ABSTRACT
BACKGROUND: Smoking is a strong risk factor for the development of Graves' ophthalmopathy (GO). Immediate early genes (IEGs) are overexpressed in patients with active GO compared to healthy controls. The aim of this study was to study the effects of tobacco smoking and simvastatin on preadipocytes and orbital fibroblasts (OFs) in the adipogenic process. METHODS: Cigarette smoke extract (CSE) was generated by a validated pump system. Mouse 3T3-L1 preadipocytes or OFs were exposed to 10% CSE with or without simvastatin. Gene expression was studied in preadipocytes and OFs exposed to CSE with or without simvastatin and compared to unexposed cells or cells treated with a differentiation cocktail. RESULTS: In 3T3-L1 preadipocytes, Cyr61, Ptgs2, Egr1 and Zfp36 expression levels were two-fold higher in cells exposed to CSE than in unexposed cells. Simvastatin downregulated the expression of these genes (1.6-fold, 5.5-fold, 3.3-fold, 1.4-fold, respectively). CSE alone could not stimulate preadipocytes to differentiate. Scd1, Ppar-γ and adipogenesis were downregulated in simvastatin-treated preadipocytes compared to nontreated preadipocytes 18-, 35- and 1.7-fold, respectively. In OFs, similar effects of CSE were seen on the expression of CYR61 (1.4-fold) and PTGS2 (3-fold). Simvastatin downregulated adipogenesis, PPAR-γ (2-fold) and SCD (27-fold) expression in OFs. CONCLUSION: CSE upregulated early adipogenic genes in both mouse 3T3-L1 preadipocytes and human OFs but did not by itself induce adipogenesis. Simvastatin inhibited the expression of both early and late adipogenic genes and adipogenesis in preadipocytes and human OFs. The effect of simvastatin should be investigated in a clinical trial of patients with GO.
ABSTRACT
CONTEXT: A potentially altered protein expression profile in orbital tissue from patients with thyroid-associated orbitopathy (TAO) is suspected. OBJECTIVE: To detect for the first time changes in proteomic patterns of orbital connective tissue in TAO and compare these with control tissue using mass spectrometry. DESIGN: Proteomics cross-sectional, comparative study. SETTING: Two academic endocrine institutions. SAMPLES: A total of 64 orbital and peripheral adipose tissue samples were collected from 39 patients with TAO and 25 control subjects. METHODS: Samples were analyzed and identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry technology. MAIN OUTCOME MEASURES: Mean intensity values of all identified peptides per protein. RESULTS: Thirty-one proteins were identified, of which 16 differentiated between controls and patients with TAO. Different protein patterns between orbital and peripheral adipose tissue were observed. Compared to controls, 10 proteins were markedly up-regulated (≥ 2-fold) in the orbital tissue of untreated patients: beta IV spectrin (6.2-fold), GTP binding G protein 2 (5.6-fold), POTE ankyrin domain family member F (5.4-fold), xylulokinase (4.1-fold), kinesin family member 1A and lipocalin 1 (both 3.6-fold), semicarbazide-sensitive metalloproteinase amine oxidase 3 and polymerase I transcript release factor (both 3.4-fold), cell-cycle protein elongin A binding protein 1 (3.3-fold), annexin A2 and cavin (both 3-fold), protein pointing to cell proliferation histone H4 (2.8-fold), and ADAM metallopeptidase with thrombospondin type 1 motif 14 (2.7-fold). The highest protein up-regulations were noted in the orbital tissue of medically untreated patients. Steroid therapy markedly reduced up-regulation of these proteins, foremost in nonsmokers. CONCLUSIONS: Proteins involved in tissue inflammation, adipose tissue differentiation, lipid metabolism, and tissue remodeling were up-regulated in orbital tissue of untreated patients with TAO. Steroids decreased the expression of these proteins, whereas smoking attenuated such effect.