Orbital Connective Tissue Research Articles

An L-type calcium channel blocker nimodipine exerts anti-fibrotic effects by attenuating TGF-β1 induced calcium response in an in vitro model of thyroid eye disease

BackgroundThyroid eye disease (TED) is a vision-threatening autoimmune disorder. Orbital tissue fibrosis leading to intractable complications remains a troublesome issue in TED management. Exploration of novel therapeutic targets and agents to ameliorate tissue fibrosis is crucial for TED. Recent work suggests that Ca2+ signaling participates in tissue fibrosis. However, whether an alteration of Ca2+ signaling has a role in fibrogenesis during TED remains unclear. In this study, we aimed to investigate the role of Ca2+ signaling in the fibrogenesis process during TED and the potential therapeutic effects of a highly selective inhibitor of the L-type calcium channel (LTCC), nimodipine, through a TGF-β1 induced in vitro TED model.MethodsPrimary culture of orbital fibroblasts (OFs) were established from orbital adipose connective tissues of patients with TED and healthy control donors. Real-time quantitative polymerase chain reaction (RT-qPCR) and RNA sequencing were used to assess the genes expression associated with LTCC in OFs. Flow cytometry, RT-qPCR, 5-ethynyl-2′-deoxyuridine (EdU) proliferation assay, wound healing assay and Western blot (WB) were used to assess the intracellular Ca2+ response on TGF-β1 stimulation, and to evaluate the potential therapeutic effects of nimodipine in the TGF-β1 induced in vitro TED model. The roles of Ca2+/calmodulin-dependent protein kinase II (CaMKII) and signal transducer and activator of transcription 1 (STAT1) in fibrogenesis during TED were determined by immunohistochemistry, WB, flow cytometry and co-immunoprecipitation assay. Selective inhibitors were used to explore the downstream signaling pathways.ResultsLTCC inhibitor nimodipine blocked the TGF-β1 induced intracellular Ca2+ response and further reduced the expression of alpha-smooth muscle actin (α-SMA), collagen type I alpha 1 (Col1A1) and collagen type I alpha 2 (Col1A2) in OFs. Besides, nimodipine inhibited cell proliferation and migration of OFs. Moreover, our results provided evidence that activation of the CaMKII/STAT1 signaling pathway was involved in fibrogenesis during TED, and nimodipine inhibited the pro-fibrotic functions of OFs by down-regulating the CaMKII/STAT1 signaling pathway.ConclusionsTGF-β1 induces an LTCC-mediated Ca2+ response, followed by activation of CaMKII/STAT1 signaling pathway, which promotes the pro-fibrotic functions of OFs and participates in fibrogenesis during TED. Nimodipine exerts potent anti-fibrotic benefits in vitro by suppressing the CaMKII/STAT1 signaling pathway. Our work deepens our understanding of the fibrogenesis process during TED and provides potential therapeutic targets and alternative candidate for TED.

Potential role of IGF-1R in the interaction between orbital fibroblasts and B lymphocytes: an implication for B lymphocyte depletion in the active inflammatory phase of thyroid-associated ophthalmopathy

BackgroundThyroid eye disease (TED) is an inflammatory process involving lymphocyte-mediated immune response and orbital tissue damage. The anti-insulin-like growth factor-1 receptor (IGF-1R) antibodies produced by B lymphocytes are involved in the activation of orbital fibroblasts and the inflammatory process of orbital tissue damage in TED. The purpose of this study was to explore the role of IGF-1R in the mechanistic connection between orbital fibroblasts and B lymphocytes in TED.MethodsOrbital fibroblasts sampled from orbital connective tissues and peripheral B lymphocytes isolated from peripheral blood, which were obtained from 15 patients with TED and 15 control patients, were co-cultured at a ratio of 1:20. The level of IGF-1R expression in orbital fibroblasts was evaluated by flow cytometry and confocal microscopy. Transient B lymphocyte depletion was induced with anti-CD20 monoclonal antibody rituximab, while the IGF-1R pathway was blocked by the IGF-1R binding protein. The expression levels of interleukin-6 (IL-6) and regulated upon activation, normal T cell expressed and secreted (RANTES) in the co-culture model were quantified via ELISA.ResultsIGF-1R expression was significantly elevated in TED orbital fibroblasts compared to that of controls. A 24-h co-culture of orbital fibroblasts with peripheral B lymphocytes induced elevated expression levels of IL-6 and RANTES in each group (TED patients and controls), with the highest levels occurring in TED patients (T + T group). Rituximab and IGF-1R binding protein significantly inhibited increased levels of IL-6 and RANTES in the co-culture model of TED patients.ConclusionsIGF-1R may mediate interaction between orbital fibroblasts and peripheral B lymphocytes; thus, blocking IGF-1R may reduce the local inflammatory response in TED. Rituximab-mediated B lymphocyte depletion played a role in inhibiting inflammatory responses in this in vitro co-culture model, providing a theoretical basis for the clinical application of anti-CD20 monoclonal antibodies in TED.

