A total of 22 male subjects, aged between 51 and 80 years (mean 67.86 ± 7.75 years), with BMI ranging from 23.18 to 32.87 kg/m2 (mean 27.57 ± 2.69 kg/m2), undergoing elective cardiac surgery either for coronary bypass grafting (CAD, n = 12) or valve replacement (non-CAD, n = 10) were included in the study. Clinical records were collected in all the subjects.
Six patients had type 2 diabetes mellitus (DM) in the CAD group, 3 subjects in the non-CAD group. Patients with liver, kidney diseases or neoplastic diseases were excluded from the protocol study, as well as subjects with weight loss greater than 5% in the month preceding the evaluation, patients on steroid or on immunosuppressive therapy in the previous six months, patients with renal insufficiency with creatinine values > 1.5 mg / dl, poorly controlled thyroid disease, patients on hormonal therapy (except for the thyroid) and patients with chronic inflammatory diseases.
Written informed consent was obtained from each participant and the study was approved by the Ethics Committee of the University of Verona (Italy).
Anthropometric Variables
Each patient underwent measurement of body weight with an approximation of 0.1 kg (Salus Scale, Milan, Italy) and to height measurement using a stadiometer, with an approximation of 0.5 cm. (Salus Stadiometer, Milan, Italy).
BMI was computed as weight in kilograms divided by height in square meters (kg/m²). The measurement of WC was obtained with the patient standing upright, using a tape measure. The WC was assessed as the minimum abdominal circumference between the xiphoid process and the navel.
Blood Collection
Venous blood samples for all metabolic assessments were obtained after overnight fasting. Plasma glucose was measured with a glucose analyzer (Beckman Instruments Inc, Palo Alto, CA). The intra-assay CV was 1.5%. Plasma insulin was evaluated using a direct chemiluminescent-based two-site sandwich immunoassay (ADVIA Centaur Insulin assay; Siemens, Erlangen,Germany). The sensitivity of the assay was 0.5mU/l. We used the homeostatic model assessment (HOMA) method as an indirect index of insulin resistance, calculated as the product of the fasting plasma insulin level (mU/l) and the fasting plasma glucose level (mmol/l), divided by the constant 22.5. Serum leptin and adiponectin were measured using specific enzyme-linked immunoassay kits (respectively from DBC-Diagnostic Biochem Canada, London, ON, Canada and B-Bridge, Cupertino,CA,USA). The sensitivity of the assays was 0.5ng/ml for leptin and 0.02ng/ml for adiponectin.
Epicardial adipose tissue Collection
In the operating room, epicardial adipose tissue (EAT) specimens were obtained after median sternotomy and heparin administration prior to initiating cardio-pulmonary bypass, approximately 1 hour within induction of anesthesia. EAT fragments (approximately 1 gram) were collected during the atrial appendage cannulation procedure for extracorporeal circulation. EAT was then stored for immunohistochemistry (IHC) assay.
Immunohistochemistry
Freshly isolated EAT was fixed by immersion in 4% paraformaldehyde in 0.1M phosphate buffer, pH7.4 overnight at 4°C, and then dehydrated, cleared and paraffin embedded. Five micrometer thick serial sections were obtained and stained by hematoxylin and eosin (H/E) to assess morphology prior to immunohistochemical processing.
The following primary antibodies were used for the IHC analysis: mouse anti-human CD68 (ready to use; Thermo Fisher Scientific), mouse anti-human CD163 (1:300; Thermo Fisher Scientific), rabbit anti-human CD11c (3µg/ml; Thermo Fisher Scientific), rabbit anti-human HIF1-α (1:400; DBA Italia), rabbit anti-human Caveolin-1 (1:400, Cell Signaling). Secondary antibodies were as follows: SignalStain Boost IHC detection reagent HRP Rabbit (ready to use; Cell Signaling) and SignalStain Boost IHC detection reagent HRP Mouse (ready to use; Cell Signaling). A negative control was conducted by using a secondary antibody alone. Diaminobenzidine hydrochloride (DAB) substrate kit for peroxidase was used for IHC (Vector Laboratories).
Briefly, sections were dewaxed and subjected to antigen heat retrieval. Endogenous peroxidase activity and non specific binding were blocked by incubation with 3% hydrogen peroxide and non immune serum, respectively. Slides were then incubated sequentially with primary rabbit antibody for 16hr at 4°C or with primary mouse antibody for 1hr at 20°C, followed by incubation with the secondary antibody for 30 minutes at 20°C. DAB was used as the chromogen to visualize peroxidase activity. Sections were counterstained with hematoxylin, assemble with Entellan and overlaid with coverslips.
Masson trichrome staining for interstitial fibrosis evaluation
EAT were stained with Masson's trichrome to visualize interstitial fibrosis, according to the manufacturer’s protocol (Bio-Optica, Milano, Italy).
Briefly, sections were deparaffined, rehydrated, stained with ferric hematoxylin according to Weigert for 10 minutes, incubated with picric acid for 4 minutes, wash quickly with distilled water, stained with Ponceau B solution for 4 minutes, quickly wash with distilled water, incubate with phosphomolibdic acid for 10 minutes, stained with aniline blue according to Masson for 5 minutes, quickly wash with distilled water, dehydrate, cleared, assemble with Entellan and overlaid with coverslips.
Image Capturing and Analyses
The CD68 (+), CD163 (+) and CD11c (+) macrophage and the HIF1-α positive nuclei counts were performed manually by observing the whole area of the EAT sections using an Olympus BX51 photomicroscope equipped with a KY-F58 CCD camera (JVC) at 400x magnification. The total area of the observed section was then calculated using ImageJ software 1.49o version on the acquired EAT images and the number of macrophages and the number of nuclei HIF1-α (+) was expressed as a number / mm2 by dividing the number for the total area expressed in mm2.
For Caveolin-1 and fibrosis evaluation, slides were observed and images analyzed and stored using EVOS FL Auto Cell Imaging System with EVOS Onstage Incubator (Thermo Fisher Scientific) photomicroscope.
The quantification of caveolin-1 was achieved by calculation of the optical density (OD). OD assay was performed on the whole section of the EAT by observing images at 200x magnification, using Color Threshold of ImageJ software 1.49o version.
ImageJ software 1.49o version was also used to measure collagen deposition by calculating the percentage of blue staining, indicative of fibrosis, on total section area of EAT at 200x magnification by automated color deconvolution [20] and manual threshold correction where necessary. The percentage of fibrosis was adjusted by adipocyte size to eliminate the effects of different adipocyte sizes in measure fields [21]. The scoring was performed by one investigator who was blind to the identity of the samples.
Statistical Analysis
All results are shown as mean ± standard deviation (SD). Logarithmic transformation was performed for not normally distributed variables (HOMA index, adiponectin, insulin, glycemia, fibrosis, number of adipocytes nuclei HIF1-α (+), CD68 (+), CD163 (+) and CD11c (+) macrophages). Comparisons between CAD and non-CAD were performed using Student’s 2-tailed unpaired test. Simple Pearson correlations were used to test association between variables.
A significance level of 5% was always adopted. All analyses were performed using SPSS (software version 17.0, SPSS, Chicago, IL, USA) and R program [22] (R project).