Pamela W. M. Kleikers

The NOX toolbox: validating the role of NADPH oxidases in physiology and disease

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.

NADPH oxidases as a source of oxidative stress and molecular target in ischemia/reperfusion injury

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.

Neuroprotection after stroke by targeting NOX4 as a source of 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.

The 1027th target candidate in stroke: Will NADPH oxidase hold up?

As recently reviewed, 1026 neuroprotective drug candidates in stroke research have all failed on their road towards validation and clinical translation, reasons being quality issues in preclinical research and publication bias. Quality control guidelines for preclinical stroke studies have now been established. However, sufficient understanding of the underlying mechanisms of neuronal death after stroke that could be possibly translated into new therapies is lacking. One exception is the hypothesis that cellular death is mediated by oxidative stress. Oxidative stress is defined as an excess of reactive oxygen species (ROS) derived from different possible enzymatic sources. Among these, NADPH oxidases (NOX1-5) stand out as they represent the only known enzyme family that has no other function than to produce ROS. Based on data from different NOX knockout mouse models in ischemic stroke, the most relevant isoform appears to be NOX4. Here we discuss the state-of-the-art of this target with respect to stroke and open questions that need to be addressed on the path towards clinical translation.

VAS2870 is a pan-NADPH oxidase inhibitor

In their detailed review ‘‘NADPH oxidases as therapeutic targets in ischemic stroke’’ [1], Kahles and Brandes discuss the pathological roles of NADPH oxidases in ischemic brain injury and the therapeutic implications. In agreement with the authors, we consider inhibition of NADPH oxidases as a promising strategy to treat ischemic stroke. As described in the review, we recently reported that NOX4-deficient mice are largely protected from brain damage caused by ischemic stroke, whereas we did not observe any effects by deleting NOX1 or NOX2. Thus, we believe that NOX4 is a highly promising target for stroke therapy. To further support our findings, we treated wildtype and NOX4 knockout mice with the NADPH oxidase inhibitor VAS2870 in a therapeutically relevant time window, i.e., post-stroke. Indeed, in wild-type mice, inhibition of NADPH oxidases by VAS2870 resulted in a similar degree of protection as did deletion of NOX4. In contrast, in NOX4 knockout mice, VAS2870 did not have any additional effects in reducing ischemic brain damage. This further supports our statement that NOX4, and not other NOX isoforms, is the likely detrimental NOX isoform in ischemic stroke in mice. Unfortunately, to the best of our knowledge, there was and is no NOX4-selective inhibitor that could have been used to further support our findings. Kahles and Brandes correctly describe that VAS2870 inhibits NOX1 and NOX2 and cite our relevant publications [2–4]. In the same issue of this journal, we provided evidence that VAS2870 also inhibits NOX4 [5]. In conclusion, we believe that VAS2870 is a pan-NOX inhibitor and not selective for any NOX isoform. However, in their review, Kahles and Brandes [1] state that we concluded that ‘‘VAS2870 was a Nox4-specific inhibitor based on the fact that the compound had no effect on the small infarcts they produced in Nox4 knockout mice’’. This is not true. We have never published such a statement on the NOX isoform-specificity of VAS2870. Both in the respective paper [6] and our other publications we describe VAS2870 as an NADPH oxidase inhibitor with no relevant specificity for any NOX isoform (data on NOX3 are not available) [5]. We have published similar data on the closely related derivative of VAS2870, VAS3947 [7]. Thus, we would kindly ask the authors to revoke their statement.

A combined pre-clinical meta-analysis and randomized confirmatory trial approach to improve data validity for therapeutic target validation

Biomedical research suffers from a dramatically poor translational success. For example, in ischemic stroke, a condition with a high medical need, over a thousand experimental drug targets were unsuccessful. Here, we adopt methods from clinical research for a late-stage pre-clinical meta-analysis (MA) and randomized confirmatory trial (pRCT) approach. A profound body of literature suggests NOX2 to be a major therapeutic target in stroke. Systematic review and MA of all available NOX2-/y studies revealed a positive publication bias and lack of statistical power to detect a relevant reduction in infarct size. A fully powered multi-center pRCT rejects NOX2 as a target to improve neurofunctional outcomes or achieve a translationally relevant infarct size reduction. Thus stringent statistical thresholds, reporting negative data and a MA-pRCT approach can ensure biomedical data validity and overcome risks of bias.

Clinical relevance of cyclic GMP modulators: A translational success story of network pharmacology

Therapies that modulate cyclic guanosine‐3′‐5′‐monophosphate (cGMP) have emerged as one of the most successful areas in recent drug discovery and clinical pharmacology. Historically, their focus has been on cardiovascular disease phenotypes; however, cGMPs relevance is likely to go beyond this rather limited organ‐based set of indications. Moreover, the multitude of targets and their apparent interchangeability is a proof‐of‐concept of network pharmacology.

