Sources of Vascular Nitric Oxide and Reactive Oxygen Species and Their Regulation

Home , Kovach (surname)

Physiol Rev 99: 311–379, 2019 Published October 31, 2018; doi:10.1152/physrev.00036.2017 SOURCES OF VASCULAR NITRIC OXIDE AND REACTIVE OXYGEN SPECIES AND THEIR REGULATION

X Jesús Tejero, Sruti Shiva, and Mark T. Gladwin

Pittsburgh Heart, Lung, Blood and Vascular Medicine Institute, University of Pittsburgh, Pittsburgh, Pennsylvania; Pulmonary, Allergy and Critical Care Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania; Department of Pharmacology and Chemical Biology, University of Pittsburgh, Pittsburgh, Pennsylvania; and Department of Medicine, Center for Metabolism and Mitochondrial Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania

Tejero J, Shiva S, Gladwin MT. Sources of Vascular Nitric Oxide and Reactive Oxygen Species and Their Regulation. Physiol Rev 99: 311–379, 2019. Published October 31, 2018; doi:10.1152/physrev.00036.2017.—Nitric oxide (NO) is a small free radical with critical signaling roles in physiology and pathophysiology. The generation of sufﬁ- cient NO levels to regulate the resistance of the blood vessels and hence the mainte- Lnance of adequate blood ﬂow is critical to the healthy performance of the vasculature. A novel paradigm indicates that classical NO synthesis by dedicated NO synthases is supplemented by nitrite reduction pathways under hypoxia. At the same time, reactive oxygen species (ROS), which include superoxide and hydrogen peroxide, are produced in the vascular system for signaling purposes, as effectors of the immune response, or as byproducts of cellular metabolism. NO and ROS can be generated by distinct enzymes or by the same enzyme through alternate reduction and oxidation processes. The latter oxidoreductase systems include NO synthases, molybdopterin enzymes, and hemoglobins, which can form superoxide by reduction of molecular oxygen or NO by reduction of inorganic nitrite. Enzymatic uncoupling, changes in oxygen tension, and the concentration of coenzymes and reductants can modulate the NO/ROS production from these oxidoreductases and determine the redox balance in health and disease. The dysregulation of the mechanisms involved in the generation of NO and ROS is an important cause of cardiovascular disease and target for therapy. In this review we will present the biology of NO and ROS in the cardiovascular system, with special emphasis on their routes of formation and regulation, as well as the therapeutic challenges and opportunities for the management of NO and ROS in cardiovascular disease.

I. INTRODUCTION 311 These include oxygen radicals and peroxides, such as super- ·Ϫ II. NITRIC OXIDE GENERATION AND... 312 oxide (O2 ) and hydrogen peroxide (H2O2), nitrogen rad- · III. SUPEROXIDE AND HYDROGEN... 327 ical species, such as NO and nitrogen dioxide (NO2 ), and Ϫ IV. OTHER ROS 344 other species, such as peroxynitrite (ONOO ) and hypo- Ϫ V. CROSS-TALK BETWEEN ROS AND... 346 chlorite (ClO ). The species containing nitrogen are often VI. CONCLUDING REMARKS:... 347 treated separately as reactive nitrogen species (RNS). It is worth indicating that despite being long considered toxic I. INTRODUCTION species, most of these molecules have been shown to exert important signaling functions (249, 778, 937, 960). There- Nitric oxide (NO) is a small free radical molecule with fore, the role of many of these molecules in health and critical signaling roles. The discovery of the function of NO disease is related to their production rates, steady-state con- in the vascular endothelium as endothelium-derived relax- centrations, and the ability of the cellular antioxidant sys- ing factor led to the awarding of the 1998 Nobel Prize to tems to modulate their activity. Drs. Furchgott, Ignarro and Murad (36, 324, 449, 491, 716). The functions of NO in mammalian systems extend In general, dysregulated production of ROS/RNS, as is the beyond vascular signaling and are relevant in all organ sys- case for NO, leads to oxidative stress and deleterious con- tems, including but not limited to neuronal signaling, and sequences for living systems. However, as pointed out host defense (448, 659, 738). above, these molecules often have important signaling roles at low concentrations. For instance, the differences in re- A number of oxygen-related species of high chemical reac- sponse to NO at varying concentrations have attracted con- tivity are referred to as reactive oxygen species (ROS). siderable attention. It has been shown that low levels (pM/

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nM) are physiological and related to the activation of high donors or supplementing with NOS substrates to reverse affinity primary binding targets such as soluble guanylyl endothelial dysfunction have had limited success. The use of cyclase (sGC) and cytochrome c oxidase (433, 863). An general antioxidants for the treatment of oxidative stress emerging paradigm proposes that intermediate levels (50– has also failed in most cases. Recent advances in the field 300 nM) can activate a range of positive and negative re- have provided many clues on why these approaches have sponses from wound healing to oncogenic pathways (938). been unsuccessful. We will discuss these and other relevant Higher concentrations of NO (Ͼ1 ␮M) can lead not only to physiological and pathophysiological issues and indicate oxidative stress but also nitrative and nitrosative stress via how advances in basic biochemistry of the generation of the generation of peroxynitrite and nitrosating species (411, NO/ROS have evolved our understanding and set new di- 412, 938, 939), and in combination with oxygen, can trig- rections in the field. ger posttranslational modification of proteins, lipids, and DNA (277, 433, 938). In this review, we will present the biology of NO and ROS in the cardiovascular system with special emphasis on their The production of adequate levels of NO in the vascular routes of formation, chemistry, mode of action, and dys- endothelium is critical for the regulation of blood flow and regulation in vascular disease. The formation pathways of vasodilation, as will be discussed at length in this review NO and the mechanisms of NO signaling will be discussed (299, 565, 573, 600, 786). In this context, it has become in sect. II. The proteins and biological systems generating increasingly appreciated that oxygen levels can impact the hydrogen peroxide and superoxide are treated in sect. III. oxidation/reduction properties of different proteins and Other ROS of particular relevance in the vascular system regulate NO levels (FIGURE 1) (367, 578, 595, 931). For are discussed in sect. IV. Section V will study the cross-talk example, nitric oxide synthases (NOSs) produce NO using between NO- and ROS-generating systems. Finally, in sect.

L-arginine and molecular oxygen (O2) as substrates. Thus, VI, we will discuss the current challenges and opportunities under hypoxic or anoxic conditions, the generation of NO for the treatment of cardiovascular disease through the reg- via NOS is compromised. However, a number of proteins ulation of NO and ROS levels in pathological conditions. that are involved in oxidative processes at basal oxygen levels can become de facto reductases as oxygen is depleted. The biological role of this transition is particularly promi- II. NITRIC OXIDE GENERATION AND nent in the case of heme- and molybdopterin-containing VASCULAR FUNCTION proteins such as hemoglobin (Hb), myoglobin (Mb), and xanthine oxidase (XO) (185, 575, 578, 862, 880, 945, The generation of sufficient NO levels to regulate the resis- 990). Clinical intervention through these pathways contin- tance of the blood vessels and hence the maintenance of an ues attracting intense research. adequate blood flow is critical to the healthy performance of the vasculature (277, 299, 573, 600, 786). A number of The concept of oxygen-regulated oxidation and reduction mechanisms are involved in both the generation of NO and processes in the metabolism of NO is not only relevant to the response to NO signaling in the vasculature. In this NO generation but also to the scavenging of NO in the section, we will overview the mammalian proteins involved vasculature (FIGURE 1). In this regard, the role of globins in the generation and sensing of NO in the cardiovascular like ␣-Hb and cytoglobin (Cygb) as catalytic NO dioxyge- system. The production of NO in basal conditions is largely nases that scavenge NO is a topic of current research (25, regulated by the activity of endothelial NOS (eNOS) in the 593, 594, 898). vascular endothelium (324, 449, 716). Nevertheless, the contribution of other agents cannot be ignored. For in- The translation of our knowledge about the biology of NO stance, nitrite reduction by heme proteins can mediate hy- and ROS has encountered significant challenges. For in- poxic vasodilation and other physiological responses (367, stance, initial attempts to enhance NO levels using NO 591, 968); neuronal NOS (nNOS) and inducible NOS (iNOS) can provide compensatory NO generation or exacerbated RNS synthesis (386, 444, 547, 704, 727). In this - NO NO3 study, we will review NO synthases and other NO-generating biological systems. Fe2+-O 2 - NO2 NO A. Oxygen-Dependent Nitric Oxide Synthesis O 2 Fe2+ The canonical pathways of NO formation rely on the spe- cialized NOS enzymes. NOS are dimeric, multidomain en- FIGURE 1. Oxygen and oxidoreductase enzymes regulate nitric oxide (NO) homeostasis. The gradient in the concentration of oxygen zymes that synthesize NO from molecular oxygen and L-ar- shifts the function of globins from oxidizing, NO-scavenging proteins ginine and use iron protoporphyrin-IX (heme), tetrahydro- to nitrite-reducing, NO-generating proteins. biopterin (BH4), FAD, flavin mononucleotide (FMN), and

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NADPH as cofactors (204, 904). The architecture of these 1. eNOS proteins is complex, with a oxygenase/heme domain that binds BH4 and heme, in which the oxidation of arginine to eNOS is the constitutive NOS form in endothelial cells NO using molecular oxygen takes place, and a reductase (ECs), and as such, it is the main contributor to vascular domain evolutionarily related to cytochrome P450 reduc- NO levels in physiological conditions (446). For exam- tase (CYPOR) that binds the cofactors FAD and FMN (204, ple, the infusion of a competitive inhibitor of eNOS into 377). The reductase domain uses NADPH as electron the human systemic or coronary circulation decreases source to reduce the FAD and FMN and finally transfer the basal blood flow by ~25% (146, 722). Basal levels of NO electrons to the oxygenase/heme domain (FIGURE 2). Be- produced by eNOS not only regulate blood flow but ton- tween both domains, there is a calmodulin (CaM) binding ically inhibit platelet activation and the expression of domain that regulates the electron shuttling between the inflammatory adhesion molecules on endothelium. The reductase and oxygenase domains. To add to this complex- role of eNOS in the maintenance of optimal cardiovas- ity, the electron transfer between domains occurs between cular function is also highlighted by the experiments with the reductase domain of one monomer and the oxygenase eNOS-deficient mice. eNOS deletion has some limited domain of the other monomer; thus, only the dimer is able effects on mouse blood pressure and vasodilation, as to generate NO catalytically. adaptive processes increase the production of nNOS and prostaglandins (444, 913). However, eNOSϪ/Ϫ mice The role of NOS enzymes in vascular function and patho- show impairments in angiogenesis and wound healing biology has been extensively studied (277, 299, 569, 790). (563). In the apolipoprotein E (ApoE) knockout (KO) In this study, we will summarize the most relevant concepts atherosclerosis model, addition of the eNOS deletion ac- about the function and regulation of the NOS isoforms in celerates the atherosclerotic process (533). Notably, health and disease states. overexpression of eNOS is also detrimental (708), high- lighting the delicate balance of eNOS function. NOS enzymes comprise three main isoforms: nNOS (NOS I), iNOS (NOS II), and eNOS (NOS III). Among these iso- Although eNOS is predominantly expressed in the endo- forms, eNOS is the enzyme more abundant in vascular en- thelium (TABLE 1), and the ECs are proposed to regulate dothelial cells and has the most significant impact on vas- NO signaling in the vasculature, a number of recent stud- cular function, with a variety of pathologies related with ies using chimeric cross bone marrow transplant mouse eNOS dysfunction. However, the specific roles of iNOS and models have shown that the red blood cell also expresses nNOS in vascular disease are not to be ignored and will be a functional eNOS that contributes in part to systemic also discussed below. blood pressure responses (181, 536, 1025). This erythro-

ACHeme Oxygenase H B 4 domain

CaM binding FIGURE 2. Architecture of nitric oxide synthases FMN (NOS). A: the arrangement of the domains in the NOS Reductase monomer. The oxygenase/heme domain (red) is con- domain NADPH nected to the reductase domain by a flexible linker, FAD containing a calmodulin (CaM) binding sequence. The reductase domain includes a flavin mononucleotide (FMN)-binding domain (orange) that shuttles electrons B Heme NADPH FMN from NADPH/FAD to the heme group and a FAD-con- H4B FAD e– taining domain (yellow) that uses NADPH as an electron NADPHADP HemeHem source. B: the binding of CaM (blue) to NOS promotes FMNMN H B FAD 4 electron transfer from the FMN domain of one monomer to the heme domain of the other monomer. C: three-dimensional structure model of NOS. The figure CaM CaM is assembled from the separated structures of the human endothelial NOS oxygenase domain (PDB: NO e– e– L-Citrulline + 4D1O) (574), the CaM binding peptide bound to CaM

L-Arginine + O2 (PDB:2N8J) (740), and the structure of the neuronal NADPHADP Heme NOS reductase (PDB:1TLL) (338). FMNMN CaM FAD H4B

HemeHemem NADPH CaM FMN H4B FAD

L-Arginine + O2 L-Citrulline + NO e– e–

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Table 1. Tissue distribution of NOS isoforms in the cardiovascular system

Tissue Cell Types Cellular Location References

nNOS (NOS1) Heart ECs Plasma membrane 114, 126, 599, 841, 977, 1037 Lung VSMCs Caveolae Blood vessels Adventitial ﬁbroblasts Sarcoplasmic Cardiomyocytes reticulum iNOS (NOS2) Heart ECs Plasma membrane 126–128, 521, 525, 604, 688, 698, 867, 903, Lung VSMCs Phagosomes 984, 1064 Blood vessels Adventitial ﬁbroblasts Golgi Cardiomyocytes Mitochondria Macrophages and other leukocytes

eNOS (NOS3) Heart ECs Plasma membrane 126, 181, 283, 358, 518, 819, 878, 1025 Lung VSMCs Lipid rafts Blood vessels Cardiomyocytes Caveolae Erythrocytes Platelets

EC, endothelial cell; eNOS, endothelial NOS; iNOS, inducible NOS; NOS, nitric oxide synthase; nNOS, neuronal NOS; VSMC, vascular smooth muscle cell.

cyte eNOS can contribute to endocrine NO signaling to Because of their impact in eNOS activity, the more relevant modulate both blood pressure and myocardial injury and phosphorylation sites are Thr495, Ser1177 (Ser1179 in bo- appears to be tonically regulated by arginase through the vine eNOS) and Tyr657. Other phosphorylation sites in- availability of the eNOS substrate L-arginine (1044). clude Tyr81, Ser114, Ser615, and Ser633 (TABLE 2).

eNOS is constitutively expressed; a number of posttransla- The activation of eNOS requires Ca2ϩ and CaM; however, tional modifications impact the ability of eNOS to produce changes in resting Ca2ϩ concentrations are not strictly nec- NO, from changes in the NO production rates to, in some essary to regulate NO synthesis by eNOS. In turn, CaM cases, the complete blockade of NO synthesis (296). The rel- affinity to eNOS is regulated via phosphorylation of ative presence of these modifications is critical to enzyme ac- Thr495. Thr495 is located in the CaM-binding site of tivity. General measurements of eNOS protein such as the eNOS, and the phosphorylation of Thr495 impairs CaM determination of monomer/dimer ratio by Western blot or a binding, thus blocking Ca2ϩ/CaM-dependent activation of single phosphorylation site status show a necessarily simplistic the enzyme (741). In resting endothelial cells, Thr495 is view of the activity of eNOS in cells. A deeper analysis of the generally phosphorylated (297). The phosphorylation has main regulatory factors of eNOS activity, including individual been attributed to protein kinase C (PKC) (297, 646), and phosphorylation sites, other posttranslational modifications, the residue is dephosphorylated by protein phosphatase 1 and the levels of the cofactors and substrates, with special (PP1) (646). The equilibrium between phosphorylated

interest on tetrahydropterin/dihydropterin (BH4/BH2) ra- and dephosphorylated Thr495 is mainly modulated by 2ϩ tios and L-arginine/asymmetric dimethylarginine (ADMA) the changes in intracellular Ca levels. Thus, bradykinin concentrations are necessary for a better assessment of the and Ca2ϩ ionophores promote Thr495 dephosphoryla- eNOS function in vivo and the assessment of endothelial tion and eNOS activation. Ser1177 phosphorylation ac- dysfunction conditions. In this section, we will describe the tivates electron ﬂow through the reductase domain and, regulation of eNOS activity by posttranslational modiﬁca- hence, increases NO synthesis in functional eNOS.

