Graves' Ophthalmopathy Orbital Fibroblasts Research Articles

EZH2 as a major histone methyltransferase in PDGF-BB-activated orbital fibroblast in the pathogenesis of Graves’ ophthalmopathy

Graves’ ophthalmopathy (GO) is an extra-thyroidal complication of Graves’ disease which can lead to vision loss in severe cases. Currently, treatments of GO are not sufficiently effective, so novel therapeutic strategies are needed. As platelet-derived growth factor (PDGF)-BB induces several effector mechanisms in GO orbital fibroblasts including cytokine production and myofibroblast activation, this study aims to investigate the roles of histone lysine methyltransferases (HKMTs) in PDGF-BB-activated GO orbital fibroblasts by screening with HKMTs inhibitors library. From the total of twelve selective HKMT inhibitors in the library, EZH2, G9a and DOT1L inhibitors, DZNeP, BIX01294 and Pinometostat, respectively, prevented PDGF-BB-induced proliferation and hyaluronan production by GO orbital fibroblasts. However, only EZH2 inhibitor, DZNeP, significantly blocked pro-inflammatory cytokine production. For the HKMTs expression in GO orbital fibroblasts, PDGF-BB significantly and time-dependently induced EZH2, G9a and DOT1L mRNA expression. To confirm the role of EZH2 in PDGF-BB-induced orbital fibroblast activation, EZH2 silencing experiments revealed suppression of PDGF-BB-induced collagen type I and α-SMA expression along with decreasing histone H3 lysine 27 trimethylation (H3K27me3) level. In a more clinically relevant model than orbital fibroblast culture experiments, DZNeP treated GO orbital tissues significantly reduced pro-inflammatory cytokine production while slightly reduced ACTA2 mRNA expression. Our data is the first to demonstrate that among all HKMTs EZH2 dominantly involved in the expression of myofibroblast markers in PDGF-BB-activated orbital fibroblast from GO presumably via H3K27me3. Thus, EZH2 may represent a novel therapeutics target for GO.

Tea Polyphenols Reduce Inflammation of Orbital Fibroblasts in Graves’ Ophthalmopathy via the NF-κB/NLRP3 Pathway

ObjectiveThis study aimed to explore the effects of tea polyphenols (TP) on inflammation of orbital fibroblasts in Graves’ ophthalmopathy (GO) and to provide new ideas for GO treatment.MethodsPrimary orbital fibroblasts were extracted from orbital adipose/connective tissues of patients with and without GO. Real-time quantitative PCR (RT-qPCR) was used to detect the expression of interleukin (IL)-6, IL-1β, and monocyte chemotactic protein (MCP)-1 in non-GO and GO orbital fibroblasts. The CCK-8 assay was used to determine the appropriate concentration of TP for subsequent experiments. RT-qPCR and enzyme-linked immunosorbent assay (ELISA) were performed to investigate the effects of TP on lipopolysaccharide (LPS)-induced production of inflammatory cytokines. Nuclear factor-κB (NF-κB) expression was measured using Western blotting analysis. NOD-like receptor 3 (NLRP3) expression was detected using both Western blotting analysis and immunofluorescence staining.ResultsThe mRNA levels of IL-6, IL-1β, and MCP-1 in GO orbital fibroblasts were significantly higher than those in non-GO cells. TP treatment significantly inhibited LPS-induced production of inflammatory factors, including IL-6, IL-1β, and MCP-1. TP also inhibited the expression levels of NF-κB and NLRP3. Inflammation in the GO orbital fibroblasts was higher than that in non-GO cells. TP inhibited the production of inflammatory cytokines in GO orbital fibroblasts in vitro through the NF-κB/NLRP3 pathway.ConclusionThese findings suggest that TP may have a potential role in GO treatment.

The Role of Oxidative Stress and Therapeutic Potential of Antioxidants in Graves' Ophthalmopathy.

