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.

Reactive oxygen species (ROS) have long been suspected as being 'bad guys'. They are frequently associated with the development and progression of chronic, degenerative, cancerous and inflammatory diseases. Indeed an excess of ROS, i.e. oxidative stress, caused by an imbalance between ROS production and their removal by antioxidant systems, may be a common underlying pathogenic mechanism in these diseases. With the recent additional description of possible roles of ROS in diverse physiological signaling processes another form of imbalance deserves attention, i.e. reductive stress-the excess of reducing agents in a cell that leads to shortage of ROS. These and other phenomena [1] may explain the poor outcomes of antioxidant therapies in clinical studies where even deleterious effects of untargeted antioxidant treatment have been reported [2][3][4][5][6][7][8][9][10]. Rather than attempting to systemically scavenge ROS, it may be more effective to specifically target the different enzymatic sources of pathophysiologically relevant ROS. Nevertheless, until this has resulted in clinical benefits, the oxidative stress hypothesis remains unproven.
Several ROS producing enzyme systems exist, including xanthine oxidase [11], the mitochondrial respiratory chain [12], lipid peroxidases [13], cytochrome P450 enzymes [14], and uncoupled endothelial NO synthase [15]. However, these enzymes produce ROS secondary to their damage, which can be proteolysis but is often caused by oxidative stress itself [11,15]. Thus, there would still be the need to identify this primary source of oxidative stress. The only enzyme family known to produce ROS as their primary and sole function are NADPH oxidases. These multi-protein complexes are comprised of a catalytic, transmembrane-spanning subunit (NOX), as well as several structural and regulatory proteins localized in both the membrane and the cytosol.

The NADPH oxidase family
We are only beginning to understand the enzyme family of NADPH oxidases, their players and their interaction. The NOX family consists of seven members, NOX1-5, and two dual oxidases (Duox), Duox1 and Duox2. Of those, NOX1, 2, 4, and 5 have been implicated in vascular diseases, on which we focus in this review. All NOX isoforms have six trans-membrane spanning alpha helices with cytosolic Nand C-termini. They are differentially expressed and regulated in various tissues and have different subcellular localizations, and even different ROS products, i.e. superoxide versus hydrogen peroxide (reviewed in [16]). NOX1, NOX2, and NOX5 appear to produce mainly superoxide NOX4, mainly H 2 O 2 [17]. All NOX isoforms have been reported to bind to one or more membrane and/or cytosolic proteins. p22 phox appears to be a general binding partner for NOX1-4 in the membrane. NOX1 and 2 also bind the small GTPase, Rac. Moreover, NOX1 binds the cytosolic subunits, NOX organizer 1 (NOXO1) and NOX activator 1 (NOXA1), and NOX2 binds the respective homologues, p47 phox and p67 phox , and also the cytosolic protein, p40 phox [18,19]. NOX4 was reported to bind to the polymerase (DNA-directed) delta-interacting protein 2 (PolDip2) [20]. In addition to these established NOX binding partners, the tyrosine kinase substrate with 4/5 SH3 domains (Tks4/5) [21,22], and protein disulfide isomerase (PDI) were recently suggested to bind to both NOX1 and 4 [23]. Upon overexpression in cells, the C-terminus of NOX5 was shown to interact with Hsp90, which may also bind to NOX1 and 2 [24]. However, the physiologic relevance of Fig. 1 The vascular NOX isoform-based NADPH oxidase complexes. Cell or subcellular compartment membranes are shown in gray, core proteins in yellow, activator binding proteins in green and organizer binding proteins in blue. All the NOX isoforms shown are membrane proteins and are localized in the plasma membrane (PM). Additionally, NOX1 was found at the plasma membrane in caveolae [147], NOX2 in membranes of phagosomes, and NOX4 in mitochondrial [182] and ER-membranes [191], as well as in the nucleus [97]. Little is known about subcellular localization of NOX5 other than the plasma membrane, but a localization at the ER membrane has been reported [29,192]. NOX1, NOX2, and NOX4 are associated with p22 phox , but only NOX1 and NOX2 are regulated by the small GTPase Rac. For its activation, the NOX1 enzyme complex requires the assembly of NOX organiser 1 (NOXO1) and NOX activator 1 (NOXA1), but also forms complexes with p47 phox and p67 phox (not shown). The NOX2 enzyme complex requires binding of p47 phox , p67 phox , and optionally p40 phox that