Graves’ Autoantibodies Exhibit Different Stimulating Activities in Cultures of Thyrocytes and Orbital Fibroblasts Not Reflected by Clinical Assays

Thyroid ◽  
2021 ◽  
Author(s):  
Christine Carag Krieger ◽  
George Kahaly ◽  
Asma Azam ◽  
Joanna Klubo-Gwiezdzinska ◽  
Susanne Neumann ◽  
...  
Keyword(s):  
Orbital Fibroblasts

scholarly journals JNK and p38 Inhibitors Prevent Transforming Growth Factor-β1-Induced Myofibroblast Transdifferentiation in Human Graves’ Orbital Fibroblasts

2021 ◽  
Vol 22 (6) ◽  
pp. 2952
Author(s):  
Tzu-Yu Hou ◽  
Shi-Bei Wu ◽  
Hui-Chuan Kau ◽  
Chieh-Chih Tsai
Keyword(s):  
Growth Factor ◽  
Transforming Growth Factor ◽  
Mapk Pathway ◽  
Transforming Growth Factor Β1 ◽  
Tissue Growth ◽  
Mitogen Activated Protein Kinase ◽  
Tissue Inhibitors Of Metalloproteinases ◽  
Western Blots ◽  
Orbital Fibroblasts ◽  
Tgf Β1

Transforming growth factor-β1 (TGF-β1)-induced myofibroblast transdifferentiation from orbital fibroblasts is known to dominate tissue remodeling and fibrosis in Graves’ ophthalmopathy (GO). However, the signaling pathways through which TGF-β1 activates Graves’ orbital fibroblasts remain unclear. This study investigated the role of the mitogen-activated protein kinase (MAPK) pathway in TGF-β1-induced myofibroblast transdifferentiation in human Graves’ orbital fibroblasts. The MAPK pathway was assessed by measuring the phosphorylation of p38, c-Jun N-terminal kinase (JNK), and extracellular-signal-regulated kinase (ERK) by Western blots. The expression of connective tissue growth factor (CTGF), α-smooth muscle actin (α-SMA), and fibronectin representing fibrogenesis was estimated. The activities of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) responsible for extracellular matrix (ECM) metabolism were analyzed. Specific pharmacologic kinase inhibitors were used to confirm the involvement of the MAPK pathway. After treatment with TGF-β1, the phosphorylation levels of p38 and JNK, but not ERK, were increased. CTGF, α-SMA, and fibronectin, as well as TIMP-1 and TIMP-3, were upregulated, whereas the activities of MMP-2/-9 were inhibited. The effects of TGF-β1 on the expression of these factors were eliminated by p38 and JNK inhibitors. The results suggested that TGF-β1 could induce myofibroblast transdifferentiation in human Graves’ orbital fibroblasts through the p38 and JNK pathways.

Selenium rescues orbital fibroblasts from cell death induced by hydrogen peroxide: another molecular basis for the effects of selenium in graves’ orbitopathy

Endocrine ◽  
2017 ◽  
Vol 58 (2) ◽  
pp. 386-389 ◽  
Author(s):  
Giovanna Rotondo Dottore ◽  
Riccardo Chiarini ◽  
Maria De Gregorio ◽  
Marenza Leo ◽  
Giamberto Casini ◽  
...  
Keyword(s):  
Hydrogen Peroxide ◽  
Cell Death ◽  
Molecular Basis ◽  
Graves Orbitopathy ◽  
Orbital Fibroblasts

Erratum to: Selenium rescues orbital fibroblasts from cell death induced by hydrogen peroxide: another molecular basis for the effects of selenium in graves’ orbitopathy

Endocrine ◽  
2017 ◽  
Vol 58 (2) ◽  
pp. 390-390 ◽  
Author(s):  
Giovanna Rotondo Dottore ◽  
Riccardo Chiarini ◽  
Maria De Gregorio ◽  
Marenza Leo ◽  
Giamberto Casini ◽  
...  
Keyword(s):  
Hydrogen Peroxide ◽  
Cell Death ◽  
Molecular Basis ◽  
Graves Orbitopathy ◽  
Orbital Fibroblasts

