Downregulation of Caveolin-1 and Upregulation of Deiodinase 3, Associated with Hypoxia-Inducible Factor-1α Increase, Are Involved in the Oxidative Stress of Graves' Orbital Adipocytes.

Background: Even though the clinical features of Graves' orbitopathy (GO) are well known, its exact pathogenesis remains controversial. The imbalance of redox homeostasis in the connective tissue could play a crucial role leading to an inflammatory state and edema of soft orbital tissues, thus contributing to orbital hypoxia and increase in hypoxia-inducible factor (HIF)-1α. This oxidative stress appears to target the orbital cells such as fibroblasts and also adipocytes. This study aims to explore which pathways can lead to the aforementioned oxidative stress in GO adipose cells and therefore offers new plausible therapeutic targets. Methods: Orbital fat samples were obtained from patients with GO (Western blot [WB]: n = 8, immunohistochemistry [IHC]: n = 8) and from control patients (WB: n = 5, IHC: n = 3-5). They were processed for WB analysis and IHC of the antioxidants (catalase, superoxide dismutase 1) and for HIF-1α. The expression of caveolin-1 (Cav-1) and deiodinase 3 (DIO3), known to be regulated by HIF-1α, was also analyzed by WB and IHC, as well as the targets of Cav-1: glucose transporter type 4 (Glut-4), NADPH oxidase (NOX)-2, and endothelial nitric oxide synthase (eNOS). Triiodothyronine (T3) expression was also analyzed by IHC. Results: In GO adipocytes, the expression of catalase was reduced, whereas that of HIF-1α was strongly increased. A decreased local T3 supply was associated with DIO3 upregulation. The low expression of Cav-1 in GO adipocytes was associated not only with low expression of Glut-4 but also with an increased expression of NOX-2 and active eNOS phosphorylated on serine 1177. Conclusions: Cav-1 and DIO3, both sensitive to hypoxia and to the increase of HIF-1α, play a pivotal role in the oxidative stress in GO adipocytes. DIO3 regulates the cellular supply of T3, which is essential for the cell homeostasis. Cav-1 determines the cellular glucose supply through Glut-4 and regulates the activity of NOX-2 generating superoxide anions and that of eNOS generating nitric oxide (NO).

Protein tyrosine phosphatase 1B as a therapeutic target for Graves' orbitopathy in an in vitro model.

Graves' orbitopathy (GO) is characterised in early stages by orbital fibroblast inflammation, which can be aggravated by oxidative stress and often leads to fibrosis. Protein tyrosine protein 1B (PTP1B) is a regulator of inflammation and a therapeutic target in diabetes. We investigated the role of PTP1B in the GO mechanism using orbital fibroblasts from GO and healthy non-GO subjects. After 24 hours of transfection with PTPN1 siRNA, the fibroblasts were exposed to interleukin (IL)-1ß, cigarette smoke extract (CSE), H2O2, and transforming growth factor (TGF)-ß stimulations. Inflammatory cytokines and fibrosis-related proteins were analysed using western blotting and/or enzyme-linked immunosorbent assay (ELISA). Reactive oxygen species (ROS) release was detected using an oxidant-sensitive fluorescent probe. IL-1ß, tumor necrosis factor (TNF)-α, bovine thyroid stimulating hormone (bTSH), high-affinity human stimulatory monoclonal antibody of TSH receptor (M22), and insulin-like growth factor-1 (IGF-1) significantly increased PTP1B protein production in GO and non-GO fibroblasts. PTPN1 silencing significantly blocked IL-1ß-induced inflammatory cytokine production, CSE- and H2O2-induced ROS synthesis, and TGF-ß-induced expression of collagen Iα, α-smooth muscle actin (SMA), and fibronectin in GO fibroblasts. Silencing PTPN1 also decreased phosphorylation levels of Akt, p38, and c-Jun N-terminal kinase (JNK) and endoplasmic reticulum (ER)-stress response proteins in GO cells. PTP1B may be a potential therapeutic target of anti-inflammatory, anti-oxidant and anti-fibrotic treatment of GO.

CD40 Enhances Sphingolipids in Orbital Fibroblasts: Potential Role of Sphingosine-1-Phosphate in Inflammatory T-Cell Migration in Graves' Orbitopathy.

