Elsevier

Atherosclerosis

Volume 229, Issue 2, August 2013, Pages 475-481
Atherosclerosis

Circulating osteopontin as a marker of early coronary vascular calcification in type two diabetes mellitus patients with known asymptomatic coronary artery disease

https://doi.org/10.1016/j.atherosclerosis.2013.06.003Get rights and content

Highlights

  • Circulating osteopontin, coronary atherosclerosis and calcification in type 2 diabetes mellitus patients.

  • Predicted power of the model determined by contrast cardiac computer tomography angiography was found.

  • Circulating osteopontin is high sensitivity tool for detection of coronary arteries calcification in asymptomatic patient.

Abstract

Objective

To evaluate the interrelation between circulating osteopontin (OPN) and coronary atherosclerosis and calcification in type 2 diabetes mellitus patients (T2DM).

Design and methods

126 subjects (46 patients with T2DM) with previously documented asymptomatic coronary artery disease (CAD) were enrolled in the study. CAD was determined by contrast multispiral CT-angiography. OPN plasma levels were measured by ELISA.

Results

Analysis of the results showed that in a patient cohort the mean value of circulating OPN was 43.55 ng/mL (95% CI = 31.5–57.0 ng/mL). OPN plasma levels were correlated with Agatston score index (r = 0.418, P = 0.009), T2DM (r = 0.411, P = 0.006), gender (r = 0.395, P < 0.001 for male), TC (r = 0.405, P = 0.006), hsC-RP (r = 0.368, P = 0.008), age (r = 0.256, P = 0.001), smoking (r = 0.255, P = 0.001) and inversely to LVEF (r = −0.579, P = 0.001). Cox-regression analyzes showed that in T2DM patients upper quartile OPN compared with the lowest quartile are associated with Agatston score index (adjusted OR = 3.23, 95% CI = 1.09–5.20; P = 0.044), numerous of damaged coronary arteries (adjusted OR = 2.60, 95% CI = 1.10–9.20, P = 0.005). The findings suggest that the predictive power of the model for asymptomatic CAD patients with T2DM, the estimated AUC (area under curve) was 0.788. In this case, the concentration of OPN that had the best predict potential on the risk of coronary atherosclerosis was 48.5 ng/mL.

In conclusions, we believe that elevated OPN in plasma can be considered as an independent predictor of coronary calcification in T2DM patients with known CAD.

Introduction

Atherosclerosis remains the leading cause of cardiovascular events including sudden cardiac death in Western populations [1]. In this regard the role of inflammation across atherosclerosis evolution is widely appreciated, especially in a focus of maladaptive mechanisms that leads to ensuing plaque necrosis, fibrosis and calcification [2]. Traditionally, coronary calcification is determined by non-contrast cardiac computer tomography (CT) angiography with estimation of the Agatston score index, which takes into account the density of vascular tissue. According to the cardiac CT angiography calcium score guideline it has recommended to use five coronary artery calcium (CAC) score categories: none (0), minimal or low (1–10), mild (11–100), moderate (101–400) and extensive or high (401 or greater) [3]. There are some technical limitations to be correctly estimating of the coronary calcium accumulation using non-contrast imaging, and, however, low radiation exposure contrast-enhanced CT angiography is considered as a new current method for improving CT findings [3]. Cardiac contrast-enhanced CT angiography can provide a reasonable estimate of total coronary atheroma including various plaque compositional parameters that lets to determine calcified and noncalcified plaques (low-density noncalcified and high-density noncalcified) in asymptomatic patient cohort with suspected CAD. Currently the role of compositional parameters in measuring clinically or prognostically meaningful changes in calcified plaque over time is intensively investigated. It has found that total amount of CAC is predictor of coronary disease events beyond conventional cardiovascular risk factors. Several studies have showed that intermediate-risk patients with an elevated CAC (intermediate Framingham Risk Score and Agatston score >300 Hounsfield units) had an annual hard event rate of 2.8%, or a 10-year rate of 28%, and thus would be considered as high risk. The best estimates of the relative risk from this study indicated that Agatston score of 3 (>300 Hounsfield units) had a hazard ratio of about 4 compared with Agatston score index of 0 [4], [5], [6]. Moreover, results of Multi-Ethnic Study of Atherosclerosis represented that progression of CAC is associated with an increased risk for future hard and total ischemic events [7]. While CAC score using multi-slice cardiac CT has been validated as a useful imaging tool for risk stratification and reclassification of risk of CAD, the clinical application of CAC scoring has been supported by evidence showing that the absence of calcium reliably excludes obstructive coronary artery stenosis. However, predictive value of CAC is, in the end, determined by the patients' symptoms.