Cyclosporin A inhibits PDGF-BB induced hyaluronan synthesis in orbital fibroblasts

Orbital connective tissue changes are contributors to the pathogenesis in thyroid eye disease (TED). Activated fibroblasts respond to immune stimuli with proliferation and increased hyaluronan (HA) production. Cyclosporin A (CsA) was reported to be beneficial in the treatment of TED. PDGF isoforms are increased in orbital tissue of TED patients and enhance HA production. We aimed to study the effect of CsA on HA production and hyaluronan synthase (HAS1, 2 and 3) and hyaluronidase (HYAL1 and 2) mRNA expressions in orbital fibroblasts (OFs). Measurements were performed in the presence or absence of CsA (10 μM) in unstimulated or PDGF-BB (10 ng/ml) stimulated OFs. The HA production of TED OFs (n = 7) and NON-TED OFs (n = 6) were measured by ELISA. The levels of mRNA expressions were examined using RT-PCR. The proliferation rate and metabolic activity were measured by BrdU incorporation and MTT assays, respectively. Treatment with CsA resulted in an average 42% decrease in HA production of OFs (p < 0.0001). CsA decreased the expression levels of HAS2, HAS3 and HYAL2 (p = 0.005, p = 0.005 and p = 0.002, respectively.) PDGF-BB increased HA production (p < 0.001) and HAS2 expression (p = 0.004). CsA could reduce the PDGF-BB-stimulated HA production (p < 0.001) and HAS2 expression (p = 0.005) below the untreated level. In addition, CsA treatment caused a decrease in proliferation potential (p = 0.002) and metabolic activity (p < 0.0001). These findings point to the fact that CsA affects HA metabolism via HAS2, HAS3 and HYAL2 inhibition in OFs. In addition to its well characterized immunosuppressant properties, CsA's beneficial effect in TED may be related to its direct inhibitory effect on basal and growth factor stimulated HA production.

Expression of Collagen XIII in Tissues of the Thyroid and Orbit With Relevance to Thyroid-Associated Ophthalmopathy.

Antibodies against collagen XIII have previously been identified in patients with active thyroid-associated ophthalmopathy (TAO). Although collagen XIII expression has been described in extraocular muscles and orbital fat, its detailed localization in extraocular and thyroid tissues and the connection to autoimmunity for collagen XIII remain unclear. Our objective was to map the potential targets for these antibodies in the tissues of the orbit and thyroid. We evaluated the expression of collagen XIII in human patient and mouse orbital and thyroid tissues with immunostainings and RT-qPCR using Col13a1-/- mice as negative controls. COL13A1 expression in Graves' disease and goiter thyroid samples was compared with TGF-β1 and TNF, and these were also studied in human thyroid epithelial cells and fibroblasts. Collagen XIII expression was found in the neuromuscular and myotendinous junctions of extraocular muscles, blood vessels of orbital connective tissue and fat and the thyroid, and in the thyroid epithelium. Thyroid expression was also seen in germinal centers in Graves' disease and in neoplastic epithelium. The expression of COL13A1 in goiter samples correlated with levels of TGF-B1. Upregulation of COL13A1 was reproduced in thyroid epithelial cells treated with TGF-β1. We mapped the expression of collagen XIII to various locations in the orbit, demonstrated its expression in the pathologies of the Graves' disease thyroid and confirmed the relationship between collagen XIII and TGF-β1. Altogether, these data add to our understanding of the targets of anti-collagen XIII autoantibodies in TAO.

