Association of chemerin mRNA expression in human epicardial adipose tissue with coronary atherosclerosis

Between October 2009 and March 2010, a total of 53 Han Chinese patients who underwent elective cardiac surgery were enrolled in the study. Exclusion criteria included non ischemic cardiomyopathy, liver or renal failure, neoplastic disease, thyroid or adrenal gland disease, acute infectious disease, the aged (> 80 years) or patients taking corticosteroids, oral contraceptives, or psychotropic drugs. Patients were divided into CAD group and NCAD group according to the results of selective coronary angiography. CAD was defined as the presence of stenoses of greater than 50% of the luminal diameter for at least one of the three major coronary arteries. All CAD patients underwent elective coronary artery bypass graft (CABG) surgery. All NCAD patients received elective open-heart surgery for valvular replacement or atrial septal defect closure. These patients had no clinical signs of CAD and did not show significant coronary stenoses (≥50%) in the pre-operative coronary angiographic examination.

This study was approved by the ethics committee of Beijing Anzhen Hospital of Capital Medical University. This study complied with the declaration of Helsinki with the written informed consent obtained from all subjects prior to enrolment.

Clinical data were obtained upon admission to hospital before surgery. Demographic data, medical history, and medications being used before surgery were recorded. In addition, height, body weight, waist circumference and blood pressure were measured. Then body mass index (BMI) was calculated as weight (kg) divided by square of height (m²).

Angiographic analyses were carried out by two experienced interventional cardiologists who were blinded to the study protocol according to the result of coronary angiography within six months prior to surgery. Then Gensini score was calculated to assess the severity of coronary atherosclerosis according to the method described in the literature [16].

Peripheral venous blood samples were collected after overnight fasting on the day after admission, and serum glucose, lipid profiles, high sensitivity C-reactive protein (hsCRP), insulin and hemoglobin A₁C (HbA₁C) were analyzed in central laboratory of Beijing Anzhen Hospital. Insulin sensitivity was estimated by homeostasis model assessment of insulin resistance (HOMA-IR) calculated as fasting glucose (mmol/L) × fasting insulin (μU/mL)/22.5.

Central venous blood was drawn before cardiopulmonary bypass during the operation. Then serum was stored in aliquots at -80°C until being analyzed. Serum levels of chemerin and adiponectin were determined by commercially available enzyme-linked immunosorbent assay (ELISA) kits (Adipobiotech, Beijing, China).

Adipose biopsy samples were obtained prior to the initiation of cardiopulmonary bypass from areas that had not previously been injured mechanically or cauterized. EAT biopsies (average 0.5 g) were taken near the proximal right coronary artery, and subcutaneous adipose tissue (SAT) samples were collected from the site of the chest incision. The specimens were rinsed with normal saline and divided into two portions. After removal of visible blood vessels, one portion was frozen immediately in liquid nitrogen and stored at -80°C for RNA isolation, another was immersed in neutralized formalin for immunohistochemical analysis.

Paraffin-embedded tissue sections were deparaffined and rehydrated in descending grades of alcohol and stained with hematoxylin and eosin. Selected serial sections were subjected to immunohistochemistry with the Histostain-Plus Kit LAB-SA Detection System (Invitrogen, USA) according to the manufacturer's protocol. Briefly, sections were incubated in 3% H₂O₂ for 20 minutes followed by blocking with normal goat serum. After they were washed in PBS, sections were incubated with primary antibodies (Chemerin, 1:200, Adipobiotech, Beijing, China) overnight at 4°C in a moisture chamber. Afterward, the slides were incubated with biotinylated secondary antibodies for 10~15 minutes followed by avidin-biotin for 15 minutes. Sections were then exposed to DAB and counterstained with hematoxylin. Negative controls were carried out omitting the primary antibody. Brightfield images were observed with a light microscope and digital images were recorded. Positive staining for chemerin was brown. Expression of chemerin was quantified by calculating the integrated optical density (IOD) of positive staining tissue via Image-pro plus software. The IOD of each tissue section was calculated from eight different 400 magnified fields.

