Pathogenesis Of Thyroid-associated Ophthalmopathy Research Articles

PDK2-enhanced glycolysis promotes fibroblast proliferation in thyroid-associated ophthalmopathy.

The study aimed to investigate the role of pyruvate dehydrogenase kinase (PDK) in regulating glycolysis and proliferation of perimysial orbital fibroblasts (pOFs) during the pathogenesis of thyroid-associated ophthalmopathy (TAO). EdU and BrdU incorporation assays were performed to examine cell proliferation. Lactate production and oxygen consumption assays were conducted to evaluate glycolysis. Real-time PCR was adapted to quantify PDK mRNA levels. Capillary Western immunoassay was adapted to quantify PDK2, Akt, pAkt308 and GAPDH in protein samples. The TAO pOFs exhibited stronger proliferation activity, higher intracellular lactate concentration, and lower oxygen consumption rate than the control pOFs. The PDK inhibitor dichloroacetic acid (DCA) dose-dependently suppressed the proliferation of both TAO and control pOFs. DCA reduced lactate production and promoted oxygen consumption in the TAO pOFs but showed no significant effects on glycolysis in the control pOFs. Among four PDK isotypes, PDK2 was overexpressed in the TAO pOFs. The potential PDK signaling mediator, cytoplasmic Akt, was more abundant in TAO pOFs than control pOFs. Knockdown of PDK2 resulted in lower lactate production, stronger oxygen consumption, weaker proliferation activity, and less cytoplasmic Akt in the TAO pOFs but showed no significant effects in the control pOFs. The Akt inhibitor MK2206 suppressed proliferation in both TAO and control pOFs, and lactate production was inhibited by MK2206 in the TAO OFs but not the control pOFs. To conclude, PDK2 overexpression enhances glycolysis and promotes proliferation via Akt signaling in the TAO pOFs. These findings yield insights that PDK2 is a potential therapeutic target for TAO treatment.

Tear proteomics of orbital decompression for disfiguring exophthalmos in inactive thyroid‑associated ophthalmopathy

The progress and achievements that have been made in tear proteomics in thyroid-associated ophthalmopathy (TAO) are critical for exploring the pathogenesis of TAO and investigating potential therapeutic targets. However, the tear proteomics of orbital decompression for disfiguring exophthalmos in inactive TAO have yet to be properly investigated. In the present study, orbital decompression was performed to repair disfiguring exophthalmos in patients with inactive TAO. Tears were collected before and after orbital decompression in patients with inactive TAO. Liquid chromatography with tandem mass spectrometry (LC-MS/MS) was performed to explore the changes in tear proteomics. Bioinformatics analyses were then employed to analyze the functions of the differentially expressed proteins (DEPs) identified by LC-MS/MS. The palpebral fissure height and exophthalmia area were significantly restored after 1 month of orbital decompression such that they approached the normal levels identified in healthy eyeballs. Among the 669 proteins identified by LC-MS/MS, 83 proteins were changed significantly between the preoperative and postoperative stages in inactive TAO patients and healthy control individuals. The DEPs were predicted to be involved in numerous signaling pathways. Bioinformatics analyses revealed that pathways associated with the immune system, metabolism, programmed cell death, vesicle-mediated transport, neuronal system and extracellular matrix organization may fulfill significant roles in orbital decompression in patients with inactive TAO. Taken together, these results provided a preliminary understanding of the mechanism of orbital decompression for disfiguring exophthalmos in inactive TAO patients.

