A Review of Novel Medical Treatments for Thyroid Eye Disease.

Thyroid eye disease (TED) is the most common extrathyroidal manifestation of Graves' disease. There has been no effective medication to prevent proptosis in thyroid eye disease until 2020 when the anti-IGF-1R receptor antibody, Teprotumumab, was approved by the US Food and Drug Administration (FDA), sparking increased interest in immune-based drug development. This study aims to review the newly developed drug therapy as well as conventional treatment for TED. Treatment of TED has traditionally been high-dose steroids and orbital radiotherapy, but recently there has been a paradigm shift in the treatment of TED in the US with the introduction of the therapeutic agent teprotumumab, which dramatically reduces proptosis. However, concerns remain about the development of hearing impairment as a potentially fatal complication and long-term safety. Recently, several clinical trials are underway to assess the efficacy and safety of novel drugs targeting mTORC1, IL-6, FcRN, and IGF-1R in treating TED. With the explosive increase in interest from academia and pharmaceutical companies in TED, there is anticipation for the development of drugs that are equivalent or superior to teprotumumab while being safer.

Inhibitory effect of recombinant tyrosine­sulfated madanin­1, a thrombin inhibitor, on the behavior of MDA­MB­231 and SKOV3 cells in vitro.

Thrombin, which plays a crucial role in hemostasis, is also implicated in cancer progression. In the present study, the effects of the thrombin­targeting recombinant tyrosine­sulfated madanin­1 on cancer cell behavior and signaling pathways compared with madanin­1 wild­type (WT) were investigated. Recombinant madanin­1 2 sulfation (madanin­1 2S) and madanin­1 WT proteins were generated using Escherichia coli. SKOV3 and MDA­MB­231 cells were treated with purified recombinant proteins with or without thrombin stimulation. Migration and invasion of cells were analyzed by wound healing assay and Transwell assay, respectively. Thrombin markedly increased cell migration and invasion in both SKOV3 and MDA­MB­231 cells, which were significantly suppressed by madanin­1 2S (P<0.05). Madanin­1 2S also significantly suppressed thrombin­induced expression of phosphorylated (p)­Akt and p­extracellular signal­regulated kinase in both cell lines (P<0.05), whereas madanin­1 WT had no effect on the expression levels of these proteins in MDA­MB­231 cells. Furthermore, madanin­1 2S significantly reversed the effects of thrombin on E­cadherin, N­cadherin and vimentin expression in MDA­MB­231 cells (P<0.05), whereas madanin­1 WT did not show any effect. In conclusion, madanin­1 2S suppressed the migration and invasion of cancer cells more effectively than madanin­1 WT. It is hypothesized that inhibiting thrombin via the sulfated form of madanin­1 may be a potential candidate for enhanced cancer therapy; however, further in vivo validation is required.

Preoperative Risk Factors for Proptosis Recurrence After Rehabilitative Orbital Decompression in Graves' Orbitopathy Patients.

PURPOSE: Rehabilitative orbital decompression treats disfiguring exophthalmos in patients with Graves' orbitopathy (GO). This study aimed to identify risk factors associated with the postoperative recurrence of proptosis after orbital decompression. DESIGN: Retrospective, case-control study. METHODS: This retrospective review included patients with GO who underwent rehabilitative orbital decompression for disfiguring proptosis in an inactive state with a low clinical activity score (0-2) between January 2017 and December 2020 by a single surgeon. Exophthalmos was measured using a Hertel exophthalmometer, and recurrence was defined as an increase of 2 mm or more after decompression during the follow-up period. The association between preoperative variables and proptosis recurrence was analyzed using multivariable logistic regression. RESULTS: Of the total 217 patients, 11 (5.1%) developed recurrence of proptosis during the follow-up period (range, 3-30; mean, 15.6 months). Univariate logistic regression analysis identified thyroid-stimulating hormone receptor antibody (TRAb) and thyroid-stimulating immunoglobulin (TSI) as significant factors for recurrence, with age, sex, smoking, disease duration, orbital radiotherapy, and total thyroidectomy history being nonsignificant. TRAb remained significant in a multivariate logistic regression analysis (odds ratio, 1.06; P = .014). Receiver operating characteristic curve analysis revealed an area under the curve of 0.86 with a sensitivity of 90.9% and specificity of 82.0% at a TRAb level of 7.96 IU/L. CONCLUSION: Preoperative TRAb and TSI are valuable markers to predict proptosis recurrence after orbital decompression. These results may help surgeons to decide the optimal timing for orbital decompression to lessen the risk of postoperative recurrence of proptosis.