Purpose: Graves' orbitopathy (GO) is an autoimmune orbital disorder associated with Graves' disease caused by thyrotropin receptor autoantibodies. Orbital fibroblasts (OFs) and CD40 play a key role in disease pathogenesis. The bioactive lipid sphingosine-1-phosphate (S1P) has been implicated in promoting adipogenesis, fibrosis, and inflammation in OFs. We investigated the role of CD40 signaling in inducing S1P activity in orbital inflammation. Methods: OFs and T cells were derived from GO patients and healthy control (Ctl) persons. S1P abundance in orbital tissues was evaluated by immunofluorescence. OFs were stimulated with CD40 ligand and S1P levels were determined by ELISA. Further, activities of acid sphingomyelinase (ASM), acid ceramidase, and sphingosine kinase were measured by ultraperformance liquid chromatography. Sphingosine and ceramide contents were analyzed by mass spectrometry. Finally, the role for S1P in T-cell attraction was investigated by T-cell migration assays. Results: GO orbital tissue showed elevated amounts of S1P as compared to control samples. Stimulation of CD40 induced S1P expression in GO-derived OFs, while Ctl-OFs remained unaffected. A significant increase of ASM and sphingosine kinase activities, as well as lipid formation, was observed in GO-derived OFs. Migration assay of T cells in the presence of SphK inhibitor revealed that S1P released by GO-OFs attracted T cells for migration. Conclusions: The results demonstrated that CD40 ligand stimulates GO fibroblast to produce S1P, which is a driving force for T-cell migration. The results support the use of S1P receptor signaling modulators in GO management.

Poly (ADP-ribose) polymerase-1 (PARP-1) in Chinese patients with Graves' disease and Graves' ophthalmopathy.

We aimed to evaluate the genetic variation of poly (ADP-ribose) polymerase-1 (PARP-1) as risk factor in development of Graves' disease (GD) and Graves' ophthalmopathy (GO) among Chinese individuals. Patients with confirmed diagnosis of GD or healthy individuals with no clinical symptoms of hyperthyroiditis were enrolled at the Department of Ophthalmology, Tianjin First Center Hospital, China. Genetic polymorphism was studied in plasma DNA samples of subjects by polymerase chain reaction of restriction fragment length polymorphism to confirm our hypothesis. Cytokine levels were measured routinely on serum samples of subjects by sandwich ELISA technique. Patients with GG genotype (odds ratio (OR) 95% CI = 2.25 (1.35-3.73), p = 0.002) and carriers of G allele (OR = 2.03 (1.23-3.36), p = 0.006) were at high risk of developing ophthalmopathy. Polymorphism of del/ins of nuclear factor-κB1 gene (NFkB1) gene (OR = 7.1 (2.88-17.52), p < 0.0001) and PARP-1 C410T polymorphism was found to be associated with GO (p < 0.05). Cytokine level was significantly higher in patients with GD (p < 0.05), but no significant change in cytokines level among GO patients from baseline (p > 0.05). Our study results recommended that polymorphism of PARP-1 gene is more likely responsible for development of GD in Chinese individuals. We also observed that the polymorphism of gene-related del/ins to NFkB1 in development of GO.

Hyperthyroidism in Patients with Graves' Ophthalmopathy, and Thyroidal, Skeletal and Eye Muscle Specific Type 2 Deiodinase Enzyme Activities.

