Histone Deacetylase 4 Controls Extracellular Matrix Production in Orbital Fibroblasts from Graves' Ophthalmopathy Patients.

Background: Graves' ophthalmopathy (GO) is an autoimmune eye disease with the characteristic symptoms of eyelid retraction and proptosis. Orbital fibroblast activation induced by platelet-derived growth factor-BB (PDGF-BB) stimulation plays a crucial role in GO pathogenesis, leading to excessive proliferation and extracellular matrix production by orbital fibroblasts. Currently, GO treatment options remain limited and novel therapies including targeted drugs are needed. Histone deacetylases (HDACs) are associated with the development and progression of several cancers and autoimmune diseases by epigenetically controlling gene transcription, and HDAC inhibitors (HDACis) may have therapeutic potential. Nevertheless, the role of HDACs in orbital fibroblasts from GO is unknown. Therefore, we studied the expression of HDACs as well as their contribution to extracellular matrix production in orbital fibroblasts. Methods: Orbital tissues were obtained from GO patients (n = 18) who underwent decompression surgery with approval from the Institutional Review Board of the Faculty of Medicine (Protocol number 401/61), Chulalongkorn University (Bangkok, Thailand). Furthermore, orbital tissue was obtained from control patients (n = 3) without inflammatory or thyroid disease who underwent surgery for cosmetic reasons. Orbital fibroblast cultures were established from the orbital tissues. HDAC mRNA and protein expression in orbital fibroblasts was analyzed by reverse transcription-quantitative real-time PCR and Western blot. PDGF-BB-activated orbital fibroblast and orbital tissues were treated with HDACis or HDAC4 small-interfering RNA. Results: PDGF-BB-stimulated orbital fibroblasts had upregulated HDAC4 mRNA and protein expression. HDAC4 mRNA expression was significantly higher in GO compared with healthy control orbital fibroblasts. Histone H3 lysine 9 acetylation (H3K9ac) decreased upon PDGF-BB stimulation. Treatment with HDAC4i (tasquinimod) and HDAC4/5i (LMK-235) significantly decreased both proliferation and hyaluronan production in PDGF-BB-stimulated orbital fibroblasts. HDAC4 silencing reduced mRNA expression of hyaluronan synthase 2 (HAS2), collagen type I alpha 1 chain (COL1A1), Ki67, and α-smooth muscle actin (α-SMA), as well as hyaluronan production in PDGF-BB-stimulated orbital fibroblasts. Tasquinimod significantly reduced HAS2 and α-SMA mRNA expression in whole orbital tissue. Conclusion: Our data indicated, for the first time, that altered HDAC4 regulation along with H3K9 hypoacetylation might represent a mechanism that contributes to excessive proliferation and extracellular matrix production by orbital fibroblasts in GO. HDAC4 might represent a novel target for GO therapy.

The Topography of the Frontal Terminations of the Uncinate Fasciculus Revisited Through Focused Fiber Dissections: Shedding Light on a Current Controversy and Introducing the Insular Apex as a Key Anatomoclinical Area.

BACKGROUND: Recent studies advocate a connectivity pattern wider than previously believed of the uncinate fasciculus that extends to the ventrolateral and dorsolateral prefrontal cortices. These new percepts on the connectivity of the tract suggest a more expansive role for the uncinate fasciculus. Our aim was to shed light on this controversy through fiber dissections. METHODS: Twenty normal adult human formalin-fixed cerebral hemispheres were used. Focused dissections on the insular, orbitofrontal, ventromedial, ventrolateral, and dorsolateral prefrontal areas were performed to record the topography of the frontal terminations of the uncinate fasciculus. RESULTS: Three discrete fiber layers were consistently disclosed: the first layer was recorded to terminate at the posterior orbital gyrus and pars orbitalis, the second layer at the posterior two thirds of the gyrus rectus, and the last layer at the posterior one third of the paraolfactory gyrus. The insular apex was documented as a crucial landmark regarding the topographic differentiation of the uncinate and occipitofrontal fasciculi (i.e., fibers that travel ventrally belong to the uncinate fasciculus whereas those traveling dorsally are occipitofrontal fibers). CONCLUSIONS: The frontal terminations of the uncinate fasciculus were consistently documented to project to the posterior orbitofrontal area. The area of the insular apex is introduced for the first time as a crucial surface landmark to effectively distinguish the stems of the uncinate and occipitofrontal fasciculi. This finding could refine the spatial resolution of awake subcortical mapping, especially for insular lesions, and improve the accuracy of in vivo diffusion tensor imaging protocols.