The Effect of Rho Kinase Inhibitors on In Vitro Human Orbital Preadipocytes.

PURPOSE: To identify the effects of Rho Kinase (ROCK) inhibitor medications on human orbital adipogenesis, fibroblast proliferation, and fibrosis. METHODS: Orbital adipose tissue was obtained from patients with Graves' ophthalmopathy (GO) as well as controls (non-GO or normal) after informed consent was done. These tissue samples were cultured and adipogenesis was initiated. Levels of Rho Kinase as well as cellular mediators of orbital inflammation and fibrosis. The same cultures and measurements were then repeated with the use of a ROCK inhibitor (KD025-ROCK2) to assess for changes in adipogenesis as well as markers associated with inflammation and fibrosis. RESULTS: Rho Kinase levels in GO tissue were more highly expressed than in controls. These levels were suppressed with the use of the ROCK inhibitor KD025. There was a dose-dependent reduction in differentiation of orbital adipocytes with the use of KD025. KD025 reduced the levels of fibrosis-related gene expression. Finally, there was a significant reduction of transforming growth factor beta mediated phosphorylation signaling pathways in the KD025-treated GO tissue. CONCLUSION: This study shows that the ROCK inhibitor, KD025, helps to reduce the expression of ROCK in GO tissue along with reducing orbital adipocyte differentiation as well as cell mediators involved in fibrosis that occurs in GO.

A comparison of proptosis reduction with teprotumumab versus surgical decompression based on fat-to-muscle ratio in thyroid eye disease.

PURPOSE: To explore if orbital fat-to-muscle ratio (FMR) is predictive of whether surgical decompression or teprotumumab leads to greater proptosis reduction in thyroid eye disease (TED). METHODS: A single-center retrospective cohort study comparing surgical decompression with teprotumumab according to FMR. All TED patients completing an 8-dose course of teprotumumab between January 2020 and September 2022 and all patients undergoing bony orbital decompression from January 2017 to December 2019 were included. Subjects were excluded if they were <18 years, received both surgical decompression and teprotumumab, or lacked orbital imaging. The primary exposure variable was teprotumumab or surgical decompression. The secondary exposure variable was baseline FMR. The primary outcome measure was change in proptosis (mm). RESULTS: Thirty-eight patients, mean age 53.5 years (±11.4), were included in the teprotumumab group and 160 patients, mean age 48 years (±11.1), in the surgical group. Average proptosis reduction after teprotumumab and surgical decompression was 3 mm (±1.44) and 5 mm (±1.75), respectively. The FMR was stratified at the median of 1.80. In subjects with FMR < 1.80, teprotumumab showed equivalent proptosis reduction compared to surgical decompression, -0.33 mm (SE 1.32) p = .802. In subjects with FMR ≥ 1.80, surgical decompression led to significantly more proptosis reduction than teprotumumab, 3.01 mm (SE 0.54), p < .001. CONCLUSIONS: Baseline FMR can be used to counsel patients as to proptosis reduction with teprotumumab versus surgery. Subjects with low FMR obtain comparable proptosis reduction with teprotumumab or surgery, whereas high FMR is associated with more significant proptosis reduction following surgery over teprotumumab.

Phospholipase C-γ as a Potential Therapeutic Target for Graves' Orbitopathy.

