Inhibition of TSH/IGF-1 receptor crosstalk by Teprotumumab as a treatment modality of Thyroid Eye Disease

Context We previously presented evidence that TSH receptor (TSHR)-stimulating autoantibodies (TSAbs) bind to and activate TSHRs but do not bind to IGF1 receptors (IGF1Rs). Nevertheless, we showed that IGF1Rs were involved in thyroid eye disease (TED) pathogenesis because TSAbs activated crosstalk between TSHR and IGF1R. Teprotumumab, originally generated to inhibit IGF1 binding to IGF1R, was recently approved for the treatment of TED (Tepezza®). Objective To investigate the role of TSHR/IGF1R crosstalk in teprotumumab treatment of TED. Design We used orbital fibroblasts from patients with TED (TEDOFs) and measured stimulated hyaluronan (HA) secretion as a measure of orbital fibroblast activation by TED immunoglobulins (TED-Igs) and monoclonal TSAb M22. We previously showed that M22, which does not bind to IGF1R, stimulated HA in a biphasic dose-response with the higher-potency phase dependent on TSHR/IGF1R crosstalk and the lower-potency phase independent of IGF1R. Stimulation by TED-Igs and M22 was measured in the absence or presence of Teprotumumab Biosimilar (Tepro) or K1-70, an antibody that inhibits TSHR. Results We show: 1) Tepro dose-dependently inhibits stimulation by TED-Igs; 2) Tepro does not bind to TSHRs; 3) Tepro inhibits IGF1R-dependent M22-induced HA production, which is mediated by TSHR/IGF1R crosstalk, but not IGF1R-independent M22 stimulation; and 4) β-arrestin 1 knockdown, which blocks TSHR/IGF1R crosstalk, prevents Tepro inhibition of HA production by M22 and by a pool of TED-Igs. Conclusion We conclude that Tepro inhibits HA production by TEDOFs by inhibiting TSHR/IGF1R crosstalk and suggest that inhibition of TSHR/IGF1R crosstalk is the mechanism of its action in treating TED.

Wnt signalling inhibits adipogenesis in orbital fibroblasts from patients with Graves’ orbitopathy

Background/AimsTo investigate the role of Wnt signalling in adipogenesis using an in vitro model of Graves’ orbitopathy (GO).MethodsOrbital fat was obtained from patients with GO and non-GO participants for primary orbital fibroblast (OF) culture. Expression levels of Wnt5a, Wnt10b, β-catenin, phospho-β-catenin and cyclin D1 were compared between GO and non-GO OFs. These expression levels were also determined during adipogenesis of GO and non-GO OFs. The effects of a stimulator and inhibitor of Wnt signalling on adipogenesis of GO and non-GO OFs were investigated.ResultsWestern blotting analysis showed significant reductions in β-catenin and cyclin D1 and significant enhancement of phospho-β-catenin in OFs from patients with GO, compared with OFs from non-GO participants (p<0.05). Expression of Wnt5a, Wnt10b, β-catenin and cyclin D1 in OFs was highest on day 0, and then gradually declined after induction of adipogenic differentiation. The expression levels of PPARγ, C/EBPα and C/EBPβ were reduced in Wnt stimulator-treated OFs in a dose-dependent manner. Oil red O staining confirmed that a stimulator of Wnt inhibited adipogenesis in GO OFs.ConclusionThese results indicate that Wnt signalling inhibits adipogenesis in OFs from patients with GO and non-GO participants. Further studies are required to examine the potential of Wnt signalling as a target for therapeutic strategies.

LncRNA LPAL2/miR-1287-5p/EGFR axis modulates TED derived orbital fibroblast activation through cell adhesion factors

Background and aims The activation of orbital fibroblasts, the prime targets in thyroid eye disease, is central to its underlying pathogenesis. We aimed to investigate the mechanism of thyroid eye disease orbital fibroblast activation from the perspective of non-coding RNA regulation. Methods Immunofluorescence (IF) staining was applied to evaluate the fibrotic changes in target cells. Cell proliferation were evaluated by EDU and colony formation assays. Collagen I concentration was determined by ELISA assay. Human microarray analysis was performed on three thyroid eye disease and 3 healthy control orbital tissue samples. Results Bioinformatics analysis showed that cell adhesion signaling factors were differentially expressed in thyroid eye disease tissues, including I-CAM-1, I-CAM-4, V-CAM, and CD44, which were all upregulated in diseased orbital tissues. LncRNA LPAL2 level was also upregulated in orbital tissues and positively correlated with I-CAM-1 and I-CAM-4 expression. Stimulation of the thyroid eye disease orbital fibroblasts by TGF-β1 significantly increased the expression of I-CAM-1, I-CAM-4, and LPAL2. Knockdown of LPAL2 in orbital fibroblasts inhibited TGF-β1-induced increases in cell adhesion factor levels and orbital fibroblast activation. Microarray profiling was performed on thyroid eye disease and normal orbital tissues to identify differentially expressed miRNAs and miR-1287-5p was remarkably reduced within diseased orbital samples. miR-1287-5p was directly bound to EGFR 3’UTR and LPAL2 and LPAL2 modulated EGFR/AKT signaling through targeting miR-1287-5p. Conclusions The LPAL2/miR-1287-5p axis modulated TGF-β1-induced increases in cell adhesion factor levels and thyroid eye disease orbital fibroblast activation through EGFR/AKT signaling.