Octreotide inhibits secretion of IGF-1 from orbital fibroblasts in patients with thyroid-associated ophthalmopathy via inhibition of the NF-κB pathway.

PURPOSE: We investigated the effect of octreotide, a long-acting somatostatin (SST) analogue, on IGF-1 secretion and its possible mechanism of action in orbital fibroblasts (OFs) from patients with thyroid-associated ophthalmopathy (TAO). MATERIALS AND METHODS: OFs were isolated from the orbital fat of patients with TAO or healthy individuals. The expression level of insulin-like growth factor (IGF)-1, at the protein and mRNA level, was determined with ELISA and quantitative RT-PCR, respectively. The expression pattern of somatostatin receptor (SSTR) 2, which has the highest affinity for octreotide, was examined by flow cytometry. The activity of NF-κB pathway was determined by examining the levels of phosphorylation of IKKα/ß and p65, and degradation of IκB via western blot analysis, and by measuring the activity of NF-kB-dependent luciferase via transfection with plasmids containing luciferase and NF-κB binding site. RESULTS: OFs from patients with TAO showed significantly higher levels of IGF-1 secretion and NF-κB activity even in the absence of stimulation, compared to those from controls. Treatment with octreotide reduced the level of IGF-1 secretion in OFs from patients with TAO, but not in OFs from controls. OFs from patients with TAO expressed higher levels of SSTR2 on the cell surface, compared to controls. In addition, the expression of IGF-1 at the protein and mRNA level was dependent on the activity of NF-κB pathway in OFs from patients with TAO. Furthermore, treatment with octreotide reduced on the activity of NF-κB pathway in OFs from patients with TAO. CONCLUSION: OFs from patients with TAO showed significantly higher levels of IGF-1 secretion via up-regulation of NF-κB activity. Treatment with octreotide inhibited the secretion of IGF-1 by reducing the NF-κB pathway in OFs, which expressed higher levels of SSRT2 on the cell surface, from patients with TAO.

MicroRNA-130a Is Elevated in Thyroid Eye Disease and Increases Lipid Accumulation in Fibroblasts Through the Suppression of AMPK.

Purpose: Thyroid eye disease (TED) is a condition that causes the tissue behind the eye to become inflamed and can result in excessive fatty tissue accumulation in the orbit. Two subpopulations of fibroblasts reside in the orbit: those that highly express Thy1 (Thy1+) and those with little or no Thy1 (Thy1-). Thy1- orbital fibroblasts (OFs) are more prone to lipid accumulation than Thy1+ OFs. The purpose of this study was to investigate the mechanisms whereby Thy1- OFs more readily accumulate lipid. Methods: We screened Thy1+ and Thy1- OFs for differences in microRNA (miRNA) expression. The effects of increasing miR-130a levels in OFs was investigated by measuring lipid accumulation and visualizing lipid deposits. To determine if adenosine monophosphate-activated protein kinase (AMPK) is important for lipid accumulation, we performed small interfering RNA (siRNA)-mediated knockdown of AMPKß1. We measured AMPK expression and activity using immunoblotting for AMPK and AMPK target proteins. Results: We determined that miR-130a was upregulated in Thy1- OFs and that miR-130a targets two subunits of AMPK. Increasing miR-130a levels enhanced lipid accumulation and reduced expression of AMPKα and AMPKß in OFs. Depletion of AMPK also increased lipid accumulation. Activation of AMPK using AICAR attenuated lipid accumulation and increased phosphorylation of acetyl-CoA carboxylase (ACC) in OFs. Conclusions: These data suggest that when Thy1- OFs accumulate in TED, miR-130a levels increase, leading to a decrease in AMPK activity. Decreased AMPK activity promotes lipid accumulation in TED OFs, leading to excessive fatty tissue accumulation in the orbit.

Functionally enhanced placenta-derived mesenchymal stem cells inhibit adipogenesis in orbital fibroblasts with Graves' ophthalmopathy.