Graves’ ophthalmopathy (GO) is the most common extrathyroidal manifestation of Graves’ disease. It is characterized initially by an inflammatory process, followed by tissue remodeling and fibrosis, leading to proptosis, exposure keratopathy, ocular motility limitation, and compressive optic neuropathy. The pathogenic mechanism is complex and multifactorial. Accumulating evidence suggests the involvement of oxidative stress in the pathogenesis of GO. Cigarette smoking, a major risk factor for GO, has been shown to induce reactive oxygen species (ROS) generation and oxidative damage in GO orbital fibroblasts. In addition, an elevation in ROS and antioxidant enzymes is observed in tears, blood, and urine, as well as orbital fibroadipose tissues and fibroblasts from GO patients. In vitro and in vivo studies have examined the efficacy of various antioxidant supplements for GO. These findings suggest a therapeutic role of antioxidants in GO patients. This review summarizes the current understanding of oxidative stress in the pathogenesis and potential antioxidants for the treatment of GO.

Effect of Pirfenidone on TGF-β1-Induced Myofibroblast Differentiation and Extracellular Matrix Homeostasis of Human Orbital Fibroblasts in Graves' Ophthalmopathy.

Pirfenidone is a pyridinone derivative that has been shown to inhibit fibrosis in animal models and in patients with idiopathic pulmonary fibrosis. Its effect on orbital fibroblasts remains poorly understood. We investigated the in vitro effect of pirfenidone in transforming growth factor-β1 (TGF-β1)-induced myofibroblast transdifferentiation and extracellular matrix (ECM) homeostasis in primary cultured orbital fibroblasts from patients with Graves’ ophthalmopathy (GO). The expression of fibrotic proteins, including α-smooth muscle actin (α-SMA), connective tissue growth factor (CTGF), fibronectin, and collagen type I, was determined by Western blots. The activities of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) responsible for the ECM homeostasis were examined. After pretreating the GO orbital fibroblasts with pirfenidone (250, 500, and 750 μg/mL, respectively) for one hour followed by TGF-β1 for another 24 h, the expression of α-SMA, CTGF, fibronectin, and collagen type I decreased in a dose-dependent manner. Pretreating the GO orbital fibroblasts with pirfenidone not only abolished TGF-β1-induced TIMP-1 expression but recovered the MMP-2/-9 activities. Notably, pirfenidone inhibited TGF-β1-induced phosphorylation of p38 and c-Jun N-terminal kinase (JNK), the critical mediators in the TGF-β1 pathways. These findings suggest that pirfenidone modulates TGF-β1-mediated myofibroblast differentiation and ECM homeostasis by attenuating downstream signaling of TGF-β1.

Simvastatin and ROCK Inhibitor Y-27632 Inhibit Myofibroblast Differentiation of Graves' Ophthalmopathy-Derived Orbital Fibroblasts via RhoA-Mediated ERK and p38 Signaling Pathways.

Transforming growth factor-β (TGF-β)-induced differentiation of orbital fibroblasts into myofibroblasts is an important pathogenesis of Graves’ ophthalmopathy (GO) and leads to orbital tissue fibrosis. In the present study, we explored the antifibrotic effects of simvastatin and ROCK inhibitor Y-27632 in primary cultured GO orbital fibroblasts and tried to explain the molecular mechanisms behind these effects. Both simvastatin and Y-27632 inhibited TGF-β-induced α-smooth muscle actin (α-SMA) expression, which serves as a marker of fibrosis. The inhibitory effect of simvastatin on TGF-β-induced RhoA, ROCK1, and α-SMA expression could be reversed by geranylgeranyl pyrophosphate, an intermediate in the biosynthesis of cholesterol. This suggested that the mechanism of simvastatin-mediated antifibrotic effects may involve RhoA/ROCK signaling. Furthermore, simvastatin and Y-27632 suppressed TGF-β-induced phosphorylation of ERK and p38. The TGF-β-mediated α-SMA expression was suppressed by pharmacological inhibitors of p38 and ERK. These results suggested that simvastatin inhibits TGF-β-induced myofibroblast differentiation via suppression of the RhoA/ROCK/ERK and p38 MAPK signaling pathways. Thus, our study provides evidence that simvastatin and ROCK inhibitors may be potential therapeutic drugs for the prevention and treatment of orbital fibrosis in GO.

