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Keywords:

  • Cardiovascular disease;
  • NADPH oxidase;
  • new therapeutics;
  • aptamers

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Author Contributions
  5. Conflict of Interest
  6. References

Over 40 years ago, NADPH (nicotinamide adenine dinucleotide phosphate) oxidase 2 (Nox2) was discovered in phagocytes and found to be essential in innate immunity. More than 20 years passed before additional Nox isoforms were discovered; and since then, studies have revealed that several of these isoforms (Nox1, Nox2, Nox4, and Nox5) are found in human cardiac and vascular cells and contribute to the pathogenesis of cardiovascular diseases (CVDs). Recently, major efforts have focused on identifying inhibitors capable of ameliorating Nox-mediated CVD. In this review, we briefly discuss the role of each Nox isoform in CVD, identify steps in Nox signaling that will serve as potential targets for the design of therapeutics, and highlight innovative strategies likely to yield effective Nox inhibitors within the next decade.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Author Contributions
  5. Conflict of Interest
  6. References

NADPH (nicotinamide adenine dinucleotide phosphate) oxidases (Nox) are a family of transmembrane proteins of which there are seven isoforms: Nox 1–5 and Duox 1–2. Each Nox subunit serves as the catalytic core of a multi-subunit NADPH oxidase enzyme complex. Interestingly, the only known function of these enzymes is to generate reactive oxygen species (ROS), specifically superoxide (O2.−) or hydrogen peroxide (H2O2). They do so in response to signaling cascades initiated by osmotic or mechanical stress, hormones, vasoactive agents, and cytokines. Nox-generated ROS serve to modulate redox-sensitive targets in intracellular signaling pathways that control a wide variety of cell processes, including differentiation [1], fibrosis [2], growth [3], migration [4], proliferation [5], apoptosis [6], cytoskeletal rearrangement [7], and contraction [8]. The importance of Nox proteins is evident not only from their roles in diverse cell processes, but also their vast tissue distribution. In mammals, Nox proteins are expressed in nearly all cell types, including several relevant to the cardiovascular system: cardiomyocytes [9], vascular smooth muscle cells (VSMCs) [10], endothelial cells (ECs) [11], adventitial fibroblasts [12], and leukocytes [13]. Within the human vasculature, the crucial Nox isoforms are Nox1, Nox2, Nox4, and Nox5; these are the focus of this review.

Nox Structure and Function

The core structure of all Nox isoforms consists of six transmembrane domains and a cytosolic C-terminus (Figure 1). The third and fifth transmembrane domains contain conserved histidine residues that bind two prosthetic heme groups; and the C-terminus encodes two domains for the purpose of binding flavin adenine dinucleotide (FAD) and NADPH (reduced form). Nox use these prosthetic groups to transfer electrons from the cytosolic donor, NADPH, to FAD, then sequentially to each of the two heme groups and then to molecular oxygen on the opposite side of the membrane to generate ROS [14]. Although all Nox use this mechanism to generate ROS, the modes of activation, subunit requirements, and subcellular localizations differ between isoforms. For instance, Nox1 and 2 are constitutively associated with another transmembrane protein, p22phox; and the full activity of Nox1/p22phox or Nox2/p22phox in response to agonist stimulation requires association with several cytosolic proteins: Rac, NoxO1 (or its homolog, p47phox), and NoxA1 (or its homolog, p67phox) are required for activation of Nox1/p22phox; and Rac, p40phox, p47phox, and p67phox are required for activation of Nox2/p22phox. Following complete enzyme complex assembly, Nox1 and Nox2 generate O2.−. Conversely, Nox4 and Nox5 do not require additional cytosolic proteins for their activation. Nox4 is constitutively associated with p22phox, is active in an agonist-independent manner, and produces H2O2 via dismutation of O2.− prior to its release from the enzyme [15]; whereas Nox5 is not associated with p22phox, is activated in a calcium-dependent manner due to an additional N-terminal calcium-binding domain, and produces O2.−[16, 17]. Once ROS are produced, they must interact with their target(s) in order to participate in cell signaling. However, O2.− does not readily cross membranes and is short-lived in its effect; and although it can be dismutated to longer lasting and membrane-diffusible H2O2, both of these ROS are relatively local in their effect. Therefore, the redox-dependent effects of Nox signaling are contingent on Nox localization such that ROS are produced in a specific location to affect a specific target [18]. These differences in modes of activation, subunit requirements, type of ROS produced, and Nox localization are means by which NADPH oxidases achieve specificity in cell signaling.

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Figure 1. Nox1, 2, 4, and 5 enzyme complex structure and function. The core structure of Nox1, 2, 4, and 5 consists of six transmembrane domains and a cytosolic C-terminus. The third and fifth transmembrane domains contain conserved histidine residues that bind two prosthetic heme groups; and the C-terminus encodes two domains for the purpose of binding FAD and NADPH. Nox use these prosthetic groups to transfer electrons from NADPH (in the cytosol) to FAD, sequentially to each heme group, and finally to molecular oxygen on the opposite side of the membrane to generate superoxide (O2.−). Nox1, 2, and 4 are constitutively associated with membrane-bound p22phox. Nox1 also associates with the cytosolic subunits NoxO1 (or its homolog p47phox), NoxA1 (or its homolog p67phox), and Rac. Nox2 associates with Rac, p47phox, p67phox, and p40phox. Nox4 does not require cytosolic subunits and performs two single-electron transfers to generate two superoxide anions that are dismutated to hydrogen peroxide (H2O2) prior to release from the enzyme. Nox5 does not associate with p22phox or cytosolic subunits, but requires calcium binding to its N-terminal domain to become activated.