tions; the role of the cofactors BH4 and L-arginine and their Ser1177 is located in the C-terminal portion of eNOS in counterparts BH2 and ADMA, and the role of arginases and a helical C-terminal tail element that blocks ﬂavin reduc- L-arginine transport metabolons will be treated in sect. IIIB. tion and regulates the conformational equilibrium of the A detailed study on other regulatory mechanisms, including reductase domain (404, 631). The mechanism is similar protein/protein interactions, shear stress, and other mecha- to that described for nNOS Ser1412 (7, 947). In resting nisms, has been provided by other reviews (59, 296, 299, cells, Ser1177 is usually not phosphorylated. Phosphor- 870). ylation is induced by a variety of signals, and the kinases involved are dependent on the inducing factor (TABLE 2). 2ϩ A) PHOSPHORYLATION. A number of amino acids have been Some agonists like bradykinin and Ca ionophores can shown to be phosphorylated in human eNOS (296, 669). induce Thr495 dephosphorylation via PP1 and Ser1177

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Table 2. Posttranslational modiﬁcation sites in human eNOS

Site Kinase Effect of Modiﬁcation References

Phosphorylation Thr495 PKC Impairs CaM binding 297, 407, 646 AMP kinase Decreases NO synthesis rates Constitutive Can cause uncoupling through the reductase domain Ser1177 Akt Activates electron transfer through the reductase domain 74, 236, 321 PKA Increases NO synthesis rates AMP kinase CaMKII Tyr81 Src Slight increase in activity 320, 322 May be involved in Ca2ϩ/CaM sensitivity Ser114 Constitutive 74, 327 Ser615 PKA No change in NO synthesis 74, 647 Akt May modulate protein/protein binding interactions Ser633 PKA No change in NO synthesis 74, 647 PKG Tyr657 PYK2 Decrease in NO synthesis 292 Glutathionylation Cys382 Not determined 160 Cys689 Decrease ﬂow through the reductase domain 162 Decreased NO synthesis Can cause uncoupling through the reductase domain Cys908 Decrease ﬂow through the reductase domain 162 Decreased NO synthesis Can cause uncoupling through the reductase domain

CaM, calmodulin; NO, nitric oxide phosphorylation via activation of CaMKII at the same and the formation of superoxide with diminished NO syn- time. Shear stress activates Ser1177 via PKA-dependent thesis activity. Glutathionylation appears to be a detrimen- phosphorylation. VEGF, insulin, and estrogens promote tal modification caused by oxidative stress conditions (162, Ser1177 phosphorylation via Akt kinase. 522) and is very sensitive to the ratio of oxidized and reduced glutathione (160). Tyr657 is located in the FMN domain of eNOS in close proximity to the FMN cofactor. The mutation of Tyr657 C) S-NITROSATION. S-nitrosation of eNOS has also been re- (292) causes complete loss of NO synthesis and L-citrulline ported (777). This reaction is also dependent on eNOS my- formation, suggesting a blockade of the intramolecular ristoylation and Ser1177 phosphorylation and involves the electron transfer. Thus, Tyr657 phosphorylation effectively nitrosation of the Zn-binding cysteines (Cys96 and Cys101 inactivates eNOS. The phosphorylation of Tyr657 is cata- in the bovine eNOS) (271, 272). As a consequence of S- lyzed by proline-rich tyrosine kinase 2 (PYK2). This kinase nitrosation, the formation of the eNOS dimer is blocked, is activated in endothelial cells by several stimuli including resulting in the loss of NO synthesis. Denitrosation of angiotensin II, oxidative stress, and insulin (296). The det- Cys96-NO and Cys101-NO restores eNOS activity. Addi- rimental role of this phosphorylation on NO synthesis has tional studies using mutant eNOS Cys96Ser and spurred interest in the pharmacological inhibition of PYK2 Cys101Ser, which cannot be S-nitrosated, indicate that the for the treatment of cardiovascular disease (94, 628, 869, enzymatic activity is not altered by treatment with NO do- 983). nors, confirming a role for the modification of these thiols in the regulation of eNOS activity (271). B) GLUTATHIONYLATION. Recent reports indicate that eNOS can also be modified posttranslationally by S-glutathionylation D) OTHER POSTTRANSLATIONAL MODIFICATIONS. eNOS can be acy- (162). At least three residues have been shown to be suscep- lated at different residues. The myristoylation of the N-ter- tible: Cys689, Cys908 (162), and Cys382 (160) (TABLE 2). minal glycine targets eNOS to the membrane (284), The process can be reversed by glutaredoxin-1 and thiore- whereas the palmitoylation of Cys15 and Cys26 targets doxin (160, 907). The reaction increases eNOS uncoupling eNOS specifically to caveolae (82, 336). These modifica-

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tions are not expected to change the intrinsic NOS activity As pointed out, iNOS expression is prevalent in macro- but are important to modulate eNOS localization and phages. In these cells, iNOS induction is necessary for the function. generation of high levels of NO in the phagosome and bac- terial lysis and, therefore, is a basic tool for the innate im- Hyperglycemia can result in N-acetyl glycosylation of mune response (519, 688). However, in pathological situa- Ser1177. This modification results in a nonphosphorylable tions, increased NO synthesis can be deleterious (e.g., in Ser1177 and limits eNOS response to agonists and vasore- macrophages recruited to the atherosclerotic plaque). The laxation (257, 280, 1035). high rate of NO production by iNOS, together with superoxide formation from iNOS or other sources, can lead to In summary, we want to remark that the generation of NO the production of peroxynitrite (see sect. IVA) (222, 624, by eNOS in optimal conditions, at least as studied in vitro 981, 1034), a very toxic oxidant and nitrating agent (78, with saturating concentrations of coenzymes and substrates 709). Studies in which iNOS deletion was added to the Ϫ/Ϫ and absence of deleterious modifications, is a well-balanced ApoE atherosclerosis model indicate improved cardiovascular function in the ApoEϪ/ϪiNOSϪ/Ϫ mice compared process with a very limited production of superoxide as a Ϫ/Ϫ side reaction. However, a number of circumstances can lead with ApoE controls, indicating a detrimental role of to changes in the NO generation cycle and trigger the pro- iNOS in atherosclerosis progression (532). duction of superoxide instead of NO. These processes are Finally, it should be noted that iNOS-derived NO can also generally termed “eNOS uncoupling” as the consumption have positive effects in certain conditions. iNOS activation of reducing equivalents by eNOS is no longer coupled to the has been identified as a mechanism of protection against formation of NO and is instead driven to the generation of ischemia/reperfusion damage. This observation appears to superoxide from molecular oxygen. This process is largely be related to a preconditioning effect caused by increased deleterious and has been linked to endothelial dysfunction levels of NO (109, 483, 579, 1009). and other vascular pathologies. eNOS uncoupling will be discussed in sect. IIIB along with other mechanisms of su- 3. nNOS peroxide generation. Similar to eNOS, nNOS is a constitutively expressed form 2. iNOS of NOS activated by CaM binding. NO synthesis rates for nNOS are generally higher than eNOS, but also tightly 2ϩ Unlike eNOS and nNOS, which are constitutively ex- regulated by Ca /CaM (unlike iNOS), and nNOS is also pressed enzymes regulated via CaM binding and posttrans- very susceptible to feedback inhibition by NO (4, 902). This lational modifications, iNOS has a much higher CaM affin- enables nNOS to produce NO in a pulsatile manner instead ity, so it binds CaM at very low Ca2ϩ concentrations, and of generating sustained low levels. These features appear to thus, its activity is not regulated by Ca2ϩ/CaM but mainly be more related to its role in synaptic transmission (811, 812). Although nNOS is commonly found in neurons (120), at the level of gene transcription (169). The kinetic param- it is also found in other tissues including vascular smooth eters of iNOS lead to a catalytic activity that generates muscle cells (VSMCs), adventitial fibroblasts, ECs, and car- higher NO levels than eNOS and nNOS, is less sensitive to diomyocytes (114, 126, 599, 841, 1037) (TABLE 1). Expres- NO-dependent autoinhibition, and generates higher levels sion levels of nNOS in the vasculature are lower than for of other nitrogen species, such as nitrate (816, 904). eNOS; however, there is increasing evidence of important functions for nNOS-derived NO in vascular physiology The expression of iNOS is usually limited to airway epithe- (188). lium and neuronal cells (TABLE 1), where it is highly expressed and active under basal conditions (604, 984, 1064), Early experiments in rodents indicated an important role of and activated macrophages and hepatocytes, where it is nNOS in the regulation of cerebral blood flow (731). Selec- expressed in the setting of inflammatory stimuli (688). tive inhibition of nNOS causes increases in blood pressure and decreased response to acetylcholine in normotensive Although healthy cells in the vasculature do not present rats (139). In eNOSϪ/Ϫ mice, nNOS can be activated by significant levels of iNOS, several pathologic processes shear stress, partially compensating the loss of eNOS in the show increased iNOS activity in blood vessels (386, 704, vasculature (444, 547). Experiments with nNOS KO mice 727). As iNOS can generate higher NO levels than eNOS, indicate increased neointima formation in carotid artery this iNOS activation leads to excess NO and severe impair- ligation and balloon injury models (666), indicating that ment of vascular function. This effect is mediated by several nNOS appears to limit vascular injury independently of pathways including continuous activation of sGC and com- eNOS. To investigate the role of nNOS in atherosclerosis, Ϫ/Ϫ petition for BH4 with eNOS (386). Overall, the excess NO the nNOS deletion was incorporated in mice carrying limits the response of blood vessels to vasodilators and de- the ApoEϪ/Ϫ deletion. The double deletion indicates a pro- creases NO sensitivity (263). tective role for nNOS in the development of atherosclerosis,

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G as the mice carrying both deletions showed accelerated pro- nine (L-NNA) and its precursor N -nitro-L-arginine methyl gression of the disease (534). ester (L-NAME) were characterized as general NOS inhibitors and continue to be broadly used in research, especially The effects of nNOS in the human vascular system have L-NAME (FIGURE 3). been demonstrated by studies with nNOS-specific inhibitors. These works indicate that NO synthesis from nNOS B) 7-NITROINDAZOLE. 7-Nitroindazole (7-NI) (FIGURE 3) is one has definite roles in the vasodilatory response, including, of the earliest inhibitors showing isoform specificity. Al- but not limited to, microvascular tone and coronary flow though it was shown that all three isoforms can bind 7-NI (498, 843, 844). with very similar affinity (17), in vivo studies showed inhibition of nNOS without significant effects in blood pres- Another relevant pathway for nNOS-dependent NO signal- sure, a surrogate of eNOS inhibition (664). It appears that ing not mediated by sGC is the formation of S-nitrosothiols 7-NI may have differential properties in cell permeability, (462). This function has special relevance in brain function with limited uptake in endothelial cells (402). Thus, al- in health and neurodegenerative diseases (686). A more though 7-NI cannot be accurately described as a specific specific description of S-nitrosation and its signaling poten- nNOS inhibitor, it can behave as such in vivo. tial is presented in sect. IID. C) 1400W. N-[3-(aminomethyl)benzyl] acetamidine (FIGURE 4. Pharmacological regulation of NOS enzymes 3) is a specific inhibitor of iNOS (344). It remains one of the most widely used NOS inhibitors in research because of its There have been significant efforts to develop specific NOS cell and tissue permeability. Its selectivity seems related to inhibitors for research and pharmacological purposes (17, the irreversible effect on the faster reacting iNOS, whereas 752). the inhibition on nNOS and eNOS is reversible. A similar effect is observed in N(5)-(1-iminoethyl)-L-ornithine A) NONISOZYME-SPECIFIC NOS INHIBITORS. Early research indicated (L-NIO) (FIGURE 3) (279). that analogs of the substrate L-arginine had inhibitory G properties on the three NOS isoforms. Among these, N - D) GW273629 AND GW274150. These sulfur-substituted acetami- G monomethyl-L-arginine (L-NMMA) and N -nitro-L-argi- dine amino acids (FIGURE 3) are specific inhibitors for iNOS

FIGURE 3. Chemical structure of the nitric oxide synthase (NOS) substrate L-arginine, tetrahydrobiopterin, and selected NOS inhibitors.

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(16). With safer toxicity profiles than 1400W, GW273629 Nitrate and nitrite can enter the circulation through the and GW274150 have been used in clinical trials for mi- dietary intake of nitrate-rich foods, particularly leafy green graine (437, 965) (NCT00242866; NCT00319137). Al- vegetables (1002). In addition, NO generated from NOS though both compounds were ineffective, it is not clear if enzymes is eventually oxidized to nitrate and nitrite as well. this is due to pharmacokinetic issues (965). Mammals do not possess efficient systems for nitrate reduction, but oral commensal bacteria have enzymatic nitrate E) VAS203. The pterin 4-amino-tetrahydrobiopterin (VAS- reduction pathways able to generate significant amounts of

203) (FIGURE 3) isaBH4 analog that can inhibit all NOS nitrite from nitrate reduction in the saliva (467, 602). Ni- proteins by replacing the BH4 cofactor (1008). VAS203 has trate is not only acquired from dietary sources but is also shown efficacy in the treatment of traumatic brain injury concentrated from the blood into the saliva via the salivary and has been used in phase II clinical trials (897) glands, pumped through the sialin transporter (761). After (NCT02012582). consumption of nitrate-rich foods (beet root, spinach, kale, etc.), the concentration of nitrate and nitrite in saliva can The structural similarities between isoforms (and particu- reach levels as high as 10 mM and 2 mM, respectively (601). larly between eNOS and nNOS) have made the develop- A portion of this nitrite is eventually absorbed into the ment of specific eNOS and nNOS inhibitors particularly circulation. Studies using antiseptic killing of the mouth challenging. Structure-based inhibitors have allowed the microbiome, or systemic NOS inhibition, suggest that development of new inhibitors with improved isoform spec- about half of basal plasma nitrite comes from the salivary ificity (337, 752). Cell permeability and other pharmacoki- reduction of dietary nitrate and the other half from the netic considerations have precluded the clinical use of novel oxidation of NO produced mainly by eNOS (485, 557, inhibitors; however, newer compounds have been devel- 763, 865). NO oxidation to nitrite is proposed to occur via oped; for instance, novel-specific nNOS inhibitors have the oxidase activity of plasma ceruloplasmin (865). been tested in animal studies (29, 254, 255, 1051). The further development of new clinically available specific in- Once nitrite accumulates in the plasma, the reduction of hibitors for NOS, in particular for the iNOS and nNOS nitrite to NO in the vasculature is mediated by several pro- isoforms, is to be expected. teins, including deoxygenated Hb (deoxyHb) (185). In- creased physiological levels of nitrite in plasma from a nitrate-rich diet can lower blood pressure (554). The effect is B. Nitrite-Dependent Nitric Oxide Synthesis dependent on the generation of nitrite from oral bacteria, as antiseptic mouthwash can eliminate both the increase in circulating nitrite and the blood pressure decrease (485). Despite early indications to the contrary (124, 329), nitrate Subsequent studies have further validated the link between and nitrite have been often overlooked as inert NO oxida- nitrite levels and improved cardiovascular function (486, tion metabolites circulating in plasma and in cells. How- 502). ever, during the last two decades, a novel paradigm of nitrite as a source of NO, especially in hypoxia, has emerged Numerous metal-containing proteins have been shown to (184, 185, 216, 364, 366, 369, 600, 603, 861, 968). Indeed, catalyze the reduction of nitrite to NO. These proteins gen- it has become increasingly accepted that the routes for the erally contain a heme or molybdopterin cofactor. The pres- production of NO in vivo are not only oxidative (as in ence of many of these proteins in diverse components of the NOS) but also reductive (particularly by nitrite-reducing vasculature, including blood cells, blood vessels, and heart proteins such as globins and molybdopterin enzymes). tissue, makes them relevant for the generation of NO in These pathways can work synergistically to maintain NO vascular biology. In the subsequent sections, we will discuss levels in response to changes in oxygen tension. We foresee the known mechanisms for nitrite reduction that are rele- this theme of synergistic oxidative and reductive pathways vant for cardiovascular function. We also note that, to date, to be potentially relevant to the generation of other reactive existing evidence indicates a role for Hb, Mb, and XO on species in health and disease. biological NO generation, whereas the role of the other possible mechanisms in vivo are still a matter of discussion. A few years after the discovery of the role of NO as the endothelial-derived relaxing factor, increasing evidence 1. Inorganic nitrite reduction supported the presence of nonenzymatic NO generation (89, 605, 1075). These studies and observations of arterial- Along with the role of different proteins in the reduction of to-venous gradients of nitrite in the human circulation in- nitrite, a number of nonenzymatic processes can reduce dicated a potential role for nitrite as a NO source in vivo nitrite to NO in vivo. In general, these systems require con- (369). Subsequent studies have extended this notion to a certed electron and proton donation, which is optimal at more global paradigm that encompasses the role of nitrate lower pH and hypoxic conditions, thus making them par- and nitrite as part of a cycle of NO generation that includes ticularly relevant in ischemic events (1074, 1075). The sim- both enzymatic and nonenzymatic processes. plest mechanism involves the disproportionation of nitrite,