Finite element model of ocular adduction with unconstrained globe translation

Details of the anatomy and behavior of the structures responsible for human eye movements have been extensively elaborated since the first modern biomechanical models were introduced. Based on these findings, a finite element model of human ocular adduction is developed based on connective anatomy and measured optic nerve (ON) properties, as well as active contractility of bilaminar extraocular muscles (EOMs), but incorporating the novel feature that globe translation is not otherwise constrained so that realistic kinematics can be simulated. Anatomy of the hemisymmetric model is defined by magnetic resonance imaging. The globe is modeled as suspended by anatomically realistic connective tissues, orbital fat, and contiguous ON. The model incorporates a material subroutine that implements active EOM contraction based on fiber twitch characteristics. Starting from the initial condition of 26° adduction, the medial rectus (MR) muscle was commanded to contract as the lateral rectus (LR) relaxed. We alternatively modeled absence or presence of orbital fat. During pursuit-like adduction from 26 to 32°, the globe translated 0.52 mm posteriorly and 0.1 mm medially with orbital fat present, but 1.2 mm posteriorly and 0.1 mm medially without fat. Maximum principal strains in the optic disk and peripapillary reached 0.05–0.06, and von-Mises stress 96 kPa. Tension in the MR orbital layer was ~ 24 g-force after 6° adduction, but only ~ 3 gm-f in the whole LR. This physiologically plausible simulation of EOM activation in an anatomically realistic globe suspensory system demonstrates that orbital connective tissues and fat are integral to the biomechanics of adduction, including loading by the ON.

SOX9 Induces Orbital Fibroblast Activation in Thyroid Eye Disease Via MAPK/ERK1/2 Pathway.

To evaluate the expression of sry-box transcription factor 9 (SOX9) in orbital fibroblasts (OFs) of thyroid eye disease (TED) and to find its potential role and underlying mechanism in orbital fibrosis. OFs were cultured from orbital connective tissues obtained from patients with TED (n = 10) and healthy controls (n = 6). SOX9 was depleted by small interfering RNA or overexpressed through lentivirus transduction in OFs. Fibroblast contractile activity was measured by collagen gel contraction assay and proliferation was examined by EdU assay. Transcriptomic changes were assessed by RNA sequencing. The mRNA and protein levels of SOX9 were significantly higher in OFs cultured from patients with TED than those from healthy controls. Extracellular matrix-related genes were down-regulated by SOX9 knockdown and up-regulated by SOX9 overexpression in TED-OFs. SOX9 knockdown significantly decrease the contraction and the antiapoptotic ability of OFs, whereas the overexpression of SOX9 increased the ability of transformation, migration, and proliferation of OFs. SOX9 knockdown suppressed the expression of phosphorylated ERK1/2, whereas its overexpression showed the opposite effect. Epidermal growth factor receptor (EGFR) is one of the notably down-regulated genes screened out by RNA sequencing. Chromatin immunoprecipitation-qPCR demonstrated SOX9 binding to the EGFR promoter. A high expression of SOX9 was found in TED-OFs. SOX9 can activate OFs via MAPK/ERK1/2 signaling pathway, which in turn promotes proliferation and differentiation of OFs. EGFR was a downstream target gene of SOX9. SOX9/EGFR can be considered as therapeutic targets for the treatment of orbital fibrosis in TED.

Functional anatomy of the orbit in strabismus surgery: Connective tissues, pulleys, and the modern surgical implications of the "arc of contact" paradigm.

Oculomotricity is a multidimensional domain characterised by a delicate interplay of anatomical structures and physiological processes. This manuscript meticulously dissects the nuances of this interplay, bringing to the fore the integral role of the extraocular muscles (EOMs) and their intricate relationship with the myriad orbital connective tissues as it harmoniously orchestrates binocular movements, ensuring synchronised and fluid visual tracking. Historically, the peripheral oculomotor apparatus was conceptualised as a rudimentary system predominantly driven by neural directives. While widely accepted, this perspective offered a limited view of the complexities inherent in ocular movement mechanics. The twentieth century heralded a paradigm shift in this understanding. With advances in anatomical research and imaging techniques, a much clearer picture of the gross anatomy of the EOMs emerged. This clarity challenged traditional viewpoints, suggesting that the inherent biomechanical properties of the EOMs, coupled with their associated tissue pulleys, play a pivotal role in dictating eye movement dynamics. Central to this revised understanding is the "arc of contact" paradigm. This concept delves deep into the mechanics of eye rotation, elucidating the significance of the point of contact between the EOMs and the eyeball. The arc of contact is not just a static anatomical feature; its length and orientation play a crucial role in determining the effective torque generated by a muscle, thereby influencing the amplitude and direction of eye rotation. The dynamic nature of this arc, influenced by the position and tension of the muscle pulleys, offers a more comprehensive model for understanding ocular kinematics. Previously overlooked in traditional models, muscle pulleys have now emerged as central players in the biomechanics of eye movement. These anatomical structures, formed by dense connective tissues, guide the paths of the EOMs, ensuring that their pulling angles remain optimal across a range of gaze directions. The non-linear paths resulting from these pulleys provide a more dynamic and intricate understanding of eye movement, challenging two-dimensional, linear models of orbital anatomy. The implications of these revelations extend beyond mere theoretical knowledge. The insights garnered from this research promise transformative potential in the realm of strabismus surgery. Recognising the pivotal role of muscle pulleys and the "arc of contact" paradigm allows for more precise surgical interventions, ensuring better post-operative outcomes and minimising the risk of complications. Surgical procedures that previously relied on basic mechanical principles now stand to benefit from a more nuanced understanding of the underlying anatomical and physiological dynamics. In conclusion, this manuscript serves as a testament to the ever-evolving nature of scientific knowledge. Challenging established norms and introducing fresh perspectives pave the way for more effective and informed clinical interventions in strabismus surgery.