can further support the activity. In contrast to NOX1 and NOX2, NOX4 and NOX5 do not depend on any of the 'classical' cytosolic NADPH oxidase subunits. Recently, the protein polymerase (DNA-directed) delta-interacting protein 2 (Poldip2) was identified to bind and to increase the activity of NOX4. Further, protein disulfide isomerase (PDI) [23] and a p47 phox analogue tyrosine kinase substrate with 4/5 SH3 domains (Tks4/5) have been reported to bind and activate NOX1 and NOX4 [21,22]. NOX4 is the only isoform that produces hydrogen peroxide instead of superoxide [17]. The NOX5 protein contains four N-terminal calcium-binding sites that regulate activation of the enzyme. Activity of NOX5 can be further supported by the binding of Hsp90 or Calmodulin to the C-terminus of the protein [24]
With respect to activity regulation, there are fundamental differences between the individual NOX catalytic subunits. Most seem to be dynamically switched on and off by either regulatory subunits (NOXA1 for NOX1 [25-27], p67phox for NOX2 [28], and calmodulin for NOX 5 [29, 30]) or intramolecularly by the N-terminal EF hands that bind free intracellular calcium (NOX5 and Duox1/2 [31]). In contrast, NOX4 is constitutively active, and modulation of its expression may thus be a major activity regulator.
The tools to validate the role of NADPH oxidase in health and disease During the validation of the involvement of a protein in a biological process or disease mechanism pharmacological GAUUUGCCUGGAAGAACCCtt-3 0 n.d. [131] The There are an increasing number of reports using siRNAs approaches directed against NOX4 (Table 1). Unfortunately, only a few of those siRNAs have been properly The NOX toolbox 2333 validated regarding their overall and NOX isoform specificity. The necessity for confirming specificity was impressively underlined in a recent study [47], which showed that out of nine tested NOX4-directed siRNAs only six down-regulated murine NOX4 mRNA levels. Moreover, five of those six also down-regulated NOX1 mRNA levels. Another problem with investigating the role of NOX4 using siRNAs is the lack of specific antibodies against NOX4. Many if not all publications thus rely primarily on the down-regulation of NOX4 mRNA (see Table 1). These reports may need to be re-evaluated, as it was also recently shown that NOX4 is highly regulated at the post-transcriptional level, and therefore mRNA levels may not necessarily reflect protein levels and ROS formation [48,49].

Antibodies against NOX
The lack of specific, freely available and validated antibodies against NOX1 and NOX4 represents one of the biggest roadblocks in the field. As described above, the validation of both siRNA-mediated down-regulation and genetic NOX1 and NOX4 KO models depends on the quality of the antibodies used for the characterization. Furthermore, as long as the tissue distribution of NOX1 and NOX4 remains unclear, it is very difficult to predict or estimate specific versus off-target effects of potential therapeutic interventions. Several groups and companies have attempted to generate polyclonal antibodies directed against different NOX1 and NOX4 peptides or recombinant proteins ( Table 2). As these are polyclonal rabbit antibodies, the access and the amount were always limited. Also, several different protein sizes have been detected for NOX4 by different antibodies in the same tissues. This may be due to unspecificity of some antibodies, but also caused by the high sensitivity of the NOX4 protein to lysis conditions that may result in degradation and dephospho- These mechanisms most likely account, at least in part, for the acute effects of increased NOX1 activity mediating endothelial dysfunction and the chronic effects that are discussed to cause hypertension. NOX2-derived superoxide is a major signaling molecule in innate immunity mediating host defense. NOX4 is unlikely to directly interfere with the NO/cGMP-signaling pathway as it releases hydrogen peroxide (H 2 O 2 ) and not superoxide. However, in high concentrations, H 2 O 2 causes acute cytotoxicity. This mechanism is suggested to be involved in NOX4-mediated effects after acute ischemic stroke, acute effects of pressure overload in heart, and bleomycin-induced cytotoxicity. The lower chronic activity of NOX4 seems to be involved in angiogenesis and wound healing, and thus rather protective The NOX toolbox 2335 slices [33], a significant decrease in ROS production was observed after VAS2870 treatment. In a mouse brain ischemia reperfusion model, NADPH oxidase activity was inhibited by in vivo treatment with VAS2870 [33], and in a zebrafish model of wound healing, DUOX was