Abstract
Purpose:
TGF-ß can induce an epithelial to mesenchymal transition (EMT) in lens, that arises from the aberrant growth and differentiation of lens epithelial cells. Studies in other models of EMT have shown that TGF-ß-driven EMT is dependent on the expression of the reactive oxygen species (ROS) producing enzyme NADPH oxidase 4, Nox4. We investigated the role of this enzyme in TGF-ß-induced lens EMT and determined whether it was required for this process.
Methods:
Rat lens epithelial explants were treated with a dose of TGF-β2 (1 ng/ml) known to result in an EMT. The development of the EMT phenotype (formation of myofibroblasts, cell loss, and capsular wrinkling) was documented using phase contrast microscopy. Over the culture period, the onset of Nox4 expression was determined by immunolabeling and was correlated with the localization of the common marker for EMT, α-smooth muscle actin. Nox4-produced ROS were visualized microscopically using the fluorescent probe, DHE. Nox4 was also inhibited pharmacologically, with a non-toxic concentration (5 μM) of VAS2870, in order to determine whether Nox4 activity was required for TGF-ß driven EMT.
Results:
Here we demonstrate for the first time in rat lens epithelial explants, that TGF-β treatment induces both Nox4 expression and activity (increase in DHE/ROS-labeling). Increased Nox4 expression was first detected at 6-8 hours following TGF-β treatment and was maintained in explants at 48 hours. At 8 hours post TGF-ß treatment, Nox4 was observed in cell nuclei, while at later stages in the EMT process, at 48 hours, Nox4 was predominately co-localized with α-smooth muscle actin. The inhibition of Nox4 expression and activity using VAS2870, inhibited EMT progression, as determined by reducing cell loss, abolishing α-smooth muscle actin expression, as well as the abrogation of lens capsular wrinkling
Conclusions:
TGF-β drives the expression of the ROS-producing enzyme Nox4 in the rat explant system. Nox4 contributes to the development of TGF-ß-induced EMT, as Nox4 inhibition impairs the EMT process. Experimental findings here may facilitate the prevention of the development of posterior capsular opacification in patients undergoing cataract surgery.