Therapeutic potential of NADPH oxidase 1/4 inhibitors.

The NADPH oxidase (NOX) family of enzymes produces ROS as their sole function and is becoming recognized as key modulators of signal transduction pathways with a physiological role under acute stress and a pathological role after excessive activation under chronic stress. The seven isoforms differ in their regulation, tissue and subcellular localization and ROS products. The most studied are NOX1, 2 and 4. Genetic deletion of NOX1 and 4, in contrast to NOX2, has revealed no significant spontaneous pathologies and a pathogenic relevance of both NOX1 and 4 across multiple organs in a wide range of diseases and in particular inflammatory and fibrotic diseases. This has stimulated interest in NOX inhibitors for therapeutic application. GKT136901 and GKT137831 are two structurally related compounds demonstrating a preferential inhibition of NOX1 and 4 that have suitable properties for in vivo studies and have consequently been evaluated across a range of disease models and compared with gene deletion. In contrast to gene deletion, these inhibitors do not completely suppress ROS production, maintaining some basal level of ROS. Despite this and consistent with most gene deletion studies, these inhibitors are well tolerated and slow or prevent disease progression in a range of models of chronic inflammatory and fibrotic diseases by modulating common signal transduction pathways. Clinical trials in patients with GKT137831 have demonstrated excellent tolerability and reduction of various markers of chronic inflammation. NOX1/4 inhibition may provide a safe and effective therapeutic strategy for a range of inflammatory and fibrotic diseases. LINKED ARTICLES: This article is part of a themed section on Redox Biology and Oxidative Stress in Health and Disease. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.12/issuetoc.

Pharmacological inhibition of NOX reduces atherosclerotic lesions, vascular ROS and immune-inflammatory responses in diabetic Apoe(-/-) mice.

AIMS/HYPOTHESIS: Enhanced vascular inflammation, immune cell infiltration and elevated production of reactive oxygen species (ROS) contribute significantly to pro-atherogenic responses in diabetes. We assessed the immunomodulatory role of NADPH oxidase (NOX)-derived ROS in diabetes-accelerated atherosclerosis. METHODS: Diabetes was induced in male Apoe(-/-) mice with five daily doses of streptozotocin (55 mg kg(-1) day(-1)). Atherosclerotic plaque size, markers of ROS and immune cell accumulation were assessed in addition to flow cytometric analyses of cells isolated from the adjacent mediastinal lymph nodes (meLNs). The role of NOX-derived ROS was investigated using the NOX inhibitor, GKT137831 (60 mg/kg per day; gavage) administered to diabetic and non-diabetic Apoe(-/-) mice for 10 weeks. RESULTS: Diabetes increased atherosclerotic plaque development in the aortic sinus and this correlated with increased lesional accumulation of T cells and CD11c(+) cells and altered T cell activation in the adjacent meLNs. Diabetic Apoe(-/-) mice demonstrated an elevation in vascular ROS production and expression of the proinflammatory markers monocyte chemoattractant protein 1, vascular adhesion molecule 1 and IFNγ. Blockade of NOX-derived ROS using GKT137831 prevented the diabetes-mediated increase in atherosclerotic plaque area and associated vascular T cell infiltration and also significantly reduced vascular ROS as well as markers of inflammation and plaque necrotic core area. CONCLUSIONS/INTERPRETATION: Diabetes promotes pro-inflammatory immune responses in the aortic sinus and its associated lymphoid tissue. These changes are associated with increased ROS production by NOX. Blockade of NOX-derived ROS using the NOX inhibitor GKT137831 is associated with attenuation of these changes in the immune response and reduces the diabetes-accelerated development of atherosclerotic plaques in Apoe(-/-) mice.

Critical role of Nox4-based NADPH oxidase in glucose-induced oxidative stress in the kidney: implications in type 2 diabetic nephropathy.

Molecular mechanisms underlying renal complications of diabetes remain unclear. We tested whether renal NADPH oxidase (Nox) 4 contributes to increased reactive oxygen species (ROS) generation and hyperactivation of redox-sensitive signaling pathways in diabetic nephropathy. Diabetic mice (db/db) (20 wk) and cultured mouse proximal tubule (MPT) cells exposed to high glucose (25 mmol/l, D-glucose) were studied. Expression (gene and protein) of Nox4, p22(phox), and p47(phox), but not Nox1 or Nox2, was increased in kidney cortex, but not medulla, from db/db vs. control mice (db/m) (P < 0.05). ROS generation, p38 mitogen-activated protein (MAP) kinase phosphorylation, and content of fibronectin and transforming growth factor (TGF)-ß1/2 were increased in db/db vs. db/m (P < 0.01). High glucose increased expression of Nox4, but not other Noxes vs. normal glucose (P < 0.05). This was associated with increased NADPH oxidase activation and enhanced ROS production. Nox4 downregulation by small-interfering RNA and inhibition of Nox4 activity by GK-136901 (Nox1/4 inhibitor) attenuated d-glucose-induced NADPH oxidase-derived ROS generation. High d-glucose, but not l-glucose, stimulated phosphorylation of p38MAP kinase and increased expression of TGF-ß1/2 and fibronectin, effects that were inhibited by SB-203580 (p38MAP kinase inhibitor). GK-136901 inhibited d-glucose-induced actions. Our data indicate that, in diabetic conditions: 1) renal Nox4 is upregulated in a cortex-specific manner, 2) MPT cells possess functionally active Nox4-based NADPH, 3) Nox4 is a major source of renal ROS, and 4) activation of profibrotic processes is mediated via Nox4-sensitive, p38MAP kinase-dependent pathways. These findings implicate Nox4-based NADPH oxidase in molecular mechanisms underlying fibrosis in type 2 diabetic nephropathy.