PERK mediates oxidative stress and adipogenesis in Graves’ orbitopathy pathogenesis

2021 ◽  
Author(s):  
JaeSang Ko ◽  
Ji-Young Kim ◽  
Min Kyung Chae ◽  
Eun Jig Lee ◽  
Jin Sook Yoon
Keyword(s):  
Oxidative Stress ◽  
Hydrogen Peroxide ◽  
Endoplasmic Reticulum ◽  
Transcription Factor ◽  
Er Stress ◽  
Adipogenic Differentiation ◽  
Ros Generation ◽  
Mrna Levels ◽  
Graves Orbitopathy ◽  
Orbital Fibroblasts

We examined endoplasmic reticulum (ER) stress-related gene expression in orbital tissues from patients with Graves’ orbitopathy (GO) and the effects of silencing protein kinase RNA-like endoplasmic reticulum kinase (PERK) in primary orbital fibroblast cultures to demonstrate the therapeutic potential of PERK-modulating agents in GO management. The expression of ER stress related genes in orbital tissue harvested from individuals with or without GO was studied using real-time polymerase chain reaction. The role of PERK in GO pathogenesis was examined through small-interfering RNA (siRNA)-mediated silencing in cultured primary orbital fibroblasts. Intracellular reactive oxygen species (ROS) levels induced in response to cigarette smoke extract (CSE) or hydrogen peroxide were measured using 5-(and 6)-carboxy-20,70-dichlorodihydrofluorescein diacetate staining and flow cytometry. Cells were stained with Oil Red O, and adipogenesis-related transcription factor expression was evaluated through western blotting after adipogenic differentiation. PERK, activating transcription factor 4 (ATF4), and CCAAT-enhancer-binding protein (C/EBP)-homologous protein(CHOP)mRNA levels were significantly higher in GO orbital tissues than in non-GO orbital tissues. PERK silencing inhibited CSE- or hydrogen peroxide-induced ROS generation. After adipogenic differentiation, GO orbital fibroblasts revealed decreased lipid droplets and downregulation of C/EBPα, C/EBPβ, and peroxisome proliferator-activator gamma (PPARγ) in PERK siRNA-transfected cells. The orbital tissues of patients with GO were exposed to chronic ER stress and subsequently exhibited enhanced unfolded protein response (especially through the PERK pathway). PERK silencing reduced oxidative stress and adipogenesis in GO orbital fibroblasts in vitro. Our results imply that PERK-modulating agents can potentially be used to manage GO.

Anti-inflammatory and Antioxidant Effects of Selenium on Orbital Fibroblasts of Patients With Graves Ophthalmopathy

2021 ◽  
Vol Publish Ahead of Print ◽  
Author(s):  
Bo-Yeon Kim ◽  
Sun-Young Jang ◽  
Dug-Hyun Choi ◽  
Chan-Hee Jung ◽  
Ji-Oh Mok ◽  
...  
Keyword(s):  
Graves Ophthalmopathy ◽  
Anti Inflammatory ◽  
Orbital Fibroblasts ◽  
Antioxidant Effects

scholarly journals Integrative Analysis of Proteomics and DNA Methylation in Orbital Fibroblasts From Graves’ Ophthalmopathy

2021 ◽  
Vol 11 ◽  
Author(s):  
Sita Virakul ◽  
Poorichaya Somparn ◽  
Trairak Pisitkun ◽  
Peter J. van der Spek ◽  
Virgil A. S. H. Dalm ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Extracellular Matrix ◽  
Rna Polymerase Ii ◽  
Cellular Proliferation ◽  
Effector Function ◽  
Inactive Disease ◽  
Poor Correlation ◽  
Graves Ophthalmopathy ◽  
Orbital Fibroblast ◽  
Orbital Fibroblasts

