A novel NADPH oxidase inhibitor targeting Nox4 in TGFβ-induced lens epithelial to mesenchymal transition

Many of the small molecule-based inhibitors of NADPH oxidase activity are largely inadequate to substantiate broad claims, often exhibiting a lack of Nox-isoform-specificity, and sometimes only acting as scavengers of reactive oxygen species (ROS). In the present study, we use a newly developed highly selective Nox4 inhibitor, GLX7013114, to modulate TGFβ-induced lens epithelial to mesenchymal transition (EMT). Rat lens epithelial explants were pre-treated with 0.3 μM of GLX7013114, and then treated with 200 pg/ml of TGF-β2 to induce lens EMT. ROS production was visualized microscopically using the superoxide fluorogenic probe, dihydroethidium (DHE). The EMT process was documented using phase-contrast microscopy, and molecular EMT markers were immunolabeled. qPCR was also performed to observe changes in EMT-associated genes. TGFβ-induced ROS was evident at 8 h of culture and its intensity was found to be significantly reduced when GLX7013114 was applied, comparable to ROS levels measured in untreated explants. Using phase-contrast microscopy to follow TGFβ-induced EMT over 5 days in the presence of the inhibitor, lens epithelial cells in explants became myofibroblastic by day 2 and underwent progressive apoptosis to reveal a bare lens capsule by day 5. Explants treated with TGFβ and GLX7013114 had some increased cell survival; however, these differences were not significant. For the first time, Nox4 inhibition by GLX7013114 was shown to reduce the TGFβ-induced gene expression of α-smooth muscle actin (αSMA), collagen 1a and fibronectin. GLX7013114, given that it appears to block aspects of TGFβ-induced EMT, including ROS production, may be a new useful Nox4-selective inhibitor for further studies. •Nox4 is involved in TGFβ-induced EMT in lens.•GLX7013114 is a new inhibitor preferentially targeting Nox4 over other Nox isoforms.•Selective Nox4 inhibition suppresses TGFβ-induced ROS production in lens.•GLX7013114 can downregulate TGFβ-induced expression of mesenchymal genes in lens epithelial cells.