Molecular biomarkers of Graves' ophthalmopathy

Abstract

Graves' ophthalmopathy (GO), a complication of Graves' disease (GD), is typified by orbital inflammation, ocular tissue expansion and remodeling and, ultimately, fibrosis. Orbital fibroblasts are key effectors of GO pathogenesis exhibiting exaggerated inflammatory and fibroproliferative responses to cytokines released by infiltrating immune cells. Activated orbital fibroblasts also produce inflammatory mediators that contribute to disease progression, facilitate the orbital trafficking of monocytes and macrophages, promote differentiation of matrix-producing myofibroblasts and stimulate accumulation of a hyaluronan-rich stroma, which leads to orbital tissue edema and fibrosis. Proteomic and transcriptome profiling of the genomic response of ocular and non-ocular fibroblasts to INF-γ and TGF-β1 focused on identification of translationally-relevant therapeutic candidates. Induction of plasminogen activator inhibitor-1 (PAI-1, SERPINE1), a clade E member of the serine protease inhibitor (SERPIN) gene family and a prominent regulator of the pericellular proteolytic microenvironment, was one of the most highly up-regulated proteins in INF-γ- or TGF-β1-stimulated GO fibroblasts as well as in severe active GD compared to patients without thyroid disease. PAI-1 has multifunctional roles in inflammatory and fibrotic processes that impact tissue remodeling, immune cell trafficking and survival as well as signaling through several receptor systems. This review focuses on the pathophysiology of the GO fibroblast and possible targets for effective drug therapy.

Keywords: Biomarkers; Fibrosis; Graves' disease; Inflammatory cytokines; Orbitopathy; PAI-1; Plasmin cascade; SERPINs; TGF-β; Tissue remodeling.

Figure 1.

Two-dimensional electrophoretic separations of de novo-synthesized ³⁵S-methionine-labeled cellular proteins from control and INF-γ-stimulated GO fibroblasts (A) confirmed a significant up-regulation (>16- to >40-fold) in the induced expression of the various distinct isoelectric variants of PAI-1 described previously (Higgins and Smith, 1993). PAI-1 map coordinates were confirmed using combined immunoblotting and 2-D gel separation criteria established previously (Higgins and Ryan, 1992). Individual protein spots were detected by fluorography and quantified with a Zeiss MOPS III digital image analyzer (Smith et al., 1992). I An approximately equal number of the resolved INF-γ-responsive GO fibroblast protein complement partitioned between the up- and down-regulated sets (B).2IndividuMM-dimensional electrophoretic protein maps derived from 2 individual strains of gamma- spot 55s and 65s-69s induction by interferon gamma in human orbital fibroblasts. Cells were incubated in control

Figure 2.

PAI-1 is a critical factor in the regulation of pericellular proteolysis and tissue fibrosis. Plasminogen activators (urokinase, uPA; tissue-type, tPA) are the physiologically relevant plasmin-generating proteinases that impact ECM homeostasis through a complex and interdependent proteolytic cascade. uPA-stimulated conversion of plasminogen to plasmin leads to an increased downstream activation of matrix metalloproteinases (MMPs). Collectively, plasmin and MMPs dictate the locale and extent of ECM remodeling. Increased PAI-1 expression and/or activity facilitates ECM accumulation and attenuates ECM degradation which, if prolonged or chronic, results in the onset and progression of fibrotic disease (reviewed in Flevaris and Vaughan, 2016; Rabieian et al., 2018; Milenkovic et al., 2017; Higgins et al., 2018).