BackgroundGraves’ ophthalmopathy (GO) is a frequent extrathyroidal complication of Graves’ hyperthyroidism. Orbital fibroblasts contribute to both orbital tissue inflammation and remodeling in GO, and as such are crucial cellular elements in active GO and inactive GO. However, so far it is largely unknown whether GO disease progression is associated with functional reprogramming of the orbital fibroblast effector function. Therefore, the aim of this study was to compare both the proteome and global DNA methylation patterns between orbital fibroblasts isolated from active GO, inactive GO and healthy controls.MethodsOrbital fibroblasts from inactive GO (n=5), active GO (n=4) and controls (n=5) were cultured and total protein and DNA was isolated. Labelled and fractionated proteins were analyzed with a liquid chromatography tandem-mass spectrometer (LC-MS/MS). Data are available via ProteomeXchange with identifier PXD022257. Furthermore, bisulphite-treated DNA was analyzed for methylation pattern with the Illumina Infinium Human Methylation 450K beadchip. In addition, RNA was isolated from the orbital fibroblasts for real-time quantitative (RQ)-PCR. Network and pathway analyses were performed.ResultsOrbital fibroblasts from active GO displayed overexpression of proteins that are typically involved in inflammation, cellular proliferation, hyaluronan synthesis and adipogenesis, while various proteins associated with extracellular matrix (ECM) biology and fibrotic disease, were typically overexpressed in orbital fibroblasts from inactive GO. Moreover, orbital fibroblasts from active GO displayed hypermethylation of genes that linked to inflammation and hypomethylated genes that linked to adipogenesis and autoimmunity. Further analysis revealed networks that contained molecules to which both hypermethylated and hypomethylated genes were linked, including NF-κB, ERK1/2, Alp, RNA polymerase II, Akt and IFNα. In addition, NF-κB, Akt and IFNα were also identified in networks that were derived from the differentially expressed proteins. Generally, poor correlation between protein expression, DNA methylation and mRNA expression was observed.ConclusionsBoth the proteomics and DNA methylation data support that orbital fibroblasts from active GO are involved in inflammation, adipogenesis, and glycosaminoglycan production, while orbital fibroblasts from inactive disease are more skewed towards an active role in extracellular matrix remodeling. This switch in orbital fibroblast effector function may have therapeutic implications and further studies into the underlying mechanism are thus warranted.

scholarly journals Cell density-dependent stimulation of PAI-1 and hyaluronan synthesis by TGF-β in orbital fibroblasts

2016 ◽  
Vol 229 (2) ◽  
pp. 187-196 ◽  
Author(s):  
Erika Galgoczi ◽  
Florence Jeney ◽  
Annamaria Gazdag ◽  
Annamaria Erdei ◽  
Monika Katko ◽  
...  
Keyword(s):  
Cell Density ◽  
Inverse Correlation ◽  
Fold Increase ◽  
Dermal Fibroblasts ◽  
Negative Regulator ◽  
Dependent Manner ◽  
Density Dependent ◽  
Cell Densities ◽  
Orbital Fibroblasts ◽  
Pai 1

During the course of Graves’ orbitopathy (GO), orbital fibroblasts are exposed to factors that lead to proliferation and extracellular matrix (ECM) overproduction. Increased levels of tissue plasminogen activator inhibitor type 1 (PAI-1 (SERPINE1)) might promote the accumulation of ECM components. PAI-1 expression is regulated by cell density and various cytokines and growth factors including transforming growth factorβ(TGF-β). We examined the effects of increasing cell densities and TGF-β on orbital fibroblasts obtained from GO patients and controls. Responses were evaluated by the measurement of proliferation, PAI-1 expression, and ECM production. There was an inverse correlation between cell density and the per cell production of PAI-1. GO orbital, normal orbital, and dermal fibroblasts behaved similarly in this respect. Proliferation rate also declined with increasing cell densities. Hyaluronan (HA) production was constant throughout the cell densities tested in all cell lines. In both GO and normal orbital fibroblasts, but not in dermal fibroblasts, TGF-β stimulated PAI-1 production in a cell density-dependent manner, reaching up to a five-fold increase above baseline. This has been accompanied by increased HA secretion and pericellular HA levels at high cell densities. Increasing cell density is a negative regulator of proliferation and PAI-1 secretion both in normal and GO orbital fibroblasts; these negative regulatory effects are partially reversed in the presence of TGF-β. Cell density-dependent regulation of PAI-1 expression in the orbit, together with the local cytokine environment, may have a regulatory role in the turnover of the orbital ECM and may contribute to the expansion of orbital soft tissue in GO.

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