Arsenic trioxide inhibits transforming growth factor-β1-induced fibroblast to myofibroblast differentiation in vitro and bleomycin induced lung fibrosis in vivo

Background: Idiopathic pulmonary fibrosis (IPF) is a progressive disease of insidious onset, and is responsible for up to 30,000 deaths per year in the U. S. Excessive production of extracellular matrix by myofibroblasts has been shown to be an important pathological feature in IPF. TGF-beta 1 is expressed in fibrotic lung and promotes fibroblast to myofibroblast differentiation (FMD) as well as matrix deposition. Methods: To identify the mechanism of Arsenic trioxide's (ATO)'s anti-fibrotic effect in vitro, normal human lung fibroblasts (NHLFs) were treated with ATO for 24 hours and were then exposed to TGF-beta 1 (1 ng/ml) before harvesting at multiple time points. To investigate whether ATO is able to alleviate lung fibrosis in vivo, C57BL/6 mice were administered bleomycin by oropharyngeal aspiration and ATO was injected intraperitoneally daily for 14 days. Quantitative real-time PCR, western blotting, and immunofluorescent staining were used to assess the expression of fibrotic markers such as alpha-smooth muscle actin (alpha-SMA) and alpha-1 type I collagen. Results: Treatment of NHLFs with ATO at very low concentrations (10-20nM) inhibits TGF-beta 1-induced alpha-smooth muscle actin (alpha-SMA) and alpha-1 type I collagen mRNA and protein expression. ATO also diminishes the TGF-beta 1-mediated contractile response in NHLFs. ATO's down-regulation of profibrotic molecules is associated with inhibition of Akt, as well as Smad2/Smad3 phosphorylation. TGF-beta 1-induced H2O2 and NOX-4 mRNA expression are also blocked by ATO. ATO-mediated reduction in Smad3 phosphorylation correlated with a reduction of promyelocytic leukemia (PML) nuclear bodies and PML protein expression. PML-/- mouse embryonic fibroblasts (MEFs) showed decreased fibronectin and PAI-1 expression in response to TGF-beta 1. Daily intraperitoneal injection of ATO (1 mg/kg) in C57BL/6 mice inhibits bleomycin induced lung a-1 type I collagen mRNA and protein expression. Conclusions: In summary, these data indicate that low concentrations of ATO inhibit TGF-beta 1-induced fibroblast to myofibroblast differentiation and decreases bleomycin induced pulmonary fibrosis.