BACKGROUND: Placenta-derived mesenchymal stem cells (PD-MSCs) have unique immunomodulatory properties. Phosphatase of regenerating liver-1 (PRL-1) regulates the self-renewal ability of stem cells and promotes proliferation. Graves' ophthalmopathy (GO) is an autoimmune inflammatory disease of the orbit and is characterized by increased orbital levels of adipose tissue. Here, we evaluated the therapeutic mechanism for regulation of adipogenesis by PRL-1-overexpressing PD-MSCs (PD-MSCsPRL-1, PRL-1+) in orbital fibroblast (OF) with GO patients. METHODS: PD-MSCs isolated from human placenta were transfected with the PRL-1 gene using nonviral transfection method. Primary OFs were isolated from orbital adipose tissue specimens from GO patients. After maturation as adipogenic differentiation, normal and GO-derived OFs were cocultured with naïve and PD-MSCsPRL-1. We analyzed the protein levels of adipogenesis markers and their signaling pathways in OFs from GO patients. RESULTS: The characteristics of PD-MSCsPRL-1 were similar to those of naïve cells. OFs from GO patients induced adipocyte differentiation and had significantly decreased a lipid accumulation after coculture with PD-MSCsPRL-1 compared to naïve cells. The mRNA and protein expression of adipogenic markers was decreased in PD-MSCsPRL-1. Insulin-like growth factor-binding proteins (IGFBPs) secreting PD-MSCsPRL-1 downregulated the phosphorylated PI3K/AKT/mTOR expression in OFs from GO patients. Interestingly, IGFBP2, - 4, - 6, and - 7 expression in PD-MSCsPRL-1, which was mediated by integrin alpha 4 (ITGA4) and beta 7 (ITGB7), was higher than that in naïve cells and upregulated phosphorylated FAK downstream factor. CONCLUSION: In summary, IGFBPs secreting PD-MSCPRL-1 inhibit adipogenesis in OFs from GO patients by upregulating phosphorylated FAK and downregulating PI3K/AKT/mTOR signaling pathway. The functional enhancement of PD-MSCs by nonviral gene modification provides a novel therapeutic strategy for the treatment of degenerative diseases.

Prostaglandin F2α Agonists Negatively Modulate the Size of 3D Organoids from Primary Human Orbital Fibroblasts.

Purpose: To elucidate the molecular etiology of deepening of the upper eyelid sulcus (DUES) induced by prostaglandin (PG) analogs, a three-dimensional (3D) tissue culture system was employed using human orbital fibroblasts (HOFs). Methods: During adipogenesis, changes in HOF 3D organoid sizes, as well as their lipids stained by BODIPY and expression of the extracellular matrix (ECM) by immunolabeling and/or quantitative PCR, were studied in the presence or absence of either 100-nM bimatoprost acid or 100-nM prostaglandin F2α. Results: The size of the 3D organoids increased remarkably during adipogenesis, but such increases were significantly inhibited by the presence of PG analogs. Staining intensities by BODIPY and mRNA expression of peroxisome proliferator-activated receptor gamma were significantly increased upon adipogenesis but were not influenced by the presence of PG analogs. Unique changes in ECM expression observed with or without adipogenic differentiation were significantly modified by the presence of PG analogs. Conclusions: Our present study indicates that PG analogs have the potential to modulate the ECM network within HOF 3D organoids. Thus, a 3D tissue culture system may be a suitable strategy for understanding the disease etiology of DUES.

Gypenosides Protect Orbital Fibroblasts in Graves Ophthalmopathy via Anti-Inflammation and Anti-Fibrosis Effects.

Purpose: To investigate the effect of Gypenosides (Gyps) on the inflammation and fibrosis in orbital fibroblasts (OFs) in Graves ophthalmopathy (GO). Methods: Bioinformatics analyses were performed to identify the enriched genes and signaling pathways related to Gyps function. For ex vivo experiments, OFs were cultured from orbital connective tissues from patients with GO. OF proliferation was estimated by Cell Counting Kit-8 assay. Effects of Gyps treatment on interleukin (IL)-1ß-induced inflammation and transforming growth factor-ß1 (TGF-ß1)-induced fibrosis were evaluated by real-time quantitative PCR (RT-qPCR), enzyme-linked immunosorbent assay (ELISA), and Western blotting. OFs were treated with IL-1ß or TGF-ß1 in the absence or presence of Gyps pretreatment, and the levels of related mRNA or proteins were evaluated by RT-qPCR or ELISA. Results: Eight inflammation-related target genes and nine fibrosis-related target genes were screened out. These genes were mainly enriched in pathways corresponding to inflammation and fibrosis, respectively. IL-1ß-induced upregulation of inflammatory cytokines, and TGF-ß-induced upregulation of fibrotic mediators in OFs were downregulated by Gyps. Moreover, Gyps reduced the activation of Toll like receptors 4/nuclear factor-κ B signaling and TGF-ß1/SMAD2/SMAD4 signaling in GO OFs. Conclusions: Gyps could protect GO-derived OFs against IL-1ß-induced inflammation and TGF-ß1-induced fibrosis. Thus Gyps might have therapeutic potential on inflammation and fibrosis in GO.

