL-cholesterol and oxLDL were independently of each other related to femoral plaque.
In the total group of subjects with carotid plaques, we found that elevated oxLDL concentrations were associated with concomitant femoral plaques detected in approximately 55% of these subjects. This might explain the discrepancy between our findings and those of other investigators with regard to the carotid echographic measures.11–13⇓⇓ Previously reported associations of oxLDL with carotid atherosclerosis might thus have been confounded by the presence of concomitant femoral plaques in the populations studied.
In the AIR study, elevated oxLDL concentrations were associated with femoral plaques, but not with carotid plaques, and were not independently related to IMT in 58-year-old men.10 Associations between risk factors and atherosclerosis can have territorial specificity (carotid, coronary, femoral).18–20⇓⇓ Taken together with a substantial but incomplete overlap in atherosclerosis prevalence between different arterial territories, this territorial specificity can be the basis of spurious associations in studies only studying a single vascular territory.
The age range of the ASKLEPIOS cohort (35 to 55 years) represents a transitional period. Many study participants show an anatomically fragmented atherosclerosis, restricted to focalized lesions and characterized by a heterogeneously “distributed” phenotype: these subjects have either IMT thickening, or plaque, or either carotid disease or femoral disease. We therefore assume that correlations with oxLDL should be better in older populations characterized by more generalized atherosclerotic disease.
The strong association between oxLDL and femoral plaques might have several explanations. oxLDL can be detected in human atherosclerotic plaques.5,7⇓ Recently, circulating oxLDL has been associated with coronary calcification deposits and with fibro-calcific remodeling.11,28⇓ One could hypothesize that some oxLDL will diffuse back from atheroma into the bloodstream, in which it can be measured.7,10⇓ Thus, leakage of oxLDL from atheroma in subjects with high total plaque burden (associated with femoral plaques)29 might have a significant impact on total circulating oxLDL. Furthermore, it is known that femoral arterial disease is strongly associated with conditions, particularly smoking, characterized by high oxidative stress.30 oxLDL concentrations negatively correlate with LDL particle size.13,31⇓ Small dense LDL particles are more susceptible to oxidation and more easily penetrate into the subendothelial space. Based on vascular ultrasound data analysis with respect to LDL size,32,33⇓ it has been suggested that the femoral artery is more vulnerable than the carotid artery to the oxidative charge on small dense LDL. In contrast, hypertension is the major factor contributing to carotid vascular damage and leading to complications such as stroke.18,19⇓
The lack of independent association between oxLDL and femoral (and also carotid) IMT could be explained by the fact that IMT contrary to plaque is not only a surrogate of atherosclerosis, and also reflects adaptation to other factors such as hypertension (normalization of wall stress).34
A limitation of this study, and most other current population studies, is that subclinical coronary atherosclerosis was not assessed. It has been reported that persons with femoral plaque have a higher probability of coronary atherosclerosis.29,35⇓ Thus, higher oxLDL concentrations in persons with femoral plaque may also reflect more extensive coronary atherosclerosis.
We conclude that femoral plaque, but not carotid atherosclerosis, is independently associated with elevated circulating oxLDL in a general population aged ≤55 years. Our data suggest that associations of oxLDL with carotid atherosclerosis are potentially confounded by the presence of concomitant underlying atherosclerosis in other arterial territories.
The authors thank the laboratory technicians of AZ St-Jan, Bruges, and the residents and general practitioners of Erpe-Mere and Nieuwerkerken for their help in completing the study.22
Sources of Funding
This research was funded by the FWO research grant G042703 (the Asklepios Study).
Original received January 21, 2008; final version accepted May 15, 2008.
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