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Nox, Oxidative Stress, and Cardiovascular Disease (CVD)

In the context of physiological conditions, Nox produce ROS at levels that maintain physiologic cell processes such as cell signaling and the respiratory burst in neutrophils. However, under pathological conditions, Nox become overexpressed and overly stimulated, leading to the production of excess oxidants (molecules that readily accept electrons in chemical reactions), which can cause oxidative stress. Traditionally, “oxidative stress” refers to a disturbance in the normal redox state of a cell or tissue, meaning that there is either an increase in the production or a decrease in the removal of oxidants. This increase in oxidants results in an environment conducive to the creation of free radicals (compounds that contain unpaired electrons) that are highly reactive. Not only do Nox create oxidants and free radicals directly via the generation of H2O2 (an oxidant) and O2.− (an oxidant and free radical), they also create an environment in which additional oxidants and free radicals are likely to be produced. If cellular levels of oxidants exceed the antioxidant capacity of the cell, dysregulated cell signaling and oxidant-mediated injury to macromolecules can occur. Oxidative stress is implicated in a wide range of pathologies, but perhaps none more so than CVD. In humans, oxidative stress is involved in hypertension (HTN), atherosclerosis, myocardial infarction (MI), myocardial hypertrophy, restenosis, arrhythmias, and heart failure [19–23]. Although multiple cellular processes and enzymes can produce ROS and oxidative stress (including the mitochondrial electron transport chain, cyclooxygenase, lipoxygenase, heme oxygenase, cytochrome P450, xanthine oxidase, NO synthase, and peroxidase), Nox are the primary enzymes responsible for inducible ROS formation in the cardiovascular system [18, 24, 25].

Nox-derived ROS contribute to CVD by multiple mechanisms (Figure 2). First, ROS cause macromolecular damage of lipids, proteins, and DNA, which can lead to cell death [26]. In the context of atherosclerosis, this cell death contributes to plaque erosion and thrombosis [27]. Second, O2.− and H2O2 participate in cell signaling by oxidizing (and consequently activating or deactivating) redox-sensitive cysteine residues on target proteins that regulate cellular function, such as ion channels, phosphatases, kinases, cytoskeletal proteins, matrix metalloproteinases (MMPs), and transcription factors [28]. For example, Nox-derived ROS modify the cysteine residue in the active site of protein tyrosine phosphatases (PTPs), thereby inactivating PTPs resulting in an increase in tyrosine phosphorylation of intermediates in signaling cascades, including the mitogen-activated protein kinases (MAPKs) [29]. In VSMCs, MAPK phosphorylation causes migration and proliferation, thereby leading to neointima formation and vascular occlusion [30]. Third, O2.− reacts with nitric oxide (NO), and as such reduces NO bioavailability, leading to: impaired vasodilation [31]; increased platelet adhesion and activation [32]; increased production of inflammatory molecules [33]; increased microvascular permeability [34]; vascular inflammation [31]; leukocyte infiltration of the endothelium [35, 36]; and plaque instability [37]. Fourth, the reaction between O2.− and NO produces peroxynitrite (ONOO), a powerful oxidant that causes lipid peroxidation, protein oxidation, protein nitration, and cell death [38]. Lastly, ROS also oxidize low-density lipoprotein (ox-LDL), which contributes to atherogenesis by causing endothelial dysfunction, vascular inflammation, and foam cell formation [39]. These findings clearly demonstrate the contribution of ROS to the pathogenesis of CVD. In addition, there have been numerous studies demonstrating the specific contribution of each vascular Nox to CVD. These studies described below not only reveal an important role for Nox1, 2, 4, and 5 in CVD, but also highlight the pressing need for targeted Nox therapeutics.

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Figure 2. Role of NADPH oxidases in CVD. Risk factors for CVDs cause vascular injury and the release of cytokines and growth factors that increase the expression and activation of NADPH oxidases (Nox), which generate the ROS hydrogen peroxide (H2O2) and superoxide (O2.−) to cause oxidative stress. These ROS have the potential to cause oxidative macromolecular damage, oxidation of cell signaling intermediates, decreased nitric oxide (NO) bioavailability, increased peroxynitrite (ONOO−), and increased oxidized low-density lipoprotein (ox-LDL). Subsequent vascular inflammation and dysregulation lead to multiple CVD outcomes. ECM: extracellular matrix. CHF: congestive heart failure.

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Nox1 is expressed in VSMCs and to a lesser extent in ECs, and contributes to the pathogenesis of atherosclerosis, restenosis, and HTN. Increased expression and activation of Nox1 in VSMCs causes increased degradation of extracellular matrix, cellular migration, and proliferation that contribute to neointimal hyperplasia, a precursor to restenosis and atherosclerosis [40]. Furthermore, Nox1-deficient mice show a dramatic decrease in VSMC migration, proliferation, and the development of neointima in response to vascular injury [40]. Moreover, in a mouse model of atherosclerosis (apolipoprotein E (ApoE) knockout and a high-fat diet), the deletion of Nox1 results in reduced levels of vascular ROS, aortic plaque area, and macrophage content of lesions [41]. Additionally, in a murine model of HTN (infusion of angiotensin II, Ang II), the overexpression of Nox1 in VSMCs results in increased blood pressure and aortic hypertrophy as compared to control mice receiving Ang II [42, 43]. Moreover, mice that are deficient in Nox1 are protected from Ang II–induced HTN, and exhibit increased NO bioavailability, a marked reduction in aortic VSMC hypertrophy, and a decrease in extracellular matrix deposition [44, 45]. These studies indicate that Nox1 is a desirable therapeutic target for the treatment of CVD.