Differentially expressed circular RNAs in orbital adipose/connective tissue from patients with thyroid-associated ophthalmopathy

Our study aimed to investigate the differentially expressed circRNAs and their potential roles in orbital adipose/connective tissue from patients with thyroid-associated ophthalmopathy (TAO). The orbital adipose/connective tissue samples from three TAO patients and three control individuals were collected for RNA sequencing after depletion of ribosomal RNA. Differentially expressed mRNAs and up-regulated circRNAs were used for co-expression analysis. Functional and pathway enrichment analysis were conducted for the up- and down-regulated mRNAs in the circRNA-mRNA co-expression network. Meanwhile, circRNA-miRNA interaction network was established by miRanda software. The expression levels of mRNAs and circRNAs in control and TAO samples were determined by qRT-PCR. Among all the 16,329 circRNAs predicted from RNA sequencing data, 163 circRNAs (95 down-regulated and 68 up-regulated) were differentially expressed in TAO samples. Besides, 607 differentially expressed mRNAs were identified. The co-expression analysis showed circRNA_14940 was correlated with CCND1 and TNXB, while circRNA_10135 was correlated with PTGFR, and circRNA_14936 was correlated with TNFRSF19. The up-regulated CCND1 participated in Wnt signaling pathway. The down-regulated TNXB was involved in the ECM-receptor interaction, focal adhesion, and PI3K-Akt signaling pathway. PTGFR participated in neuroactive ligand-receptor interaction and calcium signaling pathway. TNFRSF19 was involved in cytokine-cytokine receptor interaction. In the interaction network, circRNA_14936 could interact with hsa-miR-10392–3p, and circRNA_12367 could interact with hsa-miR-1228–3p. Moreover, the expression changes of MMP2, TNXB, PTGFR, CCND1, and TNFRSF19, as well as circRNA_14936, circRNA_14940, and circRNA_12367 were validated by qRT-PCR. In conclusion, the differentially expressed circRNAs might participate in pathogenesis of TAO, and we speculated that circRNA_14940-CCND1-Wnt signaling pathway might be an important regulatory axis.

Slit2 May Underlie Divergent Induction by Thyrotropin of IL-23 and IL-12 in Human Fibrocytes.

IL-23 and IL-12, two structurally related heterodimeric cytokines sharing a common subunit, divergently promote Th cell development and expansion. Both cytokines have been implicated in the pathogenesis of thyroid-associated ophthalmopathy (TAO), an autoimmune component of Graves disease. In TAO, CD34+ fibrocytes, putatively derived from bone marrow, can be identified in the orbit. There they masquerade as CD34+ orbital fibroblasts (OF) (CD34+ OF) and cohabitate with CD34- OF in a mixed fibroblast population (GD-OF). Slit2, a neural axon repellent, is expressed and released by CD34- OF and dampens the inflammatory phenotype of fibrocytes and CD34+ OF. In this study we report that thyrotropin (TSH) and the pathogenic, GD-specific monoclonal autoantibody, M22, robustly induce IL-23 in human fibrocytes; however, IL-12 expression is essentially undetectable in these cells under basal conditions or following TSH-stimulation. In contrast, IL-12 is considerably more inducible in GD-OF, cells failing to express IL-23. This divergent expression and induction of cytokines appears to result from cell type-specific regulation of both gene transcription and mRNA stabilities. It appears that the JNK pathway activity divergently attenuates IL-23p19 expression while enhancing that of IL-12p35. The shift from IL-23p19 expression in fibrocytes to that of IL-12p35 in their derivative CD34+ OF results from the actions of Slit2. Thus, Slit2 might represent a molecular determinant of balance between IL-23 and IL-12 expression, potentially governing immune responses in TAO.

Teprotumumab in Thyroid-Associated Ophthalmopathy: Rationale for Therapeutic Insulin-Like Growth Factor-I Receptor Inhibition.

Thyroid-associated ophthalmopathy (TAO) is an autoimmune component of Graves' disease for which no currently available medical therapy provides reliable and safe benefit. Based on insights generated experimentally over the past several decades, the insulin-like growth factor-I receptor (IGF-IR) has been implicated in the pathogenesis of TAO. Furthermore, an IGF-IR inhibitor, teprotumumab, has emerged from 2 clinical trials as a promising treatment for active, moderate to severe TAO. This brief review intends to provide an overview of the rationale underlying the development of teprotumumab for this disease. It is possible that teprotumumab will soon take its place in our therapeutic armamentarium for active TAO.