Simvastatin Inhibits CYR61 Expression in Orbital Fibroblasts in Graves' Ophthalmopathy through the Regulation of FoxO3a Signaling.

Graves' ophthalmopathy (GO), which is characterized by orbital tissue inflammation, expansion, and fibrosis, is the ocular manifestation in 25% to 50% of patients with Graves' disease. As the pathology of GO is driven by autoimmune inflammation, many proinflammatory cytokines/chemokines, including TNF-α, IL-1β, IL-6, and CCL20, are crucial in the pathogenesis of GO to activate the orbital fibroblasts. Cysteine-rich protein 61 (CYR61), which is known to regulate cell proliferation, adhesion, and migration, plays a proinflammatory role in the pathogenesis of many inflammatory diseases, such as rheumatoid arthritis. CYR61 was considered a potential biomarker of GO in recent studies. Statins, which are cholesterol-lowering drugs, were found to reduce the risk of GO, probably through their anti-inflammatory and immunomodulatory effects. In this study, we established a link between CYR61 and statins in the pathogenesis and potential treatment for GO. Firstly, our data showed the overexpression of CYR61 in the orbital tissue (n = 4) and serum specimens (n = 6) obtained from the patients with inactive GO. CYR61 could induce the production of IL-6 and CCL20 in cultured GO orbital fibroblasts. The expression of CYR61 in cultured GO orbital fibroblasts was upregulated via TNF-α stimulation. Secondly, we pretreated cultured GO orbital fibroblasts using simvastatin, a statin, followed by TNF-α stimulation. The data revealed that simvastatin could inhibit TNF-α-induced CYR61 expression by modulating the activity of transcription factor FoxO3a. Our results provided insights into some cellular mechanisms that may explain the possible protective effects of simvastatin against the development of GO.

MicroRNA-146a downregulates the production of hyaluronic acid and collagen I in Graves' ophthalmopathy orbital fibroblasts.

The present study aimed to investigate the effect of microRNA (miR)-146a on the secretion of hyaluronic acid (HA) and collagen I in Graves' ophthalmopathy (GO) orbital fibroblasts, and identify potential novel targets for the clinical treatment of GO. Orbital fibroblasts were extracted from orbital connective tissue, and primary cells were identified via immunohistochemistry. The levels of HA and collagen I in orbital fibroblasts of non-GO controls and patients with GO were examined via reverse transcription-quantitative PCR (RT-qPCR). miR-146a was overexpressed or inhibited in primary orbital fibroblasts via lentiviral infection, and the levels of HA and collagen I following miR-146a overexpression or inhibition were detected via ELISA and RT-qPCR. The results indicated that the mRNA expression of HA and collagen I was higher in orbital fibroblasts from patients with GO compared with the non-GO cohort. Overexpression of miR-146a reduced, and inhibition of miR-146a increased the production of HA and collagen I in GO orbital fibroblasts. In conclusion, it was demonstrated that miR-146a downregulated the secretion of HA and collagen I in GO orbital fibroblasts in vitro, which may affect glycosaminoglycan aggregation and collagen deposition in GO.

Expression and clinical significance of connective tissue growth factor (CTGF) in Graves' ophthalmopathy

AimsTo examine the expression of connective tissue growth factor (CTGF) in human cultured orbital fibroblasts from patients with Graves' ophthalmopathy (GO) and investigate whether a correlation exists between the presence...

Alteration of Connective Tissue Growth Factor (CTGF) Expression in Orbital Fibroblasts from Patients with Graves' Ophthalmopathy.

