Atherosclerosis is the main cause of cardiovascular disease, but the extent of atherosclerosis in individual patients is difficult to estimate. A biomarker of the atherosclerotic burden would be very valuable. The aim of the present study was to evaluate the association of plasma osteoprotegerin (OPG) to clinical and subclinical atherosclerotic disease in a large community-based, cross-sectional population study. In the Copenhagen City Heart Study, OPG concentrations were measured in 5,863 men and women. A total of 494 participants had been hospitalized for ischemic heart disease or ischemic stroke, and compared to controls, this group with clinical atherosclerosis had higher mean OPG (1,773 vs 1,337 ng/L, p <0.001) and high-sensitivity C-reactive protein (2.3 vs 1.6 mg/L, p <0.001). In a multivariate model with age, gender, body mass index, hypertension, diabetes, hypercholesterolemia, smoking status, estimated glomerular filtration rate, high-sensitivity C-reactive protein, and OPG, OPG remained significantly associated with clinical atherosclerosis (p <0.01); high-sensitivity C-reactive protein, in contrast, did not (p = 0.74). In the control group without clinical atherosclerosis, OPG was independently associated with hypertension, diabetes, hypercholesterolemia, smoking, and subclinical peripheral atherosclerosis as measured by ankle brachial index. For each doubling of the plasma OPG concentration, the risk for subclinical peripheral atherosclerosis increased by 50% (p <0.001) after multivariate adjustment. In conclusion, OPG appears to be a promising biomarker of atherosclerosis that is independently associated with traditional risk factors of atherosclerosis, subclinical peripheral atherosclerosis, and clinical atherosclerotic disease such as ischemic heart disease and ischemic stroke.
Atherosclerosis is the principal cause of cardiovascular and cerebrovascular disease and is the main cause of death worldwide. Atherosclerosis has many risk factors, among which hypertension, diabetes, hypercholesterolemia, and smoking are considered the traditional risk factors. However, in daily clinical practice, the total burden of the atherosclerotic risk factors can be difficult to estimate. A patient with known hypercholesterolemia for 10 years may well be at higher risk than a patient with hypercholesterolemia for 1 year. Similarly, a patient with stage 2 hypertension might be at higher risk than a patient with only stage 1 hypertension. Also involved are the complex interaction among all the risk factors, their unknown durations, and whether prevention against them has been initiated or not. A biomarker of the atherosclerotic burden would therefore be valuable in daily clinical practice. High-sensitivity C-reactive protein (hsCRP) has been suggested as such a biomarker, but the reported data show variable results, and given the existing data, a better biomarker is still sought.
Osteoprotegerin (OPG) is a glycoprotein that binds the receptor activator for nuclear factor κB ligand, thereby inhibiting nuclear factor κB’s regulatory effects on inflammation, skeletal, and vascular systems; additionally, OPG might be involved in vascular calcification. OPG has been shown to be present in atherosclerotic plaques, and high plasma concentrations of OPG have been associated with coronary artery atherosclerosis. However, the incremental value of OPG compared to traditional atherosclerotic risk factors and hsCRP needs to be further studied. We therefore set out to evaluate the association of OPG to subclinical and clinical atherosclerotic disease in a large population-based study.
Methods
The present study included 5,863 men and women (aged 20 to 95 years) from the fourth Copenhagen City Heart Study, a longitudinal cohort study of cardiovascular disease and risk factors. At the first examination, from 1976 to 1978, a random sample of 19,329 predominantly Caucasian citizens living within a well-defined area of the inner Copenhagen city boundary was drawn from the Central Office of Civil Registration and invited to take part in the study. At the fourth examination, from 2001 to 2003, a total of 12,600 individuals were invited in a random order. The present study population consisted of subjects who had been invited to the previous examinations (n = 11,600), supplemented by a random sample of subjects from the younger age strata (n = 1,000). Of the 6,038 participants examined, 175 were excluded because of missing data on plasma OPG measurements. All subjects gave informed consent to participate, and the study was performed in accordance with the second Declaration of Helsinki and approved by the regional ethics committee.