BACKGRUOUND: Phospholipase C-γ (PLC-γ) plays a crucial role in immune responses and is related to the pathogenesis of various inflammatory disorders. In this study, we investigated the role of PLC-γ and the therapeutic effect of the PLC-specific inhibitor U73122 using orbital fibroblasts from patients with Graves' orbitopathy (GO). METHODS: The expression of phospholipase C gamma 1 (PLCG1) and phospholipase C gamma 2 (PLCG2) was evaluated using polymerase chain reaction in GO and normal orbital tissues/fibroblasts. The primary cultures of orbital fibroblasts were treated with non-toxic concentrations of U73122 with or without interleukin (IL)-1ß to determine its therapeutic efficacy. The proinflammatory cytokine levels and activation of downstream signaling molecules were determined using Western blotting. RESULTS: PLCG1 and PLCG2 mRNA expression was significantly higher in GO orbital tissues than in controls (P<0.05). PLCG1 and PLCG2 mRNA expression was significantly increased (P<0.05) in IL-1ß, tumor necrosis factor-α, and a cluster of differentiation 40 ligand-stimulated GO fibroblasts. U73122 significantly inhibited the IL-1ß-induced expression of proinflammatory molecules, including IL-6, IL-8, monocyte chemoattractant protein-1, cyclooxygenase-2, and intercellular adhesion molecule-1 (ICAM-1), and phosphorylated protein kinase B (p-Akt) and p38 (p-p38) kinase in GO fibroblasts, whereas it inhibited IL-6, IL-8, and ICAM-1, and p-Akt and c-Jun N-terminal kinase (p-JNK) in normal fibroblasts (P<0.05). CONCLUSION: PLC-γ-inhibiting U73122 suppressed the production of proinflammatory cytokines and the phosphorylation of Akt and p38 kinase in GO fibroblasts. This study indicates the implications of PLC-γ in GO pathogenesis and its potential as a therapeutic target for GO.

Yes-Associated Protein Mediates the Transition from Inflammation to Fibrosis in Graves' Orbitopathy.

Background: In Graves' orbitopathy (GO), localized orbital inflammation within the fixed orbit often leads to a fibrotic phenotype resulting in restrictive myopathy or refractory proptosis. However, the molecular pathways related to the transition from inflammation to fibrosis in GO are less understood. Yes-associated protein (YAP) and its homolog, transcriptional coactivator with PDZ-binding motif (TAZ; a Hippo pathway effector), are critical mechanosensors of mechanical stimuli and activate signaling cascades for cell proliferation, differentiation, and transformation. In this study, we aimed to examine the role of YAP in both inflammatory and fibrotic GO pathogenesis. Methods: Based on RNA sequencing performed on freshly obtained orbital adipose tissue from patients with GO and healthy individuals, Gene Ontology analysis and gene set-enrichment analysis were performed to analyze gene-expression differences between GO and normal orbital tissues. The role of YAP in GO-related inflammation and fibrosis was studied in primary cultured orbital fibroblasts. The effects of interleukin-1ß (IL-1ß)-induced inflammation and transforming growth factor-beta (TGF-ß)-induced fibrosis on YAP expression were evaluated using real-time polymerase chain reaction and Western blotting analyses. The effects of YAP on inflammatory and fibrotic responses were also examined by YAP silencing or treatment with pharmacological YAP inhibitors. Results: RNA sequencing revealed enhanced YAP expression in GO orbital tissues. Gene Ontology analysis indicated that "response to mechanical stimulus"-related genes were overexpressed in GO orbital tissues, along with those enriched for the "adipose proliferation," "inflammatory responses," and "hormone stimulus responses" terms. IL-1ß did not enhance YAP expression, and YAP silencing decreased IL-1ß-induced IL-6 expression while increasing prostaglandin-endoperoxide synthase 2 expression, leading to paradoxical pro-inflammatory effects. Conversely, TGF-ß enhanced YAP expression, and YAP silencing and pharmacological YAP inhibitor (cerivastatin, verteporfin, TED-347, and CA3) treatment significantly reduced TGF-ß-induced myofibroblast differentiation and collagen formation. Conclusion: YAP, a mechanotransducer responding to mechanical stimuli, was strongly expressed in GO orbital tissues, and YAP was induced by TGF-ß in orbital fibroblasts. Our study establishes YAP as a novel mediator of GO pathobiology, potentially mediating the transition from early inflammation to chronic fibrosis in GO. The finding that YAP inhibition suppressed TGF-ß-induced fibrotic response suggests YAP as a therapeutic target against the fibrotic mechanism of GO.

