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The 1998 Nobel Prize in Medicine and Physiology for the discovery of nitric oxide, a nitrogen containing reactive oxygen species (also termed reactive nitrogen or reactive nitrogen/oxygen species) stirred great hopes. Clinical applications, however, have so far pertained exclusively to the downstream signaling of cGMP enhancing drugs such as phosphodiesterase inhibitors and soluble guanylate cyclase stimulators. All clinical attempts, so far, to inhibit NOS have failed even though preclinical models were strikingly positive and clinical biomarkers correlated perfectly. This rather casts doubt on our current way of target identification in drug discovery in general and our way of patient stratification based on correlating but not causal biomarkers or symptoms. The opposite, NO donors, nitrite and enhancing NO synthesis by eNOS/NOS3 recoupling in situations of NO deficiency, are rapidly declining in clinical relevance or hold promise but need yet to enter formal therapeutic guidelines, respectively. Nevertheless, NOS inhibition in situations of NO overproduction often jointly with enhanced superoxide (or hydrogen peroxide production) still holds promise, but most likely only in acute conditions such as neurotrauma (Stover et al., J Neurotrauma 31(19):1599-1606, 2014) and stroke (Kleinschnitz et al., J Cereb Blood Flow Metab 1508-1512, 2016; Casas et al., Proc Natl Acad Sci U S A 116(14):7129-7136, 2019). Conversely, in chronic conditions, long-term inhibition of NOS might be too risky because of off-target effects on eNOS/NOS3 in particular for patients with cardiovascular risks or metabolic and renal diseases. Nitric oxide synthases (NOS) and their role in health (green) and disease (red). Only neuronal/type 1 NOS (NOS1) has a high degree of clinical validation and is in late stage development for traumatic brain injury, followed by a phase II safety/efficacy trial in ischemic stroke. The pathophysiology of NOS1 (Kleinschnitz et al., J Cereb Blood Flow Metab 1508-1512, 2016) is likely to be related to parallel superoxide or hydrogen peroxide formation (Kleinschnitz et al., J Cereb Blood Flow Metab 1508-1512, 2016; Casas et al., Proc Natl Acad Sci U S A 114(46):12315-12320, 2017; Casas et al., Proc Natl Acad Sci U S A 116(14):7129-7136, 2019) leading to peroxynitrite and protein nitration, etc. Endothelial/type 3 NOS (NOS3) is considered protective only and its inhibition should be avoided. The preclinical evidence for a role of high-output inducible/type 2 NOS (NOS2) isoform in sepsis, asthma, rheumatic arthritis, etc. was high, but all clinical development trials in these indications were neutral despite target engagement being validated. This casts doubt on the role of NOS2 in humans in health and disease (hence the neutral, black coloring).

Reactive oxygen species (ROS) are ubiquitous metabolic products and important cellular signaling molecules that contribute to several biological functions. Pathophysiology arises when ROS are generated either in excess or in cell types or subcellular locations that normally do not produce ROS or when non-physiological types of ROS (e.g., superoxide instead of hydrogen peroxide) are formed. In the latter scenario, antioxidants were considered as the apparent remedy but, clinically, have consistently failed and even sometimes induced harm. The obvious reason for that is the non-selective ROS scavenging effects of antioxidants which interfere with both qualities of ROS, physiological and pathological. Therefore, it is essential to overcome this "antidote or neutralizer" strategy. We here review the most promising alternative approach by identifying the disease-relevant enzymatic sources of ROS, target these selectively, but leave physiological ROS signaling through other sources intact. Among all ROS sources, NADPH oxidases (NOX1-5 and DUOX1-2) stand out as their sole function is to produce ROS, whereas most other enzymatic sources only produce ROS as a by-product or upon biochemical uncoupling or damage. This qualifies NOXs as the main potential drug-target candidates in diseases associated with dysfunction in ROS signaling. As a reflection of this, the development of several NOX inhibitors has taken place. Recently, the WHO approved a new stem, "naxib," which refers to NADPH oxidase inhibitors, and thereby recognized NOX inhibitors as a new therapeutic class. This has been announced while clinical trials with the first-in-class compound, setanaxib (initially known as GKT137831) had been initiated. We also review the differences between the seven NOX family members in terms of structure and function in health and disease and then focus on the most advanced NOX inhibitors with an exclusive focus on clinically relevant validations and applications. Therapeutically relevant NADPH oxidase isoforms type 1, 2, 4, and 5 (NOX1, NOX2, NOX4, NOX5). Of note, NOX5 is not present in mice and rats and thus pre-clinically less studied. NOX2, formerly termed gp91