Graves’ ophthalmopathy (GO) is a disfiguring and sometimes blinding disease, which is characterized by inflammation and swelling of orbital tissues, with fibrosis and adipogenesis being predominant features. The aim of this study is to investigate whether the expression levels of fibrosis-related genes, especially that of connective tissue growth factor (CTGF), are altered in orbital fibroblasts of patients with GO. The role of oxidative stress in the regulation of CTGF expression in GO orbital fibroblasts is also examined. By a SYBR Green-based real time quantitative PCR (RT-QPCR), we demonstrated that the mRNA expression levels of fibronectin, apolipoprotein J, and CTGF in cultured orbital fibroblasts from patients with GO were significantly higher than those of age-matched normal controls (p = 0.007, 0.037, and 0.002, respectively). In addition, the protein expression levels of fibronectin, apolipoprotein J, and CTGF analyzed by Western blot were also significantly higher in GO orbital fibroblasts (p = 0.046, 0.032, and 0.008, respectively) as compared with the control. Furthermore, after treatment of orbital fibroblasts with a sub-lethal dose of hydrogen peroxide (200 μM H2O2), we found that the H2O2-induced increase of CTGF expression was more pronounced in the GO orbital fibroblasts as compared with those in normal controls (20% vs. 7%, p = 0.007). Importantly, pre-incubation with antioxidants including N-acetylcysteine (NAC) and vitamin C, respectively, resulted in significant attenuation of the induction of CTGF in GO orbital fibroblasts in response to H2O2 (p = 0.004 and 0.015, respectively). Taken together, we suggest that oxidative stress plays a role in the alteration of the expression of CTGF in GO orbital fibroblasts that may contribute to the pathogenesis and progression of GO. Antioxidants may be used in combination with the therapeutic agents for effective treatment of GO.

Forkhead Transcription Factor FOXO1 Is Regulated by Both a Stimulatory Thyrotropin Receptor Antibody and Insulin-Like Growth Factor-1 in Orbital Fibroblasts from Patients with Graves' Ophthalmopathy.

Activation of thyrotropin receptor (TSHR) and/or insulin-like growth factor (IGF-1) receptor (IGF-1R) enhances HA production and adipogenesis in orbital fibroblasts from patients with Graves' ophthalmopathy (GO) and recapitulates the tissue remodeling characteristic of the orbit in GO. A functional relationship between TSHR and IGF-1R has long been postulated, and recently bidirectional crosstalk between the receptors in GO fibroblasts was demonstrated. Because the transcription factor Forkhead box O-1 (FOXO1) was recently shown to be a critical downstream mediator of TSH and IGF-1 effects on thyrocyte proliferation, studies were designed to determine whether FOXO1 might similarly act as a common mediator of M22, a stimulatory TSHR antibody (TSAb), and IGF-1 in GO orbital fibroblasts. FOXO1 mRNA and protein were measured in orbital tissue specimens derived from normal individuals and patients with GO. In addition, the control of FOXO1 cellular localization was investigated using quantitative Western blotting of fractionated cell lysates from orbital fibroblasts treated with M22 and/or IGF-1 with or without specific TSHR, IGF-1R, or PI3K/AKT1/2 inhibitors. Significantly lower levels of both FOXO1 mRNA and protein were found in GO orbital tissue specimens compared with normal orbital tissues (M = 39%, p = 0.043; M = 46.4%; p = 0.028, respectively). In addition, treatment of GO orbital cultures with M22, IGF-1, or M22 plus IGF-1 increased cytoplasmic FOXO1 compared with control (1.63-fold, p = 0.008; 1.68-fold, p = 0.001; 1.61-fold, p ≤ 0.001, respectively) and decreased nuclear FOXO1 (M = 28%, p = 0.002; M = 38%, p ≤ 0.001; M = 35%, p = 0.007, respectively). These effects were inhibited by co-treatment with the respective, but not the opposite, receptor antagonist. AKT inhibition of M22 or IGF-1-treated cultures was found to increase nuclear (1.4-fold, p = 0.026; 1.3-fold, p = 0.001, respectively) and decrease cytoplasmic (24.2%, p = 0.001; 36%, p = 0.004, respectively) FOXO1 localization. These data point to FOXO1 as an important mediator of TSAb and IGF-1 action via their cognate receptors in GO orbital fibroblasts. These findings provide a link between the low FOXO1 protein levels demonstrated in GO orbital tissue and the tissue remodeling characteristic of GO, and suggest novel therapy for GO aimed at increasing nuclear expression of FOXO1 in GO target cells.