Progress in the pathogenesis of thyroid-associated ophthalmopathy and new drug development.

Thyroid-associated ophthalmopathy (TAO) is the most common extrathyroidal manifestation of toxic diffuse goiter (Graves' disease), also known as Graves' ophthalmopathy/orbitopathy. As an organ-specific autoimmune disease, the pathogenesis of TAO is still unclear. In recent years, great progress has been made in revealing the mechanism of TAO. Various biological and immunosuppressive agents have emerged in an endless stream, showing encouraging results. Strengthening the basic research, establishing ideal animal models, deeply understanding the pathogenesis, and developing novel targeted drugs are of great significance to guide the clinical diagnosis and management of TAO and improve the prognosis of patients.

The role of cell mediated immunopathogenesis in thyroid-associated ophthalmopathy.

Currently, thyroid-associated ophthalmopathy (TAO) lacks effective treatment due to our lack of clarity in its immunopathogenesis. Orbital fibroblasts play a key role in altering inflammation and immune response in TAO, and are considered as the key target and effector cells in its pathogenesis. The orbit infiltrating CD34+ fibrocytes add on to the process by expressing high levels of autoantigens and inflammatory cytokines, while also differentiating into myofibroblasts or adipocytes. This review focuses on the role of orbital fibroblasts and CD34+ fibrocytes in the pathogenesis of TAO, highlighting the basis of emerging treatments.

Defective Regulatory B Cells Are Associated With Thyroid-Associated Ophthalmopathy.

To investigate the change in IL-10-producing regulatory B cells (Breg), which suppress peripheral immune responses, in patients with thyroid-associated ophthalmopathy (TAO). Peripheral blood mononuclear cells (PBMCs) were isolated from healthy controls (n = 54), patients with Graves disease (n = 26), and patients with TAO (N=125), and stimulated with CpG/CD40L. The frequency of IL-10-producing Bregs and the expression of IL-10 in response to TSH stimulation were measured by flow cytometry. CD4+ T cells were cultured with Breg-depleted PBMCs to elucidate the function of Bregs in patients with TAO. The potential immunoregulatory mechanism was also investigated by Western blot and chromatin immunoprecipitation assays. Patients with active TAO had higher baseline levels of Bregs in their peripheral blood than both healthy controls and inactive patients. TSH promoted Bregs. Bregs from patients with TAO were defective in suppressing the activation of interferon (IFN)-γ+ and IL-17+ T cells in vitro. Regulatory B cells in patients with TAO are functionally defective, suggesting that the defective Bregs might be responsible for the pathogenesis of TAO.

The expression of interleukin-15 and interleukin-17 in tears and orbital tissues of Graves ophthalmopathy patients.

The objective of this study was to study the levels of interleukin (IL)-15 and IL-17 in tears and orbital tissues of the patients with the Graves ophthalmopathy (GO) (active and inactive stages) and in controls. Twenty-four active GO patients (CAS ≥ 3/7) were collected in our research. All patients were treated with corticosteroids during the therapy process (4-15 months) and developed into the inactive phase (CAS ≤ 2/7). The tear samples were gathered from inactive and active stages of patients with GO and sex-matched and age-matched controls (volunteers, n = 24) for the enzyme-linked immunosorbent assay measurement of IL-15 and IL-17. The orbital tissues were collected from inactive stage of patients with GO and healthy volunteers for the analysis of IL-15 and IL-17 positive cells using the immunohistochemical analysis. In comparison with the volunteers, significant upregulation of IL-15 and IL-17 were identified in tears of active (P < 0.05) and inactive patients with GO ( P < 0.01). Compared with inactive patients with GO, the levels of IL-15 and IL-17 in the tears were obviously higher than those in the active patients with GO. In addition, higher expression of IL-15 and IL-17 positive cells were found in the orbital organization of inactive patients with GO compared with volunteers. The differences in IL-15 and IL-17 expression between GO patients (active and inactive stages) and controls suggested both IL-15 and IL-17 expression were significantly correlated with thyroid-associated ophthalmopathy pathogenesis and development.