Graves' ophthalmopathy is characterized by hyperthyroidism, which is associated with higher serum T3 levels than T4 due to deiodinase enzymes.The effect of Graves' patient's sera (n=52) with elevated thyroid hormone and TSH receptor or thyroid peroxidase antibody (anti-TPO) levels was investigated on thyroidal, skeletal and eye muscle type 2 deiodinase enzyme (DII) activities. DII activities were measured with 125I-T4 substrate, while thyroid hormone and antibody levels with immunoassays.In Graves' ophthalmopathy, sera with elevated FT4 or FT3 levels reduced DII activites remarkably in all tissue fractions. Thyroidal DII activities were lower than those using eye muscle fraction (0.6±0.22 vs 1.14±0.43 pmol/mg/min, P<0.006). Effect of sera with increased FT3 levels demonstrated also reduced DII activities in patients with Graves' ophthalmopathy after methimazole therapy compared to those who had no ophthalmopathy (2.88±2 vs 20.42±11.82 pmol/mg/min, P<0.006 for thyroidal fraction, 4.07±2.72 vs 29.22±15.46 pmol/mg/min, P<0.004 for skeletal muscle, 5.3±3.47 vs 37.87±18.82 pmol/mg/min, P<0.003 for eye muscle). Hyperthyroid sera with TSH receptor antibodies resulted in increased DII activities, while sera with anti-TPO antibodies were connected to lower DII activities in Graves' ophthalmopathy.In summary, the actions of hyperthyroid sera derived from patients with Graves' disease were tested on tissue-specific DII activities. Elevated FT4 level-induced DII inactivation is present in Graves' ophthalmopathy, which seems to be also present at the beginning of methimazole therapy. Stimulating TSH receptor antibiodies increased DII activities via their nongenomic effects using sera of hyperthyroid Graves' ophthalmopathy, but anti-TPO antibodies could influence DII activities via altering FT4 levels.

Divergent Sp1 protein levels may underlie differential expression of UDP-glucose dehydrogenase by fibroblasts: role in susceptibility to orbital Graves disease.

UDP-glucose dehydrogenase (UGDH) catalyzes the formation of UDP-glucuronate. Glucuronate represents an integral component of the glycosaminoglycan, hyaluronan, which accumulates in orbital Graves disease. Here we report that orbital fibroblasts express higher levels of UGDH than do those from skin. This is a consequence of greater UGDH gene promoter activity and more abundant steady-state UGDH mRNA. Six Sp1 sites located in the proximal 550 bp of the UGDH gene promoter appear to determine basal promoter activity, as does a previously unrecognized 49-bp sequence spanning -1436 nucleotides (nt) and -1388 nt that negatively affects activity. Nuclear Sp1 protein is more abundant in orbital fibroblasts, and its binding to specific sites on DNA is greater than that in dermal fibroblasts. Mutating each of these Sp1 sites in a UGDH gene promoter fragment, extending from -1387 to +71 nt and fused to a luciferase reporter, results in divergent activities when transfected in orbital and dermal fibroblasts. Reducing Sp1 attenuated UGDH gene promoter activity, lowered steady-state UGDH mRNA levels, and reduced UGDH enzyme activity. Targeting Sp1 and UGDH with specific siRNAs also lowered hyaluronan synthase-1 (HAS-1) and HAS-2 levels and reduced hyaluronan accumulation in orbital fibroblasts. These findings suggest that orbital fibroblasts express high levels of UGDH in an anatomic-specific manner, apparently the result of greater constitutive Sp1. These high UGDH levels may underlie susceptibility of the orbit to localized overproduction of hyaluronan in Graves disease.

Topical cyclosporine in thyroid orbitopathy-related dry eye: clinical findings, conjunctival epithelial apoptosis, and MMP-9 expression.

OBJECTIVES: To evaluate the effects of topical cyclosporine A (CsA) 0.05% (Restasis) on the signs and symptoms of dry eye, on apoptosis, and on MMP-9 expression in conjunctiva epithelial cells in thyroid orbitopathy (TO)-related dry eye patients. METHODS: Prospective, clinical study. Twenty-four eyes of 12 consecutive TO patients with dry eye findings instilled CsA twice daily for 2 months. Ocular surface disease index, Schirmer tear test, tear breakup time (TBUT), conjunctival apoptosis index, and conjunctival MMP-9 expression were evaluated before and after 2 months treatment. Conjunctival biopsies were harvested from all eyes at baseline and after 2 months treatment. Apoptosis was detected by the terminal deoxynucleotidyl transferase-mediated dUTP-nick end labeling (TUNEL) assay. MMP-9 expression was determined by immunohistochemistry. RESULTS: After 2 months of topical CsA treatment, the mean OSDI score was significantly decreased from 58.08 +/- 6.28 to 36.41 +/- 11.75 (P = 0.001). At baseline, the mean Schirmer tear test score was 8.92 +/- 5.52 mm. It was increased to 11.25 +/- 4.71 mm after treatment (P > 0.05). The mean TBUT increased significantly from 3.92 +/- 2.18 sec to 9.16 +/- 3.34 sec (P = 0.001). The mean percentage of apoptosis index at baseline was 72.10 +/- 35.82%. This was significantly decreased to 53.29 +/- 34.46% after treatment (P = 0.008). The mean percentage of MMP-9 expression of the conjunctival epithelial cells was significantly decreased from 48.12 +/- 28.58% to 26.66 +/- 25.13% following treatment (P = 0.005). CONCLUSIONS: Topical CsA treatment appears to improve the signs and symptoms of dry eye and inhibits apoptosis and MMP-9 expression in conjunctival epithelial cells in TO-related dry eye patients after 2 months of treatment.