Nox2, also known as gp91phox, was the first Nox isoform to have been discovered. This protein is expressed at high levels in leukocytes, where its ROS production provides for the respiratory burst necessary for killing microbes within phagosomes. However, Nox2-dependent ROS production in leukocytes also contributes to the development of CVD. Furthermore, Nox2 is expressed in VSMCs, ECs, cardiomyocytes, and fibroblasts; and its activity in each of these cell types plays an important role in the development of CVD. Like Nox1, Nox2 also contributes to atherogenesis and HTN. In atherosclerotic human coronary arteries, Nox2 is most abundantly expressed in macrophages, and levels of the Nox2/p22phox mRNAs positively correlate with the severity of atherosclerosis [20]. In ECs, Nox2 activity is necessary for the upregulation of inflammatory cytokines, such as TNF-α; the adhesion molecules necessary for leukocyte infiltration, including VCAM-1, E-selectin, and ICAM-1; and proinflammatory transcription factors, such as NFκB, all factors involved in vascular inflammation and atherogenesis [46, 47]. Furthermore, Nox2/ApoE double knockout mice have reduced O2.−, increased NO, decreased neointimal formation, and decreased atherosclerotic lesion of the abdominal aorta compared to ApoE knockout alone [48]. A role for Nox2 in HTN has also been suggested by the finding that mice deficient in Nox2 are protected from Ang II–induced increases in blood pressure, vascular hypertrophy, and endothelial dysfunction [49]. However, the contribution of Nox2-derived O2.− in HTN is controversial based on an alternate study which found that Ang II caused a similar increase in blood pressure in wild-type and Nox2 knockout mice, despite that mice deficient in Nox2 had a lower baseline blood pressure and were protected from Ang II–mediated aortic hypertrophy [50]. Additional studies in Nox2-deficient mice have demonstrated that it plays a critical role in cardiac dysfunction. Nox2 knockout mice are less vulnerable to Ang II–induced cardiac hypertrophy and interstitial cardiac fibrosis [9]. Although Nox2 is absent in all cell types in this model, these effects are presumably due to the absence of Nox2 in cardiomyocytes. This presumption is supported by further studies, demonstrating that cardiomyocyte-specific deletion of Rac1 (a required subunit for Nox2) also results in diminished cardiac hypertrophy in response to Ang II [51]. Finally, Nox2 also appears to play a role in MI. Studies reveal that Nox2 expression is higher in infarcted than noninfarcted myocardium from patients who have died from MI [52] and that Nox2 deletion in mice protects against post-MI hypertrophy, fibrosis, and left ventricular dysfunction [53]. Therefore, like Nox1, Nox2 also appears to be an attractive therapeutic target for the treatment of CVD. However, Nox2 inhibition in leukocytes could potentially lead to immune dysfunction, as is seen in X-linked chronic granulomatous disease (CGD), an immune disorder whereby defects in Nox2 lead to the absence of the respiratory burst in neutrophils and recurrent infections [54]. So, although Nox2 appears to be a desirable therapeutic target, the potential side effect of immunodeficiency highlights the importance of therapeutics that selectively target pathological Nox2 activation in cardiovascular cell types while leaving physiologic Nox2 activation in neutrophils intact.

Nox4 is expressed in ECs, VSMCs, cardiomyocytes, and fibroblasts, and plays multiple and sometimes contradictory roles in the development of CVD. For instance, in ECs, Nox4 promotes the expression of endothelial surface adhesion molecules, adhesion of monocytes, and the transmigration of leukocytes into the vasculature [55]. In addition, Nox4 has been implicated in promoting the migration, proliferation, and differentiation of both VSMCs and ECs [56, 57]. However, a transgenic model of Nox4 overexpression in ECs reveals Nox4-dependent vasodilation and lower blood pressure, under both unstimulated conditions and in response to Ang II compared to wild-type littermates [58]. As indicated above, H2O2 is the primary product of Nox4, and H2O2 has been described as a vasodilator in multiple vascular beds, including human coronary arterioles [59]. Nox4 overexpression in cardiomyocytes protects against contractile dysfunction and hypertrophy, whereas Nox4 deletion promotes these deleterious cardiac effects [60]. And although renal Nox4 expression is known to be upregulated in rats with aldosterone/salt-induced HTN [61], this increased Nox4 expression may not be the cause of HTN in this model, but instead may be in response to HTN to counteract the prohypertensive effects mediated by Nox1 and 2. Although the function of Nox4 in CVD remains incompletely understood, available data suggest that Nox4 may have a complex role in CVD development. In contrast to the other Nox isoforms, therapeutic approaches to increase Nox4 activity may be protective in some situations of CVD.

Nox5 is the most recently characterized vascular Nox homolog, and its importance in CVD is not fully understood. The deficiency in our knowledge of Nox5 is due in part to the unusual finding that Nox5 is not expressed in rodents [62], and thus, studies have not been able to examine the effect of modulating Nox5 expression in murine models of CVD. In addition, the presence of multiple Nox5 splice variants also complicates its study [63]. Nox5 is expressed in ECs and VSMCs in human vasculature and responds to agonists that contribute to CVD. In ECs, Nox5 is activated by Ang II and endothelin-1 [64], and its overexpression leads to increased proliferation and thus also to angiogenesis [16]. In human aortic VSMCs, Nox5 is activated by platelet-derived growth factor (PDGF), and depletion of Nox5 by small interfering RNA (siRNA) leads to decreased PDGF-induced ROS production and proliferation of VSMCs [65]. Furthermore, expression of Nox5 mRNA and protein is increased in human coronary arteries with atherosclerosis as compared to disease-free vessels, with expression of Nox5 predominantly in ECs in early lesions and in VSMCs in advanced lesions [66]. These findings in human vascular cells suggest that Nox5 contributes to the pathogenesis of CVD; however, more work will be necessary to confirm its specific role in activating vascular cells to determine whether it will serve as an appropriate therapeutic target.

