Hypoxia increases adipogenesis and affects adipocytokine production in orbital fibroblasts-a possible explanation of the link between smoking and Graves' ophthalmopathy.

AIM: To assess the effects of hypoxia on human orbital fibroblasts (OF) on adipogenesis and adipocytokine production. METHODS: Human OF were derived from tissues obtained from patients with Graves' ophthalmopathy (GO) and from patients without known thyroid diseases undergoing blepharoplasty. The OF were cultured separately under normoxic and hypoxic conditions. Comparisons of adipocytokine concentrations using multiplex ELISA and lipid accumulation in the cells using Oil Red O staining were subsequently performed. RESULTS: There was increased adipogenesis in OF from GO subject when exposed to hypoxic culture conditions. This was not observed in OF from normal controls. Hypoxia led to an increase in leptin and a decrease in MCP-1 secretion in OF cultures. CONCLUSION: Hypoxia induces adipogenesis in OF and may represent a mechanism by which smoking contributes to deterioration of GO. We also found novel changes to leptin and MCP-1 production in OF cultures exposed to hypoxia suggesting important roles of these cytokines in the disease process.

Ethnic differences in the clinical presentation of Graves' ophthalmopathy.

Ethnic differences in a number of eye conditions have been described. The literature on ethnic differences in Graves' ophthalmopathy (GO) is limited. There is some evidence to suggest Asian patients with GO may manifest milder phenotypic features of GO, with less proptosis and evidence of extraocular muscle involvement and restriction. The reasons for these differences are likely to be multifactorial and include orbital and lid anatomy, genetic background and autoimmune responses including TSH -receptor antibodies. These differences should be kept in mind when evaluating and managing patients with GO.

Effects of mutations involving cysteine residues distal to the S281HCC motif at the C-terminus on the functional characteristics of a truncated ectodomain-only thyrotropin receptor anchored on glycosylphosphatidyl-inositol.

BACKGROUND: Cysteine (Cys) residues pair to form disulfide bonds that are important in maintaining structure and function of the thyrotropin receptor (TSHR). There are 11 Cys residues in the ectodomain (ECD). Cys 41 at the N-terminus and Cys 283 at the SHCC motif have been identified as important for ligand binding. The present study evaluated the effects of mutating Cys distal to the S281HCC motif at the C-terminus of the ECD on the functional characteristics of TSHR. METHODS: We introduced (i) individual Cys and (ii) consecutive cumulative Cys mutations into the starting template SHCS-TSHR, a truncated TSHR-ECD moiety previously shown to behave like the wild-type TSHR. Each mutant receptor was evaluated for relative specific binding (RSB), calculated as a measure of TSH-binding ability after normalization with receptor surface expression. RESULTS: In the first approach, RSB was severely affected when Cys 390 and Cys 398 were individually switched to serine. Failed receptor trafficking occurred with Cys 408 mutation. These findings were likely results of altered receptor conformation due to illegitimate disulfide bridge formation. Only SHCS-301 TSHR bound TSH in a specific manner, and it formed the base for sequential Cys mutations. Through this second approach, both Cys 301 and 390 could be removed simultaneously without hindering TSH binding significantly. Cys 398, however, was shown to be critical. Its absence resulted in huge loss of TSH binding. Leaving Cys 283 and 398 as the only Cys pair in the C-terminus alone could support 40% of the total ligand-binding capacity. CONCLUSIONS: From these data, we proposed Cys 398 as a stable disulfide bond partner of Cys 283, corroborating with a model based on evolutionary history of TSHR across species. This pairing of Cys 283 and Cys 398 also provides an objective alternative to conventional hypotheses on Cys coupling based on other predictive models.

Cysteine 390 mutation of the TSH receptor modulates its ectodomain as an inverse agonist on the serpentine domain with decrease in basal constitutive activity.

Mutations of individual cysteine residues at codon 301, 390, 398 and 408 of the thyrotropin receptor (TSHr) to serine resulted in cell surface expression of only C301S and C390S mutants. C390S mutation was a silencing mutation with decreased basal constitutive activity. Although the C301S and C390S mutants did not show any significant TSH binding, they generated cyclic AMP upon TSH stimulation. These mutants were also able to interact with stimulating and blocking anti-TSHr antibodies. In fact, C390S receptor is a more sensitive tool for blocking antibody detection than wild type receptor. Introduction of C390S to activating mutations in the ectodomain (S281N), exloop (I486F) and transmembrane (D633H) segments could not mute/nullify receptor activation. These data indicate that the C390S ectodomain behaves as a more effective inverse agonist on the noisy transmembrane segment and suggest that the basal and activated states of the receptor operate through two independent pathways.

Severe Graves' ophthalmopathy after retrobulbar anesthesia for cataract extraction in a patient with mild stable thyroid eye disease.

It has been hypothesized that the distinct anatomic localization of the Graves' triad may be partially explained by pressure and trauma. While there are reports of local trauma clearly contributing to the pathogenesis of pretibial myxedema, direct evidence for a similar mechanism in Graves' ophthalmopathy (GO) has been lacking. We describe a 65-year-old male patient with stable mild Graves' ophthalmopathy of 24 years' duration in whom a retrobulbar block was administered prior to cataract removal. Three weeks after the procedure, he complained of rapidly progressive bilateral diplopia. In 6 months, there was moderate exophthalmos, exposure keratitis, almost complete ophthalmoplegia, and decreasing visual acuity requiring surgical decompression. Postdecompression, inflammatory signs and vision improved but there was complete ophthalmoplegia. The eye signs remained unchanged for the next 4 months but there was exacerbation of the disease within a week of receiving radioiodine despite concomitant steroid administration. Orbital irradiation was finally administered with rapid improvement in extraocular eye muscle function. We hypothesize that local inflammatory and immune responses stimulated by trauma and/or pressure in the retrobulbar compartment, triggered the development of severe ophthalmopathy in this patient. Thyroid-stimulating immunoglobulin (TSI) levels remained markedly elevated despite the clinical improvement suggesting that the beneficial effects of radiotherapy in this case were not mediated by suppressing TSI production.