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

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.

Fucoxanthin improves functional recovery of orbitopathy in Graves’ disease by downregulating IL-17 mRNA expression in a mouse model

Purpose: To explore the efficacy of fucoxanthin (FX), a carotenoid, against inflammation via inhibition of IL-17 mRNA expression, and its anti-oxidant activity in Graves’ orbitopathy (GO)-induced mice model.Methods: The effects of FX on IL-6, IL-8, IL-17, MCP-1, and TNF-α, in orbital fibroblast tissues extracted from GO-induced BALB/c mice was investigated. Anti-oxidative stress markers, 8-hydroxy-2’- deoxyguanosine (8-OHdG) and malondialdehyde (MDA) levels were quantified in tear samples collected from GO-induced FX treated mice.Results: FX administration in cultured human orbital fibroblast cells revealed almost complete cell viability and no cell apoptosis. FX resulted in IL-1β induced Beclin-1 and Atg-5 silencing, in cultured human orbital fibroblasts. BALB/c mice immunized with Ad-TSHR289 indicated elevated levels ofthyroid peroxidase and thyroglobulin antibodies in the serum sample. FX predominantly downregulated the mRNA expression of IL-17, and also reduced increased 8-OHdG and MDA in the tear secretion of GO-induced mice.Conclusion: FX may be an effective and useful molecule for the treatment of GO, through its antiinflammatory and anti-oxidative potential, but it requires further investigation to ascertain its therapeutic effectiveness. Keywords: Anti-inflammatory, Anti-oxidant, Fucoxanthin, Graves’ disease, Graves’ orbitopathy, IL-17

Functionally enhanced placenta-derived mesenchymal stem cells inhibit adipogenesis in orbital fibroblast with graves’ ophthalmopathy

Background: Placenta-derived mesenchymal stem cells (PD-MSCs) have unique immunomodulatory properties, and Phosphatase of regenerating liver-1 (PRL-1) regulates 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 contents involving adipose tissue. Because the mechanism of inhibiting adipogenesis in orbital fibroblast (OF) with GO patients remains uncertain, the major objective is to investigate mechanisms alleviating adipogenesis by PRL-1 overexpressing PD-MSCs (PD-MSCsPRL-1, PRL-1+) in OF derived from GO patients. Methods: primary OFs from patients with GO were isolated from orbital adipose tissue specimens. After maturation as adipogenic differentiation, normal and GO-derived OF were cocultured with naïve and PD-MSCsPRL-1. Western blotting were conducted for evaluating molecular mechanisms for inhibiting adipogenesis in GO. Results: The characterizations of PD-MSCsPRL-1 were similar to naïve. OF with GO patients stimulated adipocyte differentiation were significantly decreased lipid accumulation by cocultivation with PD-MSCsPRL-1 comparing with naïve. The mRNA and protein expression of adipogenic markers was declined in PD-MSCsPRL-1. The expression of pPI3K/AKT/mTOR protein in OF with GO patients was downregulated by cocultivation with PD-MSCsPRL-1 secreted IGFBPs. Interestingly, IGFBP2, 4, and 7 expressions through integrin alpha 4 (ITGA4) and beta 7 (ITGB7) in PD-MSCsPRL-1 were higher than naïve and upregulated pFAK downstream factor. Conclusion: Taken together, secreted IGFBPs by PD-MSCsPRL-1 via upregulating FAK and blocking IGF inhibit adipogenesis of OF with GO patients, providing novel therapeutic approach using functionally enhanced MSCs potential for degenerative diseases.

Pharmacological Effects of Verticine: Current Status

Verticine is the major bioactive constituent of Fritillaria as a kind of Traditional Chinese Medicine. Pharmacological researches have reported various benefits of verticine, including anticancer, anti-inflammatory, protecting against acute lung injury, tracheobronchial relaxation, antitussive, expectorant, sedative, and analgesic activities, in addition to inhibiting proliferation of cultured orbital fibroblast, angiotensin converting enzyme (ACE), and acetylcholinesterase (AChE) and inhibiting hERG potassium channels. The underlying mechanisms of verticine are still under investigation. This review will comprehensively summarize the metabolism, biological activities, and possible mechanism of verticine.