Expression profiles of epicardial and subcutaneous adipose tissue. Homo sapiens

The epicardial adipose tissue (EAT) is a visceral adipose tissue in close contact with coronary vessels whose increase is associated with coronary artery disease (CAD). Our goal was to identify candidate molecule(s) characterizing EAT which could intervene in the pathogenesis of CAD. An approach combining microarrays and bioinformatic sequence analysis tools for predicting secreted proteins (TargetP) was applied to paired biopsies of subcutaneous adipose tissue (SAT) and EAT, obtained from patients with or without CAD (NCAD). Results were validated in 3 independent groups of subjects by RT-qPCR, western blot, immuno histochemistry and explants secretion. sPLA2-IIA ranked first among genes coding for potentially secreted proteins with the highest overexpression in EAT in both CAD and NCAD. RT-qPCR confirmed its increased expression in EAT ( p 50%. Group 3 was composed of 15 non diabetic patients with CAD. EAT and SAT samples were obtained within 20 min after the start of surgery respectively near the proximal tract of the right coronary artery and the site of chest incision. Samples were frozen in liquid nitrogen and stored at -80 °C. In groups 1 and 2, total RNA obtained from SAT and EAT samples of 7 subjects (4 CAD and 3 NCAD) was prepared using the RNeasy total RNA Mini kit (Qiagen). RNA concentration and integrity were assessed using the Agilent 2100 Bioanalyzer (Agilent Technologies, Massy, France). For microarray experiments, 1 µg of total RNA from each total RNA sample preparation was amplified by MessageAmp RNA Kit (Ambion, Austin, TX, USA) and 3 µg of amplified RNA was labeled with cyanin dyes (Cy) using the CyScribe First-Strand cDNA labeling kit (Amersham Biosciences, Orsay, France). (available at http://cmgm.stanford.edu/pbrown/protocols/index). Using pangenomic Agilent® cDNA microarrays, we compared the expression profiles of EAT and SAT from CAD and NCAD subjects. To this purpose we used a common reference pool that was generated by mixing equal amounts of total RNA extracted from adipose tissue samples of patients undergoing plastic surgery. Amplified RNA (aRNA) from the reference pool was labeled with Cy3, and aRNA from the 7 subjects (either EAT or SAT) was labeled with Cy5. A total of 13 pangenomic microarray hybridizations were performed for this differential comparison. After scanning the 13 arrays, the images were analyzed and data were normalized using the Stanford Microarray Database Online resources (http://genomewww5.stanford.edu/MicroArray/SMD/).