Overexpression of scavenger receptor and infiltration of macrophage in epicardial adipose tissue of patients with ischemic heart disease and diabetes

We included 45 patients with IHD who underwent coronary artery bypass surgery (IHD group) and 23 patients without IHD who underwent aortic and/or mitral valve replacement surgery (control group) due this is the only way to obtain EAT in patients without IHD. The group with patients with IHD was divided according to T2DM status: those with T2DM (n = 23) (IHD-T2DM group) and those without T2DM (n = 22) (IHD-NoT2DM group). The IHD group was defined by the presence of at least one coronary stenosis ≥ 50% of luminal diameter by coronary angiogram. The control group had chronic valve heart disease with or without stenosis less than 50% in any vessel requiring valve replacement but not IHD and without T2DM. T2DM was defined as having a history of diabetes diagnosed and/or treated with medication, fasting blood glucose ≥ 126 mg/dL and/or glycated hemoglobin (HbA1c) ≥ 6.5. Diabetic treatment of IHD patients with T2DM was: only diet (n = 6, 26.1%), oral anti-diabetic (n = 12, 52.1%), oral anti-diabetic and insulin (n = 4, 17.4%) and only insulin (n = 1, 4.3%). No patient was taking thiazolidinediones. In the IHD-T2DM patient group, the duration of diabetes was 6.7 ± 2.2 years. Dyslipidemia was defined as having a history of diagnosed and/or treated with medication for elevated triglycerides, low HDL-cholesterol or high LDL-cholesterol. The presence of greater than or equal to 50% luminal diameter stenosis in at least one major epicardial artery by coronary angiogram defined the IHD. Single vessel and multi-vessel disease were defined as the presence of this stenosis in one major epicardial artery and in two or more major epicardial arteries, respectively. Calculation of the Gensini score was initiated by giving a severity score to each coronary stenosis as follows: 1 point for ≤ 25% narrowing, 2 points for 26 to 50% narrowing, 4 points for 51 to 75% narrowing, 8 points for 76 to 90% narrowing, 16 points for 91 to 99% narrowing, and 32 points for total occlusion. Thereafter, each lesion score is multiplied by a factor that takes into account the importance of the lesion’s position in the coronary circulation (5 for the left main coronary artery, 2.5 for the proximal segment of the left anterior descending coronary artery, 2.5 for the proximal segment of the circumflex artery, 1.5 for the mid-segment of the left anterior descending coronary artery, 1.0 for the right coronary artery, the distal segment of the left anterior descending coronary artery, the posterolateral artery, and the obtuse marginal artery, and 0.5 for other segments). Finally, the Gensini score was calculated by summation of the individual coronary segment scores [28]. Patients with coronary disease with lesions that required revascularization surgery and those patients in the control group who had chronic valve heart disease without IHD or DM were included in the study. Moreover, the decision that these patients were included in the study was taken in the clinical session by cardiac surgeons and cardiology experts. Patients with acute inflammatory disease, severe infectious diseases and/or cancer and women who were taking hormone replacement were excluded from the study. All patients gave written informed consent, and the study protocol was approved by the local Clinical Research Ethics Committee and was carried out in accordance with the Declaration of Helsinki.

EAT biopsy samples (average 0.2 to 0.5 g) were obtained with a precise surgical technique during the heart surgery, near the proximal right coronary artery and located between the myocardium and the visceral pericardium. Pericardial adipose tissue is located on the external surface of the parietal pericardium and is vascularized from non-coronary sources [29]. The sample was collected approximately 1 h after the beginning of general anesthesia. All the tissues were washed in physiological saline, immediately frozen in liquid nitrogen and maintained at − 80 °C until RNA analysis.

