Decreased adiponectin and increased inflammation expression in epicardial adipose tissue in coronary artery disease

Eleven coronary artery samples (left anterior descending artery, LAD) combined with the surrounding adipose tissue were obtained from human remains voluntarily supplied by the Department of Anatomy, Tongji Medical College, HUST. Twelve internal mammary arteries, six radial arteries and 14 greater saphenous veins were obtained as bridge vessels from patients who were scheduled to undergo CABG surgery, before starting cardiopulmonary bypass (CPB) surgery. All collected vessels were fixed and embedded in paraffin for sectioning and immunochemical staining. Thirty-four patients who had been planning cardiac surgery first received selective coronary angiography (CAG). They were divided into a CAD group (n = 23) and a non-CAD control group according to the results of the CAG. Biopsy samples of epicardial adipose tissue (average 0.5-1.0 g) from all subjects and of subcutaneous fat from around the greater saphenous vein of CAD subjects (who planned to use this vein as a bridge vessel) were taken prior to heparin administration for CPB surgery. Each single tissue sample from one subject was divided into two parts, one part was fixed and embedded in paraffin for sectioning and immunochemical staining, and the other was shock-frozen and immediately stored in liquid nitrogen for total RNA extraction. Pectoral subcutaneous adipose tissues were also obtained from four patients without heart diseases with body mass indexes (BMI) ranging from 19 to 23 kg/m² who were undergoing non-cardiac surgery, and these tissue samples were cryopreserved in profound hypothermia as described above.

The paraffin-embedded sections of vessels were stained with hematoxylin and eosin (H&E). The eleven coronary artery samples were then divided into CAD group and control group according to the results of pathological examination. For all vessels and adipose tissue samples, 5-μm-thick whole paraffin-embedded cut sections on polylysine-coated glass slides were incubated overnight at 4°C with phosphate buffered saline (PBS) containing 0.5% bovine serum albumin (Sigma-Aldrich, St. Louis, MO, USA) (PBS/BSA) to block nonspecific binding. The specimens were then incubated with rat polyclonal anti-CD68 antibody (Abcam, Cambridge, MA, USA; 1:50 in PBS/BSA) overnight at 4°C in a moist incubation chamber. After washing three times in PBS, specimens were incubated with the secondary antibody against the rat polyclonal antibody produced in goat (Boster, Wuhan, China; 1:500 in PBS/BSA). The slides were again washed in PBS three times and incubated with SABC complex diluted 1:500 for 30 min at room temperature. After a new wash in PBS, the reaction was developed with chromogen solution consisting of 0.03% hydrogen peroxide. Color intensity was monitored under a light microscope and compared with positive controls included in each reaction. The specimens were then washed under running water for 10 min, counterstained with Harris hematoxylin for 20 seconds, again washed under running water, dehydrated in ethanol, cleared in xylene, and mounted in Permount resin. The immunostained sections were analyzed under Olympic MX-50 optical microscope. Positive immunostaining for the specific antibody was analyzed quantitatively in five fields of the lesion area at large magnification (4 × 100) using a graded grid divided into 10 × 10 subdivisions and comprising an area of 0.0625 mm².

Total RNA from frozen adipose tissues stored in liquid nitrogen was extracted using TRIzol reagent (Invitrogen, CA, USA) following the manufacturer's instructions. The reverse transcription reactions were conducted with Transcriptor First Strand cDNA Synthesis Kit (Roche, Indianapolis, IN, USA). The oligonucleotide primer sequences were designed by Premier Primer 5.0 software as Table 1. β-actin was used as an internal control. The synthesized first-strand cDNA samples were subjected to real-time PCR with SYBR Green PCR Master Mix (Toyobo Bio-Technology, Shanghai, China) and PCR was performed using ABI Prism 7700 Sequence Detector (Applied Biosystems, Tokyo, Japan). The integrity of PCR products was confirmed by dissociation curve analysis using 2.0 software (Applied Biosystems). The threshold cycle (C_T) values were determined and relative gene expression was calculated using the formula 2^-ΔΔCT with pectoral subcutaneous adipose tissues obtained from non-CAD patients during non-cardiac surgery.