A Small Molecule Antagonist Inhibits Thyrotropin Receptor Antibody-Induced Orbital Fibroblast Functions Involved in the Pathogenesis of Graves Ophthalmopathy

Graves ophthalmopathy (GO) is an autoimmune disorder characterized by increased adipogenesis and hyaluronan (HA) production by orbital fibroblasts. Circulating autoantibodies (thyroid-stimulating antibodies [TSAbs]) directed at the thyrotropin receptor (TSHR) on these cells stimulate or augment these cellular processes. A recently developed drug-like small molecule inverse agonist of TSHR, NCGC00229600, termed 1, binds to TSHR and blocks basal and stimulated signal transduction. The purpose of this article was to determine whether 1 might inhibit HA production and relevant signaling pathways in orbital fibroblasts cultured in the presence of monoclonal TSAbs or bovine TSH (bTSH). Primary cultures of undifferentiated GO orbital fibroblasts (n = 13) were untreated or treated with a TSAb (M22 or MS-1) or bTSH in serum-free medium, with or without 1 or a TSHR neutral antagonist, NCGC00242595, termed 2, which does not inhibit basal signaling but does inhibit stimulated signaling. cAMP production, Akt phosphorylation (Ser473pAkt in media and immunoblotting for pAkt/total Akt), and HA production were analyzed. Compound 1 inhibited basal cAMP, pAkt, and HA production and that stimulated by M22 in undifferentiated orbital fibroblasts. Inhibition of HA production was dose-dependent, with a half-maximal inhibitory dose of 830 nM. This compound also inhibited MS-1- and bTSH-stimulated cAMP, pAkt, and HA production. Compound 2 did not inhibit basal HA production but did inhibit M22-stimulated HA production. Because cAMP, pAkt, and HA production are fibroblast functions that are activated via TSHR signaling and are important in the pathogenesis of GO, small molecule TSHR antagonists may prove to be effective in the treatment or prevention of the disease in the future.

A Stimulatory Thyrotropin Receptor Antibody Enhances Hyaluronic Acid Synthesis in Graves' Orbital Fibroblasts: Inhibition by an IGF-I Receptor Blocking Antibody

Graves' ophthalmopathy (GO) is characterized by expanded volume of the orbital fat and extraocular muscle tissues and elevated levels of TSH receptor autoantibodies (TRAb). The expansion of orbital tissues involves accumulation of hyaluronic acid (HA) within the orbit. The objective of the study was to determine whether a monoclonal stimulatory TRAb (M22) impacts HA synthesis in GO orbital cells and, if so, whether this might be blocked by an IGF-I receptor (IGF-IR)-blocking antibody (1H7) or inhibitors of various downstream signaling cascades. GO orbital fibroblast cultures (n = 6) were treated with M22, bovine TSH (bTSH), or IGF-I in serum-free medium. Some cultures also received 1H7, LY294002, rapamycin, or protein kinase A inhibitor. HA production and phosphorylated Akt levels in media or immunoblotting for phosphorylated Akt were measured. M22 or bTSH stimulated HA synthesis (2.1-fold with 100 ng/ml M22 and 1.9-fold with 10 U/liter bTSH; P < 0.05 each). M22-induced HA synthesis was inhibited by LY294002 or rapamycin but not by protein kinase inhibitor. HA synthesis stimulated by M22 or IGF-I was inhibited by 1H7 (mean 36.6 ± 5.6% and mean 45.8 ± 7.6%, respectively; P < 0.05 each). Similarly, M22- or IGF-I-stimulated Akt phosphorylation was inhibited by 1H7 (mean 54 ± 9.6 and 36.1 ± 8.8%, respectively; P = 0.01 each). The stimulatory TRAb M22 increases HA production in undifferentiated GO orbital fibroblasts via phosphoinositide 3-kinase/phosphorylated AKT/mammalian target of rapamycin activation. Blockade of IGF-IR inhibits both HA synthesis and Akt phosphorylation induced by M22 or IGF-I in these cells, suggesting that TSH receptor and IGF-IR signaling may be closely linked in the GO orbit.