Characteristics of Hyaluronan Synthesis Inhibition by 4-Methylumbelliferone in Orbital Fibroblasts.

Purpose: Hyaluronan (HA) overproduction by orbital fibroblasts (OFs) is a major factor in the pathogenesis of Graves' orbitopathy (GO). 4-methylumbelliferone (4-MU) is an inhibitor of HA synthesis in different cell types in vitro and has beneficial effects in animal models of autoimmune diseases. Methods: HA production and mRNA expression of HA synthases (HAS1, HAS2, and HAS3) and hyaluronidases (HYAL1 and HYAL2) were measured in the presence and absence of 4-MU in unstimulated and transforming growth factor-ß-stimulated fibroblasts from GO orbital (n = 4), non-GO orbital (n = 4), and dermal origin (n = 4). Results: The 4-MU treatment (1 mM) for 24 hours resulted in an average 87% reduction (P < 0.001) of HA synthesis, decreased the expression of the dominant HAS isoform (HAS2) by 80% (P < 0.0001), and increased the HYAL2 expression by 2.5-fold (P < 0.001) in control OFs, GO OFs, and dermal fibroblasts (DFs) regardless of the origin of the cells. The proliferation rate of all studied cell lines was reduced to an average 16% by 4-MU (P < 0.0001) without any effects on cell viability. HA production stimulated by transforming growth factor-ß was decreased by 4-MU via inhibition of stimulated HAS1 expression in addition to the observed effects of 4-MU in unstimulated cases. Characteristics of HA synthesis inhibition by 4-MU did not differ in OFs compared with DFs. Conclusions: 4-MU has been found to inhibit the HA synthesis and the proliferation rate in OFs in vitro, adding it to the list of putative therapeutic agents in a disease the cure of which is largely unresolved.

4-Methylumbelliferone suppresses hyaluronan and adipogenesis in primary cultured orbital fibroblasts from Graves' orbitopathy.

PURPOSE: In Graves' orbitopathy (GO), hyaluronan secreted by orbital fibroblasts contributes to orbital tissue expansion. The goal of this research was to evaluate the potential benefit of 4-methylumbelliferone (4-MU), a hyaluronan synthase (HAS) inhibitor, in primary cultured orbital fibroblasts from Graves' orbitopathy. METHODS: We assessed the viability of orbital fibroblasts using a live/dead cell assay. Hyaluronan synthesis was evaluated by enzyme-linked immunosorbent assay (ELISA) and quantitative real-time PCR (qPCR). Adipogenesis was assessed by Oil Red O staining and qPCR of adipogenic transcription factors. RESULTS: In orbital fibroblasts treated with 4-MU (up to 1000 µM), cell viability was preserved by 90%. 4-MU significantly inhibited HAS gene expression and hyaluronan production (*P < 0.05). With respect to adipogenesis, 4-MU suppressed the accumulation of lipids and reduced the number of adipocytes, while decreasing expression of adipogenic transcription factors. CONCLUSIONS: 4-MU represents a promising new therapeutic agent for GO based on its ability to inhibit hyaluronan production and adipogenesis, without decreasing cell viability.

Role of Proprotein Convertase Subtilisin/Kexin Type 9 in the Pathogenesis of Graves' Orbitopathy in Orbital Fibroblasts.