Cre-loxP System-Based Mouse Model for Investigating Graves' Disease and Associated Orbitopathy.

Background: Graves' disease (GD), one of the most common forms of autoimmune thyroid disorders, is characterized by hyperthyroidism caused by antibodies (Abs) against the extracellular A-subunit of the thyrotropin receptor (TSHR). Various approaches have been used to create mouse models of GD, including transfected fibroblasts and immunization with plasmids or adenoviruses expressing human TSHR A-subunit (hTSHR A-subunit). These models, however, require repeated immunization and produce inconsistent results. In this study, we established a novel Cre-loxP system-based mouse model that is able to generate the hTSHR A-subunit, mimicking human GD, and characterized the histological changes in Graves' orbitopathy (GO) progression after a single injection. Materials and Methods: A Cre-loxP system-based mouse model was constructed by inserting the CAG-loxP-STOP-loxP-hTSHR A-subunit cassette into the Rosa26 locus of the mouse genome. Conditional expression of the hTSHR A-subunit was successfully achieved by intramuscular injection of the transactivator of transcription-Cre recombinase (GD mice). Blood tests for anti-TSHR Abs and the total thyroxine (T4) level were performed. Magnetic resonance imaging (MRI) was used to monitor morphological changes in the eyes. A histological examination of the thyroid gland and retrobulbar tissues was performed to observe pathological changes. Results: Twenty-four (8 control and 16 GD) mice were investigated. All GD mice exhibited higher levels of TSHR Abs compared with the control group. Moreover, more than 80% of the mouse models showed elevated T4 levels accompanied by thyroid goiter. MRI analysis revealed an increased volume of retrobulbar tissue, while immunohistochemical staining of orbital tissues exhibited macrophage infiltration and muscle fibrosis in the GD mice, contrasting with the control group. Conclusions: Our novel mouse model for GD, which showed the histological features of GO, was successfully established using the Cre-loxP system. This animal model offers improved insights and contributes to advancing methodological developments for GD and GO.

The Role of Adipsin, Complement Factor D, in the Pathogenesis of Graves' Orbitopathy.

Purpose: Graves' orbitopathy (GO) is an orbital manifestation of autoimmune Graves' disease, and orbital fibroblast is considered a target cell, producing pro-inflammatory cytokines and/or differentiating into adipocytes. Adipose tissue has been focused on as an endocrine and inflammatory organ secreting adipokines. We investigated the pathogenic role of a specific adipokine, adipsin, known as complement factor D in Graves' orbital fibroblasts. Methods: The messenger RNA (mRNA) expression of multiple adipokines was investigated in adipose tissues harvested from GO and healthy subjects. Adipsin protein production was analyzed in primary cultured orbital fibroblasts under insulin growth factor (IGF)-1, CD40 ligand (CD40L) stimulation, and adipogenesis. The effect of blocking adipsin with small interfering RNA (siRNA) on pro-inflammatory cytokine production and adipogenesis was evaluated using quantitative real-time PCR, Western blot, and ELISA. Adipogenic differentiation was identified using Oil Red O staining. Results: Adipsin gene expression was significantly elevated in GO tissue and increased after the stimulation of IGF-1 and CD40L, as well as adipocyte differentiation in GO cells. Silencing of adipsin suppressed IGF-1-induced IL-6, IL-8, COX2, ICAM-1, CCL2 gene expression, and IL-6 protein secretion. Adipsin suppression also attenuated adipocyte differentiation. Exogenous treatment of recombinant adipsin resulted in the activation of the Akt, ERK, p-38, and JNK signaling pathways. Conclusions: Adipsin, secreted by orbital fibroblasts, may play a distinct role in the pathogenesis of GO. Inhibition of adipsin ameliorated the production of pro-inflammatory cytokines and adipogenesis in orbital fibroblasts. Our study provides an in vitro basis suggesting adipsin as a potential therapeutic target for GO treatment.

