The study group consisted of 41 patients with coronary heart disease. In all patients MI was defined by a positive troponin level, ECG changes and obstructive coronary lesions confirmed by angiography. Inclusion criteria were male gender, age at the time of myocardial infarction less than 50 years, more than 6 months and less than 3 years from the time of MI to the time of the trial performance (an average of 20.5 months), and coronary artery disease in the stable period (absence of unstable AP). Exclusion criteria were malignancy, heart failure, an acute inflammation state 14 days before the study, a history of diabetes mellitus, anticoagulant therapy, and the use of any other drugs except ACE inhibitors, beta blockers, antiplatelet drugs and statins. We divided the patients into three groups (one, two or three vessel disease) according to the number of diseased vessels seen on the coronarography at the time of MI. Significant stenosis was determined as stenosis above 50%.
The control group consisted of 25 healthy male volunteers, who did not differ from the patients regarding age and had no manifested atherosclerotic disease.
All subjects were informed about the study protocol and gave their informed consent. The study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki, and was approved by the State Ethics Committee of Slovenia.
Clinical examination and laboratory procedures
One visit by the subjects was necessary at which their medical history was recorded, and a clinical examination, biochemical tests and ultrasound measurements were performed. All ultrasound measurements were performed in the morning. Patients were on their regular therapy and had taken their usual dose of morning medicine. Any smokers abstained from smoking on that day.
A detailed family and personal history was recorded, and a clinical examination performed according to a questionnaire. Systolic and diastolic blood pressures were measured with a mercury sphygmomanometer after a minimum of 10-min rest in the sitting position. The average of three measurements was taken. Anthropometric parameters were determined, and BMI was calculated as weight in kilograms divided by the square of the height in meters, and WHR was calculated as waist size divided by hip size.
Blood for laboratory analysis was collected in the morning after a 12-hour overnight fast. Samples were drawn from the antecubital vein. Blood for the analysis of glucose and lipids was collected without additives, centrifuged and samples of serum were analysed. Concentrations of glucose, total cholesterol, high density lipoprotein (HDL) cholesterol and triglycerides were determined by standard colorimetric assays from the fresh samples of serum (Ektachem 250 Analyzer, Eastman Kodak Company, Rochester, USA). Low density lipoprotein (LDL) cholesterol was calculated using Friedewald's formula. High sensitivity CRP (hsCRP) was measured with a fully automated, latex-enhanced nephelometric immunoassay (N High Sensitivity CRP, Dade Behring Marburg, Germany).
Concentrations of VEGF, angiogenin and interleukin-8 (IL-8were measured by Cytometric Bead Array (BD Biosciences Pharmingen, San Diego, CA, USA) containing microparticles dyed to different fluorescence intensities. The anaphylatoxin-captured beads were incubated with standards (purified from human plasma) or test samples followed by a wash and incubation with phycoerythrin-conjugated detection antibodies to form sandwich complexes. Flow cytometric analysis was performed using a FACSCalibur flow cytometer (Becton Dickinson Immunocytometry Systems, BDIS, BD Biosciences, San Jose, CA, USA). Data were acquired and analysed using the Beckton Dickinson Cytometric bead array (CBA) software.
Serum tumour necrosis factor- α (TNF-α) (Quantikine® HS Human TNF- α Immunoassay, R&D Systems, USA), interleukin-6 (IL-6) (Quantikine® Human IL-6 Immunoassay, R&D Systems, USA) and the serum cell adhesion molecules ICAM-1, VCAM-1, selectin-P, selectin-E (Parameter® ICAM-1, Parameter® VCAM-1, Parameter® selectin-P, Parameter® selectin-E, R&D Systems UK) were determined by enzyme-linked immunosorbent assays.