Intestinal permeability is associated with aggravated inflammation and myofibroblast accumulation in Graves' orbitopathy: the MicroGO study.

Graves' disease (GD) and Graves' orbitopathy (GO) result from ongoing stimulation of the TSH receptor due to autoantibodies acting as persistent agonists. Orbital pre-adipocytes and fibroblasts also express the TSH receptor, resulting in expanded retro-orbital tissue and causing exophthalmos and limited eye movement. Recent studies have shown that GD/GO patients have a disturbed gut microbiome composition, which has been associated with increased intestinal permeability. This study hypothesizes that enhanced intestinal permeability may aggravate orbital inflammation and, thus, increase myofibroblast differentiation and the degree of fibrosis. Two distinct cohorts of GO patients were studied, one of which was a unique cohort consisting of blood, fecal, and retro-orbital tissue samples. Intestinal permeability was assessed by measuring serum lipopolysaccharide-binding protein (LBP), zonulin, TLR5, and TLR9 ligands. The influx of macrophages and accumulation of T-cells and myofibroblast were quantified in orbital connective tissue. The NanoString immune-oncology RNA targets panel was used to determine the transcriptional profile of active fibrotic areas within orbital sections. GO patients displayed significantly higher LBP serum concentrations than healthy controls. Within the MicroGO cohort, patients with high serum LBP levels also showed higher levels of zonulin and TLR5 and TLR9 ligands in their circulation. The increased intestinal permeability was accompanied by augmented expression of genes marking immune cell infiltration and encoding key proteins for immune cell adhesion, antigen presentation, and cytokine signaling in the orbital tissue. Macrophage influx was positively linked to the extent of T cell influx and fibroblast activation within GO-affected orbital tissues. Moreover, serum LBP levels significantly correlated with the abundance of specific Gram-negative gut bacteria, linking the gut to local orbital inflammation. These results indicate that GO patients have enhanced intestinal permeability. The subsequent translocation of bacterial compounds to the systemic circulation may aggravate inflammatory processes within the orbital tissue and, as a consequence, augment the proportion of activated myofibroblasts, which actively secrete extracellular matrix leading to retro-orbital tissue expansion. These findings warrant further exploration to assess the correlation between specific inflammatory pathways in the orbital tissue and the gut microbiota composition and may pave the way for new microbiota-targeting therapies.

Sagging Eye Syndrome--an Overlooked Diagnosis

Background: Sagging Eye Syndrome (SES) is a relatively unknown cause for binocular distance diplopia. SES presents with an acquired comitant or non-comitant small angle esotropia and/or hypotropia commonly occurring in the elderly population. Inferior displacement of the lateral rectus secondary to age-related degeneration of orbital connective tissue and extraocular muscles appears to be responsible for the ocular misalignment. SES patients often present with new onset or progressively worsening distance diplopia, typically warranting neuroimaging. However, through understanding SES, eye care practitioners may identify these cases, avoiding unnecessary imaging studies. Case Report: A 90-year-old male presented with complaints of intermittent horizontal diplopia at distance despite being prescribed prism for presumed decompensating phoria at his most recent eye exam. The patient noted progressing diplopia which appeared worse in right gaze. Full binocular examination revealed a non-comitant esotropia worse at distance than near. External exam revealed prominent deep superior sulci, blepharoptosis, and orbital fat loss bilaterally. Due to the progressive nature and incomitancy of the diplopia, a neuro-ophthalmology consult was obtained, and the patient was diagnosed with SES. A clinical diagnosis was made based on history, adnexal features, and motility patterns distinct to SES, obviating the need for confirmatory imaging. Conclusion: Patients presenting with new onset diplopia secondary to undiagnosed SES may prompt expensive and time-consuming investigations. It is critical that eye care practitioners accurately recognize the signs, symptoms, and clinical features of SES to avoid unwarranted imaging and patient anxiety. This case report reviews the clinical presentation, exam findings, and distinct picture of SES required for diagnosis and necessary to differentiate this condition from more serious neurologic conditions. Treatment and management will be discussed.

