Patients and Research Design
In this study, 36 patients with newly diagnosed type 2 diabetes, according to the American Diabetes Association criteria , were enrolled from April to September 2017 at Nanjing Drum Tower Hospital Affiliated to Nanjing University Medical School (Nanjing, Jiangsu province, China). A further 22 control subjects who participated in health examinations at the hospital were randomly recruited. Medical history, physical examination, blood biochemistry, and clinical diagnosis were evaluated in all participants. Subjects were excluded from this study for the following reasons: history of taking antihypertensive drugs, hypolipidemic agents, estrogen supplements, herbal drugs, or antioxidants for 3 months before enrolling in the study, medical history of cardiovascular events; acute illness or infection, recent surgery or vascular intervention, ketosis, ketoacidosis, and other diseases requiring continuous medical treatment and cigarette smoking.
Patients with diabetes were treated with continuous subcutaneous insulin infusion by receiving Novolin-R (Novo Nordisk, Bagsvaerd, Denmark) with an insulin pump. Initial insulin dose was adjusted every day depending on the values from the seven-point glucose profile obtained 1 day before and was divided into 50% of basal and 50% of bolus injection. The glycemic control target was defined as fasting plasma glucose below 6.1 mmol/l and prandial plasma glucose below 8.0 mmol/l. Insulin treatment was discontinued 2 weeks later after the target goal was achieved and blood samples were re-collected for further detections.
This study was approved by the Ethics Review Committee of Nanjing Drum Tower Hospital Affiliated to Nanjing University Medical School (No. 2017-068). All patients signed written informed consent before enrollment.
Patients’ height was measured by a portable stadiometer with shoes off, to the nearest centimeter. The weight was measured on a digital scale with identical light clothing on, to the nearest 0.1 kg. BMI was calculated as weight divided by height squared. Blood pressure was measured using a mercury sphygmomanometer after the subject rested for at least 5 min. The mean of three measurements was recorded.
Blood Sample Analysis
Fasting blood samples were collected from the antecubital vein for fasting plasma glucose, insulin, glycosylated hemoglobin (HbA1c), triglycerides (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), urea nitrogen, creatinine, and uric acid measurements. The fasting plasma glucose levels were assessed by the hexokinase method using TBA-200FR analyzer (Toshiba Medical Systems, Tokyo, Japan). Insulin concentration was evaluated by a chemiluminescence immunoassay (Cobas e601; Roche, Basel, Switzerland). HbA1c was quantitated by high-performance liquid chromatography (Bio-Rad Laboratories, Inc., Hercules, CA, USA). TG, TC, HDL-C, and LDL-C concentrations were assessed by specific immunoassays (Cobas e601; Roche, Basel, Switzerland). Serum free fatty acids (FFAs) were detected via an enzymatic method (DiaSys Diagnostic Systems GmbH, Holzheim, Germany). Homeostasis model assessment of insulin resistance (HOMA-IR) was derived as follows: [fasting serum insulin (mU/l) × fasting plasma glucose (mmol/l)]/22.5 . Serum 1.5-anhydroglucitol (1.5-AG) was measured using an enzymatic assay kit (GlycoMark, Japan).
Quantification of EPCs by Flow Cytometry
Peripheral blood mononuclear cells (PBMCs) were isolated with vacutainer cell preparation tubes (CPTs) (Becton Dickinson, Franklin Lakes, NJ, USA) from fasting blood samples. After centrifugation at 1800×g for 30 min, cells in the resulting supernatants were collected, washed with phosphate-buffered saline (PBS), and gradually frozen within 8 h in freezing medium containing 10% dimethyl sulfoxide (DMSO) and 90% fetal bovine serum. Samples were stored in liquid nitrogen until analysis of EPCs.
PBMCs were stained with the following antibodies: fluorescein isothiocyanate (FITC)-labeled anti-CD34 (BD Biosciences, USA), phycoerythrin (PE)-labeled anti-CD133 (Becton Dickinson, Franklin Lakes, NJ, USA), and allophycocyanin (APC)-labeled anti-KDR (Becton Dickinson, Franklin Lakes, NJ, USA). In brief, 100 μl of PBMC samples was incubated at 4 °C for 20 min in the dark with 5 μl of FITC-CD34, 10 μl of PE-CD133, and APC-KDR. Cells labeled with FITC-, PE-, and APC-conjugated isotypic monoclonal antibodies were used as controls to determine the background of fluorescence. After staining, cells were washed in 1 ml PBS and eventually re-suspended in 500 μl PBS. Analysis was then performed within 30 min. Absolute cell numbers were measured using an LSR II Fortessa flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA); data were analyzed with FACSDiva software (Becton Dickinson, Franklin Lakes, NJ, USA) or FlowJo 9.6.4 software (Tree Star Inc., Ashland, OR, USA). All results from flow cytometry were expressed as number of cells/106 total cytofluorimetric events.
Cytokine Detection and Oxidative Activity Analysis
Serum levels of tumor necrosis factor alpha (TNFα), interferon gamma (IFNγ), and interleukin-1 beta (IL-1β) were determined with a CBA Flex set (BD Biosciences, San Jose, CA). The plasma levels of VEGF, malonyldialdehyde (MDA), glutathione (GSH), and superoxide dismutase (SOD) were respectively determined in duplicate using commercially available ELISA kits according to the manufacturer’s guidelines (USCN Business Co. Ltd., Wuhan, China). The plasma levels of reactive oxygen species (ROS) were determined using the ROS colorimetric assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).
Data were expressed as mean ± standard deviation (SD) or n (%), unless otherwise specified. Differences in continuous variables were determined by the Student’s t test, paired sample t test, or Mann–Whitney U test, and differences in categorical variables were determined by the χ2 analysis or Fisher’s exact test. Correlation analyses were assessed by Spearman’s rank correlation test. P values < 0.05 were considered statistically significant.