Most diseases are defined by a symptom, not a mechanism. Consequently, therapies remain symptomatic. In reverse, many potential disease mechanisms remain in arbitrary search for clinical relevance. Reactive oxygen species (ROS) are such an example. It is an attractive hypothesis that dysregulation of ROS can become a disease trigger. Indeed, elevated ROS levels of various biomarkers have been correlated with almost every disease, yet after decades of research without any therapeutic application. We here present a first systematic, non-hypothesis-based approach to transform this field as a proof of concept for biomedical research in general. We selected as seed proteins 9 families with 42 members of clinically researched ROS-generating enzymes, ROS-metabolizing enzymes or ROS targets. Applying an unbiased network medicine approach, their first neighbours were connected, and, based on a stringent subnet participation degree (SPD) of 0.4, hub nodes excluded. This resulted in 12 distinct human interactome-based ROS signalling modules, while 8 proteins remaining unconnected. This ROSome is in sharp contrast to commonly used highly curated and integrated KEGG, HMDB or WikiPathways. These latter serve more as mind maps of possible ROS signalling events but may lack important interactions and often do not take different cellular and subcellular localization into account. Moreover, novel non-ROS-related proteins were part of these forming functional hybrids, such as the NOX5/sGC, NOX1,2/NOS2, NRF2/ENC-1 and MPO/SP-A modules. Thus, ROS sources are not interchangeable but associated with distinct disease processes or not at all. Module members represent leads for precision diagnostics to stratify patients with specific ROSopathies for precision intervention. The upper panel shows the classical approach to generate hypotheses for a role of ROS in a given disease by focusing on ROS levels and to some degree the ROS type or metabolite. Low levels are considered physiological; higher amounts are thought to cause a redox imbalance, oxidative stress and eventually disease. The source of ROS is less relevant; there is also ROS-induced ROS formation, i.e. by secondary sources (see upwards arrow). The non-hypothesis-based network medicine approach uses genetically or otherwise validated risk genes to construct disease-relevant signalling modules, which will contain also ROS targets. Not all ROS sources will be relevant for a given disease; some may not be disease relevant at all. The three examples show (from left to right) the disease-relevant appearance of an unphysiological ROS modifier/toxifier protein, ROS target or ROS source.

The theory that oxidative stress (OS) is at the root of several diseases is extremely popular. However, so far, no antioxidant has been recommended or offered by healthcare systems neither has any been approved as therapy by regulatory agencies that base their decisions on evidence-based medicine. This is simply because, so far, despite many preclinical and clinical studies indicating a beneficial effect of antioxidants in many disease conditions, randomised clinical trials have failed to provide the evidence of efficacy required for drug approval. In this review, we discuss the levels of evidence required to claim causality in preclinical research on OS, the weakness of the oversimplification associated with OS theory of disease and the importance of the narrative in its popularity. Finally, from a more translational perspective, we discuss the reasons why antioxidants acting by scavenging ROS might not only prevent their detrimental effects but also interfere with essential signalling roles. We propose that ROS have a complex metabolism and are generated by different enzymes at diverse sites and at different times. Aggregating this plurality of systems into a single theory of disease may not be the best way to develop new drugs, and future research may need to focus on specific oxygen-toxifying pathways rather than on non-specific ROS scavengers. Finally, similarly to what is nowadays required for clinical trials, we recommend making unpublished data available in repositories (open data), as this will allow big data approaches or meta-analyses, without the drawbacks of publication bias.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.