Sarcopenia as a potential risk factor for senile blepharoptosis: Nationwide Surveys (KNHANES 2008-2011).

As the world's population is aging, sarcopenia is recognized as essential to assess people's lifelong condition and do appropriate early intervention. Senile blepharoptosis is also a problem in old age deteriorating visual function and causing a cosmetic decline. We investigated the association between sarcopenia and the prevalence of senile blepharoptosis, using a nationwide representative survey in Korea. A total of 11,533 participants were recruited. We used the body mass index (BMI)- adjusted appendicular skeletal muscle (ASM) definition as the muscle mass index (MMI, ASM [kg] divided by BMI [kg/m2]). The association between blepharoptosis prevalence and MMI was analyzed using multivariate logistic regression. Sarcopenia, defined as the lowest MMI quintile group in both men and women, was also associated with the prevalence of blepharoptosis (ORs 1.92, 95% CI 1.17-2.16; p < 0.001). These associations remained statistically significant after adjusting for various factors related to blepharoptosis using multivariate analysis (ORs 1.18, 95% CI 1.04-1.34; p = 0.012). Moreover, MMI was found to have a proportional relationship with eyelid lifting force (levator function), which is closely related to the occurrence and severity of ptosis. Sarcopenia is related to the prevalence of senile blepharoptosis, and patients with lower MMI were more likely to have blepharoptosis. These results suggest that sarcopenia can affect visual function and aesthetics.

Differential effects of teprotumumab treatment based on fat-to-muscle ratio in patients with thyroid eye disease.

PURPOSE: To characterize the distribution of fat-to-muscle ratio (FMR) across patients with thyroid eye disease (TED) and to assess the association between FMR and therapeutic response to teprotumumab. METHODS: A retrospective cohort study of patients completing a full course of teprotumumab for TED between January 2020 and March 2022 at a single tertiary referral center. Patients without baseline orbital imaging were excluded. Quantitative analysis of FMR was performed by manual segmentation of patients' imaging using OsiriX software. The primary outcome measure was change in clinical measurement of proptosis. Linear regression modelled change in proptosis against FMR. Statistical significance was set at p < .05. RESULTS: Twenty-two patients (3 M:19F) were included with a mean age of 49.4 ± 15.5 years. The FMR ranged from 1.11 to 6.54, mean 3.15 ± 1.30. The data did not deviate from a normal distribution (Shapiro-Wilk test for normality, p = .18). Pre- and post-treatment average proptosis measurements were 21.72 ± 3.56 mm and 18.81 ± 3.07 mm, respectively. Univariable linear regression demonstrated a 0.78 ± 0.36 mm greater reduction in proptosis for every 1 unit decrease in FMR (p = .038). CONCLUSIONS: Contrary to the traditional dichotomous characterization of TED into type 1 and type 2 phenotypes, orbital FMR may represent a continuum of disease manifestation, more closely following a normal rather than bimodal distribution. Furthermore, pre-treatment FMR is associated with response to teprotumumab; those with lower FMR experiencing a greater reduction in proptosis. This has implications for patient selection and counselling regarding the expected treatment outcome.

Therapeutic effect of ibrutinib, a selective Bruton's tyrosine kinase inhibitor, in orbital fibroblasts from patients with Graves' orbitopathy.

