Endocrine Abstracts

Objectives: Differentiation of orbital fibroblasts (OFs) into myofibroblastic phenotype contributes to tissue remodelling in thyroid eye disease (TED). Our study aimed to investigate the impact of TGF-β1 induced myofibroblast differentiation on hyaluronan turnover, with a particular focus on the expression of its key enzymes.

Methods: OF cultures were established from tissue samples acquired from decompression surgeries of patients with TED (TED-OFs; n = 4) and during other ophthalmic surgeries (enucleation) for non-orbital eye diseases as controls (non-TED-OFs; n=5). To induce myofibroblast differentiation, the medium was supplemented with TGF-β1 (5ng/ml). Measurements were performed after 24 and 72 hours. Proliferation rate was determined by the detection of BrdU incorporation. Hyaluronan content of the supernatant and the amount of pericellular hyaluronan were measured using an ELISA-like technique. The mRNA expression of myofibroblast markers and enzymes playing a pivotal role in hyaluronan metabolism were determined by real-time PCR.

Results: Upregulation in the mRNA expression of alpha-1 type-1 collagen, α-smooth muscle actin and fibronectin indicated that OFs underwent myofibroblast transdifferentiation after stimulation by TGF-β1. After 72 hours, proliferation rate of both untreated and treated cultures declined; the decrease in the proliferative capacity was less marked in TGF-β1 treated (i.e. myofibroblast) cells (p <0.0005). In parallel the amount of hyaluronan in the pericellular coat, but not in the supernatant of myofibroblasts, increased compared to untreated cells by 72 hours (non-TED-OFs p <0.0001, TED-OFs p <0.0001). TGF-β1 was a potent stimulator of hyaluronan synthase-1 (HAS-1) expression (non-TED-OFs p < 0.0001, TED-OFs p <0.0001 at both time points), while no significant increase was found in the expression of HAS2 and HAS3. The expression of both type 1 hyaluronidase (HYAL-1) and cell migration inducing protein (CEMIP, a recently discovered hyaluronidase) diminished following myofibroblast differentiation at 72h (non-TED-OFs p <0.0001, TED-OFs p <0.0001 and non-TED-OFs p <0.001, TED-OFs p <0.001, respectively). The expression of transmembrane protein 2 (TMEM2), the regulator of hyaluronan catabolism through CEMIP was elevated after 72 hours of TGF-β1 treatment (non-TED-OFs p <0.01, TED-OFs P < 0.05).

Conclusions: Orbital fibroblasts undergoing myofibroblast transdifferentiation are characterized by decreased hyaluronan turnover due to the downregulated expression of two hyaluronidases. The accumulation of hyaluronan in the pericellular coat leads to an increase in oedema due to its large water binding capacity, which can negatively affect the course of TED. Our results suggest that hyaluronidases could be potential targets in the treatment of TED.