Nitric oxide (NO) is synthesized in neurons and is a potent relaxor of vascular and nonvascular smooth muscle. The uterus contains abundant NO-synthesizing nerves which could be autonomic and/or sensory. This study was undertaken to determine: 1) the source(s) of NO-synthesizing nerves in the rat uterus and 2) what other neuropeptides or transmitter markers might coexist with NO in these nerves. Retrograde axonal tracing, utilizing Fluorogold injected into the uterine cervix, was employed for identifying sources of uterine-projecting neurons. NO-synthesizing nerves were visualized by staining for nicotinamide adenine dinucleotide phosphate (reduced)-diaphorase (NADPH-d) and immunostaining with an antibody against neuronal/type I NO synthase (NOS). NADPH-d-positive perikarya and terminal fibers were NOS-immunoreactive (-I). Some NOS-I/NADPH-d-positive nerves in the uterus are parasympathetic and originate from neurons in the pelvic paracervical ganglia (PG) and some are sensory and originate from neurons in thoracic, lumbar, and sacral dorsal root ganglia. No evidence for NOS-I/NADPH-d-positive sympathetic nerves in the uterus was obtained. Furthermore, double immunostaining revealed that in parasympathetic neurons, NOS-I/NADPH-d-reactivity coexists with vasoactive intestinal polypeptide, neuropeptide Y, and acetylcholinesterase and in sensory nerves, NOS-I/NADPH-d-reactivity coexists with calcitonin gene-related peptide and substance P. In addition, tyrosine hydroxylase(TH)-I neurons of the PG do not contain NOS-I/NADPH-d-reactivity, but some TH-I neurons are apposed by NOS-I varicosities. These results suggest NO-synthesizing nerves in the uterus are autonomic and sensory, and could play significant roles, possibly in conjunction with other putative transmitter agents, in the control of uterine myometrium and vasculature.

Several low molecular weight compounds are capable of activating soluble guanylyl cyclase. Recent evidence suggests that some of these are formed under physiological conditions: the nitric oxide radical, carbon monoxide and the hydroxyl radical. Thus, multiple signal transduction pathways appear to exist that form a family of guanylyl cyclase activating factors and thereby regulate the intracellular cyclic guanosine 3′, 5′-monophosphate level.

Oxidative stress, a risk factor for several cardiovascular disorders, interferes with the NO/sGC/cGMP signalling pathway through scavenging of NO and formation of the strong intermediate oxidant, peroxynitrite. Under these conditions, endothelial and vascular dysfunction develops, culminating in different cardio-renal and pulmonary-vascular diseases. Substituting NO with organic nitrates that release NO (NO donors) has been an important principle in cardiovascular therapy for more than a century. However, the development of nitrate tolerance limits their continuous clinical application and, under oxidative stress and increased formation of peroxynitrite foils the desired therapeutic effect. To overcome these obstacles of nitrate therapy, direct NO- and haem-independent sGC activators have been developed, such as BAY 58-2667 (cinaciguat) and HMR1766 (ataciguat), showing unique biochemical and pharmacological properties. Both compounds are capable of selectively activating the oxidized/haem-free enzyme via binding to the enzyme's haem pocket, causing pronounced vasodilatation. The potential importance of these new drugs resides in the fact that they selectively target a modified state of sGC that is prevalent under disease conditions as shown in several animal models and human disease. Activators of sGC may be beneficial in the treatment of a range of diseases including systemic and pulmonary hypertension (PH), heart failure, atherosclerosis, peripheral arterial occlusive disease (PAOD), thrombosis and renal fibrosis. The sGC activator HMR1766 is currently in clinical development as an oral therapy for patients with PAOD. The sGC activator BAY 58-2667 has demonstrated efficacy in a proof-of-concept study in patients with acute decompensated heart failure (ADHF), reducing pre- and afterload and increasing cardiac output from baseline. A phase IIb clinical study for the indication of ADHF is currently underway.