Insights Into Local Orbital Immunity: Evidence for the Involvement of the Th17 Cell Pathway in Thyroid-Associated Ophthalmopathy.

Unique features of local immunity in thyroid-associated ophthalmopathy (TAO) may affect disease progression. To investigate the association between the orbital immune microenvironment and TAO development. TAO and control orbital connective tissues were collected. Single-cell sequencing examined orbital lymphocytic infiltrates. Multicolor flow cytometry explored the phenotypes of different cell subsets and in vitro models for cell functional studies. Coculture experiment and western blotting assay were used to determine underlying mechanism of the enhanced T helper 17 (Th17) cell pathway. The TAO orbital microenvironment was composed of natural killer cells, dendritic cells, macrophages, T cells, plasma cells, and CD34+ orbital fibroblasts, but few B cells. Increases in CD3+CD8- IL-17A-producing and RAR-related orphan receptor (ROR)γt-expressing T cells and in CD3+CD8- IL-13-producing and GATA3-expressing T cells suggested Th17 and Th2 cell responses in TAO orbits. Increased interferon-γ (IFN-γ)-producing and RORγt+Tbet+ T cells indicated a Th1-like phenotype of orbital-infiltrating Th17 cells. Higher IL-23R and IL-1R expression and lower IL-21R expression were also observed on Th17 cells in TAO orbits. Multivariate analyses revealed that the Th17 pathway [IL-17A (P = 0.001), IFN-γ (P = 0.009), RORγt (P = 0.003), IL-23R (P = 0.033), IL-21R (P = 0.019)], and Th2 pathway [IL-13 (P = 0.015), GATA3 (P = 0.012)] were associated with TAO. IL-17A, IL-23R, and IL-1R correlated with clinical activity score and visual acuity. CD34+ orbital fibroblasts exhibited distinct cell surface marker expression and promoted IL-23R and IL-1R expression on T cells to facilitate the Th17-cell phenotype through prostaglandin E2-EP2/EP4-cAMP signaling. Our study addresses the importance of retroorbital immunity and suggests possible means of disrupting TAO pathogenesis.

The insulin-like growth factor-I receptor and its role in thyroid-associated ophthalmopathy.

Thyroid-associated ophthalmopathy (TAO), an autoimmune component of Graves' disease, remains a disfiguring and potentially blinding condition. Here, the author reviews the role of insulin-like growth factor-I receptor pathway in TAO and how it might be therapeutically targeted. The recent literature is reviewed. TAO involves reactivity of orbital connective tissues and their remodeling. While many of the details concerning the pathogenesis of TAO remain to be determined, several insights have come to light recently. Among them is the apparent involvement of IGF-IR. This receptor protein, a membrane-spanning tyrosine kinase receptor can form both physical and functional complexes with the thyrotropin receptor (TSHR). This is notable because TSHR is the established primary autoantigen in Graves' disease. IGF-IR activity is critical to signaling downstream from both IGF-IR and TSHR. In addition, antibodies against IGF-IR have been detected in patients with Graves' disease and in rodent models of TAO. Evidence has been put forward that these antibodies may act directly on IGF-IR, perhaps in some manner activating the receptor. These experimental observations have led to the development of a novel therapy for active TAO, utilizing a monoclonal anti-IGF-IR inhibitory antibody which had been produced originally as treatment for cancer. The agent, teprotumumab was recently evaluated in aclinical trial and found to be highly effective and relatively well-tolerated. It is currently undergoing assessment in a follow-up trial. Should the current study yield similarly encouraging results, it is possible that teprotumumab will emerge as a paradigm-shifting medical therapy for TAO.

Decreased Expression of TIM-3 on Th17 Cells Associated with Ophthalmopathy in Patients with Graves' Disease.