Selective BD2 Inhibitor Exerts Anti-Fibrotic Effects via BRD4/FoxM1/Plk1 Axis in Orbital Fibroblasts From Patients With Thyroid Eye Disease.

We investigated the therapeutic potential of ABBV744, a bromodomain and extra-terminal (BET) inhibitor with selectivity for the second bromodomain (BD2) in thyroid eye disease (TED). The anti-fibrotic effects of ABBV744 and its underlying mechanism were explored in cultured orbital fibroblasts (OFs) from patients with TED. Immunohistochemistry (IHC) and real-time quantitative polymerase chain reaction (RT-qPCR) assays were conducted on orbital connective tissues from TED and controls. RT-qPCR, Western blot, Cell-counting Kit-8 (CCK-8), and 5-ethynyl-2'-deoxyuridine (EdU) cell proliferation assays were conducted on OFs isolated from patients with TED. The expression of BRD4 was upregulated in the orbital tissues of patients with TED relative to controls and in TED OFs stimulated with TGF-β1. Further, we showed that BRD4 modulated the profibrotic process through the interaction with Forkhead Box M1 (FoxM1) and its downstream molecule Polo-like kinase 1 (Plk1) in cultured TED OFs. Inhibition of BRD4 both by BD2 selective inhibitor ABBV744 and pan-BET inhibitor JQ1 exerted anti-fibrotic effects, whereas ABBV744 displayed superior anti-fibrotic effects and acceptable safety compared to JQ1. We conclude that BDR4 may modulate the profibrotic process in OFs of patients with TED via the FoxM1/Plk1 axis, and that selectively targeting BD2 domain of BRD4 may therefore be a potential therapeutic option for treating patients with TED.

Serelaxin Alleviates Fibrosis in Thyroid-Associated Ophthalmopathy via the Notch Pathway.

Fibrosis is the late stage of thyroid-associated ophthalmopathy (TAO), resulting in serious complications. Effective therapeutic drugs are still lacking. We aimed to explore the mechanism of TAO fibrosis and to find a targeted drug. High-throughput RNA sequencing was performed on orbital connective tissues from twelve patients with TAO and six healthy controls. Protein-protein interaction (PPI) network was constructed using the Search Tool for the Retrieval of Interacting Genes (STRING) database and we identified the hub gene by Cytoscape software. Additionally, the RNA sequencing results were validated by quantitative real-time polymerase chain reaction (qRT-PCR). Bioinformatic prediction identified the functions of differentially expressed genes (DEGs). Further orbital connective tissue and serum samples of the TAO and control groups were collected for subsequent experiments. Histologic staining, Western blotting (WB), qRT-PCR, enzyme-linked immunosorbent assays (ELISAs), gene overexpression through lentiviral infection or silencing gene by short interfering RNA (siRNA) were performed. We found that the relaxin signaling pathway is an important regulatory pathway in TAO fibrosis pathogenesis. Serelaxin exerts antifibrotic and anti-inflammatory effects in TAO. Furthermore, the downstream Notch pathway was activated by serelaxin and was essential to the antifibrotic effect of serelaxin in TAO. The antifibrotic effect of serelaxin is dependent on RXFP1.

RETRACTED ARTICLE: Relationship between smoking and serum levels of eye muscle and orbital connective tissue antibodies in patients with Graves ophthalmopathy

Over the past three decades, several studies have quantified the risk of smoking in the development of ophthalmopathy in patients with Graves' hyperthyroidism, with an overall odds ratio of approximately 3.0. Smokers also have a greater risk of more advanced ophthalmopathy than non-smokers. We studied 30 patients with Graves' ophthalmopathy (GO) and 10 patients with upper eyelid signs as the only manifestation of ophthalmopathy, whose eye signs were assessed using the clinical activity score (CAS), NOSPECS classes and upper eyelid retraction (UER) score, half of whom were smokers and half of whom were non-smokers. Serum levels of eye muscle (CSQ, Fp2, G2s) and orbital connective tissue type XIII collagen (Coll XIII) antibodies are valuable markers of ophthalmopathy in patients with Graves' disease. Still, their relationship to smoking has not been investigated. These antibodies were measured by enzyme-linked immunosorbent assay (ELISA) in all patients as a component of their clinical management. Mean serum antibody levels of all four antibodies were significantly greater in smokers than in non-smokers in patients with ophthalmopathy but not in those with upper eyelid signs only. As determined using one-way ANOVA and Spearman's correlation test, there was a significant correlation between smoking severity, assessed as pack-years, with mean Coll XIII antibody level, but not with levels of the 3 eye muscle antibodies. These results suggest that in patients with Graves' hyperthyroidism who smoke, the orbital inflammatory reactions are more advanced than in those with Graves' hyperthyroidism who do not smoke. The mechanism of this enhanced Autoimmunity against orbital antigens in smokers is unclear and worthy of further study.