PURPOSE: Bruton's tyrosine kinase (BTK) is an essential protein in B-cell antigen receptor (BCR) signaling pathway and is known to be related to pathogenetic effect on B-cell related malignancies and various autoimmune diseases. In this study, we investigated the therapeutic effect of ibrutinib, an orally bioavailable BTK inhibitor in the pathogenesis of Graves' orbitopathy (GO) in in vitro model. METHODS: Expression of BTK in orbital tissues from GO and normal control subjects were evaluated by real-time polymerase chain reaction (PCR). Primary cultured orbital fibroblasts from each subject were exposed to ibrutinib and stimulated with interleukin (IL)-1ß or insulin like growth factor (IGF)-1. Production of inflammatory cytokines was evaluated by real time PCR and enzyme-linked immunosorbent assays (ELISA). The downstream transcription factors were also determined by western blot assays. RESULTS: The expression of BTK in GO tissues were significantly higher than in healthy controls. After stimulation of GO orbital fibroblasts with IL-1ß or IGF-1, BTK mRNA and phosphorylated (p)- BTK protein expression was also enhanced. Ibrutinib reduced the expression of BTK mRNA and proteins of p-BTK, and inhibited the IL-1ß- and IGF-1-induced production of proinflammatory cytokines including IL-6, IL-8 and COX-2 in both GO and normal cells. Ibrutinib also significantly attenuated phosphorylation of Akt, p38, c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and nuclear factor kappa-light-chain-enhancer of activated B-cells (NF-κB) in IL-1ß stimulated GO cells and Akt, JNK, and NF-κB in IL-1ß stimulated normal cells. CONCLUSIONS: BTK expression is enhanced in GO tissue and orbital fibroblasts. Ibrutinib, a BTK inhibitor suppresses proinflammatory cytokine production as well as phosphorylation of Akt and NF-κB protein. Our results suggest the potential role of BTK in GO inflammatory pathogenesis and possibility of a novel therapeutic target of GO.

Comparison of total energy intakes estimated by 24-hour diet recall with total energy expenditure measured by the doubly labeled water method in adults.

BACKGROUND/OBJECTIVES: The doubly labeled water (DLW) method is the gold standard for estimating total energy expenditure (TEE) and is also useful for verifying the validities of dietary evaluation tools. In this study, we compared the accuracy of total energy intakes (TEI) estimated by the 24-h diet recall method with TEE obtained using the doubly labeled water method. SUBJECTS/METHODS: This study involved 71 subjects aged 20-49 yrs. Over a 14-day period, three 24-h diet recalls per subject (2 weekdays and 1 weekend day) were used to estimate energy intakes, while TEE was measured using the DLW method. The paired t-test was used to determine the significance of differences between TEI and TEE results, and the accuracy of the 24-h recall method was determined by accuracy predictions percentage, root mean square error, and bias. RESULTS: Average study subject age was 33.4 ± 8.6 yrs. The association between TEI and TEE was positive and significant (r = 0.463, P < 0.001), and the difference between TEI (2,084.3 ± 684.2 kcal/day) and TEE (2,401.7 ± 480.3 kcal/day) was also significant (P < 0.001). In all study subjects, mean TEI was 12.0% (307.5 ± 629.3 kcal/day) less than mean TEE, and 12.2% (349.4 ± 632.5 kcal/day) less in men and 11.8% (266.7 ± 632.5 kcal/day) less in women. Rates of TEI underprediction for all study subjects, men, and women, were 60.5%, 51.4%, and 66.7%, respectively. CONCLUSIONS: This study shows that 24-h diet recall underreports energy intakes. More research is needed to corroborate our findings and evaluate the accuracy of 24-h recall with respect to additional demographics.

MicroRNA-155 acts as an anti-inflammatory factor in orbital fibroblasts from Graves' orbitopathy by repressing interleukin-2-inducible T-cell kinase.

