Nuove sperimentazioni in vitro sulla terapia dell'oftalmopatia basedowiana

Nuove sperimentazioni in vitro sulla terapia dell'oftalmopatia basedowiana L’oftalmopatia basedowiana (OB) è una sindrome autoimmune caratteristicamente associata alle malattie autoimmuni della tiroide, in particolare al morbo di Basedow. L’OB è caratterizzata da una proliferazione incontrollata dei fibroblasti orbitari e da una aumentata produzione di glicosaminoglicani, in particolare acido ialuronico (HA). La terapia della OB grave è basata sull’uso di glucocorticoidi ad alte dosi. Nella OB lieve, invece, non ci sono terapie disponibili standardizzate e l’uso di glucocorticoidi ad alte dosi non è giustificato a causa del cattivo rapporto benefici/effetti collaterali. Per questo motivo sono in via di sperimentazione nuove terapie che prevedano l’impiego di farmaci possibilmente privi di effetti collaterali rilevanti. La presente tesi riporta i risultati di studi in vitro finalizzati a valutare l’effetto di alcuni farmaci potenzialmente candidati, su colture primarie di fibroblasti orbitari provenienti da pazienti con OB e da soggetti senza OB. I farmaci in questione sono: 1) Quercetina, un flavonoide con proprietà anti-ossidanti. A questo proposito si segnala che lo stress ossidativo è uno dei meccanismi implicati nella patogenesi dell’OB e che è stato precedentemente dimostrato un effetto anti-proliferativo della quercetina su fibroblasti di altra origine. 2) Enalapril, un farmaco anti-ipertensivo che è stato dimostrato avere un effetto anti-proliferativo su fibroblasti di altra origine, possibilmente dovuto ad una attività inbitoria del TGF- β , una citochina coinvolta nella sintesi di HA. 3) Selenio, una molecola con proprietà anti-ossidanti che è già stata dimostrata essere efficace nella terapia della OB lieve. I risultati hanno mostrato una azione anti-proliferativa sui fibroblasti orbitari da parte della quercetina e dell’enalapril, con inibizione della produzione di HA. Il trattamento con selenio dei fibroblasti ha determinato un aumento della vitalità cellulare, precedentemente ridotta da stress ossidativo, e sono in corso ulteriori studi volti a caratterizzare i meccanismi responsabili di questa osservazione. Tali risultati, sia pur preliminari, pongono le basi per un possibile impiego delle suddette sostanze nella pratica clinic

Graves Orbitopathy: from cell biology to molecular targeting

Context. Graves’ orbitopathy (GO) is an autoimmune disease persisting when immunosuppression is achieved. Orbital fibroblasts from GO patients display peculiar phenotypes even if not exposed to autoimmunity, possibly reflecting genetic or epigenetic mechanisms, furthermore the extent to which mononuclear cells and TSH-receptor autoantibodies (TRAb) contribute to Graves’ orbitopathy is not completely defined. Design. The design entailed the use of primary cultures of orbital fibroblasts from GO and control patients in order to understand any genetic or epigenetic mechanism involved in the development of the disease, and the investigation about the relationship between the immunohistochemical phenotype of orbital infiltrating cells and GO features in a large number of patients. Methods To understand the genetic and/or epigenetic mechanism underlying the GO peculiar phenotypes of fibroblasts, cell proliferation, hyaluronic acid (HA) secretion and HA synthases (HAS) were measured. Next Generation Sequencing and gene expression analysis of the whole genome were performed, as well as Real Time-PCR (RT-PCR) of selected genes and global DNA methylation assay. To assess the relationship between the immunohistochemical phenotype and GO feature, we conducted an observational cohort study in 76 consecutive patients with GO (16 men and 60 women) who underwent orbital decompression over a period of 18 consecutive months. An ophthalmological evaluation was performed in all patients, as well as immunohistochemistry for CD3, CD4, CD8, CD56 (T-cell markers), CD25 (T and B-cell marker), CD20, CD19 (B-cell markers), and CD138 (plasmacell marker) in specimens collected at decompressive surgery. Main Outcome Measure. Exome sequencing, gene expression of the whole genome, methylome analysis and immunohistochemical assay. Results. Cell proliferation was higher in GO than in control fibroblasts. Likewise, HA in the cell medium was higher in GO fibroblasts. HAS-1 and HAS-2 did not differ between GO and control fibroblasts, whereas HAS-3 was more expressed in GO fibroblasts. No relevant gene variants were detected by whole genome sequencing. However, 58 genes were found to be differentially expressed in GO compared with control fibroblasts and RT-PCR confirmed the findings in ten selected genes. We postulated that the differential gene expression was related to an epigenetic mechanism, reflecting diverse DNA methylation, which we therefore measured. In support of our hypothesis, global DNA methylation was significantly higher in GO fibroblasts. Regarding the immunohistochemical assay, having established cutoff values for each marker, cell infiltrates were found in 60 patients (78.9%; CD3: 39.4%, CD4 55.2%, CD8 50%, CD56: 0%, CD25: 28.9%, CD20: 51.3%, CD19: 25%, CD138: 26.3%). Eleven (14.4%) stained exclusively for CD138 (plasmacells). Patients with CD4-positive mononuclear cells had a significantly greater GO clinical activity score (CAS) (mean difference 1.07, 95% CI − 0.33 to − 1.82, P = 0.004 by univariate, P = 0.05 by multivariate analysis). CAS as well as the remaining GO features were not affected significantly by the mononuclear cell subpopulations in multivariate analyses. Conclusions. We propose that, following an autoimmune insult, DNA methylation elicits differential gene expression and sustains the maintenance of GO. From the immunohistochemical point of view mononuclear cell infiltrates are present in the majority of GO patients, with a small percentage represented exclusively by plasmacells. CD4 cells exert a major role on GO activity. These findings may represent a further advancement in the comprehension of GO pathogenesis