Purpose: Thyroid-associated Ophthalmopathy (TAO) is one of the most common orbital immunological diseases in adults. CD4+ helper T (Th) cells play important roles in the pathogenesis of TAO. But the mechanisms regulating CD4+ T cell activity is unclear. This study examines T cell immunoglobulin domain and mucin domain 3 (TIM-3) expression in helper T cell type 1 (Th1), Th17, and regulatory T cells in sufferers of TAO.Methods: Participants were divided into 3 groups: patients with TAO, patients with Graves’ disease but without orbitopathy (GD), and healthy control patients (HC). Peripheral blood samples were collected for each patient in the designated group. Flow cytometry methods assessed the frequency of Th1 (CD4+IFN-γ+), Th17 (CD4+IL-17+), regulatory T cells (CD4+CD25hiCD127lo), and TIM-3 protein expression. Mean fluorescence intensity (MFI) measured the magnitude of TIM-3 expression and the percentage of TIM-3+ cells for each patient.Results: Compared to the GD group, TAO patients possessed higher frequencies of Th1 and Th17 cells in peripheral blood samples. The percentage of TIM-3+ Th1 and Th17 cells was significantly lower in the TAO patients than the GD group. Across all patients sampled, TIM-3+ cell percentage negatively correlated with Th1 cell frequency. Th1 and Th17 cells exhibited significantly decreased expression of TIM-3 in TAO patients compared to healthy controls. Regulatory T cells showed little TIM-3 expression and we observed no significant differences in frequency between groups.Conclusion: These results suggest a role for TIM-3 in the regulation of Th1 and Th17 cells and the pathogenesis of Graves’ ophthalmopathy.

40 YEARS OF IGF1: IGF1 receptor and thyroid-associated ophthalmopathy

Thyroid-associated ophthalmopathy (TAO) is a vexing and poorly understood autoimmune process involving the upper face and tissues surrounding the eyes. In TAO, the orbit can become inflamed and undergo substantial remodeling that is disfiguring and can lead to loss of vision. There are currently no approved medical therapies for TAO, the consequence of its uncertain pathogenic nature. It usually presents as a component of the syndrome known as Graves' disease where loss of immune tolerance to the thyrotropin receptor (TSHR) results in the generation of activating antibodies against that protein and hyperthyroidism. The role for TSHR and these antibodies in the development of TAO is considerably less well established. We have reported over the past 2 decades evidence that the insulin-like growth factorI receptor (IGF1R) may also participate in the pathogenesis of TAO. Activating antibodies against IGF1R have been detected in patients with GD. The actions of these antibodies initiate signaling in orbital fibroblasts from patients with the disease. Further, we have identified a functional and physical interaction between TSHR and IGF1R. Importantly, it appears that signaling initiated from either receptor can be attenuated by inhibiting the activity of IGF1R. These findings underpin the rationale for therapeutically targeting IGF1R in active TAO. A recently completed therapeutic trial of teprotumumab, a human IGF1R inhibiting antibody, in patients with moderate to severe, active TAO, indicates the potential effectiveness and safety of the drug. It is possible that other autoimmune diseases might also benefit from this treatment strategy.

Update on thyroid-associated Ophthalmopathy with a special emphasis on the ocular surface.

Thyroid-associated ophthalmopathy (TAO) is a condition associated with a wide spectrum of ocular changes, usually in the context of the autoimmune syndrome, Graves’ disease. In this topical review, we attempted to provide a roadmap of the recent advances in current understanding the pathogenesis of TAO, important aspects of its clinical presentation, its impact on the ocular surface, describe the tissue abnormalities frequently encountered, and describe how TAO is managed today. We also briefly review how increased understanding of the disease should culminate in improved therapies for patients with this vexing condition.

Elevated Serum Tetrac in Graves Disease: Potential Pathogenic Role in Thyroid-Associated Ophthalmopathy.

