Abstract
Purpose: :
Smad3 signaling plays a central role in TGFβ-mediated corneal fibrosis, which is characterized by inflammation, expression of fibrotic markers, and the proliferation and transformation of fibroblasts. Gene therapy and small chemicals targeting Smad3 signaling have shown great potential for fibrosis management. Here we investigated two Smad3 chemical inhibitors in cultured corneal fibroblasts for their anti-fibrotic potencies.
Methods: :
Corneal fibroblasts were isolated from human donor cornea and cultured with one of two Smad3 inhibitors, halofuginone and SIS3, under TGFβ treatment. Halofuginone has previously been shown to be tolerated well in animals and humans with minimal toxicity; SIS3 also targets Smad3 with great potency yet its toxicity and clinical tolerance remain unknown. Activation of Smad3 signaling and other pathways, including p38MAP and Akt, were studied using phosphorylation-specific antibodies for Western blot experiments. The expression of fibrosis-related molecules such as alpha smooth muscle actin (α-SMA), fibronectin and collagen I were investigated by Western blot, immunostaining and quantitative reverse-transcription PCR (qRT-PCR). The contractile properties of the corneal fibroblasts were evaluated with a collagen gel contraction assay.
Results: :
We confirmed that TGFβ-induced Smad3 phosphorylation was significantly suppressed by each Smad3 inhibitor in corneal fibroblasts. Treating cells with SIS3 also activated the p38MAP pathway, although the underlying mechanism remains unclear. The TGFβ-induced expression of a fibrotic marker (α-SMA) and extracellular matrix proteins (fibronectin and collagen I) were effectively suppressed by halofuginone and SIS3 from the Western blot and qRT-PCR experiments. Corneal fibroblasts treated with halofuginone or SIS3 in the presence of TGFβ and then immunostained for α-SMA or stress fibers displayed reduced immunostaining signals. Lastly, Smad3 inhibitor-treated corneal fibroblasts embedded in collagen gels contracted to a much lesser extent compared to controls treated with or without TGFβ.
Conclusions: :
Our in vitro study has shown the effectiveness of halofuginone and SIS3 in suppressing the contraction of corneal fibroblasts and their expressions of fibrotic markers and extracellular matrix proteins. Our study presents opportunities for Smad3 inhibitors in ophthalmology for anti-fibrotic clinical use.
Keywords: cornea: stroma and keratocytes • gene/expression • cornea: basic science