To investigate the role of microRNA (miR)-155 in inflammation in an in-vitro model of Graves' orbitopathy (GO). The expression levels of miR-155 were compared between GO and non-GO orbital tissues. The effects of inflammatory stimulation of interleukin (IL)-1ß and tumour necrosis factor alpha (TNF-α) on miR-155 expression on GO and non-GO orbital fibroblasts (OFs) were investigated. The effects of miR-155 mimics and inhibitors of inflammatory proteins and IL-2-inducible T-cell kinase (ITK) expression were examined, along with those related to the knockdown of ITK with siITK transfection on inflammatory proteins. We also examined how ITK inhibitors affect miR-155 expression in GO and non-GO OFs. The expression levels of miR-155 were higher in GO orbital tissues than in non-GO tissue. The overexpression of miR-155 was induced by IL-1ß and TNF-α in OFs from GO and non-GO patients. IL-1ß-induced IL-6 (ICAM1) protein production was significantly reduced (increased) by miR-155 mimics and inhibitors. The mRNA and protein levels of ITK were downregulated by overexpressed miR-155 via miR-155 mimics. Knockdown of ITK via siITK transfection induced a decrease in the expression levels of ITK, IL-17, IL-6, IL-1ß, and TNF-α protein. The expression of miR-155 was significantly downregulated by treatment with ITK inhibitors and Bruton's tyrosine kinase (BTK)/ITK dual inhibitors in a time-dependent manner. Our results indicated a potential relationship between miR-155 and ITK in the context of GO OFs. The overexpression of miR-155 repressed ITK expression and relieved inflammation. Thus, miR-155 appears to have anti-inflammatory effects in GO OFs. This discovery provides a new concept for developing GO treatment therapeutics.

Therapeutic Potential of Targeting Periostin in the Treatment of Graves' Orbitopathy.

Periostin is a matricellular protein that is ubiquitously expressed in normal human tissues and is involved in pathologic mechanism of chronic inflammatory and fibrotic disease. In this study we investigate periostin in the pathogenesis of Graves' orbitopathy (GO) using human orbital adipose tissue obtained from surgery and primary cultured orbital fibroblasts in vitro. POSTN (gene encoding periostin) expression in Graves' orbital tissues and healthy control tissues was studied, and the role of periostin in GO pathologic mechanism was examined through small-interfering RNA (siRNA)-mediated silencing. POSTN gene expression was significantly higher in Graves' orbital tissues than healthy control tissues in real-time PCR results, and immunohistochemical staining revealed higher expression of periostin in Graves' orbital tissues than normal tissues. Silencing periostin using siRNA transfection significantly attenuated TGF-ß-induced profibrotic protein production and phosphorylated p38 and SMAD protein production. Knockdown of periostin inhibited interleukin-1 ß -induced proinflammatory cytokines production as well as phosphorylation of NF-κB and Ak signaling protein. Adipocyte differentiation was also suppressed in periostin-targeting siRNA transfected GO cells. We hypothesize that periostin contributes to the pathogenic process of inflammation, fibrosis and adipogenesis of GO. Our study provides in vitro evidence that periostin may be a novel potential therapeutic target for the treatment of GO.

Potential Therapeutic Role of Bone Morphogenic Protein 7 (BMP7) in the Pathogenesis of Graves' Orbitopathy.