The sources and biological impact of 3,3',5,5' tetraiodothyroacetic acid (TA4) are uncertain. CD34+ fibrocytes express several proteins involved in the production of thyroid hormones. They infiltrate the orbit in Graves disease (GD), an autoimmune process known as thyroid-associated ophthalmopathy. It appears that the thyrotropin receptor plays an important role in the pathogenesis of thyroid-associated ophthalmopathy. To quantify levels of TA4 in healthy participants and those with GD, determine whether fibrocytes generate this thyroid hormone analogue, and determine whether TA4 influences the actions of thyroid-stimulating hormone and thyroid-stimulating immunoglobulins in orbital fibroblasts. Patients with GD and healthy donors in an academic medical center clinical practice were recruited. Liquid chromatography-tandem mass spectrometry, autoradiography, real-time polymerase chain reaction, hyaluronan immunoassay. Serum levels of TA4 are elevated in GD. TA4 levels are positively correlated with those of thyroxine and negatively correlated with serum levels of triiodothyronine. Several cell types in culture generate TA4 from ambient thyroxine, including fibrocytes, HELA cells, human Müller stem cells, and retinal pigmented epithelial cells. Propylthiouracil inhibits TA4 generation. TA4 enhances the induction by thyrotropin and thyroid-stimulating immunoglobulins of several participants in the pathogenesis of thyroid-associated ophthalmopathy, including interleukin 6, hyaluronan synthase 1, prostaglandin endoperoxide H synthase 2, and haluronan production. TA4 may be ubiquitously generated in many tissues and enhances the biological impact of thyrotropin and thyroid-stimulating immunoglobulins in orbital connective tissue. These findings may identify a physiologically important determinant of extrathyroidal thyroid-stimulating hormone action.

A study on the establishment of co-culture system of peripheral blood mononuclear cells with orbital fibroblasts and the secretion of IL-6 and IL-17A induced by phytagglutinin in thyroid associated ophthalmopathy

Background The pathogenic mechanism of thyroid associated ophthalmopathy (TAO) is still unclear, which is considered to be an autoimmune disease.It is confirmed that interleukin-17A (IL-17A) plays an important role in the occurrence and development of many autoimmune diseases.It is unclear that whether IL-17A participates in the pathogenesis of TAO. Objective This study was to explore whether IL-17A secreted by co-culture system of peripheral blood mononuclear cells (PBMCs) and orbital fibroblasts (OFs) participates in the pathogenesis of TAO and its possible mechanism. Methods Periphery blood and orbital connective tissue were obtained from 12 patients with TAO and 8 patients who received prosthesis implantation for eyeball atrophy in Xiangya Hospital during April to December 2014.PBMCs were isolated by density gradient centrifugation, and OFs were cultured by explant culture method.The purity of T leukomonocyte in PBMCs was tested by flow cytometry, and OFs were identified by Giemsa staining and immunochemistry.OFs and PMBCs were incubated into 96-well plate in a 1∶20 proportion to establish co-culture system.Different concentrations of phytagglutinin (PHA) (0, 1.0, 2.5, 5.0, 10.0 μg/ml) was added for 72 hours, and IL-6, IL-17A levels in the co-culture system supernatant and IL-17A receptor (IL-17RA) of the total cell membranes in the co-culture system were assayed by ELISA.The differences of IL-6, IL-17A, IL-17RA levels in co-culture system were compared between the TAO group and control group. Results The mean purity of T leukomonocyte in PBMCs was (81.10±0.21)% in the TAO group and (80.05±0.38)% in the control group respectively, with no significant difference between them(t=0.923, P>0.05). Cultured OFs showed the positive response for Vimentin expression and Giemsa staining.After stimulated by 1.0 μg/ml PHA, the proliferation of both PBMCs and OFs were increased in the co-culture system.Apoptosis exist in PBMCs and the number of OFs decreased when PHA was higher than 1.0 μg/ml.The growth of PBMCs and OFs was faster in the TAO group than that in the control group in the same concentration of PHA.The contents of IL-6, IL-17A and IL-17RA in co-culture system were significantly different among various concentrations of PHA subgroups (IL-6: Fgroup=12.561, P=0.000; Fconcentration=23.356, P=0.001.IL-17A: Fgroup= 12.037, P=0.000; Fconcentration =19.206, P=0.000.IL-17RA: Fgroup=16.216, P=0.000; Fconcentration=4.627, P=0.018). The production of IL-6, IL-17A and IL-17RA reached peak in both TAO group and the control group after 1.0 μg/ml PHA stimulated.However, the concentrations of IL-6, IL-17A and IL-17RA reduced with the increase of PHA concentration.The concentrations of IL-6, IL-17A and IL-17RA in co-culture system were significantly higher in the TAO group than those in the control group under the stimulation of the same concentration of PHA (all at P<0.05). Conclusions The co-culture system of PBMCs and OFs stimulated with PHA can be the imitation of TAO pathogenesis in vitro, and PHA can amplify its immune reaction to imitate TAO pathogenic processes intuitively.The IL-6, IL-17A and IL-17RA secreted by PBMCs and induced by PHA are increased in TAO patients, implying that IL-17A participates in the pathogenesis of TAO through magnifying cellular immune response and inflammatory reaction. Key words: Cell culture; Thyroid-associated ophthalmopathy; Peripheral blood mononuclear cells; Orbital fibroblasts; T lymphocytes; Interleukin-6; Interleukin-17A; Human