Total RNA was extracted from adipose tissue samples using Trizol reagent (Invitrogen, USA). The concentration and purity of isolated RNA were assessed by measuring the optical density at 260 nm (OD260) and 280 nm (OD280), and the integrity of RNA was also determined by visualisation of 18S and 28S ribosomal bands. 1 μg of RNA from each sample was reverse transcribed using oligo(dT)₁₅ and M-MLV Reverse Transcriptase (Promega, USA), according to the manufacturer's instructions. An aliquot of 2 μL of the resulting cDNA was used for semiquantitative PCR. PCR was carried out in a volume of 25 μL containing 12.5 μL 2 × PCR-Mix, 1 μL 10 μmol/L each primer, and sterile water. The primers were designed using the Primer 5.0 software program, with their sites spanning introns, so quantitation of the reaction was not affected by the presence of genomic DNA. The primers used for amplification were shown in Table 1. Apart from the optimal annealing temperature and number of cycles of amplification, other conditions were the same in all PCR reactions. PCR was performed under the following conditions: 3 minutes for an initial denaturation at 94°C, followed by different cycles (30 s for denaturation at 94°C, 30 s for annealing and 50 s for extension at 72°C), and 5 minutes for a final extension at 72°C. The number of PCR cycles in each system was chosen within linear phase in our preliminary trial to ensure the accuracy of semiquantitative analysis. A 5 μl aliquot of PCR products was then analyzed using 1.5% agarose gel electrophoresis. Band intensity for the target gene was quantified by densitometry using Scion Image software and normalized to β-actin mRNA level to determine relative mRNA expression of the target gene.

Variables such as serum adiponectin, hsCRP and triglyceride with skewed distribution were log transformed.

Continuous variables with normal distribution were expressed as mean ± SD, means were compared by paired or unpaired Student's t-test, as appropriate. Whereas for data with skewed distribution, Mann-Whitney U test was used. Categorical variables were presented as percentages and were analyzed by Chi-squared test. Associations between the levels of adipokines in EAT and serum and clinical variables were determined with the Pearson or Spearman correlation coefficients. Partial correlation analysis was performed to evaluate adjusted association between chemerin mRNA level in EAT and Gensini score. All statistical analyses were performed using SPSS 16.0 software, the P-value < 0.05 was considered statistically significant.

The baseline characteristics of the two groups were shown in Table 2. In the CAD group (n = 37), 30 were men, and mean age was 59.1 ± 8.0 years. In the NCAD group (n = 16), 12 were men, and mean age was 54.3 ± 9.4 years. As expected, the majority of patients undergoing CABG surgery had multi-vessel lesions, moreover, obesity and hypertension were more prevalent in the CAD group than in the NCAD group. There were no significant difference in age, gender, diabetes, left ventricular ejection fraction, blood pressure, triglycerides, total cholesterol, LDL cholesterol, HbA₁C, serum creatinine, urea nitrogen, uric acid, medications treatment such as angiotensin converting enzyme inhibitors/angiotensin II type 1 receptor blockers (ACEIs/ARBs), oral hypoglyceimic agents, insulin and antibiotics between the two groups.

However, patients in the CAD group presented higher levels of fasting glucose, HOMA-IR, hsCRP, and lower HDL cholesterol level compared to those in the NCAD group. As for current medications therapy, aspirin, nitrates, beta-blockers, statins, and calcium channel blockers were prescribed more often in the CAD patients than in the NCAD patients.