Effects of Thyrotropin and Thyrotropin-Receptor-Stimulating Graves' Disease Immunoglobulin G on Cyclic Adenosine Monophosphate and Hyaluronan Production in Nondifferentiated Orbital Fibroblasts of Graves' Ophthalmopathy Patients

Orbital fibroblasts are involved in the pathogenesis of Graves' ophthalmopathy (GO) by producing hyaluronan (HA), synthesized by three types of hyaluronan synthases (HAS1, HAS2, and HAS3). Thyrotropin receptors (TSHR) expressed in orbital fibroblasts activate the cyclic adenosine monophosphate (cAMP) pathway. Only sparse data are available at present supporting a role for TSHR activation in the regulation of HA in GO orbital fibroblasts. We hypothesize that TSHR activation, via cAMP signaling, results in induction of HAS1-3 mRNA expression and HA production by nondifferentiated GO orbital fibroblasts. Cultured nondifferentiated orbital fibroblasts obtained during orbital decompression surgery from 15 GO patients were stimulated with recombinant human TSH (rhTSH), TSHR-stimulating Graves' disease immunoglobulin G (GD-IgG) or forskolin (FSK), or interleukin-1beta (IL-1beta). FSK significantly stimulated cAMP production, HAS1 and HAS3 mRNA expression, and HA secretion in orbital fibroblasts. IL-1beta slightly induced cAMP production, but induced HAS mRNA expression of all three isoforms and HA secretion. In contrast, the effects of rhTSH and GD-IgG on cAMP were modest and absent, respectively, and on HAS mRNA and HA synthesis were completely absent. The strong increase in cAMP synthesis by FSK in nondifferentiated GO orbital fibroblasts results in increased HA synthesis, but TSHR activation by rhTSH or GD-IgG does not result in altered HA synthesis. Our results do not support a predominant role for GD-IgGs in the accumulation of orbital glycosaminoglycans; cytokines like IL-1beta seem largely responsible for excessive glycosaminoglycan production by nondifferentiated orbital fibroblasts in early immunopathogenesis of GO.

Increased oxidative DNA damage, lipid peroxidation, and reactive oxygen species in cultured orbital fibroblasts from patients with Graves’ ophthalmopathy: evidence that oxidative stress has a role in this disorder

We investigated the oxidative stress in orbital fibroadipose tissues and cultured orbital fibroblasts from patients with Graves' ophthalmopathy (GO). The content of 8-hydroxy 2'-deoxyguanosine (8-OHdG), an important biomarker of oxidative DNA damage, was measured in orbital fibroadipose tissues and cultured orbital fibroblasts from patients with GO and compared with age-matched normal controls. A product of lipid peroxidation, malondialdehyde (MDA), and intracellular reactive oxygen species (ROS) in cultured orbital fibroblasts was also determined. There was no significant difference in the 8-OHdG content of orbital fibroadipose tissues between patients with GO and age-matched normal controls (P=0.074). However, the levels of 8-OHdG and MDA in GO orbital fibroblasts were significantly higher than those of normal controls (P=0.0026 and P<0.001, respectively). In addition, GO orbital fibroblasts had higher contents of superoxide anions and hydrogen peroxide compared with those of normal controls (P=0.0133 and 0.0025, respectively). Orbital fibroblasts represent the most abundant cell type among orbital connective tissues and exhibit great differences in their phenotypes. Increased oxidative DNA damage and lipid peroxidation, as well as higher intracellular ROS levels in GO orbital fibroblasts may have a role in the pathogenesis of GO.

Oxidative stress and Graves' ophthalmopathy: in vitro studies and therapeutic implications.