Background: The proprotein convertase subtilisin/kexin type 9 (PCSK9) has been implicated in the pathogenesis of inflammatory diseases. We sought to investigate the role of PCSK9 in the pathogenesis of Graves' orbitopathy (GO) and whether it may be a legitimate target for treatment. Methods: The PCSK9 was compared between GO (n=11) and normal subjects (n=7) in orbital tissue explants using quantitative real-time PCR, and in cultured interleukin-1ß (IL-1ß)-treated fibroblasts using western blot. Western blot was used to identify the effects of PCSK9 inhibition on IL-1ß-induced pro-inflammatory cytokines production and signaling molecules expression as well as levels of adipogenic markers and oxidative stress-related proteins. Adipogenic differentiation was identified using Oil Red O staining. The plasma PCSK9 concentrations were compared between patients with GO (n=44) and healthy subjects (n=26) by ELISA. Results: The PCSK9 transcript level was higher in GO tissues. The depletion of PCSK9 blunted IL-1ß-induced expression of intercellular adhesion molecule 1 (ICAM-1), IL-6, IL-8, and cyclooxygenase-2 (COX-2) in GO and non-GO fibroblasts. The levels of activated nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and phosphorylated forms of Akt and p38 were diminished when PCSK9 was suppressed in GO fibroblasts. Decreases in lipid droplets and attenuated levels of peroxisome proliferator-activated receptor gamma (PPARγ), CCAAT/enhancer-binding protein ß (C/EBPß), and leptin as well as hypoxia-inducible factor 1α (HIF-1α), manganese superoxide dismutase (MnSOD), thioredoxin (Trx), and heme oxygenase-1 (HO-1) were noted when PCSK9 was suppressed during adipocyte differentiation. The plasma PCSK9 level was significantly higher in GO patients and correlated with level of thyrotropin binding inhibitory immunoglobulin (TBII) and the clinical activity score (CAS). Conclusions: PCSK9 plays a significant role in GO. The PCSK9 inhibition attenuated the pro-inflammatory cytokines production, oxidative stress, and fibroblast differentiation into adipocytes. PCSK9 may serve as a therapeutic target and biomarker for GO.

Integrative Analysis of Proteomics and DNA Methylation in Orbital Fibroblasts From Graves' Ophthalmopathy.

Background: Graves' 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. Methods: Orbital 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. Results: Orbital 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. Conclusions: Both 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.

IgG4-positive Cell Quantification Distinguishes Between Inflammatory and Noninflammatory Diseases of the Orbit.

IgG4-related ophthalmic disease (IgG4-ROD) is a rare inflammatory disorder often refractory to corticosteroids and prone to recurrence. IgG4-ROD may frequently lack the characteristic histopathological features seen in other organs. Thus, the criteria for diagnosis of IgG4-ROD relies on elevated IgG4 cells seen on biopsied tissue. Proposed threshold levels of IgG4 to diagnose IgG4-ROD are currently based on a limited understanding of this cell type's presence in the orbit. This study seeks to describe the population of IgG4 in inflammatory and noninflammatory orbital tissues. A tertiary care center's pathology database was searched with keywords "orbit" or "orbital" from 1995 to 2013. Specimens meeting the selection criteria were evaluated, and regions of highest inflammation were identified and immunostained with IgG4 and CD138 antibodies. Immunohistochemical quantification proceeded as previously established by the international consensus criteria. Eighteen cases without a history of orbital inflammation were included as controls and were evaluated as above. Specimens from 68 inflammatory and 18 noninflammatory orbits met the selection criteria. Pathologist interreader correlation coefficient on quantification was >0.70 (P<0.001). The mean IgG4+/high powered field (HPF) and IgG4+/CD138 was 10.3 and 0.1 in inflammatory tissues and 0.5 and 0.01 in noninflammatory tissues, respectively. The spearman rho correlation coefficient between IgG4/HPF and IgG4+/CD138+ was >0.95 (P<0.0001). The mean IgG4/HPF in our study reached previously proposed threshold values for diagnosis of IgG4-ROD, illustrating the need for further discussion regarding diagnostic criteria of IgG4-ROD.

TSHR Signaling Stimulates Proliferation Through PI3K/Akt and Induction of miR-146a and miR-155 in Thyroid Eye Disease Orbital Fibroblasts.