318 Physiol Rev • VOL 99 • JANUARY 2019 • www.prv.org Downloaded from www.physiology.org/journal/physrev by ${individualUser.givenNames} ${individualUser.surname} (130.070.008.131) on November 29, 2018. Copyright © 2019 the American Physiological Society. All rights reserved. SYNTHESIS AND REGULATION OF VASCULAR NO AND ROS a process accelerated at acidic pH as it requires the proto- A) Hb. The ability of deoxyHb to reduce nitrite has been nation of nitrite to nitrous acid (HNO2). Two molecules of extensively documented (124, 248, 366, 447). Initial re- nitrous acid, then disproportionate, to form N2O3 and wa- ports by Brooks demonstrated that deoxyHb reacts with ter (Eqs. 1–4) nitrite, generating nitrosyl Hb and methemoglobin, consis- 2NOϪϩ 2Hϩ → 2HNO (1) tent with electron transfer from a ferrous Hb to nitrite 2 2 (124). The studies by Huang et al. (445) confirmed the ϩ → ϩ HNO2 HNO2 N2O3 H2O(2) overall stoichiometry of two molecules of deoxyHb gener- → · ϩ · ating one molecule of nitrosyl Hb and one molecule of N2O3 NO NO2 (3) methemoglobin (Eq. 7), consistent with electron transfer from a ferrous Hb to nitrite and indicated the critical influ- Ϫ ϩ ϩ → · ϩ · ϩ 2NO2 2H NO NO2 H2O(4) ence of pH in the process. By studying the same reaction using alkyl nitrites, which do not show a pH dependence in This process is prevalent in very low pH conditions, as in their reaction rates with deoxyHb, it was established that the stomach (89, 605). In fact, this mechanism has been the reaction of nitrite requires a proton and thus formally shown to be responsible for nitrite-dependent increases in requires nitrous acid (247, 248). This property also makes gastric mucosal blood flow (98) and may relate to the role of the reaction faster in low pH conditions. The overall pro- nitrite in host defense as acidified nitrite is a potent antimi- cess can be written as (Eqs. 5–7) crobial agent (99, 261). 2ϩ ϩ ϩ ϩ Ϫ → 3ϩ ϩ DeoxyHb ͑Fe ͒ H NO2 MetHb͑Fe ͒ NO Several molecules present in cells have been shown to re- ϩ OHϪ (5) duce nitrite to NO. Ascorbic acid (vitamin C) is a widely 2ϩ present cellular antioxidant that can catalyze the reduction DeoxyHb͑Fe ͒ ϩ NO → Hb Ϫ NO (6) of nitrite to NO in vitro (205). This effect is also observed with in vivo infusions of ascorbic acid and nitrite (217). 2DeoxyHb͑Fe2ϩ͒ ϩ Hϩ ϩ NO Ϫ → MetHb͑Fe3ϩ͒ Polyphenols can be assimilated through the diet and, like 2 ϩ ϩ Ϫ vitamin C, can reduce nitrite to NO (325, 733, 834). HbNO OH (7)

Dietary interventions have indicated improved vascular This scheme has been found to apply to virtually all the function linked to the consumption of polyphenols from heme protein–mediated nitrite reduction reactions studied different sources, including cocoa and dealcoholized red to date, including Hb (248, 447), Mb (247, 862), neuroglo- wine (60, 168, 225, 417, 834). Combined use of polyphe- bin (Ngb) (932, 945), Cygb (182, 571, 780), cystathionine nols and nitrite appears to have additive effects in vascular ␤-synthase (CBS) (149, 350), globin X (184), plant and protection (791). The effects of these dietary components cyanobacterial Hb (phytohemoglobins) (905, 946), flavo- can be, in part, due to the interaction with different enzy- hemoglobin (340), heme-albumin (43), cytochrome c (41), matic systems (30, 558, 824), but the link between these protoglobin (40), and microperoxidase-11 (42). compounds and NO formation deserves further investigation (600, 789). The reaction of deoxyHb with nitrite shows an increase in the instantaneous reaction rate as the reaction progresses 2. Heme proteins (447). Studies with T-state and R-state Hb–stabilizing compounds have unambiguously identified the reason for this Heme proteins have emerged as critical parts in the gener- change is the shift from T-state to R-state during the course ation of NO under hypoxia. The specific role of globins in of the reaction, as the NO formed in the reaction binds to nitrite reduction and NO homeostasis has been studied in the unreacted deoxyHb to stabilize the R-state. This process recent reviews (39, 770, 931). It should be noted that com- is referred to as R-state autocatalysis. The rate constants for Ϫ Ϫ peting reactions, namely the scavenging of NO by the fer- the reaction of Hb with nitrite change from 0.12 M 1s 1 Ϫ Ϫ rous heme [rate constants in the order of 1.7 ϫ 107 to for T-state Hb to 6.0 M 1s 1 for R-state Hb (TABLE 3) 1.5 ϫ 108 MϪ1sϪ1 (180, 967), Eq. 6] and the rate of NO (447). As the distribution of R-state and T-state Hb in vivo dioxygenation [rate constants in the order of 3.4 ϫ 107 to are regulated by oxygen concentration, this phenomenon 8.9 ϫ 107 MϪ1sϪ1 for mammalian Hb/Mb (264, 341), Eq. has important relevance for the reaction in vivo. In fact, the 8] greatly decrease the amount of bioavailable NO gener- reaction of Hb with nitrite has been modeled mathemati- ated by the reactions of nitrite with heme proteins (931). cally at different oxygen tensions. The combination of a However, nitrite-dependent NO generation in vivo cata- faster rate at higher oxygen, but increased availability of lyzed by Hb and Mb is well documented (183, 185, 365, deoxyHb at low oxygen, leads to bell-shaped dependence of 862, 975); it is very likely that the measurement of this NO the observed rates versus the oxygen concentration with the generation is due to the fact that the high concentrations of maximal rates of NO generation around 50% Hb oxygen Hb and Mb (in the mM range) can overcome the low effi- saturation, consistent with experimental data (367, 368, ciency of the nitrite reduction reaction. 794).

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lation, questioning the role of SNO-Hb in physiologic hy- Table 3. Rate constants for the reaction of nitrite with poxic vasodilation (454). Importantly, more recent studies selected deoxygenated heme proteins have shown that the ␤-93 cysteine KO mouse shows more Protein K, M؊1s؊1 Reference cardiac injury in ischemic conditions, suggesting that SNO-Hb may play a more prominent role in cardiac rather Hemoglobin than vascular function (1060). Human (T-state)a 0.12 447 Human (R-state)a 6 447 With regards to the nitrite reduction hypothesis, human Myoglobin studies show a signiﬁcant gradient of nitrite from the arte- Horse hearta 2.9 945 rial to venous circulation, concomitant with the production Sperm whalea 5.6 945 of NO (detected as iron-nitrosyl Hb) in the venous circulation (185, 369). In vitro experiments in isolated aortic rings Neuroglobin demonstrate that nitrite mediates vasodilation in conditions Human (S-S)a 0.12 945 of Hb deoxygenation (185, 196). Consistent with these ex- Human (SH)a 0.062 945 periments, infusions of physiological nitrite concentrations Cytoglobin mediate vasodilation in humans, which is enhanced during b Human 0.14 571 exercise and not affected by NOS inhibition. This effect is c Human 1.14 182 accompanied by iron-nitrosyl Hb formation, consistent Cystathionine-␤-synthase with nitrite reductase chemistry (185). Humand 0.6 350

a100 mM sodium phosphate, pH 7.4, 25°C; b100 mM sodium A central controversy relevant to all theories of erythrocytic phosphate, pH 7.0, 25°C; c100 mM phosphate, pH 7.4, 37°C; NO transport is the question of how NO generated can d 100 mM HEPES, pH 7.4, 37°C. escape the red cell without reacting with Hb. Although theoretical calculations suggest that this type of NO escape should be limited, accumulating studies by many research B) PHYSIOLOGICAL IMPLICATIONS OF Hb-DEPENDENT NITRITE REDUC- groups now demonstrate the production of bioavailable TION. The functional implications of the Hb-dependent ni- NO generated from the incubation of erythrocytes with trite reductase reaction remains a subject of intense re- nitrite (185, 196, 883, 975). The production of NO gas has search, particularly in the context of viable physiological been measured by chemiluminescence in vitro from the re- models of erythrocytic NO transport (366, 424, 591). action of deoxygenated Hb, Mb, or erythrocytes with ni- There is consensus in the field that NO-erythrocytic interactions regulate vascular function, particularly that oxygen trite. More physiological biosensor experiments demon- desaturation of red blood cell Hb stimulates increased vas- strate that incubation of nitrite with erythrocytes can in- cular NO bioavailability, leading to NO-dependent hy- duce cGMP production and inhibit activation in platelets poxic vasodilation (266). However, the molecular mecha- coincubated with this reaction (591, 883, 975). The mechanism for NO release remains unclear but has been postu- nism of this regulation is more contentious. Three models ϩ by which this erythrocytic regulation of NO production lated to involve the formation of NO (684) or N2O3 as occurs have been proposed: 1) hypoxic release of ATP from intermediates (69). Additionally, NO escape may relate to the red blood cell (882) that stimulates endothelial NO the localization of its formation, particularly at the surface production by binding to endothelial purinergic receptors, of the erythrocyte (813) or via concerted reactions of NO 2) the S-nitrosation of cysteine 93 of the ␤-chain of Hb and autoxidation of oxygen to form NO and NO2 (N2O3) (SNO-Hb) (866), and 3) the reduction of nitrite by de- at partial oxygen saturations (i.e., the reactions of nitrite oxyHb (as described above) (425). with both oxygenated Hb and deoxyHb at around 50% Hb oxygen saturation) (384). More work is needed to under- Understanding the role of each of these potential mecha- stand the biophysics of NO signaling from the erythrocyte nisms is an ongoing area of interest. The SNO-Hb hypoth- during nitrite reactions with deoxygenated red blood cells esis proposes that SNO-Hb is formed when Hb is oxygen- and Hb. ated (R-state) in the lungs and remains stable. Once Hb becomes deoxygenated (T-state), the SNO-Hb reacts with C) Mb. The physiological relevance of the reaction of Hb with existing thiols to release a vasodilatory signal (471, 728, nitrite suggested that similar processes could involve the 886). In the last 25 years since this proposal, numerous closely related globin, Mb. The ability of Mb to catalyze groups have debated key elements of the hypothesis includ- nitrite reduction to NO was first confirmed in vitro. Con- ing the mechanism of SNO-Hb formation and release and sistent with the monomeric structure of Mb and its lack of physiological levels of SNO-Hb. Additionally, a genetic allosteric regulation, the reaction rate is constant through murine model in which the cysteine 93 of ␤-Hb was re- the reaction, with a bimolecular rate constant similar to that placed with alanine (and in which SNO-Hb formation was of R-state Hb (TABLE 3) (447). In vitro studies of isolated abolished) demonstrated no impairment in hypoxic vasodi- mitochondria in the presence of Mb and nitrite have shown

320 Physiol Rev • VOL 99 • JANUARY 2019 • www.prv.org Downloaded from www.physiology.org/journal/physrev by ${individualUser.givenNames} ${individualUser.surname} (130.070.008.131) on November 29, 2018. Copyright © 2019 the American Physiological Society. All rights reserved. SYNTHESIS AND REGULATION OF VASCULAR NO AND ROS that Mb-dependent NO is bioavailable and can inhibit mi- species is more abundant. Thus, both processes can syner- tochondrial respiration in a similar manner to a conven- gize to regulate the concentration of NO (593, 594). The tional NO donor (862). In the cardiovascular system, the presence of an efficient reduction system for Cygb in heart is the most relevant organ for the function of Mb as a smooth muscle cells via cytochrome b5 and cytochrome b5 nitrite reductase. In a murine myocardial infarction (MI) reductase further supports the possible relevance of these model, nitrite has been shown to decrease infarct size, and processes in vivo (25, 593). this effect was lost in Mb KO mice, which cannot reduce nitrite to NO (774). Through this mechanism, cardiac Mb is F) CBS. CBS is a pivotal enzyme for the metabolism of homo- thought to be critical for the reduction of circulating nitrite cysteine (649). Mutations in the CBS gene are associated accumulated through diet or through the therapeutic pro- with hereditary homocystinuria (531). CBS binds heme and cess of remote ischemic preconditioning, which mediates 5=-pyridoxal phosphate. The 5=-pyridoxal phosphate group cardioprotection (267, 775). is critical for the reaction of homocysteine with serine or cysteine, whereas the role of the heme domain in the catalysis of D) Ngb. Ngb is a recently discovered six-coordinate globin CBS in unknown, but this heme appears to be functional and that is evolutionarily related to Hb and Mb (134). The redox active (868). The CBS-mediated reduction of nitrite to physiological function of Ngb is unknown, although be- NO by the CBS heme has been recently reported (149, 350). cause of its generally low concentration in cells (with the The reaction proceeds with rate constants similar to these of exception of the retina in the eye), it is probably not related T-state Hb, Ngb, or Cygb (TABLE 3) (149, 350). The presence to oxygen transport and storage as Hb and Mb are (38, 39, of CBS in endothelial cells (809) suggests that CBS could play 133). It was recently demonstrated that, as observed with a role in vascular nitrite reduction in vivo. other globins, deoxygenated Ngb can also reduce nitrite to NO (932, 945). Furthermore, this process is redox regu- G) NOS. As NOS proteins contain a heme group, it is conceiv- lated by the redox state of two surface thiols. Thus, oxida- able that they can catalyze the reduction of nitrite as ob- tion of two cysteine residues to form an intramolecular served in other heme enzymes. Indeed, in anoxic conditions, disulfide bond doubles the rate of nitrite reduction (TABLE eNOS has been shown to catalyze nitrite reduction to NO 3). This redox transition occurs in a physiologically relevant (970). The contribution of eNOS to nitrite reduction could range and can be controlled by the cellular GSSG/GSH ra- be significant in specific tissues, including red blood cells tios (945). It appears that Ngb is not present in vascular and kidney (654, 998). cells, except for sympathetic nerves (898). The reaction of Ngb with nitrite may have implications for microcircula- 3. Molybdoproteins tion in the brain, where several studies have found a vaso- protective effect of Ngb (140, 503, 917). The relevance of molybdopterins in the generation of NO in the vasculature has been increasingly appreciated since this E) Cygb. Like Ngb, Cygb is a six-coordinate globin discovered in reaction was first described for XO (650, 1061). As the the early 2000s (132, 493, 951). Cygb is ubiquitous in human molybdopterin group does not react directly with oxygen or tissues, mainly found in fibroblasts and cells of related lineage NO, these reactions that decrease NO release from heme such as osteoblasts and chondroblasts, but also in other cell proteins do not limit Mo-dependent nitrite reduction reac- types, including neurons (403, 687). Interestingly, it has been tions. Thus, the reduction rates by molybdoproteins can be identified in VSMCs (398, 571, 594). Cygb can reduce nitrite relevant in vivo at lower rate constant values as compared to NO at rate constants similar or slightly higher than Ngb for heme protein reduction rates. The reduction of nitrite by (182, 571) (TABLE 3). In addition, the oxygenated form of all four mammalian molybdenum-containing enzymes has Cygb reacts with NO to form nitrate in a NO dioxygenation been characterized. Recent reviews on the nitrite metabo- reaction that scavenges NO (339) (Eq. 8) lism by molybdenum enzymes are available (614, 616). 2ϩ Ϫ ϩ → 3ϩ ϩ Ϫ Fe O2 NO Fe NO3 (8) A) XO. Mammalian molybdopterin-containing enzymes catalyze different oxidation-reduction reactions including, but This reaction is common to most heme proteins (264, 341, not limited to, xanthine oxidation, sulfite oxidation, and 342, 931). In practice, this indicates that Cygb expression drug metabolism processes (839, 840). To our knowledge, can regulate the diffusion of NO and possibly prevent ex- the ability of mammalian Mo-containing enzymes to reduce cessive vasodilation at high levels of NO, not unlike the nitrite to NO was first described for XO (370, 578, 650, proposed role of ␣-Hb in vascular endothelial cells (898). 1061). Earlier work had shown the ability of XO to also This role has been proposed for Cygb in the vascular endo- reduce nitrate to nitrite (50, 308). Subsequently, several thelium (398, 593, 594). It should be noted that the scav- reports have shown that all mammalian Mo-containing en- enging of NO is more efficient at high O2 tensions, which zymes [XO, aldehyde oxidase (AO), sulfite oxidase, and the favor the formation of the ferrous/oxygenated heme spe- mitochondrial amidoxime-reducing component (mARC)] cies, whereas nitrite reduction would be prevalent when the are able to reduce nitrite at different intrinsic rates (TABLE 4) oxygen concentration is low, and the ferrous/deoxygenated (575, 880, 990).