inhibited by VAS2870 [76]. In summary, VAS2870 is a well-validated NADPH oxidase inhibitor, as it shows no intrinsic antioxidant activity, does not inhibit other flavoproteins, inhibits NADPH oxidase-mediated ROS production in cell free systems, cells, tissues and in vivo, but it is not NOX isoform-specific. Very recently thioalkylation of cysteine residues of the ryanodine receptor Ca 2? channel (RyR1) was discovered as a potential off-target effect of VAS2870 in sarcoplasmic reticulum vesicles isolated in glutathione (GSH) free buffer [193]. The authors also show binding of VAS2870 to low concentrations of GSH in vitro (10 lM). It will be interesting to know to which extent thioalkylation contributes in vivo to the mechanism of action of VAS2870 in the presence of physiological (mM) concentrations of GSH. However, for further development of the compound into a drug more extended off-target effects, ADME and safety data are required, including acute and chronic toxicity determination. So far, it has only been shown that VAS2870 does not inhibit ligand-induced platelet-derivedgrowth factor receptor (PDGFR)-tyrosine phosphorylation or PDGF-dependent phosphorylation of Erk1/2 or Akt [70].

Mechanism of action
In a cell-free system (membranes plus cytosol) VAS2870 only inhibited NOX2 activity when added prior to stimulation of the active complex formation between NOX2 and its cytosolic partners [71], whereas it showed no effect on NOX2 activity when added after stimulation of the complex formation with SDS (Fig. 3). This suggests that VAS2870 inhibits NADPH oxidase complex formation and can interfere with the association of NOX and its binding proteins. Surprisingly, the activities of NOX4 and NOX5, that are believed to be independent of cytosolic binding proteins, were also inhibited by VAS2870 when tested in native, mainly NOX4-expressing, A7r5 cells and NOX4 or NOX5 overexpressing HEK-293 cells, respectively (Fig. 3) . Thus, for all NOX isoforms, it is possible that inhibition of inter-or intramolecular conformational changes is a common mechanism of action of VAS2870. Thioalkylation of critical cysteine residues of NOX enzymes by VAS2870 was recently, e.g. the cytosolic B-loop, suggested [193], but the molecular details and binding sites of this remain to be elucidated.

Applying the tools: validated targets and possible indications
It is still early days in NOX research, and certainly with respect to translation. Nevertheless, what can already be said about validated roles of NOX and NADPH oxidase in disease? And which of these roles may be translated into therapeutic indications? Different NOX subunits have been suggested to be implicated in cancer, hypertension, lung fibrosis, stroke, heart failure, diabetes, and neurodegenerative diseases [18]. Several principal ways may be differentiated by which an excess of ROS leads to pathology: spatially confined levels of ROS (e.g., in caveolae) that interfere with nitric oxide's (NO) vasoprotective signaling, and high levels (local or systemic) that act, at least in part, independently of NO and are directly cytotoxic, cause apoptosis (Fig. 4) Thus, in any animal model involving a significant inflammatory response, NOX2 inhibition may lead to an improvement. Whether this can be exploited in light of the essential immune functions of NOX2 is an important question. Importantly, even a small residual NOX2 activity in X-linked chronic granulomatous disease (CGD) is sufficient for a functional innate immune system [85]. However, it is unknown whether a partial pharmacological inhibition of NOX2 will sufficiently suppress NOX2's non-CGD disease-related activity. In addition, chronic NOX2 inhibition might lead to paradoxical autoimmune responses [86]. Rather, one may want to optimize any NOX inhibition approach by leaving NOX2 unaffected. With respect to low and spatially confined ROS overproduction, NOX1 is a good candidate to migrate into caveolae and there cause eNOS uncoupling and endothelial dysfunction, which is often associated with increased blood pressure and enhanced platelet aggregation. Moreover, it may be an early step in the development of atherosclerosis. Indeed, basal blood pressure [39], angiotensin-induced hypertension [39,40], and endothelium-dependent relaxation in spontaneously hypertensive rats [87] depends-to some degree-on NOX1. However, whether such chronic disease indications would ever become realistic for NOX inhibition is highly questionable unless sophisticated patient stratification biomarkers would become available. Phosphorylation of vasodilator-stimulated phosphoprotein (P-VASP) could become such a marker [88].
