Alteration of Connective Tissue Growth Factor (CTGF) Expression in Orbital Fibroblasts from Patients with Graves' Ophthalmopathy.

Chieh-Chih Tsai,Yau-Huei Wei,Shi-Bei Wu,Pei-Chen Chang,Paula Soares

doi:10.1371/journal.pone.0143514

Abstract

Graves’ ophthalmopathy (GO) is a disfiguring and sometimes blinding disease, which is characterized by inflammation and swelling of orbital tissues, with fibrosis and adipogenesis being predominant features. The aim of this study is to investigate whether the expression levels of fibrosis-related genes, especially that of connective tissue growth factor (CTGF), are altered in orbital fibroblasts of patients with GO. The role of oxidative stress in the regulation of CTGF expression in GO orbital fibroblasts is also examined. By a SYBR Green-based real time quantitative PCR (RT-QPCR), we demonstrated that the mRNA expression levels of fibronectin, apolipoprotein J, and CTGF in cultured orbital fibroblasts from patients with GO were significantly higher than those of age-matched normal controls (p = 0.007, 0.037, and 0.002, respectively). In addition, the protein expression levels of fibronectin, apolipoprotein J, and CTGF analyzed by Western blot were also significantly higher in GO orbital fibroblasts (p = 0.046, 0.032, and 0.008, respectively) as compared with the control. Furthermore, after treatment of orbital fibroblasts with a sub-lethal dose of hydrogen peroxide (200 μM H2O2), we found that the H2O2-induced increase of CTGF expression was more pronounced in the GO orbital fibroblasts as compared with those in normal controls (20% vs. 7%, p = 0.007). Importantly, pre-incubation with antioxidants including N-acetylcysteine (NAC) and vitamin C, respectively, resulted in significant attenuation of the induction of CTGF in GO orbital fibroblasts in response to H2O2 (p = 0.004 and 0.015, respectively). Taken together, we suggest that oxidative stress plays a role in the alteration of the expression of CTGF in GO orbital fibroblasts that may contribute to the pathogenesis and progression of GO. Antioxidants may be used in combination with the therapeutic agents for effective treatment of GO.

Highlights

Graves’ ophthalmopathy (GO), known as thyroid-associated ophthalmopathy or thyroid eye disease, is clinically evident in 10–45% of patients with Grave’s disease [1]
By a SYBR-based RT-PCR, we first observed that the protein expression levels of fibronectin, apolipoprotein J and connective tissue growth factor (CTGF) were increased in the GO orbital fibroblasts as compared to those of normal subjects (p = 0.007, 0.037, and 0.002, respectively) (Fig 1A)
We found that the production of CTGF by GO orbital fibroblasts was increased after exposure to exogenous oxidative stress

Summary

Introduction

Graves’ ophthalmopathy (GO), known as thyroid-associated ophthalmopathy or thyroid eye disease, is clinically evident in 10–45% of patients with Grave’s disease [1]. Fibrosis represents a relative quiescent stage in the natural course of GO, it may cause much of the substantial morbidity of the patients including persistent lid retraction, restrictive strabismus, proptosis, exposure keratopathy, and optic nerve compression [4]. CTGF has been shown to be most critical It is a cysteine-rich protein (~40 kD) secreted by various cell types and is often co-expressed with transforming growth factor-β (TGF-β) [8]. They exert biological functions by binding to specific receptors and induce cell migration, proliferation, differentiation, extracellular matrix synthesis, and tissue fibrosis [9]. The role of CTGF in the fibrosis process of GO has not been clarified

Objectives

Methods

Results

Conclusion