Cyclic guanosine 3', 5'-monophosphate (cGMP) plays an integral role in the control of vascular function. Generated from guanylate cyclases in response to the endogenous ligands, nitric oxide (NO) and natriuretic peptides (NPs), cGMP influences a number of vascular cell types and regulates vasomotor tone, endothelial permeability, cell growth and differentiation, as well as platelet and blood cell interactions. Reciprocal regulation of the NO-cGMP and NP-cGMP pathways is evident in the vasculature such that one cGMP generating system may compensate for the dysfunction of the other. Indeed, aberrant cGMP production and/or signalling accompanies many vascular disorders such as hypertension, atherosclerosis, coronary artery disease and diabetic complications. This chapter highlights the main vascular functions of cGMP, its role in disease and the resulting current and potential therapeutic applications. With respect to pulmonary hypertension, heart failure and erectile dysfunction, as well as cGMP signal transduction, the reader is specifically referred to other dedicated chapters.

Soluble guanylyl cyclase (sGC) is the principal receptor for NO and plays a ubiquitous role in regulating cellular function. This is exemplified in the cardiovascular system where sGC governs smooth muscle tone and growth, vascular permeability, leukocyte flux, and platelet aggregation. As a consequence, aberrant NO-sGC signaling has been linked to diseases including hypertension, atherosclerosis, and stroke. Despite these key (patho)physiological roles, little is known about the expressional regulation of sGC. To address this deficit, we have characterized the promoter activity of human alpha(1) and beta(1) sGC genes in a cell type relevant to cardiovascular (patho)physiology, primary human aortic smooth muscle cells. Luciferase reporter constructs revealed that the 0.3- and 0.5-kb regions upstream of the transcription start sites were optimal for alpha(1) and beta(1) sGC promoter activity, respectively. Deletion of consensus sites for c-Myb, GAGA, NFAT, NF-kappaB(p50), and CCAAT-binding factor(s) (CCAAT-BF) revealed that these are the principal transcription factors regulating basal sGC expression. In addition, under pro-inflammatory conditions, the effects of the strongest alpha(1) and beta(1) sGC repressors were enhanced, and enzyme expression and activity were reduced; in particular, NF-kappaB(p50) is pivotal in regulating enzyme expression under such conditions. NO itself also elicited a cGMP-independent negative feedback effect on sGC promoter activity that is mediated, in part, via CCAAT-BF activity. In sum, these data provide a systematic characterization of the promoter activity of human sGC alpha(1) and beta(1) subunits and identify key transcription factors that govern subunit expression under basal and pro-inflammatory (i.e. atherogenic) conditions and in the presence of ligand NO.

We have used a polyclonal monospecific antibody to rat cerebellum nitric oxide synthase type I (NOS-I, 160 kD) in combination with reduced NADPH-diaphorase histochemical reaction (NADPH-d) to verify colocalization of both NOS protein and NOS enzymatic activity in the rat spinal cord autonomic system. Strong immunoreactivity (IR) of NOS-I was clearly detected in the four main thoracolumbar autonomic nuclei in spinal cord layers of Rexed's laminae VI, VII and X. In all labelled neurones, NOS-I-IR colocalized with NADPH-d activity, suggesting coexistence of brain-specific NOS-I-like protein and its associated enzyme activity. For these neurones the new term 'nitroxergic' (i.e. NO-generating) neurones appears to be justified. Spinal cord nitroxergic neurones are part of a NO-mediated signal transduction pathway for control of the sympathetic 'outflow' to various peripheral target organs.