Peripheral vein blood was drawn in the fasting state, and serum biochemistries were performed with routine laboratory techniques for fasting plasma glucose (FPG), triglycerides (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), apolipoprotein AI (apo AI), apolipoprotein B (apo B) and lipoprotein (a) (Lp(a)). Blood samples for plasma adiponectin measurement from coronary sinus were collected during right heart catheterization. Adiponectin concentrations in peripheral and coronary sinus vein plasma were determined by enzyme-linked immunosorbent assay (ELISA) according to the protocol provided by the manufacturer (ALPCO Diagnostics).

Twenty milliliters of whole blood donated by healthy volunteers was randomly obtained from the Wuhan Blood Centre, which complied with the Blood Donation Law of the P.R. China. Monocytes were separated from anticoagulated whole blood using human lymphocyte separation medium according to the manufacturer's instructions (PAN Biotech, Aidenbach, Germany). Before treatment, the separated cells were suspended in a culture bottle with high-glucose RPMI1640 medium (Gibco, Invitrogen, Australia) supplemented with 10% fetal bovine serum (Gibco) and maintained at 37°C in a humidified 5% CO₂ atmosphere overnight. After disposal of non-adherent cells, and thoroughly washing the adherent cells with phosphate-buffered saline (PBS) twice, cells were recovered using 0.5% trypsin (Gibco). For evaluating the percentage of monocytes and the viability of collected cells, immunofluorescence and trypan blue staining were performed. The concentration of cells was then adjusted to 1 × 10⁶/ml to reserve.

Isolated monocytes were equilibrated to a steady state at densities of 4,000 cells per well in 96-well culture plates for 4 hours, then the pro-inflammatory effects of stearic acid (SA) and anti-inflammatory effects of adiponection on monocytes were tested. First, monocytes were cultured in the presence of 100 ng/ml lipopolysaccharide (LPS) (Sigma-Aldrich, St. Louis, MO, USA) (positive control), SA (Sigma-Aldrich) at three different concentrations (0.1, 0.5 and 1.0 mM) or PBS (negative control) for 24 hours. Meanwhile, three different final concentrations of globular adiponectin (gAd) (5, 10 and 20 μg/ml) (Peprotech, Rocky Hill, NJ, USA) or PBS (negative control) were added as follows. After incubation for 6 hours, monocytes were stimulated by 0.5 mM SA for 24 hours. In the next experiment, cultured supernatants were collected and TNF-α and IL-6 concentrations were quantified according to the manufacturer's instructions (R&D Systems, Minneapolis, MN, USA). In these two treatments, the activity of the transcription factor NF-κB was also measured in monocytes using Histostain-Plus Kit, following manufacturer's instructions (Invitrogen, CA, USA).

Monocytes were plated into a 6-well culture plate at a density of 1 × 10⁶/well and equilibrated at 37°C for 2 hours. Three different concentrations of gAd (5, 10 and 20 μg/ml) or PBS (negative control) were added and stimulated by 0.5 mM SA as described above. The collected cells were then stained with a dual-color antibody panel composed of CD14 (eBioscience, San Diego, Calif) and TLR-4 (BD PharMingen, San Diego, CA, USA ) and analyzed by 6-fluorescent-parameter BD LSRII (BD Biosciences, Sparks, MD, USA).

Data were presented as means ± SEM. When comparing groups, one-way ANOVA was used to test for significance. A value of p < 0.05 was considered statistically significant.

CD68 is especially expressed by the mononuclear phagocytic system and highly expressed on the surface of macrophages [14]. As shown in Figure 1A, no changes in the vascular endothelium, elastic fibers or adventitia (including from greater saphenous vein) were observed in samples from either the internal mammary artery or radial artery. However, atherosclerosis, elastorrhexis, a large amount of CD68⁺ cell infiltration in the adventitia, and an obvious zone of CD68⁺ cell accumulation was observed at the interface between the adventitia and epicardial adipose tissue in samples from CAD patients. In the above five types of blood vessels, the highest density of CD68⁺ cells in coronary arteries from CAD patients is shown in Figure 1C. Meanwhile, the magnitude of CD68⁺ cell infiltration into epicardial adipose tissue was significantly higher than that observed in samples either with or without subcutaneous fat around the greater saphenous vein in patients with CAD (Figure 1B and 1D).