M1-Like Macrophages Modulate Fibrosis and Inflammation of Orbital Fibroblasts in Graves' Orbitopathy: Potential Relevance to Soluble Interleukin-6 Receptor.

Background: Graves' orbitopathy (GO) is a disfiguring and sight-threatening autoimmune disease. Previous studies have shown the infiltration of macrophages in GO orbital connective tissues. However, the immunophenotypes of macrophages and their modulatory effects on orbital fibroblasts (OFs) have not been examined so far. In this study, we sought to determine the pathophysiology of macrophages in GO. Methods: In this case-control study, orbital connective tissues collected from 40 GO patients and 20 healthy controls were immunohistochemically stained for cytokines and macrophage cell surface antigens. The polarization of orbital-infiltrating macrophages was investigated by flow cytometry and immunofluorescence. Effects of interleukin (IL)-6 combined with soluble IL-6 receptor (sIL-6R) on the proliferation, differentiation, and inflammation of different OF subsets were examined by CCK-8, Western blotting, and Luminex assays, respectively. The antigen-presenting abilities of different OF subsets under IL-6/sIL-6R signaling were studied by proteomics. Finally, the differentiation of CD8+ IL-17A-producing T cells by sIL-6R was tested. Results: GO orbital connective tissues displayed increased IL-6, sIL-6R, STAT3, and IL-17A levels. CD86+ M1-like macrophages were predominant in active GO patients, while stable GO patients tended to have more CD163+ M2-like macrophages. The expression of IL-6 was higher in M1-like macrophages, and the expression of transforming growth factor-β was higher in M2-like macrophages both in GO orbital connective tissues in situ in vivo and in cell culture system in vitro. The IL-6/sIL-6R stimulation promoted the fibrosis of both CD34+ and CD34- OFs. Monocyte chemoattractant protein-1 expression was also induced by IL-6/sIL-6R stimulation in both OF subsets. IL-6/sIL-6R stimulation enhanced the antigen processing of CD34+ OFs through upregulating the intact major histocompatibility complex I and antigen transporters. However, the protein expressions of the thyrotropin receptor and insulin-like growth factor 1 receptor could not be directly increased by IL-6/sIL-6R stimulation in CD34+ OFs. Furthermore, sIL-6R was conducive to the differentiation of CD8+ IL-17A-producing T cells. Conclusions: Our study demonstrated the immunophenotypes of orbital-infiltrating macrophages that may activate OFs depending on the IL-6/sIL-6R signaling in GO. Our preclinical findings implicate, at least in part, the molecular rationale for blocking sIL-6R as a promising therapeutic agent for GO.

Glucocorticoids Directly Affect Hyaluronan Production of Orbital Fibroblasts; A Potential Pleiotropic Effect in Graves' Orbitopathy.

Orbital connective tissue expansion is a hallmark of Graves’ orbitopathy (GO). In moderate-to-severe active GO, glucocorticoids (GC) are the first line of treatment. Here we show that hydrocortisone (HC), prednisolone (P), methylprednisolone (MP), and dexamethasone (DEX) inhibit the hyaluronan (HA) production of orbital (OF) and dermal (DF) fibroblasts. HA production of GO OFs (n = 4), NON-GO OFs (n = 4) and DFs (n = 4) was measured by ELISA. mRNA expression of enzymes of HA metabolism and fibroblast proliferation was examined by RT-PCR and BrdU incorporation, respectively. After 24 h of GC treatment (1µM) HA production decreased by an average of 67.9 ± 3.11% (p < 0.0001) in all cell cultures. HAS2, HAS3 and HYAL1 expression in OFs also decreased (p = 0.009, p = 0.0005 and p = 0.015, respectively). Ten ng/mL PDGF-BB increased HA production and fibroblast proliferation in all cell lines (p < 0.0001); GC treatment remained effective and reduced HA production under PDGF-BB-stimulated conditions (p < 0.0001). MP and DEX reduced (p < 0.001, p = 0.002, respectively) PDGF-BB-induced HAS2 expression in OFs. MP and DEX treatment decreased PDGF-BB stimulated HAS3 expression (p = 0.035 and p = 0.029, respectively). None of the GCs tested reduced the PDGF-BB stimulated proliferation rate. Our results confirm that GCs directly reduce the HA production of OFs, which may contribute to the beneficial effect of GCs in GO.