a.) Measurement of endothelial dysfunction (ED); Flow mediated (FMD) (endothelium-dependent) and glyceryl trinitrate (GTN)-induced dilation (endothelium-independent) of the right brachial artery were studied using a high-resolution B mode Diasonics VST ultrasound system with a 10 MHz linear array transducer. The subjects rested in the supine position for ten minutes before hemodynamic measurements were performed. The right brachial artery was scanned in the longitudinal section 2 to 15 cm above the elbow to find the clearest images of the anterior and posterior wall layers. The mean arterial diameter was measured at the end of the diastole, which was determined by simultaneous monitoring of the electrocardiogram (concurrent with onset of the QRS complex). At least three cardiac cycles were analysed for each scan and the measurements averaged. The flow velocity was measured at a fixed incident angle of 68° to the vessel with the range gate of 1.3 mm located in the centre of the artery. The baseline blood flow was estimated by multiplying the velocity time integral of the Doppler flow signal (corrected for incident angle) by the vessel cross-sectional area. Hyperemic flow increase was induced by inflation of a blood pressure tourniquet placed around the forearm to a pressure of 300 mmHg for 4.5 minutes. Hyperemic flow was recorded for the first 15 seconds and diameter measurements were taken 45–60 seconds after cuff deflation. The endothelium-dependent dilation was expressed as the percentage change of the diameter after reactive hyperaemia, relative to the baseline scan. Ten minutes were allowed for vessel recovery, after which a further resting scan was taken. A sublingual tablet of 0.5 mg of GTN was then administered, and 4.5 minutes later, the final scan was performed. The endothelium-independent dilation was expressed as the percentage change in diameter after GTN administration, relative to the baseline scan. The same investigator, who was blind to the subjects’ characteristics, carried out all measurements. To assess the reproducibility of the measurements, 40 subjects were selected randomly for repeated vascular studies. The correlation coefficient between the absolute differences and mean values of paired measurements was 0.92, p < 0.05.
b.) Measurement of intima-media thickness; IMT of the common carotid artery (CCA) was assessed by the B-mode ultrasound technique. Measurements of IMT were obtained from the far wall of the distal part of the CCA (immediately proximal to the carotid bulb) on both sides. All studies were performed on a single ultrasound machine (Diasonics VST ultrasound system) using a linear-array 10-MHz scan head with standardized image settings, including resolution mode, depth of field, gain, and transmit focus. All sonograms were obtained with the patient in the supine position and their head turned slightly to the contra lateral side. Each ultrasound examination was performed as an independent study, without any knowledge of the history of MI and risk factors. The IMT was measured as the distance from the leading edge of the near-field (intimal-luminal surface) and far-field (medial-adventitial) arterial wall. An average of three measurements on both sides was derived. The mean IMT was calculated as the average of the left and right CCA.
c.) Measurement of ankle-brachial pressure index (ABPI); the systolic blood pressure in both arms was taken with a blood pressure cuff and Doppler probe, after a minimum of 10-min rest in the sitting position, averaged, and divided into the systolic blood pressure in the posterior tibial or dorsalis pedis artery in the leg. The higher reading was used to determine the ABPI. ABPI was calculated separately for each leg. The mean ABPI was calculated as the average of the left and right leg.
Variables showing a normal distribution, as determined by the Kolmogorov-Smirnov test, were expressed as means and standard deviations. Differences between the two groups were tested for significance by Student’s t-test for normally-distributed variables with equal variances, or by the Welch t-test for variables with unequal variances. Correlation between normally distributed variables was tested with the Pearson correlation coefficient. The criterion for statistical significance was a p value of less than 0.05. We constructed a multiple linear regression model to predict VEGF in patients on the basis of classical coronary risk factors, inflammatory parameters and functional and structural characteristics of the arterial wall. All calculations were performed by the IBM SPSS statistics 19 computer program.
A power analysis was performed to calculate the appropriate sample size to detect significant differences between the two groups at adequate power. All calculations, except the power analysis which was performed with the PASS 11 statistical program, were performed by the IBM SPSS statistics 19 computer program.