Abstract
Purpose: :
Infiltration of white blood cells and accumulation of extracellular matrix, especially hyaluronan (HA) in orbital tissue are characteristic of Thyroid eye disease (TED). Transforming growth factor beta (TGF-β) is up-regulated in orbital tissue from patients with TED where it acts as a key inducer of fibrosis by enhancing extracellular matrix production. 15-deoxy-Δ12,14-Prostaglandin J2 (15d-PGJ2) is an endogenous Peroxisome Proliferator-Activated Receptor gamma (PPARγ) ligand that exhibits diverse biological effects, including anti-inflammatory and anti-fibrogenic activities. In this study, we report that 15d-PGJ2 inhibits TGF-β-mediated responses in orbital fibroblasts.
Methods: :
Primary orbital fibroblasts were isolated from Graves’ disease patients undergoing orbital decompression surgery. The cells were grown in RPMI 1640 media containing 10% FBS. The amount of HA in the cell culture supernatant and pericellular extraction was measured by ELISA. Activated human T cell expression of CD44 was measured by flow cytometry. T cell adhesion to orbital fibroblasts was detected by measuring fluorescent intensity of bound calcein-AM labeled T cells. A fibrotic marker, alpha smooth muscle actin (α-SMA), was detected using western blot.
Results: :
TGF-β strongly induced HA levels in conditioned medium and pericellular extracts of human orbital fibroblasts after 24 hour treatment compared to untreated control. TGF-β also enhanced fibroblast-T cell adhesion and this cell-cell adhesion was inhibited by orbital fibroblast pre-incubation with hyaluronidase or HA synthase 2 siRNA, indicating that newly synthesized HA mediated the adhesion, probably through the interaction with its cellular receptor CD44 on the T cell surface. As a key inducer of fibrosis, TGF-β significantly up-regulated α-SMA protein levels in orbital fibroblasts. Remarkably, addition of the PPARγ ligand 15d-PGJ2 to TGF-β treated orbital fibroblasts blocked HA synthesis and T cell-fibroblast adhesion in a dose-dependent manner. In addition, 15d-PGJ2 also inhibited TGF-β induced α-SMA expression.
Conclusions: :
Our data reveal that 15d-PGJ2 is a potent inhibitor of TGF-β mediated pro-inflammatory and fibrogenic activities in orbital fibroblasts. Newly synthesized HA plays an impotent role in T cell-fibroblast adhesion. The ability to block HA synthesis with inhibitors such as 15d-PGJ2, therefore, presents a potential for therapeutic application.
Keywords: extracellular matrix • cell adhesions/cell junctions • drug toxicity/drug effects