There is a strong association between fibrotic lung disease and oxidative stress. Recent studies from our laboratory implicate the reactive oxygen species-generating enzyme, NADPH oxidase-4 (N0X4) in lung fibrosis. N0X4 mediates myofibroblast differentiation, matrix synthesis, and contractility; siRNA targeting of NOX4 abrogates fibrosis in animal models of non-infectious lung injury. Other reported studies of systemic N0X4 deficiency in mice further support the concept that N0X4 is critical for in-vivo fibrogenesis; however, the mechanisms and potential cell-specific effects of N0X4 remain unclear. N0X4 is expressed in fibrotic regions of alveolar and vascular remodeling in idiopathic pulmonary fibrosis (IPF). Our preliminary data demonstrate that N0X4 expression is upregulated in lung fibroblasts (Fbs) isolated from IPF patients; N0X4 expression is induced in pleural mesothelial cells (PMCs) during the process of a mesothelial-to-mesenchymal transition (Project 1) and is regulated by matrix stiffness; miR-31 regulates integrin a5 and RhoA (Project 2), which regulates cellular contractility and reciprocally controls matrix stiffness. Exogenous N0X4 expression activates latent TGF-P, and N0X4-dependent reactive oxygen species (ROS) mediates matrix cross linking in the presence of heme peroxidases. The central hypothesis of this project is that N0X4 mediates myofibroblast differentiation and contractility in IPF, and that targeting of NOX by RNAi approaches or small molecule inhibitors protects against fibrosis in animal models. Our specific aims are to (1) determine whether lung expression of N0X4 is predictive of disease severity and/or progression in IPF; (2) determine mechanisms of N0X4-dependent H2O2 generation in activation of latent TGF-(3 and in oxidative matrix cross linking that reciprocally contributes to matrix stiffness; (3) determine whether conditional deletion of N0X4 in Fbs and/or PMCs protects against fibrosis in experimental models of lung fibrosis; and (4) determine the efficacy of targeting N0X4 with small molecule inhibitors and/or siRNA in experimental models of lung fibrosis. These studies will inform and support a potential role of N0X4 as a novel therapeutic target in IPF.