With respect to higher levels of ROS that act, at least in part, independently of NO and are directly cytotoxic or cause apoptosis, NOX4 is well validated. NOX4 is induced in ischemic stroke, in pressure overload of the heart, and in a bleomycin model of lung epithelial toxicity resulting in lung fibrosis. Whilst the interpretation of the stroke data obtained with NOX4 KO mice is straightforward and was recently confirmed in a tgNOX4 model of brain ischemia showing larger infarct sizes [194], the pressure overload and lung data are less so. In pressure overload, two models have been applied, proximal aortic or thoracic aortic constriction (TAC), and abdominal aortic banding. Both models differ in the time course by which they affect the heart. The latter, less acute model allows for angiogenesis to occur. NOX4 appears to play a double role by contributing to the cardiomyocyte damage (particularly in the acute TAC model [32]) and by facilitating subacute angiogenesis and promoting cardiac function (only observable in the subacute abdominal aortic banding). This may explain why opposing phenotypes were observed in both NOX4 KO mouse models and different disease models. In particular, the TAC model was tested in a cardiomyocytespecific KO and therefore leaves vascular cell-dependent angiogenesis by definition unaffected. Thus, NOX4 might both acutely damage the cardiomyocyte and subacutely protect the heart by promoting angiogenesis. NOX4 also promotes angiogenesis in vitro as shown using HUVEC [89,90] and ovarian cancer cells [91]. Whether these effects may be exploited by defining an optimal time window for NOX4 inhibition in situations of acute heart failure or by interfering with tumor angiogenesis remains to be seen, and it needs to be tested by TAC or cancer models in a global KO animal and by applying NOX inhibitors. The situation in the lung is similarly complicated. Here, a role of NOX4 in the pathogenesis of hypoxic pulmonary hypertension was suggested [92], but not confirmed in NOX4 KO mice [33]. Recent  . Thus, NOX4 may be relevant in the bleomycin model, but this model may not reflect the wide spectrum of human lung fibrosis (idiopathic, radiation, silicosis, systemic lupus erythematosus, dermatomyositis, sclerodermia, rheumatoid arthritis, pneumoconiosis, acute respiratory distress syndrome, chronic heart failure, drug-induced). Thus, a model-independent role of NOX4 in lung fibrosis needs to be tested in different models of the disease. Even then, the clinical challenge of a life-long therapy with a NOX4 inhibitor would remain. Importantly, all published NOX4 KO models lack a basal phenotype. This is an important observation for the characterization of NOX4 as a therapeutic target, as it indicates that NOX4 inhibition would probably not cause severe complications. The situation may be different when co-morbidities occur and protective roles of NOX4 may well cause side effects. From the current state of knowledge, such potential side effects of sub-chronic and chronic NOX4 inhibition could arise from decreased angiogenesis.
In conclusion, according to the current knowledge, acute ischemic stroke appears to be one of the most promising and safest targets for NOX inhibition. It evades the risk of chronic therapy and the rather double-edged role of NOX4 in heart failure and angiogenesis. Nevertheless, specific, isoform-selective NOX inhibitors and reliable, freely available antibodies will be key in elucidating the full therapeutic potential of NOX in species other than mouse and in different disease models.