A total of 110 consecutive patients with T2DM as defined by World Health Organization criteria and no coronary artery disease were recruited at Queen Mary Hospital from January 2008 to January 2010. Patients were excluded if they had a documented history or clinical symptoms and signs of macrovascular disease including myocardial infarction, coronary artery disease, stroke or peripheral vascular disease. Patients with dilated cardiomyopathy, New York Heart Association class III/IV heart failure, significant renal dysfunction with creatinine level > 220 umol/L, liver failure or clinical/biochemical evidence of concomitant inflammatory disease or patients who declined to participate were also excluded. As a result, 87 subjects were eligible for this study. The current study is in compliance with the Helsinki Declaration and has been approved by the Hong Kong West Cluster Ethics Committee. All patients had a written informed consent.
Baseline demographic data and cardiovascular medications were recorded in all subjects. Hypertension was defined as resting systolic or diastolic blood pressure ≥140 /90 mmHg on two occasions or the prescription of anti-hypertensive medication. Hypercholesterolemia was defined as fasting total plasma cholesterol level ≥4.9 mmol/L or the prescription of lipid-lowering medication. Smoking status was recorded as ever-smoker (past or current) or non-smoker. Body height, weight and blood pressure were measured as previously described. Body-mass index (BMI) was calculated as kg/m2. Fasting blood samples were obtained to measure serum total cholesterol, triglyceride, low-density lipoprotein-cholesterol (LDL-C), high-density lipoprotein-cholesterol (HDL-C), glucose, HbA1c, creatinine, and high sensitivity C-reactive protein (hs-CRP).
After mixing, samples were centrifuged at 3000 rpm for 20 minutes and the supernatants frozen at −80°C until assay. Oxidative stress was measured as superoxide dismutase (SOD), a potent antioxidant enzyme.
Four subpopulations of EPCs, including CD34+, CD133+, CD34+/kinase insert domain-containing receptor (KDR) + and CD133+/KDR + EPCs, were measured by flow cytometry. Fluorescence-activated cell analysis was performed to determine the number of EPCs as described previously. Briefly, 100 μl of peripheral blood was incubated with a phycoerythrin-conjugated monoclonal antibody against human KDR (Sigma, St Louis, MO, USA), followed by a fluorescein isothiocyanate (FITC)-conjugated CD34 and CD133 antibody (Beckman Coulter, Fullerton, CA, USA). FITC-labelled anti-human CD45 antibody was used for differential gating during flow analysis. FITC-labelled IgG1a (Beckman Coulter) and phycoerythrin-labelled IgG2b (Becton Dickinson, Franklin Lakes, NJ, USA) served as the isotypic control for colour compensation. Analysis was performed with an automated fluorescence-activated cell counter (Elite; Beckman Coulter) in which 1 000 000 events were counted. The absolute number of cells expressing CD34+, CD133+, CD34/KDR + CD133/KDR + per 1 000 000 events in the lymphocyte gate was calculated. The percentages of all the measured components were derived from the absolute cell count divided by the lymphocyte count.
Transthoracic echocardiography was performed in all patients using a commercially available system (Vingmed Vivid 7, General Electric Vingmed Ultrasound, Milwaukee, USA). A 3.5-MHz transducer was used to obtain images that were digitally stored in cine-loop format (5 cardiac cycles). Measurements were performed offline using EchoPAC version 108.1.5 (General Electric – Vingmed, Horten, Norway). The inter-ventricular septum thickness, posterior wall thickness, and LV dimensions were measured in M-modal according to the current recommendation. LV volume and ejection fraction was determined from apical four and two-chamber views using the modified Simpson’s biplane method of discs. Evaluation of LV diastolic function was based on the pulsed-wave Doppler of mitral valve inflow. Peak velocity in early diastole (E-wave) and late diastole (A-wave) was measured and the E/A ratio calculated. Pulsed wave tissue Doppler imaging was used to measure the early diastolic velocity (E’) with the sample volume placed at lateral annulus. In addition, E/E’ ratio was calculated as an estimation of LV filling pressure. LV diastolic dysfunction was therefore classified as previously described.
Two-dimensional speckle tracking strain analysis
Two-dimensional speckle tracking strain analysis allows detailed assessment of LV myocardial deformation by tracking natural acoustic markers (speckles) in a frame-to-frame basis within the cardiac cycle. LV deformation can be evaluated in three orthogonal directions as longitudinal, circumferential and radial strains.
Longitudinal strain, assessing the shortening/lengthening of the myocardial wall, was measured from the 3 apical views: 2-chamber view (comprising anterior and inferior walls), 4-chamber view (posteroseptal and lateral walls) and long axis view (anteroseptal and posterior walls). Each wall was subsequently divided into 3 levels (basal, mid and apical) and a total of 18 segmental strain curves were obtained. Global longitudinal strain was calculated as the mean of the peak systolic strain value of the 18 segments.
From LV mid-ventricular short-axis view, both circumferential strain (evaluating myocardial shortening/lengthening along LV curvature) and radial strain (evaluating myocardial thickening/thinning) were measured. The global value of circumferential and radial strains were derived from the average peak systolic strain value of 6 segments. Global longitudinal and circumferential strains are expressed as negative values, and a lower strain is represented by less negative values. Global radial strain is expressed as a positive value: a lower value indicates lower strain.
Impairment of the 3-orthogonal directional global strains was defined as mean ± 2 standard deviations according to the results of a recent study that assessed global strains in healthy Asian subjects using the same vendor machine as the current study. Thus in this study impaired global longitudinal strain is defined as > −17.1%; impaired global circumferential strain > −17.0% and; impaired radial strain <29.4%.
The interobserver and intraobserver variability for longitudinal, radial, and circumferential strains were 6.5% and 2.6%, 10.4% and 7.6%, 4.9% and 2.4%, respectively.
All continuous variables and categorical variables are expressed as mean ± standard deviation and frequencies or proportions, respectively. Continuous demographic variables were compared using the Mann–Whitney U test and categorical demographic variables were compared using Pearson Chi-square test or the Fisher’s exact test if at least one cell had an expected cell count below five. Correlation coefficients were performed in order to assess the association of circulating EPCs and oxidative stress with myocardial function in T2DM patients. Multivariate analyses were performed to detect the predictors for abnormal myocardial function. To avoid multi-collinearity, multi-directional strains were entered individually into the model. All statistical analyses were performed using the statistical package SPSS for windows (Version 18.0, SPSS, Chicago, USA). All P values reported are 2-sided for consistency. A P value <0.05 was considered statistically significant.