Endocrine Abstracts

Introduction: In the pathogenesis of thyroid eye disease (TED) activated fibroblasts in the orbital connective tissue show increased proliferation, hyaluronan (HA) production, and adipogenic differentiation potential. As a result of these, the orbital tissue volume expands and the intraorbital pressure increases. Mechanical stimuli, including high intraorbital pressure, may lead to the activation of mechanosensitive receptors, such as Piezo1, whose presence in orbital fibroblasts (OF) and role in the adipogenesis of OF has not been studied yet. Piezo1 can be activated in vitro by its synthetic ligand, Yoda1.

Methods: TED orbital (n = 5) and NON-TED orbital (n = 5) fibroblast lines were studied. We analysed the expression of Piezo1 by Western blot and fluorescent imaging and its functionality by intracellular Ca measurement. On days 0, 4, 8 and 12 of in vitro induction of adipogenic differentiation, the effect of Piezo1 activation on lipid accumulation was measured by Oil Red O staining (ORO). RT-PCR was performed to analyse the expression of CEBPβ, CEBPδ, CEBPα, PPARy and FABP4.

Results: Functional Piezo1 expression was confirmed in OF. During adipogenesis, lipid accumulation increased at all examined time points (P < 0.0001); the proportion of cells entering adipocyte differentiation was intrinsically higher in TED OF, compared to NON-TED cultures. Piezo1 activation by Yoda1 treatment reduced lipid accumulation measured by ORO staining in TED OFs by an average of 14,2% on day 8 (P = 0.015) and 28.5 % on day 12 (P < 0.0001). After adipogenic induction CEBPβ and CEBPδ expression increased (P = 0.027 and P = 0.029, respectively), which was inhibited by Piezo1 activation (CEBPβ P = 0.038; CEBPδ P = 0.003). PPARγ and CEBPα mRNA expressions also increased under adipogenic conditions (P < 0.0001and P < 0.0001, respectively), which was strongly inhibited by Yoda1 treatment on day 8 (TED P < 0.0001) and day 12 (P < 0.0001). FABP4 expression was enhanced throughout adipogenesis (P < 0.0001); Yoda1 decreased it by day 8 and day 12 (P = 0.046 and P = 0.019, respectively).

Conclusions: Differentiation into adipocytes is characteristic of OF derived from the orbital connective tissue of TED patients. We found that OF express functional Piezo1 receptor. Intraorbital pressure modifies the mechanical properties of the tissue and presumably activates Piezo1, which, based on our in vitro results, inhibits the adipogenesis of fibroblasts by reducing the expression of the major transcription factors of the adipogenic cascade. The mechanosensitive ion channel Piezo1 may have a role in regulation of adipogenesis in the presence of increased intraorbital pressure. The investigation of the role of the Piezo1 receptor may help to identify new treatment options.