The remaining Nox homologs—Nox3, Duox1, and Duox2—are not known to be expressed in the human vasculature; and studies manipulating these isoforms have not revealed a CVD phenotype. Therefore, these Nox isoforms are unlikely to have a substantial role in CVD pathogenesis.

Other NADPH oxidase Subunits

Since p22phox is a required subunit for the Nox1, Nox2, and Nox4 enzyme complexes, and p22phox protein level is a major determinant of the expression level of each of these Nox isoforms, it is anticipated that the effects of altering p22phox expression represent the combined effects on all three of these Nox complexes. Furthermore, general observations from multiple studies suggest that Nox4 plays a contradictory role to Nox1 and Nox2 [58–60], which may explain the conflicting results of studies that manipulate p22phox expression. For instance, targeted depletion of p22phox by siRNA decreases the expression of Nox 1, 2, and 4 in kidney and also reduce Ang II–induced HTN [67]. However, mice that overexpress p22phox in VSMCs do not develop HTN. Additionally, polymorphisms in the promoter of p22phox have seemingly opposing effects in CVD. For example, 930A>G causes increased phagocyte p22phox expression, increased ROS levels, decreased NO bioavailability, and is associated with HTN [68]. However, the same polymorphism has a protective effect against premature coronary artery disease and the incidence of MI in individuals less than 40 years of age [69]. These findings combined suggest that p22phox exerts a protective role in CVD via its association with Nox4-dependent signaling, whereas p22phox can exert detrimental effects in CVD via its association with Nox1 and/or Nox2-mediated signaling. These observations suggest that p22phox may be a poor therapeutic target in CVD.

As with p22phox, the remaining Nox enzyme complex subunits: p47phox, p67phox, and Rac, have also been evaluated to determine the role of Nox in CVD, and likewise, the outcomes of these studies are difficult to attribute to a specific Nox isoform due to the involvement of these subunits with both Nox1 and Nox2. However, these studies have been more consistent in revealing a detrimental role for p47phox [70], p67phox [71], and Rac [51] in CVD, and therefore support these subunits as potential therapeutic targets. An unresolved concern is that inhibition of p47phox, p67phox, or Rac for the treatment of CVD may also impair immune function by inhibiting Nox2 activation. However, it has been suggested by Drummond et al. that since phagocytic Nox2 activity must be two orders of magnitude lower than healthy controls to risk severe immunodeficiency; and since the activity of vascular Nox1 and Nox2 in animal studies is upregulated by less than one order of magnitude in CVD; it may be possible to titrate inhibitors targeting the shared cytosolic subunits such that vascular Nox is preferentially inhibited while immune function is preserved [72]. If so, p47phox could be an ideal therapeutic target by inhibiting both Nox1 and Nox2 in the blood vessel.

In contrast, the cytosolic subunits NoxO1 and NoxA1 interact with Nox1, and not Nox2. NoxA1 has been shown to be necessary for VSMC migration and proliferation [73], and its overexpression increases O2.− production and neointimal hyperplasia following vessel injury. Additionally, NoxA1 is required for ROS production by EC in response to ox-LDL [74]. And while the specific contribution of NoxO1 to CVD has yet to be demonstrated, it is known that NoxO1 is involved in Nox1-dependent ROS production in response to Ang II [75]. Therefore, NoxA1 and NoxO1 may serve as appropriate targets for the treatment of CVD. However, NoxO1 [76] and, to a lesser extent, NoxA1 [77] are also involved in the activation of Nox3, which is expressed in fetal tissues and in adult inner ear, including the cochlear and vestibular sensory epithelia [77, 78]. Therefore, inhibition of NoxO1 or NoxA1 may impair fetal development or auditory and vestibular function. These potential side effects must be considered when determining the feasibility of NoxO1 and NoxA1 as potential therapeutic targets. For additional reviews of Nox in CVD, see [72, 79–81].

Nox Signaling Steps and Current Inhibitors

The identification of Nox inhibitors has been a priority for both research and clinical applications. A recent report indicates that more than 350 Nox inhibitors exist [82]. These agents exert their effects by targeting specific steps of Nox activation including: (1) Nox expression; (2) initiation of signal transduction upstream of Nox activation (usually by ligand-receptor binding); (3) trafficking of Nox to the appropriate subcellular membrane; (4) activation and assembly of the NADPH oxidase complex; (5) electron transfer from NADPH to molecular oxygen; and (6) oxidation of downstream targets (Figure 3). Each of these steps offers a unique target for disrupting Nox-mediated cell signaling. The aim of this section is to present examples of inhibitors that work at each step, describe characteristics of an ideal therapeutic agent to target Nox-mediated CVD, and identify strategies that are most likely to yield effective Nox therapeutics (Table 1).

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Figure 3. Steps in NADPH oxidase (Nox) signaling that can be targeted for the treatment of Nox-mediated CVD. (1) Nox transcription and translation. (2) Ligand-receptor binding causing the signaling upstream of Nox activation. (3) Nox trafficking to the plasma membrane or to endosomes, lipid rafts, or organelles. (4) Association of cytosolic subunits to form a complete enzyme complex. (5) Transfer of electrons to create ROS. (6) ROS oxidation of downstream targets (Red: reduced, Ox: oxidized).

