Hepatic fibrosis has been considered as an irreversible Imatinib process but recent experimental and clinical data indicate that removal of the pro-fibrotic agent or condition may reverse liver fibrosis [33]. Results presented in this manuscript suggest that either T��RI inhibition or NOX4 silencing reverses the MFB phenotype, decreasing the expression of extracellular matrix genes, such as collagen I or fibronectin, down-regulating ��-SMA and vimentin expression and changing cell morphology, which loses myofibroblastic appearance. Since it has been recently proposed that NOX4 modulates ��-SMA and procollagen I (alpha1) expression in pulmonary fibrosis by controlling activation of Smad2/3 [34], we checked the effect of silencing NOX4 on TGF-�� levels and Smad2/3 phosphorylation.

Our results have indicated that NOX4 clearly acts downstream TGF-��-independently from Smads activation. It also has been described that NOX4 is critical for maintenance of smooth muscle gene expression in vascular smooth muscle cells [35], where ROS production impairs the TGF-��-induced phosphorylation of Ser103 on serum response factor (SRF) and reduces its transcriptional activity. Thus, NOX4 may play an important role associated with the ��-SMA phenotype, being not only important in fibrotic processes, but also in cardiovascular physiology. We have previously described that liver cells respond to TGF-�� in vitro undergoing EMT [25], [36]. The role of EMT is perhaps the most intriguing and controversial of recent hypothesis on the origin mechanisms of liver fibrosis [37].

Strong evidences indicate that hepatocytes from transgenic animals that overexpress Snail (a master gene involved in EMT through its capacity to repress E-cadherin gene, among others) fully undergo EMT [38] and may propagate liver fibrosis progression [39]. However, under normal genetic background, data from different experimental approaches in animals and humans show controversy [40]. Our results show that EMT occurs in hepatocytes in vitro, but NOX4 is not required for this process. However, NOX4 mediates TGF-��-induced cell death that is prevented in the presence of antioxidants. In agreement with these results, it has been recently proposed a role for NOX4 in epithelial cell death during development of bleomycin-induced lung fibrosis [41].

Using a model of NOX4-deficient mice, authors demonstrated that these animals were resistant to fibrosis due to the abrogation of TGF-��-induced apoptosis in epithelial cells. Prevention of apoptosis impaired fibrosis development, although inflammation was comparable to wild-type. A similar situation may occur in liver fibrosis, where engulfment of apoptotic bodies by HSC contributes to induce their activation [7]. Indeed, GSK-3 hepatocyte apoptosis not only would facilitate fibrosis through blocking liver regeneration, but it could play an active role.