Metformin Attenuates Inflammation and Fibrosis in Thyroid-Associated Ophthalmopathy

The pathogenesis of thyroid-associated ophthalmopathy (TAO) is still unclear, and therapeutic drugs have great limitations. As metformin has multiple therapeutic effects in many autoimmune diseases, we explored the effects of metformin on TAO in an in vitro fibroblast model. We used orbital connective tissues and fibroblasts that were obtained from TAO patients and normal controls. The activity of adenosine monophosphate-activated protein kinase (AMPK) and the levels of inflammatory or fibrotic factors were examined by immunofluorescence (IF) and immunohistochemistry (IHC). Quantitative real-time polymerase chain reaction (qPCR), cytokine quantification by enzyme-linked immunosorbent sssay (ELISA), IF, and western blotting (WB) were used to measure the expression of factors related to inflammation, fibrosis, and autophagy. To determine the anti-inflammatory and antifibrotic mechanisms of metformin, we pretreated cells with metformin, 5-aminoimidazole-4-carboxamide 1-β-D-ribofuranoside (AICAR, an AMPK activator) or compound C (CC, an AMPK inhibitor) for 24 h and used WB to verify the changes in protein levels in the AMPK/mammalian target of rapamycin (mTOR) pathway. We determined that the low activity of AMPK in the periorbital tissue of TAO patients may be closely related to the occurrence and development of inflammation and fibrosis, and metformin exerts multiple effects by activating AMPK in TAO. Furthermore, we suggest that AMPK may be a potential target of TAO therapy.

Differential Circular RNA Expression Profiling of Orbital Connective Tissue From Patients With Type I and Type II Thyroid-Associated Ophthalmopathy.

The purpose of this study was to investigate the molecular mechanism underlying thyroid-associated ophthalmopathy (TAO) clinical subtypes, to do so, we performed transcriptomic analysis to reveal the expression profile of circular RNAs (circRNAs) in TAO subtypes. High-throughput RNA-sequencing was performed in six pairs of type I and type II orbital connective tissue samples from patients with TAO. The expression levels of circRNAs and mRNAs in type I and type II samples were measured by quantitative real-time polymerase chain reaction (qRT-PCR) in another three pairs of type I and type II TAO connective tissue samples. We used bioinformatics predictions to construct a circRNA-microRNA (miRNA)-mRNA network. A protein-protein interaction (PPI) network was constructed based on differential mRNA expression, and the hub genes were determined by the Cytoscape software plugin. Functional and pathway enrichment analyses were performed to elucidate circRNA function. Lentiviral-mediated overexpression of hsa_circ_0007006 and the relationship between hsa_circ_0007006 with COL1A1 and MMP2 were evaluated by Western blotting (WB). Moreover, the differential pathways were assessed by WB. RNA sequencing results predicted a total of 7489 circRNAs and 15,803 mRNAs, with 94 upregulated and 76 downregulated circRNAs and 488 upregulated and 138 downregulated mRNAs. The qRT-PCR analysis of seven dysregulated circRNAs and two major mRNAs validated the RNA-sequencing data. The competing endogenous RNA (ceRNA) network included 7 circRNAs, 23 miRNAs, and 262 mRNAs. Functional analysis revealed several important pathways. Overexpression of hsa_circ_0007006 led to decreased expression levels of COL1A1 and MMP2. Activation of the relaxin signaling pathway differed between the two subtypes. We showed that circRNAs are differentially expressed between type I and type II TAO. We speculate that the hsa_circ_0007006-COL1A1 and MMP2-relaxin signaling pathways are important regulatory axes in the pathogenesis of this disease type and could be considered promising diagnostic and therapeutic targets in the future.

Cytokines in Thyroid-Associated Ophthalmopathy.

Thyroid-associated ophthalmopathy (TAO), also known as thyroid eye disease (TED) or Graves' orbitopathy (GO), is a complex autoimmune condition causing visual impairment, disfigurement, and harm to patients' physical and mental health. The pathogenesis of TAO has not been fully elucidated, and the mainstream view is that coantigens shared by the thyroid and orbit trigger remodeling of extraocular muscles and orbital connective tissues through an inflammatory response. In recent years, cytokines and the immune responses they mediate have been crucial in disease progression, and currently, common evidence has shown that drugs targeting cytokines, such as tocilizumab, infliximab, and adalimumab, may be novel targets for therapy. In this review, we summarize the research development of different cytokines in TAO pathogenesis in the hope of discovering new therapeutic targets.