Hypertension was defined as systolic blood pressure ≥140 mm Hg or diastolic blood pressure ≥90 mm Hg or the use of antihypertensive medication. Diabetes mellitus was defined as plasma glucose concentration ≥11.1 mmol/L, the use of insulin or other antidiabetic medicine, self-reported disease, or glycosylated hemoglobin level >7.0%. Hypercholesterolemia was defined as total plasma cholesterol ≥7 mmol/L or the use of statins. Smoking status was self-reported, and subjects were defined as smokers if they were current or former smokers. Body mass index (BMI) was calculated as weight in kilograms divided by the square of height in meters. Glomerular filtration rate (GFR) was approximated from the estimated creatinine clearance using the Cockcroft-Gault formula: [140 − age (years)] × [weight (kg)] × (1.23 if male and 1.04 if female)/[serum creatinine (μmol/L)]. Plasma hsCRP concentrations were analyzed using a nephelometric assay (Dade Behring, Rødovre, Denmark) on nonfasting venous blood samples.
Plasma OPG concentration was quantified using modified time-resolved immunofluorometric assay using commercially available monoclonal antibodies (DY085E; R&D Systems, Abingdon, United Kingdom). Wells were coated with 0.1 μg OPG antibody in 100 μl phosphate-buffered saline (PBS) overnight at 4°C. Residual protein-binding sites were blocked with 1% bovine serum albumin (Sigma-Aldrich, St. Louis, Missouri) in PBS for 1 hour at room temperature and washed in PBS containing 0.05% Tween 20 (Sigma-Aldrich). Recombinant OPG in the range of 31.3 to 4,000 ng/L was used as standard, and ethylenediaminetetraacetic acid plasma samples were diluted fourfold in PBS with 1% bovine serum albumin and incubated overnight at 4°C. Bound OPG was determined by incubation with 20 ng biotinylated anti-OPG detection antibody in PBS with 1% bovine serum albumin at room temperature for 2 hours, followed by wash and the addition of 10 ng Eu 3+ -labeled streptavidin (PerkinElmer Life Sciences, Turku, Finland) in 100 μl PBS/Tween 20 containing 25 μmol/L ethylenediaminetetraacetic acid for 1 hour at room temperature. After wash, bound europium was detected by the addition of 200 μl of enhancement solution (PerkinElmer Life Sciences), 5 minutes of vigorous shaking, and reading the time-resolved fluorescence on a DELFIA fluorometer (Victor ; PerkinElmer Life Sciences). The limit of detection was 15 ng/L. Intra- and interassay variation was <5% and <9%, respectively.
Clinical atherosclerosis was defined as ischemic heart disease or ischemic stroke. Ischemic heart disease was defined as a history of hospital admission for acute coronary artery occlusion, percutaneous coronary intervention, coronary artery bypass grafting, or angina pectoris. Ischemic stroke was defined as hospitalization for stroke excluding transient ischemic attack and excluding computed tomography–proved cerebral and subarachnoid hemorrhage.
Systolic brachial blood pressure in both arms and systolic ankle blood pressure of the posterior tibial arteries in both legs were obtained by Doppler (Huntleigh Mini Dopplex D900; Huntleigh, Cardiff, United Kingdom) in a subgroup of the study participants without clinical atherosclerosis (n = 4,872). Ankle brachial index (ABI) was calculated as the lowest ankle systolic blood pressure divided by the highest brachial systolic blood pressure and used as an indicator of peripheral atherosclerosis.