Purpose: To investigate the molecular pathways that drive thyroid stimulating hormone receptor (TSHR)-induced cellular proliferation in orbital fibroblasts (OFs) from thyroid eye disease (TED) patients. Methods: Orbital fibroblasts from TED and non-TED patients were treated with TSH and changes in gene expression and proliferation were measured. To determine the role of TSHR, TSHR-specific siRNA was used to deplete TSHR levels. Proliferation was measured by bromodeoxyuridine (BrdU) incorporation. PI3K/Akt activation was analyzed by Western blot. The PI3K inhibitor LY294002 was used to investigate PI3K/Akt signaling in OF proliferation. Expression of TSHR, inflammatory cytokines, proliferation related genes and miR-146a and miR-155 were measured by qPCR. Results: Orbital fibroblasts from TED patients proliferate significantly more than non-TED OFs in response to TSH. TSH-induced proliferation was dependent upon TSHR expression and required the PI3K/Akt signaling cascade. TSHR activation stimulated miR-146a and miR-155 expression. TED OFs produced significantly more miR-146a and miR-155 than non-TED OFs. MiR-146a and miR-155 targets, ZNRF3 and PTEN, which both limit cell proliferation, were decreased in TSH treated OFs. Conclusions: These data reveal that TSHR signaling in TED OFs stimulates proliferation directly through PI3K/Akt signaling and indirectly through induction of miR-146a and miR-155. MiR-146a and miR-155 enhance TED OF proliferation by reducing expression of target genes that normally block cell proliferation. TSHR-dependent expression of miR-146a and miR-155 may explain part of the fibroproliferative pathology observed in TED.

Human placenta-derived mesenchymal stem cells ameliorate orbital adipogenesis in female mice models of Graves' ophthalmopathy.

BACKGROUND: Graves' ophthalmopathy (GO) is a complication of Graves' disease (GD), in which orbital connective tissues become inflamed and increase in volume and orbital fibroblasts within the orbital fat and extraocular muscles differentiate into adipocytes in vitro when stimulated by hormones, several cytokines, and growth factors including TSH, IGF-1, IL-1, interferon γ, and platelet-derived growth factor. Human placental mesenchymal stem cells (hPMSCs) have immunomodulatory effects in disease pathogenesis. Although a number of studies have reported that hPMSCs can elicit therapeutic effects, these are not sufficient. Therefore, we constructed a GO animal model in order to find out the hPMSCs recovery effect. METHODS: We investigated their anti-adipogenic effects in in vitro cultures of orbital fibroblasts established from GO patients. Primary orbital fibroblasts were exposed to differentiation medium for 10 days. After being co-cultured with hPMSCs, the characteristics of orbital fibroblast were determined by Oil Red O stain and real-time PCR. Then, we explored the in vivo regulatory effects of hPMSCs in an experimental mouse model of GO. We developed the GO mouse model using immunization by leg muscle electroporation of pTriEx1.1Neo-hTSHR A-subunit plasmid. Human PMSC injection was performed into the left orbit. We also analyzed the effects of hPMSCs in the GO animal model. RESULT: We found that hPMSCs inhibited a lipid accumulation and activated factors, such as ADIPONECTIN, PPARγ, C/EBPα, and TGFß2 genes in adipogenesis-induced primary orbital fibroblasts from GO patients. Moreover, hPMSCs were highly effective at ameliorating adipogenesis in the orbital tissue of the model. CONCLUSION: These data indicate that hPMSCs recover pathogenic activation of orbital fibroblasts in animals undergoing experimental GO and confirm the feasibility of applying hPMSCs as a novel treatment for GO patients.

PH20 Inhibits TGFß1-Induced Differentiation of Perimysial Orbital Fibroblasts via Hyaluronan-CD44 Pathway in Thyroid-Associated Ophthalmopathy.