Graves' ophthalmopathy (GO) is a disorder of autoimmune origin caused by a complex interplay of endogenous and environmental factors. After recognition of one or more antigens shared by thyroid and orbit, activated T lymphocytes infiltrating the orbit trigger a cascade of events leading to production of cytokines, growth factors and oxygen reactive species. Proliferation of adipocytes and fibroblasts then follows, with an increased synthesis of glycosaminoglycans (GAG), which attract water and cause edema of orbital structures and venous congestion. Proliferation of orbital fibroblasts and adipocytes, both in the retroocular tissue and in the perimysium of extraocular muscles, are among the most important events leading to the increased volume of orbital structures (fibroadipose tissue and extraocular muscles). The contribution of oxygen reactive species to the changes occurring in the orbit is underscored by in vitro studies. Superoxide radical stimulates orbital fibroblasts to proliferate and to produce GAG. Furthermore, hydrogen peroxide induces expression of HLA-DR and heat shock protein-72, involved in antigen recognition and T-lymphocyte recruitment. Cigarette smoking, which is probably the most important environmental factor associated with GO occurrence and maintenance, might also act, among other mechanisms, by enhancing generation of oxygen reactive species and reducing antioxidant production. Substances such as nicotinamide, allopurinol and pentoxifylline reduce superoxide- or hydrogen peroxide-induced proliferation of fibroblasts, GAG production and HLA-DR or HSP-72 expression by GO orbital fibroblasts, possibly through scavenging oxygen free radicals. Two small, non-randomized and/or uncontrolled studies investigated the effects of nicotinamide, allopurinol or pentoxifylline on GO. Favorable results were reported, but data are not fully convincing and the true effectiveness of these agents needs to be verified in randomized, controlled trials enrolling a larger number of patients. It currently seems unlikely that they may find a relevant place in the limited armamentarium available for the management of severe GO.

Stimulation of adipogenesis, peroxisome proliferator-activated receptor-gamma (PPARgamma), and thyrotropin receptor by PPARgamma agonist in human orbital preadipocyte fibroblasts.

The symptoms and signs of Graves' ophthalmopathy (GO) result from both an accumulation of hydrated hyaluronan in the orbital muscles and connective tissues and an expansion of the orbital adipose tissues. Recent studies have suggested a link between the stimulation of adipogenesis within the orbit in GO and the expression in these tissues of TSH receptor (TSHR), the putative orbital autoantigen. To further investigate this association, we treated orbital fibroblasts from patients with GO with rosiglitazone, a thiazolidinedione agonist of the PPARgamma receptor that stimulates adipocyte differentiation. We found this compound to be a potent stimulator of functional TSHR expression as well as TSHR and PPARgamma mRNA levels in differentiated cultures. In addition, rosiglitazone treatment stimulated recruitment and differentiation of a subset of cells within these cultures into mature lipid-laden adipocytes. These results suggest that TSHR expression in GO orbital preadipocyte fibroblasts is linked to adipogenesis, and that ligation of the PPARgamma receptor results in differentiation of these cells. It is possible that endogenous PPARgamma ligands play a role in stimulating orbital adipogenesis in GO, and that future treatments may be aimed at antagonism of various components of the PPARgamma signaling system.

Differentiation of human orbital preadipocyte fibroblasts induces expression of functional thyrotropin receptor.

Although the autoantigen involved in Graves' hyperthyroidism is known to be the TSH receptor (TSHr), whether this antigen plays a primary role in the pathogenesis of Graves' ophthalmopathy (GO) is unclear. We sought to determine whether fibroblasts derived from orbital adipose/connective tissue are capable of differentiating into adipocytes that bear immunoreactive and functional TSHr. In addition, we assessed relative levels of TSHr gene expression in normal and GO orbital adipose/connective tissue specimens. GO and normal orbital preadipocyte fibroblasts, cultured under conditions known to stimulate adipocyte differentiation, showed evidence of adipogenesis and positive immunostaining for TSHr protein. In addition, significantly more cAMP was produced in response to TSH stimulation in the differentiated cultures than in undifferentiated cultures derived from the same individuals' cells. Other studies demonstrated relatively greater TSHr gene expression in GO than in normal orbital tissue specimens. These results indicate that orbital preadipocyte fibroblasts increase their TSHr expression with differentiation and suggest that these cells play an important role in the pathogenesis of GO. Furthermore, our studies support the concept that TSHr may be an important target antigen in this condition. Factors that stimulate adipocyte differentiation and TSHr expression in the orbit in GO have yet to be defined.