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B) AO. AO is a molybdenum-containing protein with high Table 4. Kinetic parameters for the reaction of nitrite with sequence and structural similarity to XO. Its function is yet selected molybdopterin proteins unknown, although it has been related to the metabolism of

-؊1 ؊1 ؊1 retinoic acid, neurotransmitters, and a variety of xenobiot Protein kcat,s KM,mM K,M s Reference ics (513, 756, 920). AO has been shown to reduce nitrite to Xanthine oxidase NO, albeit a slower rate than XO (TABLE 4) (568, 575, 617, Human, pH 7.4a 0.41 2.2 186 616 1001). As observed for XO, the pH dependence of nitrite Human, pH 6.3b 1.2 0.67 1800 616 reduction shows a bell-shaped pattern with a maximal rate Rat, pH 7.4a 0.55 1.9 289 616 around pH 6.5 (617). The relevance of AO-dependent ni- Rat, pH 6.3a 0.58 0.25 2300 616 trite reduction has not been studied in so much detail as for XO, but depending on their tissue abundance and the avail- Aldehyde oxidase ability of their reducing substrates, some studies suggest Human, pH 7.4a 0.47 4.1 115 616 that the magnitude of AO activity in the vasculature can be Rat, pH 7.4a 0.67 3.6 186 616 similar to XO (568, 745, 1073). Rat, pH 6.3a 0.66 0.43 1500 616 Sulfite oxidase C) SULFITE OXIDASE. The molydopterin-containg sulfite oxidase c Human, pH 7.4 0.002 1.6 1.3 990 catalyzes the oxidation of sulfite to sulfate, preventing the c Human, pH 6.5 0.004 1.7 2.4 990 accumulation of toxic levels of sulfite. Impaired sulfite oxi- mARC-1 dase function leads to severe neurological damage and Human, pH 7.4d 0.1 9.5 11 880 death (510). Like XO and AO, the molybdopterin cofactor aReaction contains 50 ␮M of reductant (aldehyde); breaction con- of sulfite oxidase is able to catalyze nitrite reduction to tains 750 ␮M of reductant (aldehyde); creaction contains 5 ␮Mof produce NO (TABLE 4) (990). Unlike other molydopterin reductant (sulfite); dreaction contains 1 mM NADH, 0.2 ␮M cyto- proteins, in sulfite reductase only the Mo4ϩ center, and not chrome b reductase and 2 ␮M cytochrome b . ϩ 5 5 the Mo5 species, is able to catalyze the reaction (990). The

reaction has a marked O2 dependence with very low NO formation in the presence of O2. This is most probably Although the architecture of these enzymes is variable, ex- related to the fast oxidation of the Mo4ϩ species to Mo6ϩ isting evidence indicates that the reduction of nitrite takes by molecular oxygen as observed in other molybdopterin place in the molybdopterin site, where Mo reduces nitrite to proteins (880, 990). NO in a single electron transfer reaction similar to that of the heme proteins (615, 616) (Eqs. 9 and 10) D) mARC 1 AND 2. Two isoforms of the mARC are expressed in 4ϩ ϩ ϩ ϩ Ϫ → 5ϩ ϩ ϩ Ϫ mammals (705). Sequence homology suggests that mARC Mo H NO2 Mo NO OH (9) proteins belong to the sulfite oxidase family. The structure 5ϩ ϩ ϩ ϩ Ϫ → 6ϩ ϩ ϩ Ϫ Mo H NO2 Mo NO OH (10) of the mARC proteins has not been elucidated, but electron paramagnetic resonance data seem to support these similar- Depending on the redox potential of the enzyme, nitrite ities (1043). mARC proteins have a not yet understood reduction may occur by either of the two reactions or only physiological function. Existing evidence suggests that they ϩ by Mo4 (Eq. 9) pathways or one specific pathway (614, may be involved in the detoxification of N-hydroxylated 616). The pH dependence of the reaction is not linear as in substrates; mARC enzymes are required for the detoxifica- the case of the heme-dependent reactions, probably because tion of some hydroxylamines (706). mARC can also cata- of the protonation of important catalytic residues at low pH lyze the reduction of the NOS reaction-intermediate NG- that offsets the effect of the increased proton concentration. hydroxy-L-arginine to L-arginine in a reaction that may be These circumstances lead to a maximal rate for XO-depen- involved in NO biosynthesis (528). dent nitrite reduction around pH 6.3 (TABLE 4) (617). As noted above, unlike nitrite reduction by heme, the Mo co- Both mARC enzymes can reduce nitrite to NO in a process factor does not bind NO. Thus, this reaction potentially has very sensitive to oxygen-mediated inhibition (TABLE 4)

a higher yield of free NO. (880). As mARC enzymes can use the cytochrome b5 reductase/cytochrome b5 system as a source of electrons, the three In the vasculature, XO has been identified in the surface of proteins can form a mitochondrial metabolon for the reduc- epithelial cells, endothelial cells (5, 798, 979, 1010), and tion of nitrite to NO under hypoxic conditions (880). erythrocytes (357, 998). To date, XO is the most relevant nonheme nitrite reductase in vivo. Many studies have 4. Other proteins shown the relevance of XO in mammalian physiology (357, 568, 577, 578, 998). The use of specific XO inhibitors such A) CARBONIC ANHYDRASE. The reduction of nitrite by carbonic as allopurinol and oxypurinol has allowed the determina- anhydrase has been described (1). Unlike heme- or molyb- tion of specific XO effects on nitrite reduction and NO denum-containing proteins, the Zn2ϩ in the active site of metabolism (357, 568, 575, 998). the carbonic anhydrase is not redox active, thus invoking a

322 Physiol Rev • VOL 99 • JANUARY 2019 • www.prv.org Downloaded from www.physiology.org/journal/physrev by ${individualUser.givenNames} ${individualUser.surname} (130.070.008.131) on November 29, 2018. Copyright © 2019 the American Physiological Society. All rights reserved. SYNTHESIS AND REGULATION OF VASCULAR NO AND ROS nitrite anhydrase mechanism as described in Eqs. 1–4. Such C. The NO Receptor sGC and the reaction would be of notable interest in vascular physiology Modulation of Vascular Tone because of the ubiquitous presence of carbonic anhydrase in erythrocytes; however, this activity remains highly contro- 1. Structure and function of sGC versial (615). The effects of NO in the vasculature at concentrations in the B) CYTOCHROME C. The electron carrier cytochrome c has also nM/pM range (397) are mainly mediated through the acti- been noted as a situational nitrite reductase. As cytochrome vation of the canonical NO receptor sGC (223, 662, 711, c is a six-coordinate heme protein with no distal site avail- 751). sGC is a cytosolic, heterodimeric protein comprising able for nitrite ligation, the reaction is only observable in two homologous subunits, ␣ and ␤. Although at least two conditions where the protein is partly unfolded, a situation isoforms for either ␣ and ␤ subunit exist (termed ␣1, ␣2 and that can be elicited by cardiolipin or other anionic phospho- ␤1, ␤2) with different tissue distributions (129), the ␣1␤1 lipids (477). In the presence of cardiolipin-containing lipo- heterodimer is the most common form. Each monomer con- somes, cytochrome c has been shown to catalyze nitrite tains four domains: an N-terminal heme-binding domain, a reduction to NO in a reaction that may be prevalent in Per-Arnt-Sim (PAS) domain, a helical/coiled coil motif, and apoptotic processes (68). a C-terminal, guanylyl cyclase domain that catalyzes the formation of cGMP from GTP (FIGURE 4). It should be C) CYP AND CYPOR. The heme P450 cytochromes (CYPs) are noted that despite the homology between the N-terminal involved in a wide range of detoxiﬁcation processes. CYP domains, only the N-terminal domain of the ␤ subunit 2B4 and microsomal rat liver CYP fractions have been char- binds heme (489). The heme domain in the ␤ subunit is the acterized as nitrite reductases (576). Notably, the associ- NO-sensing center of sGC. Remarkably, unlike most other ated CYP-reducing protein, CYPOR, can reduce nitrate to hemoproteins that bind oxygen, CO and NO, this heme nitrite, forming a metabolon that can tentatively catalyze group shows a high selectivity toward NO and can also the complete process from nitrate to NO (576). bind CO weakly but shows no afﬁnity toward molecular

H-NOX PAS Helix GTP Cyclase -subunit

-subunit HemeHeme

GTP Cyclase Domain FIGURE 4. Architecture of soluble guanylyl cyclase (sGC). Top: arrangement of sGC domains in the sequence of ␣ and ␤ subunits. Bottom: model for the interaction of the N-termini domains of the ␣ and ␤ subunits of human sGC. Domains are colored according to the top panel. Catalytic GTP cyclase domains are not shown. The model for the human sGC protein is based on the model for Manduca sexta sGC derived from chemical cross-linking, small-angle X-ray scattering, and homology modeling (312, 313, 662).

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oxygen (625). When NO binds to the heme, changes in the In addition to the presence of heme-free protein, it is in- heme coordination cause a conformational change in the creasingly appreciated that a pool of ferric sGC can also N-terminal heme domain that relieves an inhibitory inter- exist in smooth muscle cells (641, 769, 891). As noted, the action between the heme domain and the catalytic C-termi- oxidation of the heme iron is probably the intermediate step nal domain, activating cGMP synthesis (1017). The exact in the generation of free-heme sGC (312, 363, 888). The details of the conformational changes that mediate enzyme generation of ferric sGC is one of the mechanisms of sGC activation are not yet known and are the focus of ongoing desensitization, and this form of the protein, whereas non- structural studies (145, 313, 958). responsive to stimulator drugs, can be rescued by sGC activators (302, 891). Recent work indicates that CYB5R3 The activation of sGC by NO leads to an increase in cGMP (methemoglobin reductase) may mediate the reduction of synthesis of at least two orders of magnitude (895). cGMP ferric sGC to NO-responsive ferrous sGC in cells (769). exerts a number of downstream signaling effects on phos- These ﬁndings open the possibility of novel pathways to phodiesterases such as PDE5 and other targets including regulate sGC responses in blood ﬂow regulation and for cGMP-dependent kinases and cGMP-gated ion channels, targeted therapy in diseases associated with oxidative stress eliciting a vasodilatory response (673, 674, 993). and endothelial dysfunction.

Apart from changes in NO levels or downstream signaling, Posttranslational modifications of sGC have been also described. Prolonged exposure to NO has been long known to the activity of sGC itself is compromised in a number of cause a decrease in sGC activity, usually characterized as pathological conditions (355, 363, 600, 853, 888, 1022). sGC desensitization (85, 671, 831). This process is related Situations directly related to sGC that can account for a to some of the tolerance profiles observed for NO donor decrease in sGC activity include changes in the expression treatments (459). sGC desensitization is probably due to of sGC, defective incorporation or loss of sGC heme, a different concurrent mechanisms, including S-nitrosation deficient reduction of the sGC heme iron, leading to an NO and heme iron–nitrosylation. Recent reports indicate that unresponsive ferric enzyme, posttranslational modifica- several cysteine residues in sGC can undergo S-nitrosation, tions, ubiquitination and proteosomal degradation, and de- leading to decreased activity (93, 822, 823). The formation fects in cGMP synthesis. of a heme iron–nitrosylated form of sGC, which has decreased guanylyl cyclase activity, has been reported (953). Changes in sGC related to the mRNA transcript have been Ubiquitination of sGC targets the protein for proteosomal ␣ described. For example, expression of a variant 2 subunit degradation, a process that can be inhibited by sGC activa- ␣ ( 2i) containing a 31 amino acid insertion has been shown tors (643). Further research on these sGC modifications, in different tissues. This subunit can form a dimer with ␤1 and interventions to prevent their occurrence, is ongoing but produces an inactive sGC form (80). Alternatively, sev- (92). eral splice variants of the ␣1 and ␤1 monomers have been identified in vivo (626, 787, 852). These splice forms can be 2. Pharmacological regulation of sGC related to decreased sGC activity and appear to be more abundant in disease conditions such as aortic aneurysm Because of the pivotal role of sGC in the regulation of the (626). NO response, substantial efforts have been made to develop pharmacological regulators of sGC function. In theory, di- The incorporation of the heme group in the sGC ␤1 mono- rect targeting of sGC offers notable advantages, bypassing mer is regulated by the chaperone heat shock protein 90 the complex mechanisms that may limit NO generation and (Hsp90) (95, 354, 356, 818). This process is also regulated availability under pathological conditions (FIGURE 5). Two by NO, with low NO doses improving Hsp90/sGC interac- main groups of compounds, activators and stimulators, tion and heme insertion, whereas high NO blocks heme have been developed with different mechanisms of action. insertion (354, 985). The relevance of heme-free sGC in the In both cases, the target of the drugs appears to be the context of cardiovascular disease is not well characterized. NO-sensing, heme domain of the ␤ subunit (662). A large Several lines of evidence suggest the presence of pools of number of sGC agonists have been developed over the last heme-free sGC in vivo (354, 356, 799, 943), and it is rea- three decades; we discuss below a few of the more widely sonable to expect that the exacerbated ROS conditions that studied compounds. For a more complete view of the devel- exist in the origin and development of many cardiovascular opment of these and other sGC-related drugs, the reader is pathologies can promote heme oxidation and subsequent referred to more specific reviews (274, 302, 888). heme loss (312, 363, 888). Thus, a plausible therapeutic strategy can target the inactive, heme-depleted sGC by use A) STIMULATORS. Stimulators are compounds that enhance the of agonists (activators) that can bind to the empty heme site activity of functional sGC and require the presence of fer- activating the enzyme. Some examples of these drugs are rous (Fe2ϩ) heme-bound sGC. These compounds can coop- discussed in the next section. erate with endogenous NO-mediated activation.