Oxidative stress profile in graves' ophthalmopathy in Indian patients.

PURPOSE: To report the oxidative stress profile in patient of Graves' ophthalmopathy and to study the effect of hormone level normalization on oxidative stress profile. METHODS: All first time reporting patients with Graves' ophthalmopathy to Department of ophthalmology CSM Medical University (erstwhile King George's Medical University) Lucknow during the period January 2006 to December 2008 formed the cohort. Before initiating treatment a proforma directed detailed history, complete ophthalmological examination and investigations were done. Blood sample for pro/antioxidant enzyme were withdrawn for study after taking an informed consent. Patients were treated with antithyroid drugs alone to achieve a stable euthyroid status for at least 6 months following which a blood sample was again withdrawn to study the pro/anti oxidant enzyme status following treatment. RESULTS: On normalization of thyroid status the values of reactive oxygen species decreased significantly (p<0.05) and levels of antioxidants also got corrected significantly (p<0.05). However both these values remained significantly (p<0.05) altered as compared to normal persons. CONCLUSION: We demonstrated that even after normalization of thyroid hormone level, the oxidative stress levels remain elevated. Moreover, activity of Superoxide Dismutase (SOD), Catalase (CAT), Glutathione reductase (GSHR), Glutathione peroxidise (GPx) showed decrease which could be attributed to altered metabolism and already prevalent deficiency of essential micronutrients like zinc, copper, mercury, and selenium in the Indian population. Hence, this gives way to the thought that the supplementation of these nutrients may have a role as an adjuvant to hormonal therapy in patients with Graves' ophthalmopathy.

Expression of cyclooxygenase-2 in orbital fibroadipose connective tissues of Graves' ophthalmopathy patients.

OBJECTIVE: The aim of this study is to evaluate the expression of cycloocygenase-2 (COX-2) in orbital fibroadipose connective tissue in Graves' ophthalmopathy (GO) patients, and investigate the associations between COX-2 expression and GO characteristics. METHODS: The orbital fibroadipose connective tissues of 23 cases demonstrating moderate or severe GO, and eight control subjects without any history of thyroid or autoimmune disease were analyzed for COX-2 mRNA expression. Real-time relative quantitative PCR was performed to assess transcripts of COX-2 using the LightCycler. The disease activity was evaluated by the clinical activity score (CAS). The clinical features of GO were evaluated by total eye score (TES) and the cases were divided into two groups; type 1 cases included higher degrees of proptosis with orbital fat volume increase, and type 2 cases included cases with compressive neuropathy and limited extraocular muscle functions. RESULTS: The mean +/- s.d. disease duration was 5.7 +/- 7.1 years. The mean +/- s.d. CAS and TES of cases were 1.60 +/- 1.04 and 7.5 +/- 1.8 respectively. The mean +/- s.d. expression of COX-2 was 0.023 +/- 0.013 and 0.010 +/- 0.002 in GO cases and controls (P = 0.008), and 0.015 +/- 0.073 and 0.029 +/- 0.135 in type 1 and type 2 cases respectively (P = 0.007). COX-2 expression showed a statistically significant positive correlation with TES (r = 0.634, P = 0.001), and a negative correlation with the disease duration (r = -0.621, P = 0.002). CONCLUSIONS: COX-2 is expressed at higher levels in orbital fibroadipose tissues of GO cases. This showed a positive correlation with increasing severity of orbital disease suggesting possible relation with COX-2 expression and orbital inflammation in GO.