Purpose: Hyaluronan (HA) is a potential regulator of TGFß1-induced differentiation in perimysial orbital fibroblasts (pOFs). Our study aimed to explore the effects of PH20 (a hyaluronidase) and HA on TGFß1-induced differentiation in pOFs cultured from thyroid-associated ophthalmopathy (TAO) and control subjects. Methods: TAO and control pOFs (passages 3-6) were incubated with TGFß1 ± PH20 or TGFß1 ± HA for 72 hours and processed for various analyses, including HA electrophoresis; αSMA immunofluorescence; and quantification of αSMA (encoded by ACTA2), CD44 (a cell surface HA receptor), and SMAD2/3 (signaling molecule in the TGFß1 pathway). Results: TGFß1 induced myogenic differentiation (marked by αSMA upregulation) of pOFs. After TGFß1 treatment, more HA accumulated in the TAO group than in the control group. PH20 mainly digested medium to small HA and increased the percentage of high molecular weight HA (HMW-HA) in total HA. Both PH20 and HMW-HA inhibited TGFß1-induced differentiation in the TAO group, but neither showed significant effects in the control group. CD44 level negatively correlated with ACTA2 level in the TAO group, but no correlation was detected in the control group. Both PH20 and HMW-HA upregulated CD44 expression and inhibited SMAD2/3 expression in the TAO group, and the inhibitory effects were partially reversed by CD44 blockage. CD44 level in the control group was not affected by PH20 or HMW-HA treatment, and CD44 blockage showed no significant effects on control pOF differentiation. Conclusions: PH20 inhibits TGFß1-induced differentiation of TAO pOFs via HA-CD44-SMAD2/3 signaling, and the HA-CD44 signaling plays a divergent role in TAO and control pOFs.

Basic Fibroblast Growth Factor Induces Adipogenesis in Orbital Fibroblasts: Implications for the Pathogenesis of Graves' Orbitopathy.

BACKGROUND: Basic fibroblast growth factor (bFGF) has been implicated in the pathogenesis of Graves' orbitopathy (GO). It stimulates several processes, including hyaluronan synthesis, involved in orbital tissue volume expansion and may act synergistically with platelet-derived growth factor (PDGF)-BB. PDGF-BB is known to stimulate adipogenesis in orbital fibroblasts, but the effect of bFGF on adipogenesis in orbital fibroblasts is so far unknown. This study was conducted to determine whether (i) bFGF induces adipogenesis in orbital fibroblasts, (ii) bFGF and PDGF-BB together exert an additive or synergistic effect on adipogenesis, and (iii) treatment directed at bFGF- and PDGF-BB signaling may potentially be of interest for the treatment of GO. METHODS: Orbital fibroblasts from GO patients and controls were cultured in adipocyte differentiation medium with or without bFGF and/or PDGF-BB at different concentrations. Adipogenesis was determined by Oil Red O staining and messenger RNA expression of the late adipocyte differentiation markers cell death-inducing DFFA-like effector C (CIDEC) and adiponectin (ADIPOQ). To demonstrate involvement of FGF-receptor and PDGF-receptor signaling, experiments were also conducted in the presence of dasatinib (inhibitor of PDGF-receptor) or nintedanib (inhibitor of PDGF-receptor and FGF-receptor). RESULTS: bFGF significantly stimulated adipogenesis by orbital fibroblasts, as shown by increased Oil Red O staining and CIDEC and ADIPOQ expression after 14 days of differentiation. Furthermore, an additive effect of bFGF/PDGF-BB co-stimulation on adipogenesis was observed at the lowest concentration (12.5 ng/mL) of the growth factors tested. Nintedanib completely inhibited bFGF-, PDGF-BB-, and bFGF/PDGF-BB-induced adipogenesis, while dasatinib only fully abrogated PDGF-BB-induced adipogenesis. CONCLUSION: bFGF induces adipogenesis in orbital fibroblasts and as such may contribute to GO. The additive effect of bFGF and PDGF-BB on adipogenesis, along with the observed inhibitory effects of dasatinib and nintedanib, point at independent receptor-mediated effects. This supports the hypothesis that multi-target directed therapy might be more efficient in the treatment of GO.

CD34- Orbital Fibroblasts From Patients With Thyroid-Associated Ophthalmopathy Modulate TNF-α Expression in CD34+ Fibroblasts and Fibrocytes.