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Normal Endothelial dysfunction

Blood flow

Endothelial cells L-arginine L-citrulline L-arginine L-citrulline

eNOS OxidativeOxidative sstresstress eNOS

BH4 BH eeNOSNOS uuncouplingncoupling 2 eNOS eNOS

•- NO O2

ONOO •- sGC Fe (II) heme sGC Fe (III) heme (Reduced) Oxidation and heme loss (Oxidized)

GTP cGMP Vasodilation

Smooth muscle cells

FIGURE 5. Soluble guanylyl cyclase (sGC) function in healthy and endothelial dysfunction states. Oxidative

stress conditions cause oxidation of BH4 to BH2, superoxide production in the endothelial cells, and promote oxidation and heme loss in smooth muscle cell sGC.

The benzyl indazole YC-1 (FIGURE 6) was the first devel- the treatment of pulmonary arterial hypertension and oped sGC stimulator (524). The effects of YC-1 on sGC chronic thromboembolic pulmonary hypertension (351, activation were found to be synergistic with NO donors 352). and required heme-bound sGC (310, 436, 896). The compound presented some limitations and a lack of spec- Another second-generation stimulator compound is BAY ificity with cGMP-independent effects and inhibition of 1021189 (vericiguat) (301) (FIGURE 6), which has completed PDE5 (309, 326, 991). Based on YC-1 structure, a num- Phase IIB trials (287, 349, 744) and is currently in Phase III ber of optimized compounds have been developed such as clinical trials for the treatment of heart failure with reduced BAY 41–2272 and BAY 41–8543 (889). These com- ejection fraction (301) (NCT02861534). pounds are more specific than YC-1 and despite some poor pharmacokinetic properties, have been extensively B) ACTIVATORS. sGC activators are compounds that can trigger used in research (105, 353, 670, 760, 764, 852, 889). the catalytic activity of sGC independently of the redox state of the sGC heme or even in the absence of bound heme. Further studies led to the development of another YC-1– In general, these compounds work as heme analogs, replac- related compound, BAY 63–2521 (riociguat) (83, 655) (FIG- ing the heme group and triggering a similar conformational URE 6). Riociguat (commercialized as Adempas) was ap- change to that of NO-bound heme in the native enzyme, proved in 2013 by the Food and Drug Administration for thus activating cGMP formation (302).

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ample, as described above in sect. IIB2, S-nitrosation of Hb is an active area of research and SNO-Hb has been considered a reservoir of NO activity. In this regard, decreased levels of SNO-Hb have been reported in conditions such as pulmonary hypertension and hypothesized to contribute to disease pathogenesis (586, 729).

Accumulating studies demonstrate that beyond simply rep- resenting a reservoir of NO activity, S-nitrosation is a mechanism of enzymatic regulation. S-nitrosation of specific cysteine residues can modulate enzymatic activity and function. One prototypical example of this paradigm is the type 2 ryanodine receptor (RyR2). The RyR2 is a Ca2ϩ release channel that releases Ca2ϩ from the sarcoplasmic reticulum to mediate cardiac excitation/contraction coupling. The magnitude and duration of Ca2ϩ release from through the RyR2 determines contractility of the myocyte. Although the RyR2 contains many surface-exposed thiols, physiological S-nitrosation of specific cysteine residues activate the protein and con- FIGURE 6. Chemical structure of selected soluble guanylyl cyclase tribute to maintainance of healthy excitation contraction cou- (sGC) stimulators and activators. pling (1038). A lack of this S-nitrosation has been associated with cardiac arrhythmias as well as heart failure (131, 372, 373). Similar to the RyR2, a multitude of enzymes have now High-throughput screening assays led to the discovery of a been shown to be activated or inhibited by S-nitrosation. Al- novel series of sGC regulators that do not require the sGC though review of all these proteins is beyond the scope of this heme. The first developed drug in this category was the manuscript, the S-nitrosation of mitochondrial complex I and compound BAY 582667 (cinaciguat) (890) (FIGURE 6). its cardioprotective signaling is discussed in sect. IIIC3, and the Apart from the recovery of sGC synthesis, it was noted that S-nitrosation of eNOS as a regulator of its activity is outlined BAY 582667 could limit the degradation of heme-oxidized in sect. IIA1. The role of S-nitrosation in regulating cardiovas- and heme-free sGC (643). Cinaciguat showed promising cular function has been reviewed elsewhere (586, 677, 914). results in animal studies and acute decompensated heart failure (553), but the trials were stopped because of the In the following section, we review the chemical mecha- development of low blood pressure (269). nisms by which S-nitrosothiols are formed. It is important to note that physiological levels of S-nitrosothiols are deter- Another sGC stimulator that has reached clinical use is mined not only by the rate of S-nitrosation but also by HMR 1766 (ataciguat) (827) (FIGURE 6). Phase II clinical protein denitrosation. Although S-nitrosothiols degrade trials are currently evaluating the effect of ataciguat on through transnitrosation reactions (outlined below) and in- aortic valve calcification (NCT02481258). teraction with other low molecular–weight thiols, the existence of denitrosating enzymes has been reported. The best D. S-Nitrosothiols and the Modulation of characterized enzyme in this class is S-nitrosoglutathione Vascular Function reductase (GSNOR), a member of the aldehyde dehydrogenase enzyme group, which catalyzes the denitrosation of 1. S-nitrosation and vascular function glutathione and thus regulates the equilibrium of S-nitrosation (592). Although activation of sGC is considered the predominant mechanism of NO-dependent signaling, it is now recog- 2. Formation of S-nitrosothiols nized that posttranslational modification of cysteine residues by S-nitrosation comprises a significant alternative The formation of S-nitrosothiols is, on occasion, described mechanism of NO signaling. The first protein shown to be as the reaction of NO with a thiol (R-SH) group to generate S-nitrosated was albumin (885), and its existence was first a protein-bound nitrosothiol (R-SNO). However, it should proposed to represent a reservoir of NO bioactivity. How- be noted that NO is not a nitrosating species and does not ever, S-nitrosated albumin is now considered to be a prod- react directly with thiols. Formation of S-nitrosothiols in- uct of more detrimental NO “scavenging” by the protein volves the reaction of the deprotonated thiol (R-SϪ) and a (332, 956). It is now well recognized that a plethora of formal nitrosonium ion (NOϩ)(Eq. 11) proteins of various function and localization are S-nitrosated physiologically or in pathological conditions. For ex- R-SϪ ϩ NOϩ → R-SNO (11)

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In physiological conditions, the thiolate is generally pro- Finally, the reaction of an existing S-nitrosothiol with a vided by glutathione or cysteine, yielding GSNO or Cys- thiolate group can result in transnitrosation (Eq. 17) NO, respectively. The nature of the nitrosating species can R-SNO ϩ R’-SϪ ↔ R-S- ϩ R’-SNO (17) vary, generating different possible mechanisms for the reaction. Most of these routes require the formation of the ni- · Transnitrosation is a fundamental reaction of S-nitrosothi- trogen dioxide radical intermediate (NO2 )(Eqs. 12a, 12b) ols and allows for the formation of GSNO or S-nitrosated · ϩ ↔ · NO O2 O2NO (12a) proteins to mediate the formation of other S-nitrosothiols O NO· ϩ NO· → 2NO · (12b) and probably serve as a reservoir for S-nitrosothiols in the 2 2 cellular environment. Altogether, we want to note that the formation of the S-nitrosothiols is a complex process, and The reaction of NO · with a thiolate in the presence of NO 2 existing evidence indicates the requirement of signiﬁcant can generate the S-nitrosothiol via a thiyl (RS·) radical (Eqs. concentrations of NO and usually the presence of superox- 13a, 13b) ide and/or peroxynitrite. · ϩ Ϫ → · ϩ Ϫ NO2 R-S R-S NO2 (13a) The formation of S-nitrosothiols and their functional roles R-S· ϩ NO· → R-SNO (13b) in vivo is a growing ﬁeld, with protein S-nitrosation identi-

· · fied in most physiological pathways. It should be considered The reaction of NO2 with another molecule of NO2 or NO that the stability and precision of this modification is under generates dinitrogen tetroxide (N2O4) or dinitrogen triox- intense study to determine its parallels with other protein ide (N2O3), respectively, which are strong nitrosating spe- modifications such as phosphorylation (1023, 1024). Re- cies and can react directly with the thiolate (691, 692, 1018) cent studies of the proteome of eNOSϪ/Ϫ mice compared (Eqs. 14a–d) with wild type mice identify a more limited set of S-nitro- · ϩ ↔ NO2 NO N2O3 (14a) sated proteins (125, 378). Altogether, it appears that the detection of S-nitrosated proteins in the presence of exoge- NO · ϩ NO · ↔ N O (14b) 2 2 2 4 nous NO or R-SNO species can lead to nonphysiological ϩ Ϫ → ϩ Ϫ N2O3 R-S R-SNO NO2 (14c) S-nitrosation and more sensitive methods, monitoring pro- N O ϩ R-SϪ → R-SNO ϩ NO Ϫ (14d) tein S-nitrosation levels in vivo are necessary for accurate 2 4 3 detection of these modifications. Peroxynitrite (sect. IVA), formed by the reaction of NO and · superoxide, is another source of NO2 , either through ho- III. SUPEROXIDE AND HYDROGEN · molytic decomposition, via peroxynitrous acid, into NO2 PEROXIDE GENERATION AND · and OH radicals (Eqs. 15a–b) or via reactions with CO2 VASCULAR FUNCTION (Eq. 15c) or metal centers (Eq. 15d) ONOOϪ ϩ Hϩ → ONOOH (15a) The presence of ROS in the vasculature has historically been considered detrimental and a consequence of abnormal → · ϩ · ONOOH NO2 OH (15b) generation and/or an inability of the endogenous reductant Ϫ ϩ → · ϩ Ϫ· and antioxidant systems to scavenge these reactive species. ONOO CO2 NO2 CO3 (15c) The excess of superoxide, or other ROS, in the cell causes Ϫ ϩ nϩ ϪϾ · ϩ (nϩ1) ϭ ONOO M NO2 M O(15d) the modification of different parts of the cellular machinery and is a known player in the genesis and progression of · These reactions leading to the formation of NO2 are well cardiovascular disease (227, 408, 409, 570, 672, 700, 893). characterized (123, 548, 585, 1018). Global kinetic analy- Conversely, the signaling properties of many of these mol- sis of the reactions involved suggests that the main source of ecules are increasingly recognized. Indeed, the generation of · NO2 in vivo may be the decomposition of peroxynitrite superoxide and hydrogen peroxide, among others, has been (585). This result is consistent with the unfavorable rates shown to mediate critical processes such as angiogenesis, · for the direct formation of NO2 from oxygen and NO in hypoxic adaptation, and energy homeostasis (96, 249, 291, vivo, (1018) although the exact mechanisms of formation 720, 778, 825, 960). In this section, we will discuss the main remain a topic of current research (123, 872). sources of superoxide and hydrogen peroxide generation in the vasculature and their endogenous and pharmacological An additional pathway involves the formation of a ferric regulation. Among these, NADPH oxidases (NOXs) ap- nitrosyl heme which can be in transient equilibrium with a pear to be the most important sources quantitatively. In ferrous-nitrosonium species (Eqs. 16a–c) healthy situations, the generation of ROS by eNOS, mito- Fe3ϩ ϩ NO ↔ Fe3ϩ Ϫ NO (16a) chondria, or XO is limited, and in the case of mitochondria, it is probably more related to signaling pathways (289, Fe3ϩ Ϫ NO ↔ Fe2ϩ Ϫ NOϩ (16b) 290). However, there is significant evidence that not only Fe2ϩ Ϫ NOϩ ϩ R-SϪ → Fe2ϩ ϩ R-SNO (16c) NOXs but also the generation of ROS by uncoupled eNOS,

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mitochondria, or XO has particular relevance in the origin and development of cardiovascular disease. Loop C

A. NOXs

NOXs are multiprotein enzyme complexes that catalyze the electron transfer to molecular oxygen to form ROS, more commonly superoxide, but also hydrogen peroxide (79, 252, 383, 546, 912). To date, seven isoforms have been described [NOX 1–5 and dual oxidases (DUOX) 1 and 2]. Each isoform consists of a core, catalytic subunit with a Cytosol transmembrane domain and a variable number of regulatory subunits. The core subunit deﬁnes the name of the NOX complex. Thus, NOXs complexes show differences in Loop B their molecular architecture, activator proteins, localization, and function. NADPH is the preferred substrate for all NOX isoforms, with the NOX2 isoform being particularly NADPH speciﬁc (48, 63, 177, 1047).

The putative mechanism of all NOX proteins can be described as an electron transfer chain in which electrons are transferred from NADPH to the FAD cofactor in the flavoprotein/dehydrogenase domain and from FAD to the heme moieties (Eqs. 18, 19), where molecular oxygen is reduced FIGURE 7. Structure of the NADPH oxidase (NOX) core protein to superoxide. The specific mechanism of superoxide gen- membrane and cytosolic domains. The sequence comprises six eration is still unknown. According to one hypothesis, the transmembrane helices and a cytosolic flavoprotein/dehydroge- 2ϩ nase domain. The transmembrane helices are indicated by colors: I, reduced (Fe ) heme moiety binds oxygen to form a fer- red; II, orange; III, yellow; IV, green; V, cyan; and VI, dark blue. The rous/oxy species that decays to ferric heme and superoxide location of loops B and C is indicated. The dehydrogenase domain is (Eq. 20) (245, 980). Routes that do not involve the forma- shown in purple. The cofactors heme and FAD are shown as red and tion of a stable heme/oxy complex are also possible (Eq. 21) yellow sticks, respectively. Figure was drawn with PyMOL based on (456). Notably, the reaction of NOX1–3 and 5 generates the structures for the transmembrane (PDB: 5O0T) and dehydrogenase (PDB: 5O0X) domains of NOX5 (613). superoxide, whereas NOX4 and DUOX1/2 produce hydrogen peroxide (26, 936). Existing evidence suggests that the primary species generated is superoxide in all isoforms, and NOX core subunits 1–4 are structurally similar with a N- the formation of hydrogen peroxide is mediated by the per- terminal, membrane domain and a C-terminal flavoprotein, oxidase-like domain in DUOX or by the dismutation of dehydrogenase domain with high sequence homology to the superoxide molecules in NOX4 (Eq. 22). The mechanism of ferredoxin/NADP reductase family (490). NOX5 has a sim- NOX4 is still controversial, but elegant work by Takac et ilar architecture but has an additional N-terminal domain al. indicates that the E-loop (sequence between helices V before the transmembrane domain with four EF hand mo- and VI) of NOX4 shows notable differences with the other ϩ tifs that are responsive to Ca2 . The reductase domain of NOX isoforms (925). This loop in NOX4 seems to provide NOX5 also includes a CaM-binding motif not found in the steric hindrance to slow down superoxide release, forcing ϩ NOX1–4 proteins. When Ca2 is bound, the EF hands fold two superoxide molecules to react with each other in a over the reductase CaM element to relieve autoinhibition reaction that appears to be catalyzed, in part, by a histidine (63, 944). DUOX1 and 2 have a similar architecture to residue (925). NOX5 and include an additional transmembrane domain ϩ ϩ ϩ → ϩ ϩ NADPH FAD H NADP FADH2 (18) connected to a putative extracellular peroxidase domain ϩ 3ϩ → ϩ 2ϩ (262). The ability of this domain to act as a peroxidase is FADH2 2Fe FAD 2Fe (19) still under debate (638, 639). 2ϩ ϩ → 2ϩ Ϫ Fe O2 Fe O2 (20a) 2ϩ Ϫ → 3ϩ ϩ Ϫ· Fe O2 Fe O2 (20b) Recently, the crystal structure of a NOX core subunit (NOX5) has been determined (613). This structure is con- Fe2ϩ ϩ O → Fe3ϩ ϩ O Ϫ· (21) 2 2 sistent with previous evidence suggesting the presence of six Ϫ· ϩ ϩ → ϩ 2O2 2H O2 H2O2 (22) transmembrane ␣ helices with five connecting loops (FIG- URE 7) (613). The transmembrane domain includes four The architecture of the NOX/DUOX core subunits has been conserved histidines that serve as binding sites for two heme described in detail elsewhere (13, 79, 383, 546) (FIGURE 7). moieties bound between helices III and V (288, 613), and

