A total of 153 subjects were enrolled in this study. This included 113 asymptomatic patients who had undergone a cardiac evaluation with 64-slice multidetector computed tomography (MDCT) either as a health check or for cardiac evaluation of high-risk patients with prediabetes or drug-naïve patients with type 2 diabetes at the Seoul National University Bundang Hospital [SNUBH] from March 2005 to May 2010.
The other 40 subjects were symptomatic patients with coronary artery stenosis ≥50% in three major coronary arteries. Elective coronary artery bypass surgery was performed and samples of whole adipose tissue (subcutaneous, visceral (preperitoneal), and/or pericardial fat), internal mammary artery, and blood were obtained as a part of a study to identify candidate biomarkers of atherosclerosis.
We identified their risk factors from medical history, demographics, baseline clinical profile, and concomitant medications. This study was conducted according to the Declaration of Helsinki and was approved by ethics committees of SNUBH (SNUBH IRB#B-1203/147-006, #A111218-CP02) and all subjects provided their written informed consent.
Measurement of anthropometric and biochemical parameters
Body weight, height, and blood pressure were measured at enrollment in the study. Body mass index was calculated as the weight in kilograms divided by the square of the height in meters (kg/m2). Serum concentrations of total cholesterol, triglycerides, high-density lipoprotein-cholesterol, low-density lipoprotein (LDL)-cholesterol, aspartate aminotransferase, alanine aminotransferase, creatinine, glycated hemoglobin (HbA1c), high-sensitivity C-reactive protein (hs-CRP), and plasma glucose were measured after a ≥12-hour fast. We defined diabetes mellitus as a fasting plasma glucose concentration of ≥126 mg/dL, an HbA1c level ≥6.5%, or taking antidiabetic medicine. Hypertension was defined as systolic blood pressure >140 mmHg, or diastolic blood pressure >90 mmHg, or taking antihypertensive medicine. Dyslipidemia was defined as an LDL-C concentration of ≥130 mg/dL, or hypertriglyceridemia (triglyceride concentration ≥200 mg/dL), or taking lipid-lowering medicine. Serum C1q concentration was measured using an enzyme-linked immunosorbent assay (ELISA) kit (HK356, Hycult Biotech, Uden, The Netherlands) using samples that had been frozen at −80°C until analyzed. ELISA kits were used to measure serum total APN concentration (EZHADP-61 K, Millipore, Billerica, MA, USA) and HMW-APN (EZHMWA-64 K, Millipore) in the same samples.
CAD was detected using 64-slice MDCT during a routine health examination. Subjects with a heart rate >70 beats per min received 10–30 mg of intravenous esmolol (Jeil Pharm, Seoul, Korea) before MDCT imaging. CT angiography was performed with a 64-slice MDCT scanner (Brilliance 64, Philips Medical Systems, Best, The Netherlands) using the standard scanning protocol described previously .
Definition of coronary artery stenosis
Coronary artery stenosis was identified as percent of luminal narrowing of at least 1 major coronary artery and was evaluated with MDCT or coronary angiography. Coronary artery stenosis was categorized into three classifications: normal (any coronary artery stenosis <25%), mild to moderate (any coronary artery stenosis ≥25%), and severe (three coronary artery stenosis ≥50%; coronary artery bypass surgery). The presence of CAD was defined as any coronary artery stenosis ≥50% regardless of number of involved coronary artery.
Blood and tissue samples
Serum was obtained from peripheral blood at the time of patient enrollment. Fat tissues from subcutaneous, visceral, and pericardial areas, and samples of the internal mammary artery were obtained from 40 patients during coronary artery bypass surgery and were irrigated with saline. All of the samples were stored at −80°C until analyzed.
Immunoblotting assay to detect the C1q–APN complex
Human serum was mixed with C1q antibody (SC-53544, Santa Cruz Biotechnology, Dallas, TX, USA) in 1× kinase buffer (20 mM HEPES pH 7.4, 5 mM MgCl2, 1 mM dithiothreitol) and agitated overnight at 4°C. Protein G (17-0618-01, GE Healthcare, Buckinghamshire, UK) was added to the mixture, and the mixture was rotated for 4 h at 4°C. After three washes, the C1q-immunocomplexes were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blotting using anti-APN antibody (ab18851, Abcam, Cambridge, UK).
Deparaffinized adipose tissue sections and blood vessels were fixed in 4% paraformaldehyde in phosphate-buffered saline. The sections of fat tissues and vessels were stained with hematoxylin–eosin. Immunostaining was performed overnight at 4°C with mouse anti-C1q (ab71089, Abcam,) or with rabbit anti-APN (NB100-65810, Novus Biologicals, Littleton, CO, USA) antibodies. The sections were then washed and incubated in Alexa Fluor 488 goat anti-rabbit IgG (Moleaulr Probes, Eugene, OR) and Alexa Fluor 594 goat anti-mouse IgG (Molecular Probes, Eugene, OR) as the secondary antibody for 1 h at 37°C. The slides were examined under a Zeiss Axio Imager A1 fluorescent microscope (Carl Zeiss, Cambridge, UK).
All values are expressed as mean and standard deviation (SD) or number (percent). Clinical parameters that were not normally distributed were log-transformed to reduce the skewness; we present the results as the anti-logarithm for ease of interpretation. Differences in parameters between groups were analyzed using χ
2-test, Student’s t-test, or one-way analysis of variance (ANOVA). When significantly different values were observed in an ANOVA, Bonferroni post hoc analysis was applied to identify the significantly different values. Simple and multivariate logistic regression analysis was used to determine the associations between parameters and CAD. A p-value <0.05 was considered significant. All analyses were performed using SPSS 17.0 for Windows.