Purpose: We investigated a role of bone morphogenic protein 7 (BMP7), a member of the TGF-ß superfamily on pathogenic mechanism of Graves' orbitopathy (GO). The therapeutic effects of BMP7 on inflammation and fibrosis were evaluated in cultured Graves' orbital fibroblasts. Methods: Expression of BMP7 was compared in cultured orbital tissue explants from GO (n = 12) and normal control (n = 12) subjects using real-time PCR. Orbital fibroblasts were cultured from orbital connective tissues obtained from GO (n = 3) and normal control patients (n = 3). Cells were pretreated with recombinant human BMP7 (rhBMP7) before stimulation with TGF-ß, IL-1ß, and TNF-α. Fibrosis-related proteins and inflammatory cytokines were analyzed by Western blotting. The activation of signaling molecules in inflammation and fibrosis was also analyzed. Results: The expressions of BMP7 mRNA were lower in GO orbital tissues than control. Fibrosis-related proteins, fibronectin, collagen 1α, and α-SMA induced by TGF-ß were suppressed by treating rhBMP7, and rhBMP7 upregulated TGF-ß induced SMAD1/5/8 protein expression, whereas downregulated SMAD2/3. Increased pro-inflammatory molecules, IL-6, IL-8, and intercellular adhesion molecule-1 (ICAM-1) by IL-1ß or TNF-α were blocked by rhBMP7 treatment, and the expression of phosphorylated NFκB and Akt was suppressed by rhBMP7 treatment. Conclusions: BMP7 transcript levels were downregulated in Graves' orbital tissues. Exogenous BMP7 treatment showed inhibitory effects on the production of profibrotic proteins and proinflammatory cytokines in orbital fibroblasts. Our results provide a molecular basis of BMP7 as a new potential therapeutic agent through the opposing mechanism of profibrotic TGF-ß/SMAD signaling and proinflammatory cytokine production.

Longitudinal association of thyroid-stimulating immunoglobulin levels with clinical characteristics in thyroid eye disease.

OBJECTIVES: The clinical course of thyroid eye disease (TED) is heterogeneous and predicting patients who may develop the severe sequelae of the disease is difficult. In this study, we evaluated the longitudinal association between changes in serum thyroid-stimulating hormone (TSH) receptor antibody (TRAb) levels and course of disease activity and severity over time. DESIGN: This was a multicentre, prospective, observational study. SETTING: Fifteen tertiary care oculoplastic service centres in Korea. PARTICIPANTS: Seventy-six patients with newly diagnosed TED were included and followed up for 12 months. METHODS: We evaluated clinical characteristics and serum TRAb levels at baseline, 6 and 12 months of TED diagnosis. Additionally, we analysed longitudinal associations between the serum TRAb levels and clinical activity score (CAS), no signs or symptoms, only signs, soft tissue involvement, proptosis, extraocular muscle involvement, corneal involvement, sight loss (NOSPECS) score and proptosis. RESULTS: Thyroid-stimulating immunoglobulin (TSI) and TSH-binding inhibitory immunoglobulin (TBII) levels decreased during the 1-year follow-up, whereas disease activity measured using CAS decreased mainly in the first 6 months. Disease severity measured using NOSPECS score and proptosis remained unchanged. Moreover, inter-person differences in TBII levels were associated with CAS, NOSPECS score and proptosis over time, whereas inter-person differences in TSI levels were associated with NOSPECS score. Subgroup analysis of patients with a baseline CAS≥4 demonstrated that within-person changes in TSI levels affected the CAS and NOSPECS score. CONCLUSIONS: Follow-up measurement of serum TSI and TBII levels may help evaluate TED prognosis and enable accurate clinical decision-making.

Thyroid eye disease: From pathogenesis to targeted therapies.

Thyroid eye disease (TED) is the most common extrathyroidal manifestation of autoimmune Graves' hyperthyroidism. TED is a debilitating and potentially blinding disease with unclear pathogenesis. Autoreactive inflammatory reactions targeting orbital fibroblasts (OFs) lead to the expansion of orbital adipose tissues and extraocular muscle swelling within the fixed bony orbit. There are many recent advances in the understating of molecular pathogenesis of TED. The production of autoantibodies to cross-linked thyroid-stimulating hormone receptor and insulin-like growth factor-1 receptor (IGF-1R) activates OFs to produce significant cytokines and chemokines and hyaluronan production and to induce adipocyte differentiation. In moderately severe active TED patients, multicenter clinical trials showed that inhibition of IGF-1R with teprotumumab was unprecedentedly effective with minimal side effects. The emergence of novel biologics resulted in a paradigm shift in the treatment of TED. We here review the literature on advances of pathogenesis of TED and promising therapeutic targets and drugs.