Osteopontin (OPN) is a secreted molecule belonged to non-collagenous matrix phosphorylated sialoproteins that is highly expressed by several types' cells, such as macrophages, endothelial cells, smooth muscle cells and epithelial cells, and interacted with wide types of integrins [8], [9]. OPN as a multifunctional protein plays a key role in the pathology of several chronic inflammatory diseases including atherosclerosis. Biological effect of OPN is realized thereby regulating of several macrophage functions, such as migration, survival, and tissue accumulation [8], [9], [10]. Results obtained in animal model using ultrastructural analysis through transmission electron microscopy have been suggested that OPN was dramatically over-expressed by vasculature in the presence of oxidized low-density lipoproteins [11]. This mechanism is important to induce atherosclerosis in subintima and vasculature [12]. Moreover, specific histochemical techniques revealed that OPN is detected in vasculature near calcium phosphate precipitates that indicated pivotal role of OPN in regulation of vascular calcification [11]. Currently, OPN is considered as a key factor in vascular remodeling, development of atherosclerosis and vascular calcification in subjects with known CAD [13], [14], [15], while in a population of young adults without symptoms of cardiovascular disease OPN is not associated with vascular markers of subclinical atherosclerosis [16]. Other recent studies reported that hyperglycemia enhance cell proliferation and the development of diabetic atherosclerosis via stimulation of OPN expression and synthesis in vasculature [17], [18]. Moreover, vascular calcification in type 2 diabetic patients is frequently accompanied by intima–media thickening beyond severe atherosclerotic lesions [18]. It has been expected that a closely interrelationship between circulating OPN level and severity of coronary artery and aorta calcification will be able to reclassification of asymptomatic subjects irrespective to conventional risk factors into high risk group for further examination [19]. However, we cannot find results of studies dedicated examining different risk group depended of metabolic disorders and the relevance of OPN in type 2 diabetes mellitus (T2DM). The aim of the study was to evaluate the interrelation between circulating OPN and coronary atherosclerosis in T2DM patients with known CAD.

One hundred twenty-six out subjects (46 patients with T2DM) who underwent cardiac computer tomography (CT) angiography and documented asymptomatic coronary artery disease (CAD) were enrolled in the study. All subjects gave their written informed consent to participation in the study. The excluding criteria were: symptomatic chronic heart failure, left ventricular ejection fraction (LVEF) ≤40%, severe diabetes mellitus, severe kidney and liver diseases that have ability to independently influence to clinical outcomes, malignancy, unstable angina, Q-wave and non-Q-wave MI within 3 months before study entry; creatinin plasma level above 440 μmol/L, GFR index < 35 mL/min/m2, brain injury within 3 months before an enrollment, body mass index above 30 kg/m2 and less 15 kg/m2, pulmonary edema, tachyarrhythmia, valvular heart disease, thyrotoxicosis, ischemic stroke, intracranial hemorrhage, acute infections, surgery, trauma, all ischemic events during the previous 3 months, and inflammatory conditions within 1 month, and incident of neoplasm were ruled out by careful medical history and physical examination; pregnancy; an implanted pacemaker, any disorders that accordingly investigators opinion can stop to participate the patients to the study, and refusal to participate and give consent to this study.

Echocardiography in B-mode was performed accordingly to the Recommendation of American Society of Echocardiography on the scanner ACUSON (Siemens, Germany) using a transducer with a frequency of 2.5–5 MHz. End-diastolic and end-systolic LV volumes were obtained using a two-dimensional reference sector according to the Simpson's method, and LV ejection fraction (LVEF) was calculated by accordingly conventional methods [19].

Coronary vessel-wall, and plaque geometrical, and compositional parameters were measured on contrast-enhanced spiral computer tomography (CT) angiography [20]. Contrast-enhanced spiral CT was performed on a “Somatom Volume Zoom” scanner (Siemens, Erlangen, Germany) with 2 rows of detectors (32 × 2 CT system) during the end-expiratory breath-hold. After noncontrast localization image acquisition, injection of nonionic contrast “Omnipak” (Amersham Health, Ireland) was used to determine the optimal coronary arterial image. Standardized calcium scores were obtained with beam energy of 120 kV, full rotation time of 350 ms, and tube current of 300 mA. The images were reconstructed in 0.6-mm axial slices. Scans were electrocardiogram-gated and were triggered at either 40% or 75% phase contingent on heart rate.