By performing real-time quantitative RT-PCR, the expression levels of adiponetin, IL-6, TNF-α and TLR4 mRNA in different adipose tissues were examined. As shown in Figure 2, the expression patterns of these genes in epicardial adipose tissue in non-CAD patients were similar to those in subcutaneous fat around the greater saphenous vein in CAD patients. Comparing the two types of samples more closely, the gene levels in epicardial adipose tissue in CAD patients were rather different. Adiponectin mRNA was poorly expressed, but IL-6, TNF-α and TLR4 were highly expressed. It would seem that a strongly negative correlation would be expected between the levels of adiponectin mRNA and the other three genes.

It has been reported that the plasma level of adiponectin in coronary circulation is significantly related to adiponectin expression in epicardial adipose tissue [15]. To investigate whether decreased adiponectin expression in epicardial fat can be used as a sensitive predictor of coronary atherosclerosis in non-obese patients, we examined the coronary sinus vein plasma levels of adiponectin and routine biochemical values, including TG, LDL-C, HDL-C, TC, FPG, apo AI, apo B and Lp(a), in peripheral vein of elderly male with body mass index (BMI) < 25.0, who received coronary angiography, a routine examination for patients over the age of 60 years undergoing cardiac surgery to exclude CAD. Subjects were divided into two groups. Within the CAD group, nine subjects were confirmed to have significant (>75%) narrowing of at least one major coronary artery; the non-CAD group included eleven subjects with smooth coronary arteries. As summarized in Table 2, there was no statistically significant difference between the two groups for TG, LDL-C, HDL-C, TC, FPG, apo AI, apo B and Lp(a), but the adiponectin concentrations in peripheral and coronary sinus vein plasma were both significantly lower in the CAD group suggesting that decreased circulating adiponectin may play an independent role in the pathogenesis of coronary atherosclerosis.

Using density gradient centrifugation techniques as described above, we successfully enriched monocytes from human whole blood. Studies on the expression of monoclonal antibodies demonstrate that CD14 is a specific marker for monocytic cells, so immunofluorescence was performed to identify the percentage of CD14 positive cells [16]. Results confirmed that the purity of monocytes in the isolated cells was more than 90% (data not shown) and that cell viability was over 95% as determined by staining with trypan blue. TNF-α and IL-6 are critical cytokines that respond to the progression of atherosclerosis [17]. To examine the proinflammatory effect of SA on monocyte secretion of TNF-α and IL-6, we added SA into the medium to a final concentration of 0.1, 0.5 and 1.0 mM. PBS and 100 ng/ml LPS were added as negative and positive controls, respectively. Compared with negative controls, SA had a stimulatory effect on monocyte secretion of TNF-α and IL-6, and an obvious dose-response relationships was observed between SA and both TNF-α and IL-6 (Figure 3A and 3C). Adioponectin is present in trimer form, and gAd is its proteolytic cleavage product [18]. To address the antiinflammatory effects of adiponectin, different concentrations of gAd were tested. Compared with PBS, gAd can inhibit stimulated monocytes secreting TNF-α and IL-6 (Figure 3B and 3D). Consistent with these results, SA can active NF-κB and induce monocyte apoptosis, which can be inhibited by gAd (Figure 3E and 3F).

As analyzed by real time quantitative RT-PCR, epicardial adipose tissue exhibited a relatively high level of TLR4 mRNA consistent with inflammatory cytokines IL-6 and TNF-α. However, the level of adiponectin mRNA is relatively low. To understand the relationship between expression levels of TLR4 mRNA and the inflammatory cytokines, we used CD14 and TLR4 antibodies applicable for flow cytometry (FCM). FCM of monocytes was observed on samples collected from three different gAd concentration groups after stimulation with SA. We observed a significant decrease in the ratio of TLR4 positive cells as the treatment concentration of gAd increased from low to high (Figure 4). These results strongly suggest that adiponectin down-regulates the expression of TLR4 when monocytes are treated with SA.