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Table 1. Nox inhibitors
Nox signaling stepInhibitorsMode of actionCurrent therapeutic roadblocksProposed solutions
  1. ACEI, angiotensin converting enzyme inhibitor; AEBSF: 4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride; Ang II, angiotensin II; ARB, angiotensin receptor blocker; CVD, cardiovascular disease; ClC-3, chloride channel-3; DIDS, 4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid; DPI, diphenylene iodonium; H2O2, hydrogen peroxide; mRNA, messenger RNA; NFA, niflumic acid; Nox, NADPH oxidase; O2•−, superoxide; PI, phosphoinositol; siRNA, small interfering RNA; SOD, superoxide dismutase; TNF-α, tumor necrosis factor-alpha.

(1) Enzyme expression[DOWNWARDS ARROW] Nox mRNA and Protein LevelsiRNADrug deliveryEnhance delivery using aptamers or novel vectors
(2) Ligand-receptor binding[DOWNWARDS ARROW] Ang II maturationACEITargets only one of many Nox activatorsUse in combination with inhibitors that target Nox
 [DOWNWARDS ARROW] Ang II-receptor bindingARB  
 [DOWNWARDS ARROW] TNF-α-receptor bindingInfliximab/Adalimumab Conduct clinical testing for effectiveness against CVD
(3) Nox localizationPrevent Nox targeting to correct membraneLocalization motif mutationRequires gene therapyDisrupt interaction between targeting motifs and membrane lipids or adaptor proteins
 Alter composition of PI in membranePI kinase or phosphataseSide effects 
(4) Assembly and activation of Nox complexesPrevent p47phox association with Nox complexAEBSFPotential side effects from serine protease inhibitionIdentify small molecules or aptamers with favorable therapeutic properties that prevent complex assembly
  ApocyninDosing, inactive prodrug 
  PR-39Interactions with other proteins 
  Gp91 ds-tatDrug delivery, pharmacokinetics 
 Prevent Rac association with Nox complexStatinsExtent of Nox-mediated effect unknownConduct clinical testing of statins combined with alternative Nox inhibitors
  Cdc42Q6ILRequires gene therapyEnhance delivery using aptamers or novel vectors
 Prevent phosphorylation necessary for Nox complexKinase inhibitorsNonspecific; Not tested for CVDsIdentify site specific inhibitors of Nox phosphorylation
 Ca++ Channel BlockerDiltiazemExtent of Nox5-mediated effect unknownRequires further study
(5) Electron transferExtracts electronsDPINonspecific for other flavoenzymesIdentify electron acceptor specific for Nox
 Anion channel inhibitor; prevent charge compensationDIDS /NFANonspecificIdentify specific inhibitor of ClC-3
(6) ROS productionConvert O2·− to H2O2SODDrug deliveryDevelop novel antioxidants
 Convert H2O2Catalase  
 to H2O and O2Glutathione peroxidase  
 Prevent propagation of lipid peroxidationChemical antioxidant (Vitamin E and C)Pro-oxidant activity; Clinical trials fail to show protection 
  • 1
    Nox expression: This is the primary step essential for Nox signaling and has been successfully inhibited in vitro by the application of RNA interference (RNAi). This appears to be a promising therapeutic approach, having been effectively utilized in cell culture to decrease Nox expression and signaling known to be involved in CVD development. For instance, the application of siRNAs targeting Nox1 in VSMCs leads to a reduction in the induction of NO synthase [83], a decrease in Ang II–dependent signaling [84], and a reduction in VSMC migration [85]. Furthermore, siRNA-mediated Nox1 inhibition in macrophages prevents LPS-induced ROS production and the conversion of these cells to foam cells—an important process in the development of atherosclerosis [86]. Additionally, in VSMCs, siRNA-mediated inhibition of either Nox4 or p22phox abolishes ROS production, inhibits NF-κB activation, and prevents apoptotic changes [87]. Notwithstanding the promise of findings, the use of RNAi as a therapeutic intervention for humans is still in its infancy, and may take years to optimize [88]. Additionally, the delivery of RNAi requires either local or intravenous injection, which significantly diminishes the ease of use of this approach. These downfalls highlight the necessity to design future Nox inhibitors with the viability of drug delivery in mind. Despite these drawbacks, RNAi remains a viable potential approach for the therapeutic targeting of Nox.
  •  
    An alternative approach to modifying Nox expression is promoter targeting. For instance, it is known that AP-1 binding to the promoter region of Nox1 is necessary for its upregulation in response to treatment with PGF in VSMCs [89]. Furthermore, Nox2 expression is extensively regulated by repressing and activating factors at its promoter region and several other sensitive regions upstream of the start site [90]. Importantly, promoter regions that bind the activating factor PU.1 are necessary for Nox2 expression, and CGD patients with mutations in PU.1 binding sites do not express Nox2 in neutrophils, monocytes, and B lymphocytes [91]. Therefore, promoter targeting may be an effective strategy for inhibiting Nox expression.
  •  
    A possible limitation of inhibiting the expression or activity of a specific Nox is the potential for compensatory change in expression and/or activity of other Nox isoforms [92]. However, studies using siRNA and in knockout animals suggest that if compensatory expression of alternative Nox isoforms is occurring, the extent of compensation is not sufficient to overcome the inhibition.
  • 2
    Ligand-receptor binding: Although Nox isoforms can be activated by multiple upstream signals, including mechanical and osmotic stress, the most well-characterized upstream signaling required for Nox activation is ligand-receptor binding. A variety of drugs inhibit Nox function by preventing ligand-receptor interaction necessary for activation of Nox. For instance, two approaches whereby Ang II–mediated Nox activation can be prevented are angiotensin converting enzyme inhibitors (ACE inhibitors), and Ang II receptor blockers (ARBs) [93]. Additional agents that could work in this capacity are the monoclonal antibodies (mABs) infliximab and adalimumab, which bind to and inhibit TNF-α, an inflammatory cytokine that must interact with its receptor in order to activate Nox1 [94]. Patients with autoimmune disease taking infliximab or adalimumab exhibit improved lipid profiles and a decrease in their atherogenic index [95–98]. However, since Nox1 is activated by multiple ligands, a drug that targets a specific receptor ligand will offer limited protection from Nox1 activation. Therefore, inhibitors that act on Nox directly rather than on an upstream activator are more likely to be effective for the treatment of Nox-mediated CVD.
  • 3
    Nox localization: In addition to the plasma membrane, the Nox subunits have been detected in multiple subcellular locations, including membranes of the endoplasmic reticulum, mitochondria, nucleus, and intracellular vesicles [99–102]. The subcellular localization of Nox is crucial in its activation, site of ROS production, and coordination of redox-signaling events. For example, differential Nox signaling occurs based on whether it is located at the plasma membrane or within endosomes. For instance, treatment of VSMCs with TNF-α causes Nox1 activation and ROS generation within endosomes with subsequent NFκB activation [94, 103]. However, treatment of VSMCs with thrombin causes nonendosomal Nox1-dependent ROS production, which leads to transactivation of epidermal growth factor receptor and subsequent downstream events [103]. Similarly, studies have revealed that localization regulates signaling outcomes of Nox2, 4, and 5 [100, 104, 105]. However, the mechanism by which Nox traffic to the correct subcellular location in order to accomplish appropriate signaling remains poorly defined. In general, the trafficking and localization of transmembrane proteins are regulated by short cytoplasmic motifs that bind to adaptor proteins that target their cargo to the correct membrane [106, 107]. Studies have revealed Nox1, 2, 4, and 5 contain unique localization motifs [99, 100, 108]. Interfering with the binding of these trafficking motifs to their cognate adaptor proteins is expected to prevent the trafficking of Nox proteins to their correct intracellular localizations and interfere with Nox activation and/or signaling. Furthermore, the sequences within and surrounding these trafficking motifs are unique to each Nox isoform, which would allow for isoform-specific targeting. Therefore, these motifs provide an attractive therapeutic target for the development of Nox inhibitors. However, additional studies identifying and characterizing Nox trafficking motifs and adaptor protein interactions must be conducted before this therapeutic approach becomes a viable option.
