ROCK inhibitor Netarsudil fails to suppress fibrogenesis genes in thyroid eye disease orbital fibroblasts, even upregulating some markers

Thyroid eye disease (TED) is a debilitating autoimmune orbital disorder characterized by significant fibroblast activation, their differentiation into myofibroblasts, and excessive extracellular matrix production, leading to tissue remodeling and fibrosis. Current therapeutic strategies often fall short in effectively managing the fibrotic component of TED, leaving a critical gap in treatment. The RhoA/ROCK signaling pathway has been increasingly implicated in these fibrotic processes across various tissues, making its inhibition a potential therapeutic target for conditions like TED.

Researchers investigated the effects of the Rho-associated protein kinase (ROCK) inhibitor netarsudil on fibrogenesis-related gene expression. Primary orbital fibroblasts were isolated from two patients diagnosed with Thyroid Eye Disease (TED). These cells were then treated with a range of netarsudil concentrations, specifically from 2 × 10- 2 to 2 × 10- 8 mg/mL. Gene expression levels of key fibrogenesis markers, including TGF-β, α-SMA, ZEB1, Snail, Thy-1, and β-catenin, were quantitatively assessed using quantitative real-time PCR (qPCR).

At non-cytotoxic concentrations, netarsudil treatment did not significantly suppress the mRNA expression of any of the examined fibrogenic markers. Surprisingly, the study observed an upregulation in the expression of TGF-β, α-SMA, and ZEB1 following netarsudil exposure. This suggests a lack of direct antifibrotic efficacy at doses that do not harm cell viability. Higher concentrations of netarsudil were found to reduce cell viability, indicating that any apparent antifibrotic effects observed at elevated doses might be secondary to general cytotoxicity rather than specific transcriptional repression of fibrotic pathways. This finding underscores the complexity of TED fibrogenesis, implying that targeting ROCK alone may be insufficient.

Non-cytotoxic doses of netarsudil failed to suppress, and in some cases upregulated, key fibrogenesis markers like TGF-β, α-SMA, and ZEB1 in TED orbital fibroblasts.