Total RNA from frozen human EAT was isolated using RNeasy Lipid Tissue Mini Kit (Qiagen, GmbH, Germany) as previously described [30‐32]. Total RNA was reverse transcribed using random hexamers as primers and transcriptor reverse transcriptase (Roche, Mannheim, Germany). Gene expression was assessed by real time PCR using an Applied Biosystems 7500 Fast real time polymerase chain reaction system (Applied Biosystems, Darmstadt, Germany). Reactions were carried out in duplicate for all genes using specific TaqMan^® Gene Expression Assays: macrophage scavenger receptor 1 (MSR1) (Hs00234007_m1, RefSeq. NM_002445.3, NM_138715.2, NM_138716.2), chemokine (C–X–C motif) ligand 16 (CXCL16) (Hs00222859_m1, RefSeq. NM_001100812.1, NM_022059.2), oxidized low-density lipoprotein (lectin-like) receptor 1 (LOX-1) (Hs01552593_m1, RefSeq. NM_001172632.1, NM_001172633.1, NM_002543.3), collectin sub-family member 12 (CL-P1) (Hs00560477_m1, RefSeq. NM_130386.2), CD68 (Hs00154355_m1, RefSeq. NM_001040059.1, NM_001251.2), integrin subunit alpha X (CD11c) (Hs00174217_m1, RefSeq. NM_000887.4, NM_001286375.1), mannose receptor, C type 1 (CD206) (Hs00267207_m1, RefSeq. NM_00267207_m1), CD36 (Hs01567185_m1, RefSeq. NM_000072.3, NM_001001547.2, NM_001001548.2, NM_001127443.1, NM_001127444.1, NM_001289909.1, NM_001289911.1) and LDL receptor related protein 1 (LRP1) (Hs00233856_m1; RefSeq. NM_002332.2). During PCR, the threshold value for all genes studied was 0.1. The Ct value for each sample was normalized by constitutively expressed cyclophilin A signals (PPIA) (4326316E, RefSeq. NM_021130.3). SDS software 2.3 and RQ Manager 1.2 (Applied Biosystems, Foster City, CA) were used to analyze the results with the comparative Ct method (2^−ΔCt). All data were expressed as an n-fold difference relative to the calibrator.

Statistical analyses were performed with SPSS for Windows version 15 (SPSS Inc. Chicago, IL, USA). The normality of continuous variables was checked by means of the Kolmogorov–Smirnov test. Continuous variables are summarized as mean ± standard deviation, and as mean ± SEM in figures. Discrete variables are summarized as frequencies (percentages). The comparison between the results of the different groups was made with the Chi square test and Fischer exact test for categorical data, or with the analysis of variance (ANOVA) and Kruskal–Wallis for continuous variables. Logistic regression models were used in order to identify independent factors (odds ratio [OR]; 95% confidence interval) for IHD in patients with T2DM associated with mRNA expression of SRs, as well as to control for confounding factors. The inclusion of 7 patients in each group for an α of 0.05 (assuming a difference of 0.22 of CD36 mRNA expression, a standard deviation of 0.14 and a power of 0.80) has the power to detect a significance difference of 95%. Values were considered to be statistically significant when P < 0.05.

A total of 68 patients were included in our study. Among the 45 patients of the IHD Group, 23 (51.1%) had T2DM (IHD-T2DM group). Table 1 summarizes the anthropometric, clinical and biochemical characteristics of the IHD groups and controls. No significant differences were found between the groups regarding age, gender, cardiovascular risk factors and left ventricular ejection fraction. However, IHD-T2DM patients showed significantly higher levels of fasting glucose, HbA1c, insulin, HOMA-IR score and C-reactive protein than IHD-NoT2DM and controls. The levels of oxLDL and LRP1 were significantly higher in IHD-T2DM patients than in control group.

On the other hand, the characteristics of coronary artery disease were significantly different between the IHD groups. IHD-DM patients were more likely to have multi-vessel disease and major coronary stenosis than the IHD-NoT2DM patients (Table 1). Also, patients with T2DM had more severity of coronary disease measured by the Gensini score compared with those without T2DM (Table 1).

The mRNA expression of SRs in EAT in the three groups of patients is shown in Fig. 1. The mRNA expression of LOX-1, CL-P1, CD68 and CD11c in EAT were significantly higher in IHD-T2DM patients when compared with IHD-NoT2DM (P < 0.01) and control patients (P < 0.01) (Fig. 1a, b, e and g, respectively). However, mRNA expression of MSR1, CXCL16, CD36, CD206 and LRP1 showed no significant differences (Fig. 1c, d, f, h and i, respectively). The mRNA expressions of SRs were similar in IHD-NoT2DM and control patients (Fig. 1).

With respect to the distribution of M1 and M2 macrophages, significant differences were found in CD11c/CD68 ratio (M1 macrophages) between IHD-T2DM and control group, showing a higher level those IHD-DM patients (Fig. 2a). No significant differences were found in the CD206/CD68 ratio (M2 macrophages) (Fig. 2b).

The factors associated with the presence of T2DM in patients with IHD in a logistic regression model was the level of LOX-1 and CD68 mRNA expression in EAT. These receptors were identified as independent risk factors of suffering T2DM. Glucose and HbA1c were also shown to be risk factors. This regression was adjusted for sex, age, BMI, statins, FRS and CL-P1 mRNA expression (Table 2). No significant association was found between LOX-1 and T2DM duration.