We performed immunohistochemistry to illustrate chemerin expression in adipose tissue. EAT and SAT samples were collected randomly from the CAD group (n = 6) and the NCAD group (n = 6), respectively. Figure 1A showed the representative slides of EAT and SAT from one patient with CAD (Figure 1A-a and 1A-b) and one without CAD (Figure 1A-c and 1A-d). Immunohistochemical staining revealed that chemerin was expressed in both EAT and SAT of the two groups. Furthermore, as shown in Figure 1B, quantitative analysis of immunohistochemistry revealed that the amounts of chemerin protein in EAT were higher in the patients with CAD than those without CAD (70128.28 ± 13068.83 vs. 52312.03 ± 9899.90, P < 0.05). For the CAD group, significantly higher level of chemerin protein was found in EAT than in SAT (70128.28 ± 13068.83 vs. 42942.04 ± 15460.67, P < 0.01), whereas no significant difference was found between EAT and SAT in the NCAD group (52312.03 ± 9899.90 vs. 50533.71 ± 17289.54, P = 0.871).

As illustrated in Figure 2, we found significantly higher levels of chemerin, TNF-α mRNA and significantly lower level of adiponectin mRNA in EAT of CAD group compared to that of NCAD group, whereas the mRNA expression of chemR23 in EAT did not show significant difference between the two groups (chemerin 0.94 ± 0.17 vs. 0.78 ± 0.21, P < 0.01; TNF-α 0.36 ± 0.34 vs. 0.15 ± 0.19, P < 0.01; adiponectin 0.71 ± 0.15 vs. 0.83 ± 0.12, P < 0.01; chemR23 0.83 ± 0.45 vs. 0.62 ± 0.45, P = 0.140).

As for the comparison of paired EAT and SAT, 3 SAT samples of CAD group were excluded from the analysis due to insufficient adipose tissue biopsy samples or being damaged during the operation. As shown in Figure 3, in the CAD group, significantly higher level of chemerin mRNA was observed in EAT than in paired SAT (0.94 ± 0.17 vs. 0.84 ± 0.28, P < 0.05), whereas chemerin mRNA level did not seem to differ between the two adipose depots in the NCAD group (0.78 ± 0.21 vs. 0.82 ± 0.37, P > 0.05). Moreover, chemerin mRNA expression in SAT of CAD group was comparable with that in SAT of NCAD group (0.84 ± 0.28 vs. 0.82 ± 0.37, P > 0.05).

Chemerin mRNA expression in EAT was positively correlated with Gensini score (r = 0.365, P < 0.05). Furthermore, this correlation revealed by partial correlation analysis remained statistically significant (r = 0.357, P < 0.05) even after adjusting for age, gender, BMI and waist circumference. Chemerin mRNA expression in EAT was also found to be positively correlated with BMI (r = 0.305, P < 0.05), waist circumference (r = 0.384, P < 0.01), fasting glucose (r = 0.334, P < 0.05), chemR23 mRNA expression in EAT (r = 0.349, P < 0.05) and negatively correlated with adiponectin mRNA expression in EAT (r = -0.322, P < 0.05). However, no association was observed between chemerin mRNA expression in EAT and TNF-α mRNA expression in EAT (P > 0.05).

The serum samples of 33 CAD patients and 11 NCAD patients met the criteria for analysis. The two groups were also age and gender matched. There were no significant differences in the serum levels of chemerin or adiponectin between the two groups (chemerin 118.13 ± 32.02 ng/ml vs. 99.60 ± 28.66 ng/ml, P = 0.096; adiponectin 4.90 ± 1.39 μg/ml vs. 5.87 ± 1.52 μg/ml, P = 0.155). Correlation analysis revealed that serum chemerin concentration was positively correlated with BMI (r = 0.323, P < 0.05), waist circumference (r = 0.398, P < 0.01), HOMA-IR (r = 0.299, P < 0.05) and hsCRP (r = 0.340, P < 0.05). However, neither serum chemerin (r = 0.295, P = 0.120) nor serum adiponectin (r = -0.328, P = 0.083) level was found to be associated with Gensini score. Also, there was no statistically significant correlation between serum chemerin level and its expression level in EAT (P > 0.05).