  • 4
    Assembly and activation of Nox complexes: Multiple drugs inhibit Nox1 and Nox2 activation by preventing assembly of the enzyme complex. This approach has been most successful in inhibiting Nox2 because the activation of this isoform depends on its association with the cytosolic subunits p47phox, p67phox, and p40phox, and Nox2 does not associate with these cytosolic subunits under resting conditions. p47phox is constitutively associated with p67phox and p40phox in the cytosol and contains an autoinhibitory domain that prevents the translocation of these subunits to the Nox2/p22phox subcomplex. Phosphorylation of p47phox relieves the autoinhibition and allows translocation of the cytosolic subunits to form the complete Nox enzyme complex [109]. Conversely, Nox1 does not necessarily rely on complex assembly for activation because it can associate with NoxO1, the p47phox homolog, which does not possess an autoinhibitory domain, and therefore can constitutively associate with Nox1 and cause stimulus-independent ROS production [110]. However, there are circumstances under which Nox1 assembles with p47phox instead of NoxO1. For instance, it is known that in VSMCs, p47phox is the predominantly expressed homolog that associates with Nox1 [111]. Conversely, Nox4 and 5 do not utilize cytosolic subunits and therefore do not rely on complex assembly for activation.
  •  
    Aminoethyl-benzenesulfono-fluoride (AEBSF) prevents the binding of p47phox to the Nox complex [112]. However, AEBSF is also a serine protease inhibitor, and thus may have significant unwanted side effects if used therapeutically. An additional commonly used inhibitor of complex assembly in animal and tissue culture studies is apocynin, which prevents the translocation of p47phox and p67phox to Nox/p22phox [113]. However, limitations to the therapeutic use of apocynin include the need for high oral concentrations, the necessity of activating the compound by peroxidases, and nonspecific effects [114, 115]. Another inhibitor that works by interfering with subunit assembly is gp91 ds-tat. This peptide inhibitor is composed of an Nox2 sequence that specifically binds to p47phox, and thereby prevents its association with Nox2 [116]. Gp91 ds-tat reduces Ang II–induced hypertension [117] and balloon-mediated neointimal hyperplasia [118]. These findings make gp91 ds-tat an attractive therapeutic candidate; however, this agent cannot be given orally and may have unfavorable pharmacokinetics [116].
  •  
    Statins interfere with Nox complex assembly by inhibiting the geranylgeranylation of Rac1, thereby preventing its translocation and activation of the Nox enzyme complex [119, 120]. Like ACE inhibitors and ARBs, statins are attractive therapeutic agents because they have the ability to treat CVD by multiple independent mechanisms. Like statins, Cdc42Q61L prevents the association of Rac with Nox. As a Rac isoform, Cdc42Q61L acts as a dominant-negative inhibitor, binding to Nox and preventing the binding of Rac1/2 [121]. A drawback of using this Rac isoform as a therapeutic agent is that the delivery of a dominant-negative protein into human cells has yet to be optimized. In addition, Rac is important for the activation of numerous proteins other than Nox; thus, general inhibition of Rac is expected to have multiple unintentional consequences. The viability of Rac inhibition as a therapeutic strategy would be greatly enhanced by specific interference with Nox-Rac interactions while leaving the interaction of Rac with other proteins intact.
  •  
    Phosphorylation is also an important mechanism that regulates Nox complex assembly and activation [122]. As previously mentioned, p47phox must be phosphorylated in order for Nox complexes to assemble and become activated [123]. In addition, it has also been recently shown that phosphorylation of Nox2 by protein kinase C (PKC) enhances Nox2 complex assembly and activation, and PKC inhibition has been successfully used to interfere with Nox2 signaling [124]. PKC inhibitors have also been used to treat diabetic microangiopathy [125] and should be further evaluated for their effectiveness in treating Nox-mediated CVD. Collectively, these studies reveal that phosphorylation is an important mechanism by which Nox complexes are regulated and suggest that approaches to prevent subunit phosphorylation would be of therapeutic interest.
  •  
    The calcium dependence of Nox5 activation offers a unique therapeutic target. Nox5 relies on calcium binding to an N-terminal calmodulin-like domain, which enables a conformational change allowing enhanced ROS generation. Inhibition of calcium signaling is expected to interfere with Nox5 signaling. Diltiazem, a commonly used calcium channel blocker, inhibits Ang II and endothelin-1 induction of Nox5 expression in cultured ECs [64]. The extent to which the beneficial effects of calcium channel blockers, commonly used in the treatment of HTN and angina, are mediated by inhibition of Nox5 signaling is not known and requires further study.
  • 5
    Electron transfer by Nox: The generation of ROS by Nox can be inhibited by preventing electron flow through the enzyme. One commonly used example of such a drug is diphenylene iodonium (DPI), which acts by extracting electrons from the electron transport pathway of Nox [126]. While DPI effectively inhibits Nox enzymes, it is nonspecific and capable of inhibiting other electron-transporting enzymes including nitric oxide synthases, xanthine oxidase, and a variety of mitochondrial enzymes [127]. Additional work is necessary to develop an electron extractor capable of binding specifically to a particular Nox isoform. Another approach that interferes with electron transfer is anion channel inhibition. The transfer of electrons across the membrane from cytoplasmic NADPH to reduce oxygen to superoxide is electrogenic and requires charge compensation. Without accompanying charge compensation, the oxidase is inactivated [128]. It was recently proposed that the chloride-proton antiporter ClC-3 provides charge neutralization to Nox1 and Nox2-containing vesicles [129, 130]. Deficiency of ClC-3 prevents Nox activation and attenuates the neointimal hyperplasia following carotid injury in mice [131]. Similarly, anion channel inhibitors (e.g., 4, 4’-diisothiocyanostilbene-2, 2’-disulfonic acid (DIDS) or niflumic acid) inhibit superoxide generation by Nox enzymes [94]. Although general anion channel inhibitors would be expected to have multiple off-target effects, specific inhibitors of ClC-3, or similarly relevant antiporters, may be an effective strategy to reduce Nox-mediated CVD.
  • 6
    Oxidation of downstream targets by ROS: Essentially, all prior efforts in clinical studies to reduce oxidative stress in CVD have focused on removing ROS after its formation and thereby preventing the subsequent oxidation of molecules. However, prospective antioxidant trials have proven ineffective at reducing major adverse cardiovascular events [132–135]. The reasons for the failure of oral antioxidants (e.g., alpha-tocopherol (vitamin E) and ascorbic acid (vitamin C)) to modify CVD are likely multiple and include: nonspecificity, insufficient dosing, inappropriate subcellular targeting, and low rates of reaction [136]. Another likely reason for this antioxidant paradox is inappropriate patient selection, such as inclusion of those individuals with severe advanced disease, who are unlikely to respond to therapy. Similarly, enrollment in these studies did not mandate demonstration of increased oxidative stress and there was no validation of effective dosing in individual patients. This would be the equivalent of testing an antihypertensive therapy on a population without confirmation of high blood pressure at enrollment and without measuring effectiveness of the therapy in reducing blood pressure. Further clinical studies will require the development of a sensitive and reliable measurement of oxidative stress to determine patient eligibility.