328 Physiol Rev • VOL 99 • JANUARY 2019 • www.prv.org Downloaded from www.physiology.org/journal/physrev by ${individualUser.givenNames} ${individualUser.surname} (130.070.008.131) on November 29, 2018. Copyright © 2019 the American Physiological Society. All rights reserved. SYNTHESIS AND REGULATION OF VASCULAR NO AND ROS the flavoprotein domain binds the cofactor FAD and in- domain (694). However, a number of NOX4-interacting cludes a binding site for NADPH (49, 480, 613, 980). proteins have been recently identified. The protein polymerase delta–interacting protein 2 (poldip2) seems to exert reg- NOX isoforms are regulated by a large number of protein/ ulatory effects on NOX4 (212, 607), although poldip2 is protein interactions, and most isoforms are not functional involved on many other pathways (316). The membrane without the assembly of the NOX protein with several ad- protein Toll-like receptor 4 (TLR4) has been shown to in- ditional proteins. NOX1–4 are associated to another mem- crease NOX4 activity (86, 724), and Tks4/5, a homolog of brane component, p22phox (24). Although there is no struc- NOXO1, also regulates NOX4 activity (231). Additional tural data available for p22phox, sequence analysis and com- interactions with protein disulfide isomerase have also been putational models have been used to elaborate a consensus reported (465). In addition, gene splicing also appears to prediction of a three-helix transmembrane domain and a modulate NOX4 activation and play important roles in cytoplasmic C-terminal domain (637). Other models are cardiovascular disease (971). consistent with the presence of two transmembrane helices instead of three (135, 451, 930). The regulation of NOX expression is modulated by a variety of agonist species and transcription factors. We will only In the human vasculature, the most relevant isoforms ap- highlight some of the main agonists for each isoform; a pear to be NOX1, NOX2, NOX4, and NOX5 (252, 253, more detailed view is covered in recent reviews (79, 555, 555). The relative architecture of these three proteins and 737, 912). their activator proteins are shown in FIGURE 8. NOX1 activation requires p22phox, NOX-organizer 1 (NOXO1), 1. NOX2 NOX-activator 1 (NOXA1), and the GTPase Rac1 (24, 108, 348). The activator protein p47phox can replace NOX2 is the main NOX isoform in neutrophils and mac- NOXO1 in a noncanonical mode (61). Recent studies indi- rophages where it mediates the oxidative burst (198, 845). cate a possible role of EBP50 in NOX1 assembly (14). Genetic defects in the main constituents of NOX2 can trig- NOX2 activation requires p22phox, p47phox, p67phox, ger an inability of the immune system cells to kill invading p40phox, and Rac1 (276). As described for NOX1, a non- bacteria and fungi. This condition, characterized by an in- canonical mode of activation can use NOXO1 instead of creased susceptibility to infections, is known as chronic p47phox (927). The activation mechanisms of NOX4 are granulomatous disease. Most mutations causing the disease not completely understood. NOX4 appears to be constitu- (around 90%) are found in the membrane component tively active in the presence of p22phox without the partici- NOX2 (also known historically as gp91phox) and the inter- pation of cytosolic proteins (24, 627). This, in part, can be acting protein p47phox. Mutations in p22phox, p67phox, and explained by the constitutive activity of its dehydrogenase Rac2 have been also identified (428, 438).

-• O -• O2 2 NOX1 NOX2 p22NOX1NOX1 p22 NOX2NOX2

NOXO1/ p47 p40

Rac1 Rac1/ p47/ NOXA1/ p67/ Rac2 NOXO1 FIGURE 8. Putative assembly architecture of NADPH p67p6 NOXA1 oxidase (NOX)1, NOX2, NOX4, and NOX5 and their regulatory proteins.

H O 2 2 -• O2 p22 NOX4NOX4 NOX5N OX5

Ca2+ Ca2+ Ca2+ Ca2+

Poldip2

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Blood vessels NOX2

L-citrulline Endothelial NOX1NOX1 NOX4NOX4 cells NOX2NOX2 NOX5NOX5 FIGURE 9. Cellular distribution of NADPH oxidase (NOX) species in the blood vessels. Predominant forms NNOX1OX1 Smooth in each cell type are shown in bold. muscle NOX2NOX2 cells NOX4NOX4 NOX5NOX5

Adventitial NOX2NOX2 fibroblasts NOX4NOX4 NOX2

NOX2 expression has been also reported in other cell types and increased NOX2 activity is linked to vascular disease including endothelial cells and adventitial ﬁbroblasts (376, (142, 252, 478). 382, 657, 714, 715, 736, 876) (FIGURE 9, TABLE 5). Thus, the effects of NOX2 activity depend on different cell types In the vasculature, different effectors can increase NOX2 and have important links to the immune response (392, expression and activity (555, 737). A large number of 488). The current knowledge about NOX enzymes in the cytokines and hormones have been shown to induce cardiovascular system indicates that NOX2 is usually the NOX2 expression, including angiotensin II (658, 714, main player in the development of pathological conditions 802), thrombin (725), and TNF-␣ (275, 964). Circulat-

Table 5. Tissue distribution of NOX isoforms in the cardiovascular system

Tissue Cell Types Cellular Location References

NOX1 Heart ECs Plasma membrane 9, 423, 431, 652, Blood vessels VSMCs Endosomes 876, 879, 909 Adventitial fibroblasts Caveolae Cardiomyocytes NOX2 Heart ECs Plasma membrane 9, 166, 376, 382, Lung VSMCs Phagosomes 427, 468, 473, 580, 657, 714, Blood vessels Adventitial fibroblasts Endoplasmic reticulum 715, 736, 742, Cardiomyocytes 876, 950, 964 Hematopoietic stem cells Macrophages neutrophils, monocytes, dendritic cells NOX4 Heart ECs Plasma membrane 9, 10, 100, 118, Lung VSMCs Focal adhesions 166, 179, 201, 228, 265, 347, Blood vessels Adventitial fibroblasts Endoplasmic reticulum 423, 431, 540, Cardiomyocytes Nucleus 541, 556, 736, Cardiac fibroblasts Mitochondria 742, 876, 906, Hematopoietic stem cells 964 NOX5 Heart ECs Plasma membrane 81, 166, 201, Blood vessels VSMCs Endoplasmic reticulum 396, 469, 494, 848 Cardiomyocytes Cardiac fibroblasts

EC, endothelial cell; NOX, NADPH oxidase; VSMC, vascular smooth muscle cell.

330 Physiol Rev • VOL 99 • JANUARY 2019 • www.prv.org Downloaded from www.physiology.org/journal/physrev by ${individualUser.givenNames} ${individualUser.surname} (130.070.008.131) on November 29, 2018. Copyright © 2019 the American Physiological Society. All rights reserved. SYNTHESIS AND REGULATION OF VASCULAR NO AND ROS ing lipids also increase NOX2 production in ECs in vitro 2. NOX1 (159, 208, 856, 1063). The role of endothelial NOX2 in the development of cardiovascular disease is controver- The isoform NOX1 is expressed in VSMCs and in lower sial. Some studies have described that endothelial over- levels is also found in ECs (556, 909) (FIGURE 9, TABLE 5). expression of NOX2 increases endothelial dysfunction NOX1 is found in caveolae and signalosomes of vascular (88, 676). However, in other cases, an increased expres- cells (423, 431, 652). NOX1 expression is activated by sion of endothelial NOX2 has not been linked to patho- several factors including angiotensin II, PDGF, and throm- logical conditions even though an increased production bin (382, 492, 630, 695, 999). The nature of the agonist can of superoxide and macrophage recruitment was observed vary in different cellular compartments (651, 652, 1055). (243, 443). Increased NOX1 activity is associated with vascular disease, and studies have shown links between high NOX1 Global KO studies in mice indicate a negative role of NOX2 levels and hypertension (1015), diabetes (163, 380, 1003), in disease conditions. The deletion of the organizer subunit ischemia/reperfusion injury (479), and coronary artery dis- phox p47 ameliorates the hypertensive response to angioten- ease (393). NOX1-dependent signaling has been linked to sin II, decreases endothelial superoxide formation and angiogenesis. For instance, knockdown of NOX1 can de- eNOS uncoupling, and improves survival after MI (238, crease angiogenesis (343), whereas increased NOX1 pro- Ϫ Ϫ 549, 550). The mice combining the mutation of p47phox / motes tumor angiogenesis and growth (582). These findings with ApoEϪ/Ϫ appear to be protected from atherosclerotic indicate a critical role of NOX1-derived superoxide in an- lesions (253) compared with ApoEϪ/Ϫ mice (64, 976), al- giogenesis signaling via PPAR␣-, AKT-, and ERK1/2-medi- though some reports do not find significant differences in ated pathways. atherosclerosis progression or lesion size (443). It should be noted that the use of p47phoxϪ/Ϫ as a surrogate for the In agreement with these observations, the overexpression of NOX2 deletion is partly limited by the involvement of NOX1 in VSMCs increases blood pressure and hypertro- p47phox in other protein complexes, including not only the phy in mouse hypertension models (235). siRNA against noncanonical activation of NOX1, as already mentioned NOX1 or NOXO1 decreased superoxide production and above but also its interaction with protein disulfide isomer- eNOS uncoupling in diabetic mice (1049). NOXA1 has ase (214, 559). been also associated with atherosclerotic injury, with overexpression of NOXA1 increasing ROS production and increased NOXA1 levels detected in atherosclerotic lesions Other studies have directly targeted the NOX2 protein. (695). Mouse models of NOX2 deletion have generally shown phox similar benefits as those using p47 -deficient mice. The Studies of NOX1 function have also taken advantage of the Ϫ/Ϫ NOX2 mice are also protected against angiotensin II– generation of several KO lines. Mice with deletions of dependent hypertension (151, 988). The combination of NOX1 (345, 630), NOXO1 (511), and NOXA1 (293) have Ϫ Ϫ Ϫ Ϫ NOX2 / with ApoE / can decrease the atherosclerotic been developed. NOX1 deletion limits the blood pressure lesions (475), but this is not always the case (508). Other increase elicited by angiotensin II, although it is unclear if works have found a positive effect of the NOX2 deletion in basal blood pressure levels are reduced (345, 346, 630). mice treated with high fat diet. In these studies, mice carrying the deletion gain weight but show no increase in ROS The effect of NOX1 in atherosclerosis has been investigated Ϫ Ϫ Ϫ Ϫ production or endothelial dysfunction as compared with similarly to NOX2, using a Nox1 / ApoE / mouse the control animals (256, 609). model. The results of different studies are contradictory, with examples of improved outcomes for NOX1 deficient mice subjected to high fat diet (854) and diabetic mouse Finally, it is worth noting that the role of NOX2 in the models (380), whereas in other cases, NOX1 deletion did immune system and in vascular endothelial cells will not seem to have an effect (875). have, as noted, different impacts in cardiovascular disease (573). The relative contribution of these sources for The effects of the p47phox deletions, often used as a surro- the initiation and development of atherosclerotic lesions gate for the NOX2 deletion, can be partly due to changes in and other pathologies remains an issue of active research. NOX1 activity. This notion is supported by a diminished The current paradigm indicates that macrophage NOX2 effect of angiotensin II in the p47phox KO (549, 550) as is more detrimental in the development of cardiovascular compared with NOX1 (345, 630) or NOX2 deficient mod- disease (395, 876), with endothelial NOX2 probably in- els (988). volved in the initiation, but not so much in the development of the disease, and a subsequent progress of the Altogether, the role of NOX1 in cardiovascular disease atherosclerotic lesion dependent on immune cells (243, seems secondary to NOX2, but still significant, as shown in 573, 976). several models (345, 380, 630, 854). It is important to

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consider that therapies focused on NOX2 can overlook can modulate the role of VEGF-induced dimerization of compensatory effects of NOX1 in part by the sharing of a CD146 necessary for cell adhesion (1065), and NOX4-de- number of regulatory elements that can activate the produc- rived ROS can induce the reversible glutathionylation of tion of superoxide in both proteins. SERCA and increase Ca2ϩ levels, thus regulating VEGF- signaling cell migration (273). 3. NOX4 In a final note, it should be also considered that although NOX4 is the most abundant isoform in ECs, and it is also NOX4 activity has been shown to reduce fibrosis in athero- present in VSMCs and adventitial fibroblasts (10, 265, 736, sclerosis (230), it can also promote fibrosis, particularly in 832, 876, 1036). In the heart, NOX4 has been identified in the lung, but also in kidney and other tissues (110, 201, ECs, cardiomyocytes, and fibroblasts (682, 1058). The in- 416, 596, 820). The basis of these differences in NOX4 tracellular location of NOX4 suggests a role in signaling roles are not completely understood, but could be based on and may be involved in endoplasmic reticulum stress (817) the specific roles of NOX4 in different cell types and/or (FIGURE 9, TABLE 5). The expression of NOX4 is upregu- detrimental effect of eNOS activation in conditions in lated by TGF-␤ (201, 906). Other regulators include TNF-␣ which eNOS is uncoupled (323). (70, 681, 884, 1048), hypoxia (455), and PDGF (487). 4. NOX5 The role of NOX4 in vascular biology appears to be generally protective. Endothelial-targeted overexpression of NOX5 is the last discovered NOX isoform (62). In human NOX4 has beneficial effects and decreases blood pressure blood vessels, NOX5 is expressed in ECs and VSMCs (81, (779). Existing evidence supports a role of NOX4 in main- 469) (FIGURE 9, TABLE 5). taining blood pressure levels (552, 723, 779, 832). NOX5 expression can be activated by most of the factors Endothelial-specific overexpression of NOX4 in mice pro- that activate other vascular NOX enzymes such as angio- motes angiogenesis and eNOS function (193) and decreases tensin II, TNF-␣, PDGF, thrombin, and endothelin-1 (81, blood pressure (779). Comparable results were achieved 660, 681). As indicated above, the particular architecture of with NOX4 knockdown studies in cells (642). A beneficial NOX5 indicates a regulation of its activity through Ca2ϩ effect of NOX4 in ischemia/reperfusion has also been ob- levels (62, 469). Phosphorylation of Ser475, Ser490, served (989). Thr494, and Ser498 within the flavoprotein domain increases Ca2ϩ sensitivity (463, 717, 718); this phosphoryla- Ϫ Ϫ Studies with NOX4 / mice indicate protection from en- tion is mediated by different kinases, including PKC iso- dothelial dysfunction in wild-type mice compared with the forms (164). NOX4 deletion (832). In general, NOX4 shows protective Ϫ Ϫ effects in ApoE / mice (194, 229, 230, 379, 552, 838). Unlike the rest of the NOX proteins, NOX5 does not have One study has related NOX4 to hyperglycemia and insulin an homolog in rodents, which has hampered the research of resistance in mice (1012). However, NOX4 deletion in the this isoform (871). New approaches, such as mice strains Ϫ Ϫ ApoE / mouse does not ameliorate diabetes-associated incorporating the human NOX5 gene, may provide new vasculopathy (380). Other studies have found no change in information about NOX5 function (439, 517). NOX4 levels in diabetic rat models (1003). Heart-specific NOX4 overexpression has protective effects in the response Despite these difficulties, increasing data suggest a role of to chronic load stress in part due to increased HIF1␣ re- NOX5 in cardiovascular disease. Higher NOX5 expression sponse and increased angiogenesis (1058). It should be levels have been observed in patients with coronary artery noted that some aspects of NOX4 function in the cardio- disease (391), but causation has not been proven. NOX5 vascular system remain controversial. For instance, splicing expression also increases in the human heart after MI (396). of NOX4 appears to influence cardiovascular disease, with Expression of NOX5 in VSMCs and in macrophages and a shorter protein (NOX4D) exerting beneficial signaling monocytes can contribute to the development of atheroscle- functions in the nucleus, whereas full-length NOX4 is over- rotic lesions (622, 623). NOX5 also appears to modulate expressed in ischemia with detrimental effects (971). proliferation and cell migration processes (371, 719). Alto- gether, increased NOX5 activity seems to be involved in a NOX4 protective role may be due to its generation of hy- pathological response (661). Further work is required to drogen peroxide, and not superoxide (253). As hydrogen delineate the role of NOX5 in human cardiovascular dis- peroxide does not scavenge NO, changes in NOX4 activity ease. do not directly impact NO levels. The signaling mechanisms of hydrogen peroxide in ECs (941) lead to the increased 5. Pharmacological regulation of NOX enzymes activity of eNOS and other enzymes (121, 141, 193, 251, 815, 940). Consistent with this role of NOX4-derived ROS A plethora of NOX inhibitors, including small molecules in angiogenesis (642, 989), it has been shown that NOX4 and peptides, have been developed (22). Most of the inhib-