Mechanistic Pathways of Selenium in the Treatment of Graves' Disease and Graves' Orbitopathy

Based on the role of oxidative stress in the pathogenesis of Graves' hyperthyroidism and Graves' orbitopathy, the use of the antioxidant agent selenium has been proposed and several studies on the subject have been conducted, both in vitro and in vivo. Whereas a true benefit related to the use of selenium in patients with Graves' hyperthyroidism has been questioned, its use in patients with mild Graves' orbitopathy is generally believed to be beneficial because of which selenium has entered in the clinical practice for this eye condition

Selenium in Graves Hyperthyroidism and Orbitopathy

PURPOSE: To review the in vitro and in vivo studies supporting a role of selenium for the treatment of mild Graves orbitopathy.METHODS: Review of the current literature on the role of selenium in the management of Graves orbitopathy.RESULTS: Graves orbitopathy (GO) is a disfiguring and disabling disorder usually observed in patients with Graves hyperthyroidism, and more rarely in patients with hypothyroid autoimmune thyroiditis or in the absence of overt thyroid dysfunction. Noninvasive treatments include intravenous glucocorticoids and orbital radiotherapy and are generally offered to patients with moderately severe GO. In contrast, patients with mild GO are generally treated only with local measures. Thus, the benefits of intravenous glucocorticoids in mild GO are limited and do not justify the risks that the treatment carries. However, a medical treatment for mild GO is heavily wanted, as a relevant proportion of patients have a significant decrease in their quality of life, and GO can progress into more severe forms. Because of the role of oxidative stress in the pathogenesis of GO, an antioxidant approach has been proposed and the antioxidant agent selenium has been shown to be effective for GO.CONCLUSION: Studies have shown that a 6-month course of sodium selenite can improve the course of mild GO and prevent deterioration when compared with placebo

Binding of thyroglobulin (Tg) to the low-density lipoprotein receptor-associated protein (RAP) during the biosynthetic pathway prevents premature Tg interactions with sortilin

Sortilin, a Vps10p family member, is expressed by thyroid epithelial cells (TEC), where it binds to internalized thyroglobulin (Tg) molecules. Premature binding of Tg to sortilin during biosynthesis may cause intracellular retention of Tg. Such a premature interaction may be prevented by one or more inhibitor/s. Because both sortilin and Tg bind to the low-density lipoprotein receptor-associated protein (RAP), we investigated whether RAP serves such a function

Antioxidant effects of β-carotene, but not of retinol and vitamin E, in orbital fibroblasts from patients with Graves' orbitopathy (GO)

Background: Oxidative stress is involved in the pathogenesis of Graves' orbitopathy (GO) and several antioxidant agents, namely, selenium, quercetin, enalapril, vitamin C, N-acetyl-l-cysteine, and melatonin, have been shown to reduce oxidative stress and its consequences in primary culture of orbital fibroblasts. In addition, selenium is effective for the treatment of mild GO. Here, we investigated the action of three additional antioxidants in orbital fibroblasts, namely, retinol, β-carotene, and vitamin E. Methods: Primary cultures of orbital fibroblasts were established from GO patients and control subjects. To induce oxidative stress, cells were treated with H2O2, after which glutathione disulfide (GSSG) (a parameter of oxidative stress), cell proliferation, hyaluronic acid, TNFα, IFNγ, and IL1β were measured. Results: H2O2-dependent oxidative stress (augmented GSSG) was associated with increased cell proliferation and cytokine release. All the three antioxidant substances reduced GSSG in both GO and control fibroblasts.β-carotene reduced proliferation in GO, but not in control fibroblasts. IL1β was reduced by all three substances. Retinol reduced IFNγ in GO and control fibroblasts. Conclusions: Our study supports an antioxidant role of retinol, β-carotene, and vitamin E in orbital fibroblasts from patients with GO and provides a basis for a possible clinical use these substances

Action of three bioavailable antioxidants in orbital fibroblasts from patients with Graves’ orbitopathy (GO): a new frontier for GO treatment?

Objective: Oxidative stress is involved in the pathogenesis of Gravesâ\u80\u99 orbitopathy (GO) and an antioxidant approach has been advocated for GO treatment. Here, we investigated the action of three antioxidants in orbital fibroblasts, namely, vitamin C, N-acetyl-l-cysteine, and melatonin. Methods: Primary cultures of orbital fibroblasts from six GO patients and six control subjects were established. Cells were treated with H2O2 to induce oxidative stress. Cell vitality assays were performed to determine the non-cytotoxic dose of each antioxidant. The following assays were performed: glutathione disulfide (GSSG), as a measure of oxidative stress, cell proliferation, hyaluronic acid (HA), TNFα, IFNγ, and IL1β. Results: H2O2 induced oxidative stress (augmented GSSG), increased cell proliferation as well as cytokine release, but did not affect HA release. All of the three antioxidant substances reduced H2O2-dependent oxidative stress. Vitamin C reduced proliferation in GO, but not in control fibroblasts. N-acetyl-l-cysteine reduced proliferation and IFNγ in GO, and HA and IL1β in both GO and control fibroblasts. Melatonin reduced IL1β and HA in GO and control fibroblasts, and IFNγ only in GO fibroblasts. Conclusions: Our study provides evidence in support of an antioxidant role of vitamin C, N-acetyl-l-cysteine and melatonin in orbital fibroblasts. Some of the effects of these compounds are exclusive to GO fibroblasts, whereas some other are observed also in control fibroblasts. Our observations provide a basis for a possible clinical use of these substances in patients with GO