One approach to address many of the shortcomings of oral antioxidants is the overexpression of cellular antioxidants (e.g., superoxide dismutase, catalase, and glutathione peroxidases) [137, 138]. However, gene therapy for vascular disease will require significant advances to address the challenges of vector delivery to the target tissues, minimization of immune responses, and regulation of the levels and duration of transgene expression.

Additionally, a potentially important limitation of antioxidant therapy is the disruption of physiologic ROS-dependent cell signaling and intracellular redox state. In response to changes in cellular antioxidant levels, the cell is likely to modify expression and activity of antioxidant capacity to maintain a desired redox balance. Since ROS are produced by multiple enzymes, creating antioxidants that associate with cellular microdomains containing Nox may be a solution to this limitation.

Therapeutic Barriers and Innovative Solutions

Nearly all currently available inhibitors of Nox are not isoform-specific; thus, treatment with these agents inhibits the function of multiple Nox isoforms. This could lead to potentially serious undesired effects, including immune dysfunction as a result of Nox2 suppression. Additionally, available Nox inhibitors do not specifically target pathologic signaling and leave physiologic signaling intact. Ideally, newly developed Nox inhibitors would be isoform-specific, cell-specific, and preferentially target pathological modes of Nox signaling. This level of specificity could potentially be achieved by directly interfering with the interface between a particular Nox isoform and the proteins with which it must interact to become active under pathological conditions, such as kinases, trafficking adapter proteins, or the cytosolic subunits of the NADPH oxidase. Specific sequence targeting could potentially be achieved by the following three approaches: mABs, small-molecule inhibitors, or aptamers.

mABs selectively bind to specific epitopes causing either activation or inactivation of the targeted protein. Multiple antibodies have been generated to p22phox, Nox2, and Nox4 [139, 140]. However, these antibodies have not been found to exert a therapeutic effect. An important limitation to this approach is that antibodies do not readily cross cell membranes, restricting their use to Nox isoforms that are expressed at the plasma membrane. Furthermore, since regulation of Nox activity occurs primarily on the cytoplasmic side of the membrane, the number of extracellular epitopes with regulatory effects are limited. This problem is highlighted by a recent study, showing that mABs generated against an intracellular component of Nox4 inhibit activity in a cell-free system, whereas an extracellular-targeted antibody is not inhibitory [140]. Nonetheless, an antibody capable of interfering with electron transfer from the Nox heme group to molecular oxygen has the potential to inhibit ROS formation at the plasma membrane and within Nox-containing endosomes and may be a viable therapeutic option.