NOS knockout or inhibition but not disrupting PSD-95-NOS interaction protect against ischemic brain damage.

Promising results have been reported in preclinical stroke target validation for pharmacological principles that disrupt the N-methyl-D-aspartate receptor–post-synaptic density protein-95–neuronal nitric oxide synthase complex. However, post-synaptic density protein-95 is also coupled to potentially neuroprotective mechanisms. As post-synaptic density protein-95 inhibitors may interfere with potentially neuroprotective mechanisms and sufficient validation has often been an issue in translating basic stroke research, we wanted to close that gap by comparing post-synaptic density protein-95 inhibitors with NOS1−/− mice and a NOS inhibitor. We confirm the deleterious role of NOS1 in stroke both in vivo and in vitro, but find three pharmacological post-synaptic density protein-95 inhibitors to be therapeutically ineffective.

NOX4-dependent neuronal autotoxicity and BBB breakdown explain the superior sensitivity of the brain to ischemic damage

Significance Why the brain is uniquely sensitive to hypoxia and which cells are involved is incompletely understood. Here we identify that, upon ischemic stroke, in endothelial cells and neurons the reactive oxygen-forming NADPH oxidase 4 (NOX4) causes breakdown of the BBB and neuronal cell death. This mechanism is unique to the brain and not found in other forms of ischemia in the body. Genetic deletion of either cell type (endothelial or neuronal) or pharmacological inhibition of NOX4 leads to a significant reduction of infarct volume and direct neuroprotection. This mechanism explains the unique vulnerability of the hypoxic brain compared with other organs and provides a clear rationale for first-in-class neuroprotective therapies in stroke. Ischemic injury represents the most frequent cause of death and disability, and it remains unclear why, of all body organs, the brain is most sensitive to hypoxia. In many tissues, type 4 NADPH oxidase is induced upon ischemia or hypoxia, converting oxygen to reactive oxygen species. Here, we show in mouse models of ischemia in the heart, brain, and hindlimb that only in the brain does NADPH oxidase 4 (NOX4) lead to ischemic damage. We explain this distinct cellular distribution pattern through cell-specific knockouts. Endothelial NOX4 breaks down the BBB, while neuronal NOX4 leads to neuronal autotoxicity. Vascular smooth muscle NOX4, the common denominator of ischemia within all ischemic organs, played no apparent role. The direct neuroprotective potential of pharmacological NOX4 inhibition was confirmed in an ex vivo model, free of vascular and BBB components. Our results demonstrate that the heightened sensitivity of the brain to ischemic damage is due to an organ-specific role of NOX4 in blood–brain-barrier endothelial cells and neurons. This mechanism is conserved in at least two rodents and humans, making NOX4 a prime target for a first-in-class mechanism-based, cytoprotective therapy in the unmet high medical need indication of ischemic stroke.

SFRR-E Young Investigator AwardeeNOXing out stroke: Identification of NOX4 and 5as targets in blood-brain-barrier stabilisation and neuroprotection.

Stroke is the second leading cause of death with high blood pressure and female gender being the main risk factors. However, only one treatment is available and with many contraindications, which leaves more than 80% of patients untreated. Over a thousand experimental stroke treatments have remained unsuccessful in the clinic. In preclinical research, low reproducibility and publication bias have been suggested as causes of low translatability success. NADPH oxidases might be key players in stroke via their unique role as a major and/or early source of reactive oxygen species (ROS). To clarify the role of the different NOX isoforms (1, 2, 4, and 5) we analysed different KO and KI models. Previous literature claimed a role for NOX2. Using both a meta-analytical and a blinded randomised controlled trial approach, we however find that NOX2 plays only a minor role and publication bias and lack of power perturbed the published literature. We earlier showed a detrimental role of NOX4 in stroke and extend this based on cell-specific KO animals that endothelial but not vascular smooth muscle cells are the major source of NOX4 in stroke. Mice do not express the human NOX5 gene. Using a NOX5 KI model, we show that endothelial NOX5 induces hypertension and increased stroke risk, particularly in females. In human hypertension, NOX5 is upregulated, and women have a higher stroke risk. Thus NOX5 might be a missing link in this context. In conclusion, NOX4 and NOX5, but not NOX2, are promising targets for the development of new neuroprotective therapies for ischemic stroke. A priori power and sample size calculation as well as reporting of also negative data is essential with respect to preclinical validation of therapeutic targets.