332 Physiol Rev • VOL 99 • JANUARY 2019 • www.prv.org Downloaded from www.physiology.org/journal/physrev by ${individualUser.givenNames} ${individualUser.surname} (130.070.008.131) on November 29, 2018. Copyright © 2019 the American Physiological Society. All rights reserved. SYNTHESIS AND REGULATION OF VASCULAR NO AND ROS itors have shown poor specificity between isoforms, ham- the inhibitory effects of apocynin; notably, apocynin is an pering the progress in the field (174). In this section, we will antioxidant and scavenger of ROS like HOCl and hydrogen present some of the most relevant inhibitors because of their peroxide (426, 735). Thus, although apocynin remains wide use or high isoform specificity. A more comprehensive widely used in research and has led to important insights in analysis of the available inhibitors for NOX enzymes can be the function of NOX enzymes (237, 413, 581), the results found in recent reviews (22, 174, 175, 252). of experiments with apocynin need be considered with cau- tion. In particular, effects in isoforms other than NOX1 and A) DIPHENYLENE IODONIUM. Diphenylene iodonium (DPI) (FIG- NOX2 should be validated with other approaches. URE 10) is a nonspecific inhibitor of all NOX isoforms (18, 197, 246). Its mechanism of action is based on the reaction C) NOX2 DOCKING SEQUENCE-TAT. NOX2 docking sequence-tat with the FAD moiety of the NOX dehydrogenase domain (NOX2ds-tat), the first isoform-specific NOX inhibitor, is a (699). Unfortunately, this reaction is general for any flavin- peptide based on the sequence of the B-loop of NOX2 (199, containing protein, which means that DPI is also an inhib- 218, 782). NOX2ds blocks specifically the interaction be- itor of many other ROS-producing enzymes, including, but tween NOX2 and p47phox (199). The addition of the tat not limited to, NOS, mitochondrial complex I and XO (18). sequence (nine amino acids from HIV-tat) confers mem- Some reports indicate that the use of low levels of DPI may brane penetration ability to the peptide (782). The inhibitor selectively target NOX in the presence of other flavopro- shows high specificity toward NOX2 (199). The chemical teins (241); however, the lack of specificity makes unlikely nature of the compound makes oral delivery unsuitable and the general use of DPI as a NOX inhibitor in vivo. has limited its pharmacological application, however, because of its isoform specificity and lack of off-target effects B) APOCYNIN. Apocynin (FIGURE 10) is a methoxy-substituted remains widely used in research (244, 461, 476, 762, 782, catechol generally considered to be a specific NOX2 iso- 910). Notably, a similar approach with NOX4 did not re- form inhibitor. Apocynin inhibits NOX2 by reacting with phox sult in significant inhibition of NOX4 (200), which suggests cysteine residues in p47 that appear to be necessary for a strong interaction between dehydrogenase and transmem- enzyme assembly (894). It should be noted that this mech- brane domains in NOX4 and points out a critical function anism of action converts apocynin into a NOX1 inhibitor of the B-loop in this interaction (460), very probably linked when NOX1 uses p47phox for activation in the noncanoni- to the constitutive activity of this NOX isoform. cal mode. Moreover, there are several factors that question

D) NOXA1 DOCKING SEQUENCE. NOXA1 docking sequence (NOXA1ds) is a peptide developed to block the interaction between NOX1 and NOXA1. The sequence contains 11 amino acids from the putative binding region of NOXA1 to NOX1. The selected sequence shows low similarity with p67phox to prevent cross reactivity with NOX2. The peptide prevents NOX1 assembly and does not inhibit NOX2, NOX4, or NOX5 as tested in in vitro assays (772, 792).

E) ML171. ML171 (2-acetyl phenothiazine) (FIGURE 10) is one of the few small-molecule, NOX isoform–speciﬁc inhibitors reported to date. ML171 inhibits NOX1 at nano-

molar concentrations, with IC50 values at least 20-fold higher for NOX2, NOX3, NOX4, and XO (359). The mechanism of action of ML171 seems to involve only interaction with the NOX1 protein and not with its activating subunits. ML171 showed off-target effects with the inhibition of serotonin and adrenergic receptors in the micromo- lar range (359). The study of differently substituted phe- nothiazines indicates some isoform selectivity that can represent a promising pathway to novel small-molecule, selective NOX inhibitors (847).

F) VAS2870 AND VAS3947. VAS2870 and VAS3947 (FIGURE 10) are recently described NOX inhibitors based on a triazol- FIGURE 10. Chemical structures of selected NADPH oxidase opyrimidine scaffold (892, 934, 1016). VAS2870 appears (NOX) inhibitors. to inhibit all isoforms, with proven inhibitory effects at least

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for NOX1, NOX2, NOX4, and NOX5 isoforms (21, 520). tant to recognize and understand the causes of eNOS un- The mode of action of these compounds is unknown, al- coupling to develop adequate interventions. In addition, a though for NOX2, they appear to block complex assembly number of situations impinge NOS function and NO gen- (21). These inhibitors do not have a noticeable effect on eration, but not necessarily cause increased superoxide pro- XO- or eNOS-dependent ROS production, so they can be duction, despite being usually characterized as uncoupling. considered specific pan-NOX inhibitors. VAS3947, a more Factors that can trigger eNOS uncoupling are discussed soluble derivative of VAS2870, shows similar inhibitory below. properties (1016). Off-target effects for VAS2870, namely nonspecific alkylation of thiols, have been reported (915). Initial reports indicated that nNOS had the ability to produce superoxide in a reaction dependent on NADPH and 2ϩ G) GKT-136901 AND GKT-137831. GKT-136901 and GKT- Ca /CaM (750). The effect of NOS inhibitors on this pro- 137831 are pyrazolopyridines structurally related to cess was diverse, with some inhibitors efficiently suppress- VAS2870 and VAS3947. GKT-136901 and GKT-137831 ing superoxide production (e.g., L-NAME), whereas others G (FIGURE 10) are the first inhibitors suitable for oral admin- had little effect (e.g., N -monomethyl-L-arginine) (749). istration, and currently GKT-137831 is in phase 2 clinical The reaction is observed in the three NOS isoforms (750, trials (472). GKT-136901 was first characterized as a selec- 973, 1034). The effect of cofactors and NOS inhibitors was tive NOX1 and NOX4 inhibitor, although it can also in- thoroughly investigated (150, 250, 590, 749, 973, 1033). hibit NOX 2 at higher concentrations (545). Inhibitory ef- Superoxide can be generated by the oxygenase/heme do- fects against NOX5 have also been documented (680). No main, in a Ca2ϩ/CaM-dependent way, or independently by off-target effects were detected against a panel of 135 en- the reductase domain from Ca2ϩ/CaM (653, 1032). In con- zymes, including XO and eNOS (545). GKT-137831 shows ditions that promote uncoupling, the heme group is usually similar inhibitory properties with potent inhibition of the main generator of superoxide species through the for- NOX1 and NOX4 and inhibitory effects on NOX2 and mation of a ferrous/oxy complex that decays to ferric heme NOX5 at higher concentrations (35). The basis of the inhi- and superoxide (Eq. 20) (FIGURE 11) (250, 901, 973, 1033). bition by these compounds is not known, although the activity of GKT-136901 as peroxynitrite scavengers has been The loss of the dimer structure renders eNOS unable to recently reported (826). produce NO, as the heme group of one monomer must receive electrons from the FMN domain of the other mono- H) GSK2795039. The discovery of a novel, specific NOX2 in- mer. Thus, conditions that increase the monomer/dimer ra- hibitor was recently reported (435). GSK2795039 (FIGURE 10) appears to be specific toward NOX2 and does not show - • - • off-target effects toward eNOS or XO. Existing evidence O2 O2 O2 O2 suggests that GSK2795039 inhibits NOX2 through compe- tition with NADPH for the binding site of the NOX2 de- NOS hydrogenase domain (435). monomer HEME FMN FAD

O O - • B. Uncoupling of Endothelial NOS 2 2 - • - • O2 O2 O2 O2 NO production is critical for vascular function as described NOS dimer in previous sections (299, 573, 600, 786). In conditions in -CaM which the substrates and cofactors are present in saturating HEME FMN FAD concentrations, NOS enzymes catalyze the synthesis of NO L O O - • from molecular oxygen and -arginine with high efﬁciency. FAD FMN HEME 2 2 The electrons transferred from NADPH are almost quantitatively used to catalyze L-arginine oxidation, and the ratio NOS dimer - • of NADPH to L-citrulline is close to the optimal value of 1.5 O2 O2 +CaM molecules of NADPH per L-citrulline (and NO) generated - • (3, 590). However, a number of factors can alter the elec- CaM O2 O2 tron transfer in NOS so the amount of NADPH required to FAD FMN HEME

generate NO becomes much larger. This is largely due to the CaM reduction of other substrates, usually molecular oxygen, to HEME FMN FAD generate superoxide. This situation when the consumption - • of NADPH is not correlated with a stoichiometric produc- O2 O2 tion of NO is generally termed eNOS uncoupling (488, 570, FIGURE 11. Superoxide generation by nitric oxide synthase (NOS) 870). Although the superoxide generation is the common monomer and dimer in the presence or absence of calmodulin manifestation of this NOS dysfunctional state, it is impor- (CaM).

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tio decrease NO output and can increase superoxide pro- The conversion of BH4 to BH2 in the setting of endothelial duction through the reductase domain but also decrease dysfunction has been widely reported (192, 258, 550). The superoxide formation in the oxygenase domain (FIGURE accumulation of BH2 becomes deleterious as the binding 11). Therefore, it should be noted that measurement of site of BH4 in NOS can also bind this pterin. This is partic- dimer/monomer ratios as an indication of eNOS uncou- ularly problematic in the case of eNOS (190). iNOS and pling is not a reliable and reproducible method, and more nNOS show higher affinity toward BH4 than for BH2 specific methods monitoring posttranslational modifica- (around 10-fold in the absence of L-arginine) (6, 514, 753); tions and substrate levels are preferable. however, eNOS shows almost identical binding affinities for BH4 and BH2, and it is thus very sensitive to imbalances The concomitant formation of NO and superoxide can be in the concentrations of BH4 and BH2 (190). Thus, rather especially deleterious as both radicals can combine to form than the relative amount of BH2, the BH4/BH2 ratio gives a peroxynitrite at diffusion-limited rates. Peroxynitrite is a more precise estimation of the endothelial function (190, strong oxidant and nitrosating species (78). Therefore, the 192, 974). superoxide generated by the uncoupled eNOS not only in- Mechanistically, when NOS binds BH2 instead of BH4, the duces toxic effects on its own but 1) acts as a NO scavenger, G decreasing NO availability and 2) generates peroxynitrite, a ferrous/oxy heme cannot hydroxylate L-arginine to N -hydroxy-L-arginine, NO synthesis is blocked, and the oxygen more harmful species (78). Peroxynitrite can in turn oxidize is eventually oxidized to superoxide. Thus, not only is the BH to BH , further promoting eNOS uncoupling (543). 4 2 NO synthesis completely abolished but the protein also be- Notably, the deleterious effect of superoxide on the endo- comes an oxidase, generating superoxide instead of NO thelium relaxing effect of NO was discovered even before (191, 972, 1007) (TABLE 6). the chemical nature of NO as endothelium-derived relaxing factor was elucidated (385). In the following sections, we 2. L-Arginine/ADMA dissect the different causes of NOS uncoupling and overview the current status of pharmacological approaches to In saturating conditions of the NOS cofactors, the synthesis prevent or reverse this dysfunctional state. Special emphasis of NO only requires L-Arginine and O2 as substrates (904). is dedicated to the two most relevant aspects in pathophys- L-Arginine is also important for the dimerization of NOS iology, namely the dysregulation of BH4 and L-arginine lev- (51, 515) and increases BH4 binding affinity (514, 753). els. The absence of L-arginine leads to the production of superoxide in an otherwise active form of NOS; thus, L-arginine

1. BH4/BH2 availability can have an important effect on eNOS uncoupling (TABLE 6). Given the concentration of L-arginine in The binding of the cofactor tetrahydrobiopterin (BH )isa ECs [0.1–0.8 mM (1030)] and the high affinity of NOS 4 ϭ ␮ enzymes toward L-arginine [eNOS KM 2.9 M (746)], it requisite for NO synthesis by NOS enzymes. BH4 binding synergistically enhances the affinity toward the substrate was long thought that L-arginine availability would not be a functional issue for NOS in vivo. However, in recent years, L-arginine. In addition, BH4 plays a critical role in the catalytic cycle of NOS (87, 204, 377, 904, 933, 1000, 1007). it has become clear that extracellular L-arginine and ADMA concentrations, compartmentalization, and membrane The ability of BH4 to support electron delivery to the heme group at critical steps, generating a transient radical species, is specific to BH4. Other pterins are, in general, unable to support NO synthesis in mammalian NOS enzymes, or can Table 6. Relative effect of different factors on eNOS only support a NO synthesis rate much slower than BH4 superoxide and NO production (753). BH4 can be oxidized in vivo by molecular oxygen or Superoxide NO ROS to generate dihydrobiopterin (BH2), a pterin very Production Pterin Substrate Other Synthesis structurally similar to BH4 but unable to support NO synthesis (544, 753, 933). ϩϩϩ None L-arginine None ϩϩϩ None ADMA None Biological de novo synthesis of BH4 is mediated by three ϩϩϩ None None None ϩϩ enzymes that catalyze the stepwise process from GTP to BH2 L-arginine None ϩϩ BH4, namely GTP cyclohydrolase I (GCH1), pyruvoyl tet- BH2 None None ϩ ϩ rahydropterin synthase (PTPS), and sepiapterin reductase. BH4 L-arginine S1177p ϭ ϭ In this route, the reaction of GTP with GCH1 is the limiting BH4 L-arginine ϭ step. An additional salvage pathway can produce BH4 from BH4 L-arginine T495p – sepiapterin and/or BH . In this pathway, sepiapterin reduc- 2 Qualitative assessment based on data from Refs. 150, 161, 250, tase reduces sepiapterin to BH2, whereas dihydrofolate re- 334, 753, 973, 974, 1033. ADMA, asymmetric dimethylarginine; ductase can reduce BH2 to BH4 (87, 1007). eNOS, endothelial nitric oxide synthase; NO, nitric oxide.

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transporters are important for NOS function, as will be Coupled Uncoupled discussed below in sect. IIIB4 (167, 668, 1030).

ADMA (FIGURE 3) is mainly produced from the breakdown of proteins containing methylated arginine residues. The NO synthesis same processes also yield the methyl arginines NG-methyl Superoxide output monoarginine (FIGURE 3) and symmetric dimethylarginine G NO output (FIGURE 3) (668). Notably, ADMA and N -monomethyl-L- arginine (but not symmetric dimethylarginine) are NOS inhibitors and can be important regulators of NOS activity in Peroxynitrite output vivo (566, 963). The metabolism of ADMA and its effects on cardiovascular disease have been covered by recent re- BH BH 2 views (668, 790, 962). Soon after its discovery in cells and 4 tissue, ADMA was recognized as an important marker for monomer ↑ monomer ↑

cardiovascular disease progression (656, 829, 961, 1011, (Zn-S breakdown) (BH4 deficiency) 1053, 1068). More recent observations indicate that the FIGURE 12. Progression of nitric oxide (NO) synthesis, superox- ADMA/L-arginine ratios in plasma may be a better marker ide, and peroxynitrite during the development of endothelial nitric than either metabolite assessed independently (75, 589, oxide synthase (eNOS) uncoupling. 606, 790, 966).