Intersection of Chemokine and TSH Receptor Pathways in Human Fibrocytes: Emergence of CXCL-12/CXCR4 Cross Talk Potentially Relevant to Thyroid-Associated Ophthalmopathy.

Fibrocytes are monocyte progenitor cells that have been implicated in normal and pathological tissue remodeling. Among the prominent chemokine receptors expressed by these cells is CXC motif receptor 4 (CXCR4), which, with its cognate ligand CXCL motif ligand 12 (CXCL-12), directs fibrocytes to sites of fibrosis. Fibrocytes have been implicated in the pathogenesis of thyroid-associated ophthalmopathy, the ocular manifestation of Graves' disease (GD), by virtue of their unique accumulation as CD34+ orbital fibroblasts (OFs). Fibrocytes also express high levels of functional TSH receptor (TSHR). Here, we determined CXCL-12 and CXCR4 expression in fibrocytes and GD-OF and whether that pathway interacts with TSHR. CXCL-12 is highly expressed in GD-OF, whereas CXCR4 levels are dramatically higher in fibrocytes. Levels of these proteins are differentially regulated by TSH in a cell type-specific manner. Further, CXCL-12 enhances the induction by TSH of IL-6 in fibrocytes but attenuates this induction in GD-OF. In contrast, in pure CD34+ OF, the interplay between TSH and CXCL-12 reverts to that observed in fibrocytes. Our results indicate that CXCL-12 enhances TSH actions in fibrocytes but inhibits them in GD-OF, a dichotomy imposed by factors emanating from CD34- OF. They also suggest a potentially important modulatory role for CD34- OF in determining the factors that influence pathological TSHR signaling in the TAO orbit.

Orbital decompression surgery and horse chestnut seed extract improved superior orbital vein blood flow in patients with thyroid-associated ophthalmopathy.

To evaluate the efficacy and safety of orbital decomposition (OD) surgery in combination with horse chestnut seed extract (HCSE), as compared to OD alone, in patients with thyroid-associated ophthalmopathy (TAO). Sixty-two orbits from 62 TAO patients were randomly assigned to OD or OD+HCSE at 1:1 ratio (31 received OD alone, 31 received OD+HCSE). Forty-two orbits from 21 healthy subjects were used as controls. Complete ophthalmic examination and color Doppler flow imaging (CDFI) were performed before surgery and 3mo post-surgery on all 62 orbits from the TAO patients. CDFI were also performed on the 42 control orbits. The effect of OD+HCSE and OD alone on TAO orbits was compared on several endpoints, including superior ophthalmic vein blood flow (SOVBF) parameters, subjective assessment, soft tissue involvement, lid retraction, diplopia, eye movement restriction, degree of exophthalmos, and intraocular pressure. The control orbits were used as reference for the SOVBF parameters. OD surgery with or without HCSE improved SOVBF, symptoms and soft tissue involvement, decreased degree of exophthalmos and intraocular pressure in orbits of TAO patients. The OD+HCSE combination led to significantly better improvement of SOVBF than OD alone. The differences between the reductions of SOVBF in the two groups are 1.26 cm/s in max-volecity and 0.52 cm/s in min-volecity (P<0.0001). SOVBF is significantly reduced in the orbits affected with TAO, indicating that congestion may be an important factor contributing to TAO pathogenesis. OD surgery improves the SOVBF, and combination of HCSE medication and OD surgery further improved venous return than OD surgery alone.

