Gene expression and immunohistochemical study of orbital fat, extraocular muscle, and orbicularis oculi muscle in acute and chronic Graves’ ophthalmopathy -Insight to substitutive therapy for Graves’ ophthalmopathy
Date Issued
2007
Date
2007
Author(s)
Lee, Cheng-Wen
DOI
zh-TW
Abstract
Purpose: Graves’ disease (GD) is an autoimmune disease with hyperthyroidism, and about 25% to 50 % patients have Graves’ ophthalmopathy (GO). The manifestations of GO, including lid retraction, proptosis, soft tissue swelling, restrictive myopathy, and compressive optic neuropathy, can be explained by an increase in the volume of the adipose, connective tissue, and extraocular muscles within the orbit. Some patients predominantly present with proptosis, which comes from profound adipogenesis. Some patients present with diplopia and strabismus as a consequence of restrictive myopathy. And others present with both types of symptoms. The different presentation of different tissues may be due to the heterogeneity of fibroblasts. Recent studies implicate adipogenesis, accumulation of glycosaminoglycans (mainly hyaluronan), and fibrosis of muscle tissue are effects accompanied with inflammation. According to previous in vitro and in vivo studies of GO and other disease, IL-1β increases the expression of COX-2. COX-2 converts arachidonic acid directly into PGH2, then PGE2, hence inducing inflammation. On the other hand, PGH2 can be further metabolized to yield 15d-PGJ2, which binds PPAR-γ and then induces anti-inflammatory activity, together with adipogenesis. Through action of PGE2, COX-2 induces TGF-β and produces fibrosis, and activates HAS, starting accumulation of glycosaminoglycan. TGF-β can increase the formation of hyaluronan. In turn, hyaluronan and TGF-β can stimulate the production of COX-2 and prostaglandin. PPAR-γ disrupts TGF-β/Smad signal transduction and blocks profibrotic responses. Some of these responses may take part in the pathogenesis of GO. Many studies have focused on finding the alternative therapy, because sometimes the symptoms of patients in acute stage of GO are not improved by high dosage of steroids and that patients often have to suffer many side effects from long-tern usage of steroids. In this study, we try to link the results of immunohistochemical stain and gene expression of autoantigen, cytokines, genes relating with adipogenesis, and hyaluronan synthesis by studying orbital adipose tissue, orbicularis oculi muscle, and extraocular muscle from acute and chronic stage of GO, and to find out if there is increased expression of these genes. This adds more information on whether the hypothesis proposed from in vitro studies is possible or not in the pathogenesis of GO. We hope that after we have more understanding of this pathogenesis, in the future we can find an alternative therapy to steroids, such as PPAR-γ antagonist or COX-2 inhibitor, for the management of GO.
Methods: From chart review, we collected the clinical data. We collected orbital adipose tissue and orbicularis oculi muscle specimens from patients who received orbital decompression surgery due to compressive optic neuropathy in acute stage, and proptosis in chronic stage. Extraocular muscles were collected from patients who received muscle surgeries due to restrictive myopathy. Orbital adipose tissue, orbicularis oculi muscle and extraocular muscle specimens were collected from patients of non-GO receiving orbitotomy and surgeries for strabismus as control group. Specimens were processed for routine H&E stain and immunohistochemical stain with HLA-DR, CD3, CD20, and CD68. Messenger RNA was extracted for comparison of the expression of IGF-1α, IGF-1R, TSHr, IL-1β, IFN-γ, IL-6, TGF-β, COX-2, PPAR-γ, adiponectin, HAS, CXCL10, sFRP1, and CYR61.
Results: We collected orbital adipose tissue and orbicularis oculi muscle specimens from 10 patients in acute stage and 10 patients in chronic stage of GO receiving orbital decompression surgery. Extraocular muscles were collected from 5 patients who received muscle surgeries due to restrictive myopathy. Orbital adipose tissue specimens were collected from extra five GO patients in chronic stage for analysis of PPAR-γ. Orbital adipose tissues and orbicularis oculi muscles from 10 patients with non- GO and extraocular muscles from 5 patients with strabismus were collected as control group. The immunoreactive cells were more prominent in GO than in control group. Most of the HLA-DR (+) cells were CD20 (+) B lymphocytes, and then came CD3 (+) T lymphocytes. Only a few cells were CD68 (+) macrophages. As for gene expression, the levels were highest in chronic stage group and acute stage group, higher than control group in most genes at orbital adipose tissues and orbicularis oculi muscle tissues. Like other studies, we found the expression of many genes in orbital adipose tissues of GO was higher than control group (p<0.05), except adiponectin, IL-6, TGF-β, CXCL10, sFRP1, and CYR61. The expression of PPAR-γ was higher in 15 orbital adipose tissues of chronic GO than control group. In orbicularis oculi muscles, the expression of IL-6 and PPAR-γ was higher in GO group. In extraocular muscles, almost all genes, including genes of autoantigens, cytokines, COX-2, PPAR-γ, adiponectin, and other genes of hyaluronan synthesis, were up-regulated in GO group with a statistical significant difference (p<0.05).
Conclusion: In orbital adipose tissue and orbicularis oculi muscle specimens, most genes expressed more in chronic stage than acute stage. It possibly was under the anti-inflammation effect of recent and large dose of steroids used in patients in acute stage disease. Comparing GO groups with control group, almost all genes expressed higher in extraocular muscles, but the genes were totally different in orbital adipose tissues and orbicularis oculi muscles. This may be due to the heterogeneity of fibroblasts among different tissues. In diseased group, the expression of COX-2 and HAS in orbital adipose tissue and extraocular muscle was higher than control group. PPAR-γ in orbital adipose tissue, orbicularis oculi muscle, and extraocular muscle was up-regulated in GO. Also there was more expression of TGF-β1 in extraocular muscle of GO. This in vivo evidence gives us more information about the hypothesis proposed from in vitro studies. In brief, COX-2 produces 15d-PGJ2, which activates PPAR-γ and then induces anti-inflammatory activity, together with adipogenesis. Also COX-2 induces TGF-β and produces fibrosis, and activates HAS, starting accumulation of glycosaminoglycan. So besides steroid therapy, COX-2 inhibitor and PPAR-γ inhibitor are potential ways to be considered in patients of acute stage disease with severe orbital adipogenesis or restrictive myopathy. More studies will be helpful for us to get useful information on the safety and efficacy of COX-2 inhibitor or PPAR-γ antagonist in the treatment of Graves’ ophthalmopathy.
Subjects
葛瑞夫氏眼病變
眼窩脂肪組織
眼輪匝肌
眼外肌
免疫組織化學染色
定量即時聚合酶
連鎖反應
orbital adipose tissue
orbicularis oculi muscle
extraocular muscle
immunohistochemical study
Real-time PCR
Type
text
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