In this issue of the Journal, Xie and co-workers (1) demonstrate that introducing nitric oxide (NO) synthase type II (NOSII, iNOS) into tumor cells produces cytotoxicity not only in the transfected cells but also in bystander tumor cells. Here, we discuss the potential implications of these experiments in the light of present knowledge on the complex role of NO in tumor biology. The growth of solid tumors is regulated by interactions between endothelial cells of the tumor vasculature, tumorinfiltrating immune cells (such as T lymphocytes and macrophages), and the tumor cells themselves. In these cellular interactions, the unusual biologic messenger molecule and cytotoxin, NO, may play important pathobiologic roles in addition to its many physiologic functions (2). Endogenous NO production from L-arginine has been directly or indirectly demonstrated in all of these cell types. In most cases, the inducible NO synthase gene (Nos2)—one of three known human Nos genes (3)— was switched on (presumably by NF-kB-dependent mechanisms). Its gene product, the high-output NOS-II isoform, unlike its constitutively expressed low-output counterparts (NOS-I and -III), is not regulated by the intracellular concentration of free calcium (3) and is chronically active. Such continuously high exposure of cells to endogenous NO as well as exogenous NO donors will inhibit proliferation and induce cell death (4). High NO levels inhibit mitochondrial respiration, the citric acid cycle, glycolysis, and DNA replication. Locally high levels of reactive oxygen species (ROS), stemming, for example, from host immune cells or from an insufficient oxygen supply to the tumor tissue, may exacerbate these toxic effects by generating even more reactive compounds, such as peroxynitrite (ONOO). The latter compound arises from the diffusion-limited interaction of NO and O2 − and is even more reactive than NO, but it is stable enough to diffuse to and thus harm tumor cells. Apart from these tumoricidal effects, NO has facilitated tumor growth and vascularization in a few experimental models, and, in certain human carcinomas, endogenous NO production was positively associated with tumor grade (5). Thus, NO has a complex, at least dual, action on tumor growth that may depend on the local concentrations of NO, additional factors such as the presence of ROS, and the type of tumor and its susceptibility to NO. In those models where NO had a permissive effect on tumor growth, NOS activity was up to two orders of magnitude lower than that associated with NO-dependent tumor toxicity and apoptosis (5). The dual action of NO on tumor cell proliferation is reminiscent of what has been repeatedly demonstrated for ROS (6,7). Maximal growth promotion by ROS is observed when cells maintain a low but sufficient oxidant signal for the induction of growth-competence genes (8,9), as may be the case with moderate NO concentrations. At high ROS concentrations, the equilibrium balance between the cellular antioxidant defense and oxidant levels is shifted, i.e., toward lipid peroxidation and DNA fragmentation. Besides this concentration dependence, the mechanisms of action of NO may also diverge considerably (Fig. 1). Signaling functions at comparatively low NO concentrations involve soluble guanylyl cyclases as the principal molecular target and subsequent increases in the intracellular level of the second messenger molecule, cyclic guanosine monophosphate (cGMP) (10). The tumor-relevant consequences of these increases in cGMP may be the stimulation of (neo)vascularization and angiogenesis. Moreover, endogenous NO may not only promote the growth of existing tumors, but it may also be tumorigenic itself. NO can cause mutations by mediating the deamination of S-methylcytosine to thymine and also by increasing the formation of DNA strand breaks (11). Increased NO formation has been implicated in several tumor types associated with chronic infections and inflammations, such as gastric, duodenal, esophageal, bladder, and liver cancers (12,13). What are the therapeutic implications of this dual role of NO? One apparent goal may be to enhance NO synthesis to a maximum level only in tumor tissue so that cell proliferation is impaired and cell death is induced. This may be achieved in principle by simply substituting NO in the form of so-called NO donors. Indeed, NO is an effective hypoxic radiosensitizer of tumor tissue (14), while a nonselective NOS inhibitor increases tumor survival (15). Alternatively, endogenous NO formation may be induced in the tumor cell by different cytokines (tumor necrosis factor-a and interleukin 2). Tumor necrosis factor-a has been shown to induce both NO and superoxide (O2 ) production, providing an effective means of local ONOO formation (16). However, cytokines induce NOS-II expression systemically in the blood vessel wall, resulting in massive NOand cGMPmediated vasodilatation and hypotension, which has been recognized as the primary limiting factor in high-dose cytokine therapy (17). A more direct approach is to transfer cytokine or NO synthase