Analysis of Facial Features of Patients With Sagging Eye Syndrome and Intermittent Exotropia Compared to Controls

To compare the facial features of patients with sagging eye syndrome (SES) and other ophthalmic diseases, and to evaluate the diagnostic usefulness of facial features for SES. Retrospective cross-section study. We evaluated frontal facial photographs of patients >60 years of age with SES and intermittent exotropia (IXT), and control patients who visited the ophthalmology outpatient clinics of 2 institutions between June 2020 and December 2021. Three ophthalmologists evaluated each eye for sunken upper eyelid, blepharoptosis, and baggy lower eyelid, using a scoring scale. The average scores for each parameter among the 3 groups were analyzed. Patients with glaucoma, visual acuity <16/20, SES with a vertical strabismus angle of ≥6 Δ, IXT that could not be maintained in the phoria position during photography, a history of previous oculoplastic or ophthalmic surgery, and use prostaglandin analogs for cosmetic purposes were excluded. A total of 86 patients were included: 23 with SES, 28 with IXT, and 35 in the control group. All were Japanese. In all, 45 patients were male and 41 were female. The mean age was 72.7 ± 7.4 years. The sunken upper eyelid scores were significantly higher in the SES group than in the control and IXT groups (P < .001), whereas the baggy lower eyelid scores were significantly higher in the IXT group than in the control group (P < .05). Age-related orbital connective tissue degeneration may manifest as SES in the upper eyelid and as IXT in the lower eyelid.

Therapeutic Effect of Gypenosides on Antioxidant Stress Injury in Orbital Fibroblasts of Graves' Orbitopathy.

Purpose To examine the impact of gypenosides (Gyps) on oxidative stress damage of orbital fibroblasts (OFs) from Graves' ophthalmopathy (GO) patients. Methods The relationship between Gyps and GO oxidative stress was understood by bioinformatics analysis. Orbital connective tissues of GO and non-GO patients were obtained for primary OF culture. The proliferation level of OFs was measured by Cell Counting Kit-8 method, and the appropriate intervention concentration of Gyps and H2O2 was obtained. The expression of apoptosis-related protein mRNA was analyzed by RT-qPCR technique. ROS and SOD test suites were employed to detect the oxidative stress level in OFs. Flow cytometry apoptosis detection, TUNEL detection, and lactate dehydrogenase detection were used to analyze the level of apoptosis. Western blotting detection was utilized to examine the regulatory pathway of oxidative stress, apoptosis, and autophagy-related proteins. The changes of cell morphology, autophagosome, and autophagy lysosome were observed by transmission electron microscope. Results The suitable intervention concentration of Gyps is 100 μg/mL, and the suitable intervention concentration of high concentration H2O2 is 350 μM. In comparison with the blank control group, the H2O2 intervention group enhanced the expression of apoptosis-related mRNA, the expression of ROS and SOD, the apoptosis rate, the expression of autophagy activation-related protein and Nrf2/ERK/HO-1 protein, and the number of autophagosomes and autophagy lysosomes. Compared with H2O2 intervention group, the expression of apoptosis-related mRNA decreased, ROS expression decreased, SOD expression increased, apoptosis rate decreased, autophagy activation-related protein expression decreased, Nrf2/ERK/HO-1 protein expression increased, and the quantity of autophagosomes and autophagy lysosomes decreased in H2O2 + Gyps intervention group. Conclusion Gyps can decrease the oxidative stress level of OFs generated by H2O2, reduce cell autophagy, and reduce apoptosis. Gyps may regulate the oxidative stress response of OFs in GO patients via the Nrf2/ERK/HO-1 signaling pathway.

Single-cell RNA sequencing depicts the local cell landscape in thyroid-associated ophthalmopathy

SummaryThere is a specific reactivity and characteristic remodeling of the periocular tissue in thyroid-associated ophthalmopathy (TAO). However, local cell changes responsible for these pathological processes have not been sufficiently identified. Here, single-cell RNA sequencing is performed to characterize the transcriptional changes of cellular components in the orbital connective tissue in individuals with TAO. Our study shows that lipofibroblasts with RASD1 expression are highly involved in inflammation and adipogenesis during TAO. ACKR1+ endothelial cells and adipose tissue macrophages may engage in TAO pathogenesis. We find CD8+CD57+ cytotoxic T lymphocytes with the terminal differentiation phenotype to be another source of interferon-γ, a molecule actively engaging in TAO pathogenesis. Cell-cell communication analysis reveals increased activity of CXCL8/ACKR1 and TNFSF4/TNFRSF4 interactions in TAO. This study provides a comprehensive local cell landscape of TAO and may be valuable for future therapy investigation.