Comparisons between groups were performed using Student’s t tests and Fisher’s exact tests. Plasma hsCRP and OPG were positively skewed and therefore logarithmically transformed before further analysis: the mean values presented in the text are geometric mean values, unless otherwise stated. Associations with clinical atherosclerosis, peripheral atherosclerosis, and plasma OPG were tested using univariate and multivariable logistic and linear regression analyses. Linearity, variance homogeneity, and the assumption of normality were tested with plots of residuals. Linear associations were examined by scatterplots and are reported as Pearson’s product-moment correlation coefficients. The diagnostic performance of plasma OPG and hsCRP was evaluated using receiver-operating characteristic curves with calculation of areas under the curves. Trend was analyzed using linear regression by considering the groups as a continuous variable, and departure from linearity was assessed by simultaneous assessment of linear and quadratic effects. A p value <0.05 on 2-sided tests was considered significant. All analyses were performed using SAS for Windows release 9.1 (SAS Institute, Inc., Cary, North Carolina).
Results
The study cohort constituted 5,863 subjects from the general population, of whom 494 had clinical atherosclerosis ( Table 1 ). The group with clinical atherosclerosis had a higher frequency of cardiac risk factors, and the risk for acute coronary events within 10 years based alone on the cardiac risk factors according to the Prospective Cardiovascular Münster (PROCAM) algorithm was significantly higher (18% vs 9%, p <0.001).
| Variable | Controls (n = 5,369) | Patients With Clinical Atherosclerosis (n = 494) | p Value |
|---|---|---|---|
| Age (years) | 58 ± 17 | 71 ± 10 | <0.001 |
| Men | 42% | 55% | <0.001 |
| BMI (kg/m 2 ) | 25.7 ± 4.4 | 27.2 ± 4.2 | <0.001 |
| Hypertension | 47% | 77% | <0.001 |
| Diabetes mellitus | 7% | 20% | <0.001 |
| Hypercholesterolemia | 13% | 43% | <0.001 |
| Smokers | |||
| Never | 34% | 18% | |
| Former | 33% | 51% | |
| Current | 33% | 31% | <0.001 |
| GFR (ml/min) | 88 ± 29 | 75 ± 26 | <0.001 |
| hsCRP (mg/L) | 1.6 ×/÷ 3.1 | 2.3 ×/÷ 3.0 | <0.001 |
| Plasma osteoprotegerin (ng/L) | 1,337 ×/÷ 1.7 | 1,773 ×/÷ 1.6 | <0.001 |
The proportion of clinical atherosclerosis increased according to increasing quintiles of plasma OPG ( Figure 1 ) , and this remained significant after multivariate adjustment for age, gender, BMI, hypertension, diabetes, hypercholesterolemia, smoking status, GFR, and hsCRP (p <0.001).
Plasma hsCRP and OPG concentrations correlated weakly (univariate, r = 0.23; adjusted for age, r = 0.12), but plasma hsCRP concentration (p = 0.02) remained significantly associated with clinical atherosclerosis in a multivariate model including age, gender, and GFR. However, the predictive information of clinical atherosclerosis ( Figure 2 ) was higher for plasma OPG (area under the curve 0.67) compared to hsCRP (area under the curve 0.59), and in a multivariate model including all the risk factors (i.e., age, gender, BMI, hypertension, diabetes, hypercholesterolemia, smoking status, GFR, plasma hsCRP, and OPG concentration), plasma OPG was significantly (p <0.01) associated with clinical atherosclerosis, whereas plasma hsCRP was not (p = 0.74).
The same analysis in the subgroup without statin treatment yielded similar results for plasma OPG (p <0.01) and plasma hsCRP (p = 0.22).
In the control group without clinical atherosclerosis, plasma OPG concentration was positively associated with risk factors of atherosclerosis, such as hypertension (p <0.001), diabetes (p <0.001), hypercholesterolemia (p <0.001), and smoking (current and former; p <0.001).
Plasma OPG concentrations increased with increasing numbers of atherosclerotic risk factors ( Figure 3 ) , even after multivariate adjustment for age, gender, BMI, and GFR (p <0.001).
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