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| Status |
Public on Sep 24, 2007 |
| Title |
Expression profile of human preadipocytes cultured with activated macrophages medium |
| Organism |
Homo sapiens |
| Experiment type |
Expression profiling by array
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| Summary |
Extracellular matrix (ECM) remodelling occurs during tissue repair and inflammation-related pathologies with deposition of specific proteins. White adipose tissue (WAT) was recently shown to be the site of substantial interstitial fibrosis. ECM components, such as fibronectin, and their receptors integrins control cell migration, proliferation and differentiation. Adipocyte differentiation which is under the control of a specific transcriptional network is associated with decrease of fibronectin-rich matrix. Considering macrophage accumulation in white adipose tissues from obese subjects, we recently demonstrated that macrophage-secreted factors provoke an inhibition of differentiation with a proinflammatory state of human preadipocytes. The functional analysis of gene expression measurements obtained from cDNA microarray experiments reveals that themes related to the “extracellular matrix” are among the most significantly enriched in overexpressed genes in inflammatory preadipocytes. ECM remodelling, characterized by high deposition of fibronectin, collagen I and tenascin-C and important clustering of ECM receptors integrin alpha-5, is associated with increased proliferation and migration of preadipocytes. siRNA deletion of NF-kappaB in inflammatory preadipocytes decreased fibronectin network formation and their proliferation. Cyclin D1 and FAK constitute pivotal molecular targets in the synergistic promotion of migration and proliferation of the inflammatory preadipocytes. Importantly, interactions between preadipocytes and macrophages are favoured in 3D collagen I, a microenvironment mimicking fibrosis context of obese WATs.
These data suggest that inflammatory preadipocytes could contribute to the production of fibrosis components and, in the context of WAT repair, these altered properties of preadipocytes could further lead to reconstitution of new fat clusters.
Keywords: cell type comparison
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| Overall design |
Isolation of human preadipocytes:
Subcutaneous adipose tissues biopsies were obtained from non obese (BMI < 30 kg/m2) young female patients undergoing elective surgery. None of the patients had diabetes or metabolic disorders and taking medications. This study was approved by the Ethical Committees of Hôtel-Dieu (Paris, France). Minced adipose tissue was digested by collagenase treatment. The digested material was filtered and centrifuged. The resulting pellet (stroma-vascular fraction, SVF) was resuspended in erythrocyte lysis buffer (154 mM NH4Cl, 5.7 mM K2HPO4 and 0.1 mM EDTA, pH 7.0) at 250g for 10 min. After washing in PBS, the SVF cells were suspended in DMEM-10% FBS and used for cell culture at passage 2 in order to eliminate non preadipocyte cell contamination as confirmed by negative staining for macrophage markers (Ham 56 and Mac-1).
Preparation of human blood monocyte-derived macrophages and conditioned medium:
Blood from overweight (BMI > 25 kg/m2) female patients was immediately processed for plasma blood mononuclear cells (PBMC) isolation. Blood sample was layered on PBMC isolation medium (GE healthcare, little Chaford, UK). PBMCs were suspended in PBS/2% FBS/1mmol/L EDTA were incubated at room temperature for 15 min with CD14-positive selection cocktail, followed by 10 min incubation period with magnetic nanoparticles (Stemcell Technologies, Grenoble, France). The bead-coupled CD14+ cells were maintained for 24h in 2 ml of RPMI-10 % FBS at a cell density of 4x105 cells. After 8 days of differentiation, the cells were incubated in RPMI-1 % FBS for 24 h with 100 ng/ml lipopolysaccharides (LPS from E. Coli 0127:B8, Sigma St Louis, Mi, USA) prior collecting the medium (Ac CM). Control medium was RPMI-1 % FBS kept at 37°C for 24h in the absence of macrophages. The conditioned media of the macrophages obtained from 3-5 individuals were pooled and stored at –80°C until used. Distinct pools were used for each culture experiment.
Preparation of Adipose Tissue Macrophages (ATM) and conditioned medium:
Isolated human SVF cells were obtained from subcutaneous adipose tissue biopsies, as described above. SVF cells were suspended in PBS/2% FBS/1mmol/L EDTA were incubated at room temperature for 15 min with CD34-positive selection cocktail, followed by 10 min incubation period with magnetic nanoparticles (Stemcell Technologies, Grenoble, France). The CD34-negative cell fraction was incubated with CD14 positive selection cocktail. The bead-coupled CD14+ cells were maintained for 24h in 1 ml of RPMI-1 % FBS at cell density of 4x105 cells in 12-well plates to obtain macrophage conditioned media (ATM CM), which was stored at - 80°C before use.
Differentiation of human preadipocytes:
Preadipocytes were cultured for 24h in DMEM-10 % FBS at a cell density of 105 cells per well in 12-well plates. The preadipocytes were then incubated with control RPMI or Ac CM (corresponding to 1x105 activated macrophages) and DMEM/F12 induction medium (final concentration of 50 nM insulin, 100 nM dexamethasone, 0.25 mM IBMX and 100 nM rosiglitazone) for 4 days, as described in nous. Next, this medium was replaced by control RPMI or Ac CM and DMEM/F12 culture medium (final concentration of 50nM insulin and 100 nM rosiglitazone). The medium was changed every 2 days until 10-12 days. For ATM CM, the experimental conditions were as described above except that the proportion of ATM CM corresponds to 2x105 AT macrophages.
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| Contributor(s) |
Lacasa D, Clement K, Rouault C, Henegar C |
| Citation(s) |
18208606 |
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| Submission date |
Sep 12, 2007 |
| Last update date |
Mar 19, 2012 |
| Contact name |
Corneliu Henegar |
| E-mail(s) |
corneliu@henegar.info, flavien.jacques@upmc.fr
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| Organization name |
Institut national de la santé et de la recherche médicale
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| Department |
U872 Eq7
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| Lab |
Eq7 Nutriomique
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| Street address |
15 Rue de l'Ecole de Medecine
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| City |
Paris |
| State/province |
Ile de France |
| ZIP/Postal code |
75005 |
| Country |
France |
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| Platforms (2) |
| GPL5834 |
University Health Network Human 19K array version 8 (19Kv8) |
| GPL5835 |
SHGI |
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| Samples (8)
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| This SubSeries is part of SuperSeries: |
| GSE9157 |
Expression profiling of human adipose tissue in obese and lean subjects and in various clinical conditions |
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| Relations |
| BioProject |
PRJNA105305 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE9017_RAW.tar |
25.4 Mb |
(http)(custom) |
TAR (of GPR) |
| Processed data included within Sample table |
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