Significance: Oxidative stress, an excess of reactive oxygen species (ROS) production versus consumption, may be involved in the pathogenesis of different diseases. The only known enzymes solely dedicated to ROS generation are nicotinamide adenine dinucleotide phosphate (NADPH) oxidases with their catalytic subunits (NOX). After the clinical failure of most antioxidant trials, NOX inhibitors are the most promising therapeutic option for diseases associated with oxidative stress. Recent Advances: Historical NADPH oxidase inhibitors, apocynin and diphenylene iodonium, are un-specific and not isoform selective. Novel NOX inhibitors stemming from rational drug discovery approaches, for example, GKT137831, ML171, and VAS2870, show improved specificity for NADPH oxidases and moderate NOX isoform selectivity. Along with NOX2 docking sequence (NOX2ds)-tat, a peptide-based inhibitor, the use of these novel small molecules in animal models has provided preliminary in vivo evidence for a pathophysiological role of specific NOX isoforms. Critical Issues: Here, we discuss whether novel NOX inhibitors enable reliable validation of NOX isoforms' pathological roles and whether this knowledge supports translation into pharmacological applications. Modern NOX inhibitors have increased the evidence for pathophysiological roles of NADPH oxidases. However, in comparison to knockout mouse models, NOX inhibitors have limited isoform selectivity. Thus, their use does not enable clear statements on the involvement of individual NOX isoforms in a given disease. Future Directions: The development of isoform-selective NOX inhibitors and biologicals will enable reliable validation of specific NOX isoforms in disease models other than the mouse. Finally, GKT137831, the first NOX inhibitor in clinical development, is poised to provide proof of principle for the clinical potential of NOX inhibition. Antioxid. Redox Signal. 23, 406-427.

Reactive oxygen species (ROS) are cellular signals but also disease triggers; their relative excess (oxidative stress) or shortage (reductive stress) compared to reducing equivalents are potentially deleterious. This may explain why antioxidants fail to combat diseases that correlate with oxidative stress. Instead, targeting of disease-relevant enzymatic ROS sources that leaves physiological ROS signaling unaffected may be more beneficial. NADPH oxidases are the only known enzyme family with the sole function to produce ROS. Of the catalytic NADPH oxidase subunits (NOX), NOX4 is the most widely distributed isoform. We provide here a critical review of the currently available experimental tools to assess the role of NOX and especially NOX4, i.e. knock-out mice, siRNAs, antibodies, and pharmacological inhibitors. We then focus on the characterization of the small molecule NADPH oxidase inhibitor, VAS2870, in vitro and in vivo, its specificity, selectivity, and possible mechanism of action. Finally, we discuss the validation of NOX4 as a potential therapeutic target for indications including stroke, heart failure, and fibrosis.

Major contributors to atherosclerosis are oxidative damage and endoplasmic reticulum (ER) stress-induced apoptosis; both of which can be diminished by the anti-oxidative protein paraoxonase-2 (PON2). ER stress is also relevant to cancer and associated with anti-cancer treatment resistance. Hence, we addressed, for the first time, whether PON2 contributes to tumorigenesis and apoptotic escape. Intriguingly, we found that several human tumors upregulated PON2 and such overexpression provided resistance to different chemotherapeutics (imatinib, doxorubicine, staurosporine, or actinomycin) in cell culture models. This was reversed after PON2 knock-down. Remarkably, just deficiency of PON2 caused apoptosis of selective tumor cells per se, demonstrating a previously unanticipated oncogenic function. We found a dual mechanistic role. During ER stress, high PON2 levels lowered redox-triggered induction of pro-apoptotic CHOP particularly via the JNK pathway, which prevented mitochondrial cell death signaling. Apart from CHOP, PON2 also diminished intrinsic apoptosis as it prevented mitochondrial superoxide formation, cardiolipin peroxidation, cytochrome c release, and caspase activation. Ligand-stimulated apoptosis by TRAIL or TNFα remained unchanged. Finally, PON2 knock-down caused vast reactive oxygen species formation and stimulated JNK-triggered CHOP expression, but inhibition of JNK signaling did not prevent cell death, demonstrating the pleiotropic, dominating anti-oxidative effect of PON2. Therefore, targeting redox balance is powerful to induce selective tumor cell death and proposes PON2 as new putative anti-tumor candidate.

Ischemia/reperfusion injury (IRI) is crucial in the pathology of major cardiovascular diseases, such as stroke and myocardial infarction. Paradoxically, both the lack of oxygen during ischemia and the replenishment of oxygen during reperfusion can cause tissue injury. Clinical outcome is also determined by a third, post-reperfusion phase characterized by tissue remodeling and adaptation. Increased levels of reactive oxygen species (ROS) have been suggested to be key players in all three phases. As a second paradox, ROS seem to play a double-edged role in IRI, with both detrimental and beneficial effects. These Janus-faced effects of ROS may be linked to the different sources of ROS or to the different types of ROS that exist and may also depend on the phase of IRI. With respect to therapeutic implications, an untargeted application of antioxidants may not differentiate between detrimental and beneficial ROS, which might explain why this approach is clinically ineffective in lowering cardiovascular mortality. Under some conditions, antioxidants even appear to be harmful. In this review, we discuss recent breakthroughs regarding a more targeted and promising approach to therapeutically modulate ROS in IRI. We will focus on NADPH oxidases and their catalytic subunits, NOX, as they represent the only known enzyme family with the sole function to produce ROS. Similar to ROS, NADPH oxidases may play a dual role as different NOX isoforms may mediate detrimental or protective processes. Unraveling the precise sequence of events, i.e., determining which role the individual NOX isoforms play in the various phases of IRI, may provide the crucial molecular and mechanistic understanding to finally effectively target oxidative stress.

Significance: Stroke, a leading cause of death and disability, poses a substantial burden for patients, relatives, and our healthcare systems. Only one drug is approved for treating stroke, and more than 30 contraindications exclude its use in 90% of all patients. Thus, new treatments are urgently needed. In this review, we discuss oxidative stress as a pathomechanism of poststroke neurodegeneration and the inhibition of its source, type 4 nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX4), as a conceptual breakthrough in stroke therapy. Recent Advances: Among potential sources of reactive oxygen species (ROS), the NOXes stand out as the only enzyme family that is solely dedicated to forming ROS. In rodents, three cerebrovascular NOXes exist: the superoxide-forming NOX1 and 2 and the hydrogen peroxide-forming NOX4. Studies using NOX1 knockout mice gave conflicting results, which overall do not point to a role for this isoform. Several reports find NOX2 to be relevant in stroke, albeit to variable and moderate degrees. In our hands, NOX4 is, by far, the major source of oxidative stress and neurodegeneration on ischemic stroke. Critical Issues: We critically discuss the tools that have been used to validate the roles of NOX in stroke. We also highlight the relevance of different animal models and the need for advanced quality control in preclinical stroke research. Future Directions: The development of isoform-specific NOX inhibitors presents a precious tool for further clarifying the role and drugability of NOX homologues. This could pave the avenue for the first clinically effective neuroprotectant applied poststroke, and even beyond this, stroke could provide a proof of principle for antioxidative stress therapy.