Small molecule inhibitors are organic compounds of less than 0.8 kD that bind to and alter the activity of biological molecules including DNA and protein. With the increased availability of large libraries of small molecules, investigators are developing protocols for high throughput screening to identify compounds that inhibit specific Nox isoforms in intact cells [141]. Several inhibitors that are specific for Nox1 have been identified by this approach [142]. It is important to note that the percent inhibition of Nox activity that is necessary to modify CVD development and preserve physiological activity is not known. For example, animal studies showing protection from CVD have relied primarily on mice with complete genetic deletion of an NADPH oxidase subunit [40, 48]. However, as little as 5–10% respiratory burst activity in neutrophils is sufficient to allow patients with CGD to remain healthy [143]. It will be important to assess whether incomplete inhibition of Nox has similar protective effects and whether the small molecules effective in inhibiting Nox in vitro are capable of reducing CVD in vivo.

Aptamer technology is the newest approach and may be the most effective strategy in yielding Nox inhibitors that have all of the characteristics necessary to make them ideal therapeutics for CVD [144–146]. Aptamers are short (15–40 nucleotides) synthetic single-stranded DNA or RNA macromolecules that form intramolecular interactions, shaping these molecules into unique three-dimensional structures that can bind tightly to a particular target. Since there are a tremendous number of possible nucleotide sequences, allowing a vast diversity of possible three-dimensional structures, aptamers have the potential to bind to nearly any molecular target. SELEX (systematic evolution of ligands by exponential enrichment) is the process by which aptamers are subjected to repeated rounds of in vitro selection in order to select for aptamers that have interactions with specific targets such as proteins, nucleic acids, or cells. The advantages to this approach are: (1) These oligonucleotides recognize targets with specificities and affinities similar to those of antibody–antigen interactions, but unlike antibodies can be synthesized entirely in vitro[147]. (2) Since these agents can be chemically synthesized, it is possible to substitute modified nucleotides for their native counterparts, to both promote nuclease resistance and improve their in vivo properties [148]. (3) Aptamers can target specific protein isoforms, making it possible to target one Nox homolog over another [149]. (4) Aptamers can specifically recognize post-translational modified sequences, which can be used to enhance selection for activated proteins. For example, some Nox and Nox subunit proteins are known to require phosphorylation for activation [122–124]. Therefore, peptides modified with constitutively phosphorylated residues to mimic activated Nox can be used as bait and nonphosphorylated peptides then used in a counter selection. (5) The potential of using aptamers as vascular therapeutics has already been demonstrated; several vascular proteins including VEGF (vascular endothelial growth factor) and factor IXa have been successfully targeted by aptamers and have undergone FDA approval for use as therapeutics [150, 151]. (6) Aptamers can be delivered in a cell type–specific manner by specifically selecting for aptamers capable of entering one cell or tissue type and using that pool to counter-select against aptamers that are capable of entering other cell types [152]. Therefore, Nox-targeted aptamers could be selected for their ability to internalize into VSMCs but not ECs so as to inhibit VSMCs migration and proliferation while preserving EC function. This approach would provide a novel solution to the problem of late thrombosis associated with drug-eluting stents, which results in part from the inhibition of re-endothelialization caused by nonselective delivery of antiproliferative agents to both VSMCs and ECs. (7) Aptamers can be conjugated to both small-molecule inhibitors and to antibodies to improve the delivery of these agents to specific cell types [153]. Therefore, aptamers can not only serve as Nox inhibitors, but also as delivery vectors for other types of Nox inhibitors. Aptamers have many unique properties that make them ideal as a novel strategy in the treatment of CVD. Aptamer technology is still in its infancy but has the potential to significantly alter the approach of treating multiple diseases, including CVD.

Conclusions and Future Directions

Although NADPH oxidases have been studied for over 40 years, their role in CVD has been confirmed only during the past decade. Several steps of Nox activation have been identified as promising targets for therapeutic intervention, including complex assembly, subcellular localization and trafficking, post-translational modifications, and electron transfer. Each of these critical steps requires motif-dependent protein–protein interactions between Nox and associated regulatory proteins. The design of specific Nox inhibitors to target these interactions will likely include innovative strategies using mABs, small-molecule inhibitors, or aptamers. These approaches have the potential to yield novel and effective therapies for Nox-mediated CVD in the next decade.

Author Contributions

  1. Top of page
  2. Summary
  3. Introduction
  4. Author Contributions
  5. Conflict of Interest
  6. References

Concept and design was the work of Miller FJ and Streeter J. Drafting of the article was the work of Streeter J, Thiel W, and Brieger K. Critical revision of the article was the work of Streeter J and Miller FJ. Approval of the article was the work of all authors.

Conflict of Interest

  1. Top of page
  2. Summary
  3. Introduction
  4. Author Contributions
  5. Conflict of Interest
  6. References

The authors have no conflict of interest.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Author Contributions
  5. Conflict of Interest
  6. References