Gene expression specificity in relation to the main cellular fractions of the adipose tissue

In human obesity, the stroma vascular fraction (SVF) of white adipose tissue (WAT) is enriched in macrophages. These cells may contribute to low-grade inflammation and to its metabolic complications. Little is known about the effect of weight loss on macrophages and genes involved in macrophage attraction. We examined subcutaneous WAT (scWAT) of 7 lean and 17 morbidly obese subjects before and 3 months after bypass surgery. Immunomorphological changes of the number of scWAT-infiltrating macrophages were evaluated, along with concomitant changes in expression of SVF-overexpressed genes. The number of scWAT-infiltrating macrophages before surgery was higher in obese than in lean subjects (HAM56+/CD68+; 22.6 ± 4.3 vs. 1.4 ± 0.6%, P < 0.001). Typical "crowns" of macrophages were observed around adipocytes. Drastic weight loss resulted in a significant decrease in macrophage number (–11.63 ± 2.3%, P < 0.001), and remaining macrophages stained positive for the anti-inflammatory protein interleukin 10. Genes involved in macrophage attraction (monocyte chemotactic protein [MCP]-1, plasminogen activator urokinase receptor [PLAUR], and colony-stimulating factor [CSF]-3) and hypoxia (hypoxia-inducible factor-1{alpha} [HIF-1{alpha}]), expression of which increases in obesity and decreases after surgery, were predominantly expressed in the SVF. We show that improvement of the inflammatory profile after weight loss is related to a reduced number of macrophages in scWAT. MCP-1, PLAUR, CSF-3, and HIF-1{alpha} may play roles in the attraction of macrophages in scWAT. Keywords: cell type comparison Subcutaneous abdominal white adipose tissue was obtained from nine healthy female subjects (mean body mass index, BMI 28±7 kg/m2) undergoing plastic surgery in agreement with French laws on biomedical research. Mature adipocytes were collected after collagenase (type II, Gibco, Cergy pontoise, France) cell dissociation at 37°C, filtration, decantation, and centrifugation. The floating cellular layer was kept free of any detectable stromavascular element and contained only mature adipocytes filled with triglyceride droplets (tested by light microscopy). SVF cells were recovered at the bottom of the tubes after centrifugation. For measurements of mRNA levels, adipocytes and SVF cells were lysed with denaturing buffer from RNeasy kit (Qiagen, Courtaboeuf, France), then stored at –80°C until RNA preparation. Total RNA 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). We compared total RNA isolated from adipocytes and from SVF cells. The microarray experiments were performed after pooling an equal amount of total RNA from adipocyte and from SVF cell preparations and repeated six times. Amplified RNA from SVF cells was labeled with Cy3 whereas aRNA from adipocytes was labeled with Cy5. The labeled cDNA mixtures were hybridized according to the protocol described at http://cmgm.stanford.edu/pbrown/protocols/index