Th1, Th2, and Th17 Cytokine Involvement in Thyroid Associated Ophthalmopathy.

To determine serum cytokine profiles in Graves' disease (GD) patients with or without active and inactive thyroid associated ophthalmopathy (TAO), we recruited 65 subjects: 10 GD only (without TAO), 25 GD + active TAO, 20 GD + TAO, and 10 healthy controls. Liquid chip assay was used to measure serum Th1/Th2/Th17 cytokines including IFN-γ (interferon-gamma), TNF-α (tumor necrosis factor-alpha), IL-1α (interleukin-1 alpha), IL-1Ra (IL-1 receptor antagonist), IL-2, IL-4, IL-6, and IL-17 and two chemokines: RANTES (regulated upon activation, normal T cell expressed and secreted) and IP-10 (IFN-γ-induced protein 10). Serum levels of TSH (thyroid stimulating hormone) receptor autoantibodies (TRAb) were measured using an enzyme linked immunosorbent assay. Compared with healthy controls, TAO patients showed significantly elevated serum levels of IFN-γ, TNF-α, IL-1α, IL-4, IL-6, IL-17, and IP-10. Comparing active and inactive TAO, serum Th1 cytokines IFN-γ and TNF-α were elevated in active TAO, while serum Th2 cytokine IL-4 was elevated in inactive TAO. Serum Th17 cytokine IL-17 was elevated in GD but reduced in both active and inactive TAO. A positive correlation was found between TRAb and IFN-γ, TNF-α, IL-1α, IL-2, IL-4, and IL-6. Taken together, serum Th1/Th2/Th17 cytokines and chemokines reflect TAO disease activity and may be implicated in TAO pathogenesis.

Soluble CD40L promotes proliferation of orbital fibroblasts and expression of hyaluronan synthase in patients with thyroid associated ophthalmopathy

Objective To investigate the effects of soluble CD40L(sCD40L)on proliferation of orbital fibroblasts(OFs)and the expression of three types of hyaluronan synthase(HAS)in vitro,so as to explore the role of sCD40 Lin the pathogenesis of thyroid-associated ophthalmopathy(TAO).Methods OFs obtained from 5patients with TAO and 3normal controls were primarily culutred.The effect of different concentrations of sCD40L(6.25,12.5,25,50,100 and 200ng/mL)on proliferation of OFs of different sources were examined by MTS after 48 hexposure.OFs were also cultured with different concentrations of sCD40L(12.5,25,50 and 100ng/mL)for 3,6,12 and 24h,and then the expression levels of HAS1-3mRNA were determined by real-time RT-PCR.Results Treatment with sCD40 Lat concentrations higher than 25ng/mL for 48 h obviously promoted the proliferation of OFs in patients with TAO(P0.05).In contrast,treatment with sCD40 Lonly at concentrations higher than 50ng/mL for 48 hcould promote proliferation of OFs from normal control,and the effect was comparatively weak.HAS3 mRNA expression of OFs in TAO patients was increased after exposed to sCD40L(P0.05),and the increase was in a concentration-and time-dependent manner.Conclusion sCD40 Lcan promote the proliferation of OFs and expression of HAS3 mRNA in patients with TAO,which implies that sCD40 Lplays an important role in the pathogenesis of TAO.