Purpose: Orbital fibroblasts from patients with Graves' disease (GD-OF) express many different cytokines when treated with bovine thyrotropin (bTSH). The present study aimed to determine why TNF-α cannot be induced by bTSH in GD-OF. Methods: Fibrocytes and GD-OFs were cultivated from donors who were patients in a busy academic medical center practice. Real-time PCR, Western blot analysis, reporter gene assays, cell transfections, mRNA stability assays, ELISA, and flow cytometry were performed. Results: We found that bTSH induces TNF-α dramatically in fibrocytes but is undetectable in GD-OF. The induction in fibrocytes is a consequence of increased TNF-α gene promoter activity and is independent of ongoing protein synthesis. It could be attenuated by dexamethasone and the IGF-1 receptor inhibiting antibody, teprotumumab. When separated into pure CD34+ OF and CD34- OF subsets, TNF-α mRNA became highly inducible by bTSH in CD34+ OF but remained undetectable in CD34- OF. Conditioned medium from CD34- OF inhibited induction of TNF-α in fibrocytes. Conclusions: Our data indicate that CD34- OF appear to release a soluble(s) factor that downregulates expression and induction by bTSH of TNF-α in fibrocytes and their derivative CD34+ OF. We proffer that CD34- OF produce an unidentified modulatory factor that attenuates TNF-α expression in GD-OF and may do so in the TAO orbit.

Depot-specific characteristics of adipose tissue-derived stromal cells in thyroid-associated orbitopathy.

BACKGROUND: Thyroid-associated orbitopathy (TAO) causes inflammatory fibroproliferation of periocular connective tissues. We compared adipose tissue-derived stem/stromal cells (ADSCs) from three adipose depots of each patient with TAO on mesenchymal, myofibrogenic, adipogenic properties and associated hyaluronan (HA) synthesis. METHODS: ADSCs were generated from periocular (eyelid, orbital) and subcutaneous (abdominal) adipose tissues of three patients with TAO. Mesenchymal markers were characterised by reverse transcription-PCR and immunofluorescent staining. A 3-week adipogenic induction was evaluated by Nile red staining and quantitative PCR (qPCR) of peroxisome proliferator-activated receptor (PPARγ), adiponectin and hyaluronan synthase (HAS)-2. A 7-day myofibrogenic induction was assayed by immunofluorescent staining and qPCR of α-smooth muscle actin (α-SMA). RESULTS: ADSCs from all depots expressed similar levels of mesenchymal markers CD44, CD90 and CD105 (p=0.288, p=0.43 and p=0.837, respectively). After adipogenic induction, intracellular lipid increased for more than 32% and PPARγ mRNA showed more than twofold increase from all three depots. However, adiponectin and HAS-2 mRNA levels were significantly higher in the eyelid and orbital ADSCs than those from the subcutaneous ADSCs after induction (2.4×107, 3.9×106 folds vs below detection limit; 63.3-fold, 26.1-fold, vs 33% reduction, respectively; all p=0.002). Significantly more myofibroblasts and higher mRNA level of α-SMA were obtained from the orbital and eyelid compared with the subcutaneous ADSCs during myofibrogenic induction (80.2%, 70.6% vs 29.3%; 30.2-fold, 24.2-fold vs 1.7-fold, respectively; all p=0.002). CONCLUSION: ADSCs from different adipose depots of the same donors exhibited similar mesenchymal phenotypes but differed significantly in adipogenic, myofibrogenic potentials and associated HA synthesis. These depot-specific characteristics of ADSCs may contribute to site-specific adipose tissue involvement in TAO.

In Vitro Modeling of Co-Transplantation of Multipotent Stromal Mesenchymal Cells from Orbital Fat Pad and Lipoaspirate of Human Subcutaneous Adipose Tissue in Organ Culture in Collagen Gel.

The interplay of multipotent stromal cells derived from the orbital fat pads and cells of the lipoaspirate from the subcutaneous adipose tissue was studied using in vitro co-transplantation model in an organ culture in a collagen gel. Microscopy findings and intensity of apoptosis and cell proliferation in cultures of lipoaspirate with and without multipotent stromal cells showed that the cells maintained their viability, proliferation capacity, and cytokine secretion activity. Higher proliferatitive activity of cells in cocultures promotes renewal of fat transplant cells and can help to maintain its stable volume in delayed terms after transplantation.

Comparison of Morphology of Adipose Body of the Orbit and Subcutaneous Fat in Humans.

We compared histological structure of the orbital and subcutaneous adipose tissue. Quantitative morphometry showed that the mean diameter of adipocytes and their volume density in the orbital adipose tissue were significantly lower than in the subcutaneous fat, while volume density of vessels and elements of the connective tissue in the orbital adipose tissue was higher. Previously known and revealed here differences in the structure of orbital and subcutaneous adipose tissue do not answer the question, whether subcutaneous adipose tissue can be used for replacement of the orbital tissue deficit.