Coronary artery calcification was quantified by calculating of the Agatston score index as 1 for 130–199 HU, 2 for 200–299 HU, 3 for 300–399 HU, and 4 for 400 HU and greater [21]. We also determined calcified atherosclerotic plaque, high-density noncalcified plaque (HD-NCP), and low-density noncalcified plaque (LD-NCP) using follow criteria: calcified atherosclerotic plaques (CAP) were classified with hyperattenuation values 150 HU (Hounsfield units) or greater with an area ≥3 adjacent pixels, as HD-NCP with 30–149 HU, and as LD-NCP with −100 to +30 HU [22], [23], [24]. For all patients with determined atherosclerotic plaques we recommended further coronary angiography performing.

All blood samples were collected after fasting in cooling vacutainer and after that it was immediately centrifugated (4 °C for 6.000 × 15 min). After centrifugation serum was blind coded and stored at −70° until used. Osteopontin levels were measured by ELISA technique. Human OPN Quantikine ELISA Kit (R&G, United Kingdom) was used for examination. Assay range of this kit was 0.312–200.0 ng/mL with less 0.5% cross-reactivity observed with available related molecules. All determinations were done by duplicating. The mean intra-assay coefficients of variation were <10% of all cases.

Fasting plasma glucose (FPG) was quantified by the glucose oxidase procedure; HbA1c was measured by ion-exchange high-performance liquid chromatography (HPLC; Bio-Rad, Hercules, CA, USA). The homeostasis model assessment insulin resistance estimate (HOMA-IR) was calculated as a serum glucose (mg/dL) plasma insulin (mU/mL)/22.5. Serum high sensitive C-reactive protein (hs-C-RP) levels were determined by the nephelometric method. Concentrations of total cholesterol (TC) and high density lipoprotein (HDL) cholesterol were determined by a Dimension Clinical Chemistry System (Dade Behring Inc., Newark, NJ). Low density lipoprotein (LDL) cholesterol was calculated by using the formula of Friedewald W.T., Levy R.I., Fredrickson D.S. (1972). All measurements and blood sample for OPN, glucose, creatinin, hs-C-RP, glycated hemoglobin (HbA1c), TC, LDL-C, HDC-C were collected at the same visit.

All statistical analyses were performed in SPSS for Windows v. 17.0 (SPSS Inc., Chicago, IL, USA). All values were given as mean and 95% CI or median and percentiles. An independent group t-test was used for comparisons for all interval parameters meeting the criteria of normality and homogeneity of variance. For interval parameters not meeting these criteria, the non-paramentric Mann–Whitney test was used to make comparisons between the groups. Comparisons of categorical variables between the groups were performed using the Chi2 test, and the Fisher exact test. OPN concentration was normally distributed (using by Kolmogorov–Smirnov test) and it was no positively skewed. However, the data were not transformed. The potential factors (age, sex, smoking, systolic and diastolic BP, fasting plasma glucose, hypercholesterolemia, HOMA-IR, hs-C-RP, TC, LDL cholesterol, OPN, creatinin level, HbA1c) that may be associated with severity of coronary atherosclerosis was identified first by the univariate analysis (ANOVA), then Cox proportional hazards multivariate analyses were used to identify the predict factors. Effect-size of the predict factors was explained by Eta-test calculation. Receiver operating characteristic (ROC) curves were configured to establish cutoff points of OPN level that optimally predicted coronary calcification measured by Agatston score index. A calculated difference of P < 0.05 was considered significant.

Section snippets

General characteristics of study patient population

General characteristics of study patients are presented in Table 1. Significant differences between the both cohorts of patients for demographics (age, sex), conventional risk factor (premature CAD in family anamnesis, smoking, arterial hypertension), biochemical (creatinin, TC) and some hemodynamic parameters (mean systolic and diastolic BP, heart rate and LV EF) were not found. Fasting glucose and HbA1c in patients with were expectantly higher when compared with non-T2DM individuals. There is

Discussion and conclusion

Proteins in the serum of CAD patients predominantly reflected a positive acute phase, inflammatory response and alterations in lipid metabolism, transport, peroxidation and accumulation. Recent studies have been suggested that markers of stromal stem cells with osteogenic potential, such as OPN, osteonectin and osteoprotegrin, presumably can play an important role in atherogenesis and they can be reflection low-intensity proinflammatory activation [25]. It was postulated that several

Funding

This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

Limitations of the study

This study has some limitations. We believed that a greater cohort would be desirable to improve the power of the study. We also relied on clinical data to rule out infection and other inflammatory diseases before sampling, but we couldn't exclude that some patients had unrecognized the conditions responsible for the elevated OPN levels observed. Excluding criteria BMI > 30 kg/m2 seems to be a limitation the diabetic population eligible for the study. We suppose to mean that these limitations

Ethical declaration

The study was approved by the local ethics committee of State medical university, Zaporozhye, Ukraine. The conduct of the study was in keeping with the declaration of Helsinki.

Declaration of conflicting interests

None.

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