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.

The microenvironment of solid tumors plays an essential role in tumor progression. In lung cancer, the stromal cells produce a fibronectin rich extracellular matrix which is known to contribute to both tumor metastasis and drug resistance. Due to its conformational lability, fibronectin is considerably remodeled by the contractile forces of the fibrotic microenvironment within the tumor stroma. As a result, the secondary structure of fibronectin's Type III domains is disrupted and the molecule becomes highly stretched. The contribution/impact of these strained forms of fibronectin on tumor growth and metastasis is not known. In the current study we show that the partially unfolded first Type III domain of fibronectin, III-1c, activates a toll-receptor/NF-κB pathway leading to an increase in the expression of IL-8. Using a 3-D model of tumor-associated extracellular matrix, we demonstrate that lung cancer cells seeded onto this matrix activate a TLR4/NF-κB signaling pathway leading to a robust increase in the release of IL-8. Cytokine release by these cells is completely dependent on the presence of fibronectin in the extracellular matrix. These findings suggest that paracrine signaling between the tumor and the stromal myofibroblasts causes a remodeling of the matrix fibronectin into a strained conformation which supports the activation of a TLR4/NF-κB signaling pathway resulting in the upregulation of fibro-inflammatory cytokines.

Objective: Dysfunctional remodeling of the extracellular matrix contributes to the formation of TLR-dependent feed forward loops that drive chronic inflammation. We have previously shown that two Type III domains of Fibronectin, FnEDA and FnIII-1c, cooperate to induce the synergistic release of interleukin 8 (IL-8) from dermal fibroblasts. We now identify steps in the TLR4 pathway where synergy can be demonstrated as well as additional kinases functioning in fibronectin activation of TLR4 signaling. We also evaluate the ligand and cell-type specificity of this synergistic response. Approach: FnEDA, FnIII-1c, and lipopolysaccharide (LPS)-induced genes in fibroblasts were analyzed by a quantitative reverse transcription-polymerase chain reaction (qPCR) and protein was measured by an enzyme-linked immunosorbent assay (ELISA). Kinases functioning in gene expression were identified by using specific inhibitors. Activated TLR4-dependent effector molecules were identified by cell fractionation and Western blot and quantified by image analysis. Results: The addition of FnEDA and FnIII-1c to dermal fibroblasts resulted in a synergistic increase in the expression of IL-8, tumor necrosis factor alpha (TNF-a), and vascular cell adhesion molecule (VCAM-1). Synergy between these domains was detected at the level of nuclear factor kappa-light chain enhancer of activated B cells (NF-jB) and inhibitor of kappa B kinase (IKK) activation. Induction of IL-8 by fibronectin ligands was partially attenuated in the presence of inhibitors to either epidermal growth factor receptor or Src kinases. FnIII-1c also synergized with LPS to induce IL-8 in dermal fibroblasts, whereas the combined effect of FnEDA and LPS on IL-8 synthesis was additive. In contrast, synergistic responses to these ligands were not observed in THP-1 monocytic cells. Innovation: The data suggest that chronic inflammation may be driven by matrix-and pathogen-derived TLR4 ligands that work in synergy to promote an exuberant innate response. Conclusion: The data suggest that the molecular mechanism underlying synergistic responses to TLR4 ligands lies upstream of IKK activation, likely in the molecular composition of the TLR4 receptor complex that assembles in response to each ligand. In addition, synergistic responses to TLR4 activation may be both cell-type and ligand specific. Keywords: fibronectin, TLR4, IL-8, inflammation, LPS INTRODUCTIONProlonged inflammation in response to injury is a contributory factor to the development of chronic wounds. Extracellular matrix (ECM)- fibronectin has also been shown to promote fibro-inflammatory responses in several mouse models. 9,17,18 The EDA domain of fibronectin can activate TLR4 signaling as either the individual domain, proteolytic fragment or in the context of the intact molecule. 19,20 Being responsible for matrix synthesis, assembly, and turnover, stromal fibroblasts are in constant dialogue with the fibronectin matrix in its polymerized and fragmented forms. Changes in tissue mechanical forces have been shown to caus...

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