3. Other causes of eNOS uncoupling reversed by glutaredoxin-1 (160). The glutathionylation of Cys689 and Cys908 causes eNOS uncoupling. These resi- A) DISRUPTION OF THE Zn/S CLUSTER. The dimeric structure of NOS dues, located in the reductase domain, appear to disturb the is held together by interactions between the oxygenase do- electron transfer between FAD and FMN cofactors, and mains, and among these interactions, the formation of a indirectly limit the rate of reduction of the oxygenase do- Zn/4S cluster with the participation of two cysteines from main. Consistent with this hypothesis, the addition of the each NOS monomer (195). The oxidation of the cysteine inhibitor L-NAME decreases superoxide production only in residues involved in the formation of the Zn/S cluster dis- part, indicating that most of the superoxide production oc- rupts the dimer structure. Work by Zou et al. indicated that curs in the reductase domain via electron transfer from the this cluster is particularly sensitive to peroxynitrite (1070). reduced FAD and FMN to molecular oxygen (162) (TABLE This observation is not unexpected, as metal-thiol bonds 6, FIGS. 11, 12). are some of the main targets of peroxynitrite (78, 526). S-glutathionylation has been involved in the development The breakdown of the Zn/S cluster is, in most cases, sec- of angiotensin II–mediated endothelial dysfunction (328) ondary to eNOS uncoupling. As discussed above, the mo- and nitroglycerin resistance (522). Both processes appear to nomeric eNOS cannot catalyze the NO synthesis reactions, be initiated by mitochondrial ROS. Consistent with this and the monomerization renders the heme group inactive. view, the increase in ROS levels, BH depletion, and gluta- The superoxide formation from the monomer is low as 4 compared with the uncoupled dimer, and thus, this mecha- thione oxidation, with subsequent eNOS S-glutathionyla- nism does not increase the rate of superoxide formation. tion, appears to be intimately related (189, 215). However, the breakdown of the Zn/S cluster is a marker that indicates the generation of peroxynitrite in the cellular Along with glutaredoxin-1, thioredoxin has also been pro- environment. Although this is not a 100% specific marker posed to deglutathionylate eNOS, and this process is inde- of eNOS uncoupling (the superoxide source may be differ- pendent of glutathione oxidation (907). This offers thera- ent from NOS, for example, a NOX enzyme), the correla- peutical opportunities in oxidative stress situations in tion of the Zn/S cluster breakdown and eNOS uncoupling is which glutathione levels are low (432). probably elevated (TABLE 6, FIGURE 12). C) PHOSPHORYLATION. Phosphorylation of Thr495 is a negative Another modification that disrupts the Zn/S cluster is S- regulator of CaM binding to eNOS (297, 407, 646). The nitrosation of the cysteine residues Cys96 and Cys101, as Thr495-phosphorylated NOS shows decreased electron discussed in sect. IIA1. transfer from the reductase domain to the heme domain and decreased NO synthesis (588). The accumulation of elec- B) GLUTATHIONYLATION OF CYSTEINE RESIDUES. Recent reports indi- trons in the reductase domain causes an increased uncou- cate that eNOS can be modified posttranslationally by S- pling from the reductase domain, which, as discussed pre- glutathionylation (162). At least three residues have been viously, is less pronounced than when this occurs in the shown to be susceptible of modification: Cys 689, Cys908 heme but can cause significant production of superoxide (162), and Cys382 (160) (TABLE 2). The process can be (TABLE 6).

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4. Pharmacological interventions in eNOS uncoupling In most examples, BH4 is able to improve endothelial function, but the study by Cunnington et al. (202) clearly indi- The sensitivity of the eNOS system to oxidative stress con- cated that in some conditions, BH4 supplementation is not a verts eNOS into a main target in conditions of ROS imbal- practical therapeutic approach. In severe oxidative stress ance. According to the kindling hypothesis of endothelial conditions, the BH4 will be rapidly oxidized to BH2, further dysfunction, the initial formation of ROS by other sources increasing the BH2/BH4 ratio (FIGURE 12). As the treatment can promote uncoupling of NOS, amplifying the initial ex- with antioxidants has been shown to improve endothelial cess of ROS (488). The uncoupling causes eNOS activity to function in certain conditions (56, 419, 420, 422, 572), it is change from healthy (producing NO) to toxic (generating possible that the coadministration of BH4 with ascorbic superoxide) (303). As such, the uncoupling of eNOS has acid or other antioxidants can circumvent these issues. Of been noted as a target for therapeutic potential (488, 569, note, BH4 is also an antioxidant, and it is not clear if some 570, 600, 784). Different interventions have been devel- of the observed effects at the supraphysiological concentra- oped to address this misdirected reactivity. As the status of tions used are just due to short-term antioxidant effects; eNOS uncoupling changes with the severity of the cause, related pterins, such as folate, can also produce similar ef- different treatments may prove more efﬁcacious at different fects (32, 33, 860). Techniques that can assess the oxidative stages (FIGURE 12). Current strategies are discussed below. stress conditions in tissues can certainly improve the use of BH4 therapy with a more personalized approach. Pleiotro- pic effects of some drugs can improve BH availability; this A) BH4. Many studies have shown that BH4 levels correlate 4 with eNOS activity (796, 1006). Conversely, low levels of has been observed for statins such as atorvastatin (31).

BH4 are found in endothelial dysfunction and cardiovascular disease (421, 429, 440, 550, 560, 858, 899). Spear- As an alternative to BH4, supplementation using other re- headed by these observations, interventions to reverse lated compounds such as sepiapterin has been also used. eNOS uncoupling via BH4 supplementation have been nu- Sepiapterin can be converted into BH4 via the salvage path- merous, and in general, the supplementation of BH4 or way by sepiapterin reductase and dihydrofolate reductase positive modiﬁcations in the BH4 synthesis pathways have as described in the previous sections. Sepiapterin supple- been positive. A large number of studies on mice and other mentation improves endothelial function in several mouse Ϫ Ϫ animals have been described; for the sake of conciseness, we models, including the atherosclerosis model ApoE / mice will only discuss some of the recent human studies. For a (560), diabetes models (721), or obesity models (527). more complete overview of the BH4 supplementation ex- Mouse GCH1 KO mice show abolished endothelial NO periments including studies in other organisms, the reader is synthesis and increased superoxide production that can be referred to recent reviews (87, 828). reversed by sepiapterin supplementation (173). It should be noted that this pathway has BH2 as an intermediate, which

Acute supplementation of BH4, either orally or injected, could lead to detrimental effects. In conditions in which generally improves vasodilatation. This effect has been ob- dihydrofolate reductase is inactive or absent, the recovery served in a number of human studies, including healthy of eNOS coupling by sepiapterin supplementation is inef- individuals (450, 743, 987) and patients with hypertension fective (452). (429), hypercholesterolemia (318, 899, 1031), chronic heart failure (849), coronary artery disease (618, 850), Folic acid has wide effects on the metabolism, impacting heart failure with reduced ejection fraction (178), or rheu- cysteine/homocysteine metabolism, but also pathways in- matoid arthritis (619) or smokers (418, 929, 957). Studies, volving tetrahydrofolate and dihydrofolate and hence BH4 including the assessment of coronary artery function in pa- biosynthesis (598). The effects on eNOS uncoupling seem tients with atherosclerosis, did not ﬁnd an improvement related to the antioxidant properties of the folic acid metab- after BH4 treatment (1028). olites (900). Supplementation of folic acid (5-methyltetrahydrofolate) shows positive effects on eNOS function (900, Several studies monitoring endothelial function after 978) and has been shown to prevent nitroglycerin tolerance chronic BH4 administration have been conducted. In hyper- in short-term human studies (375). Folic acid supplementa- cholesterolemia patients, oral BH4 for 4 wk (400 mg twice tion studies in human blood vessel samples have shown daily) improved endothelial function (186). A treatment increases in BH4 levels and BH4/BH2 ratios (33). with oral BH4 for 2–6 wk in patients with coronary artery disease did not show improvement; and actually increased To improve endogenous BH4 synthesis, efforts have focused BH2 levels (202). In patients with hypertension, a 4–8-wk, in GCH1, the rate limiting enzyme during de novo BH4 400 mg/day treatment was able to decrease blood pressure synthesis (143). Overexpression of GCH1 in cells and (747). Studies on pulmonary hypertension also show im- mouse models improves eNOS function (19, 143). Human provement in the 6-min walking distance (788). A recent variants of GCH1 show measurable effects in blood pres- study using 400 mg daily for 1 wk improved endothelial sure, indicating the direct relationship between GCH1 and function in rheumatoid arthritis patients (619). eNOS function (1057).

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B) L-ARGININE. Several lines of evidence indicate that the avail- of L-arginine levels as a biomarker a poor indicator of out- ability of L-arginine may be jeopardized under endothelial comes (668, 857, 1029). Studies of a panel of L-arginine dysfunction, and the concentrations of inhibitory L-arginine metabolites indicate some correlations, particularly for metabolites, such as ADMA, can also increase in patholog- ADMA, but also the need to consider other factors such as ical conditions (107, 1053). In this regard, different studies age, sex, and body mass index (75, 771). The interaction of have investigated alternatives to improve L-arginine supply L-arginine levels with ADMA and other metabolites is the either via direct L-arginine supplementation, the use of L-ar- focus of current studies. As pointed out above, the ratio of ginine precursors, or through the inactivation of L-arginine L-arginine to ADMA appears to be a better indicator of consuming arginases. cardiovascular risk (28, 103, 104, 606, 1050). It has been noted that when ADMA levels are low, the effects of exog- The direct supplementation of L-arginine was shown to in- enous L-arginine are limited; however, the therapy seems to crease NO production in cells. This finding was rather sur- be more beneficial in patients with higher ADMA levels, in prising, as most cells have intracellular L-arginine concen- which the treatment increases the L-arginine/ADMA ratios and improves endothelial function (103). trations manyfold higher than the eNOS KM value, and thus, this observation has been termed the “arginine paradox” (12, 797). Further studies showed that L-arginine im- Other L-arginine–related species may allow for interventions port via the CAT1 transporter is critical for eNOS activity of L-arginine levels. For example, L-citrulline has also been (167, 634). A membrane metabolon including arginine suc- used to increase L-arginine levels (842, 1029). This supplemen- cinate lyase, argininosuccinate synthetase, hsp90, and tation can avoid the formation of detrimental high ADMA eNOS associates in the caveolae and is necessary for regular levels (632). The studies of L-citrulline in humans show mixed eNOS function (270, 294, 634). Another factor contribut- results. In some cases, L-citrulline increased NO synthesis, but ing to the arginine paradox, as will be discussed below, is no changes in blood pressure were observed (506). Other stud- the concentration of endogenous eNOS inhibitors, mainly ies have observed positive effects of L-citrulline including reduced blood pressure (20, 285, 286). ADMA. At high ADMA concentrations, L-arginine supplementation may not overcome eNOS inhibition, indicating Finally, it must be noted that arginases play an important the need to determine L-arginine/ADMA ratios to better role in the regulation of L-arginine levels (144, 259, 734, assess L-arginine availability (102, 106, 954). 804). Exacerbated arginase activity can decrease L-arginine levels and cause eNOS uncoupling (91, 144, 667, 804, In animal models of MI, L-arginine supplementation shows 1039). Thus, the use of arginase inhibitors is gaining in- mixed results. Some studies show improvement in functional creasing attention. Several human studies have used the recovery (712), whereas studies in L-arginine–treated dogs G arginase inhibitor nor-N -hydroxy-L-arginine (FIGURE 3). show detrimental outcomes (665). Further studies have ex- In these cases, arginase inhibition showed protection plored L-arginine supplementation in human patients. Again, against ischemia-reperfusion injury (530) and improved en- the results have been heterogeneous. The causes of this vari- dothelial function in patients with coronary artery disease ability are still unclear. Many studies have reported a lack of and diabetes (855) and in patients with hypercholesterol- effect on L-arginine in endothelial function markers, and on emia (529). Further studies in this field and the search for occasion, the supplementation of L-arginine has been associ- novel natural and synthetic arginase inhibitors are war- ated with higher mortality effects, such as tests of L-arginine in ranted (757). patients after MI (835). Other studies show impaired recovery in peripheral artery disease patients (1014). In contrast, most studies detect decreases in blood pressure with supplementa- C. Mitochondria tion. A recent meta-analysis indicates that doses between 4 and 24 g/day decrease blood pressure (systolic, 5 mmHg; dia- The mitochondrial electron transport chain (ETC) repre- stolic, 3 mmHg) (240). Other recent studies show improved sents a physiologically significant generator of ROS within endothelial function and insulin sensitivity in patients with vascular cells. Mitochondria generate ATP through the impaired glucose uptake and metabolic syndrome (6.6 g/day transfer of electrons from ETC complexes I and II to com- L-arginine for 2 wk) (663) and improved endothelial function plex IV, where oxygen binds and is reduced to water. and decreased homocysteine levels in patients with elevated Through electron transfer steps, protons are pumped from blood pressure and hyperhomocysteinuria (2.4 g/day for the matrix to the inner membrane space, establishing a pro- 4 wk) (781). ton-motive force that couples electron transfer to the phosphorylation of ADP to ATP at complex V (FIGURE 13). Altogether, it is not clear if the levels of L-arginine are the Although the majority of electrons entering the ETC ulti- best target for intervention. In some cases high, L-arginine mately reduce oxygen to water at complex IV, a small pro- levels have even been correlated with higher incidence of portion (~2–11%) of electrons escape from the chain at ischemic heart disease (1045). It seems that the involvement complex I or III to generate superoxide (34, 115, 678). of L-arginine in different metabolic pathways makes the use Although this superoxide, being a charged molecule, likely

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Complex I Complex II Complex III Complex IV Complex V NADH: Ubiquinone Succinate Quinol: Cytochrome c Cytochrome c ATP synthase reductase dehydrogenase reductase oxidase

+ -• + -• + + H O2 H O2 H H Intermembrane space

e– e– Cyt c e– Q cycle – – e e

Mitochondrial matrix + NADH NAD O2 2H2O ADP ATP

Succinate Fumarate

FIGURE 13. Mitochondrial electron transfer chain. Main sites of superoxide production are indicated.

does not escape the mitochondrion, manganese superoxide (FIGURE 13). In this process, ubiquinol (QH2) binds to the dismutase (MnSOD) located within the mitochondria cata- Q0 site of complex III and undergoes a one-electron oxida- lyzes the dismutation of superoxide to hydrogen peroxide, tion that produces an unstable semiquinone radical. Al- which can freely diffuse out of the organelle to mediate though in most cases this semiquinone is rapidly oxidized, cellular signaling or contribute to oxidative stress. its reaction with oxygen leads to superoxide production at this site (567, 678). Importantly, specific complex III inhib- 1. Mechanisms of ROS production itors, such as Antimycin A, can stabilize this semiquinone radical, significantly enhancing superoxide production by Mitochondrial ROS production is regulated by a number of complex III (678). Physiologically, it is unclear which fac- factors including oxygen levels, substrate availability, and mi- tors stabilize this species to regulate superoxide production. tochondrial morphology/dynamics, all of which ultimately However, a number of studies demonstrate that complex modulate the redox state of the ETC. Complex I, a 1,000-kDa III–dependent ROS production is important in physiologi- protein comprised of ~45 subunits, is the most significant site cal signaling pathways, including hypoxic sensing (997). of superoxide generation within the ETC and produces superoxide by two distinct mechanisms depending on the reduction Although complexes I and III are considered the predomi- status of the ETC and strength of the proton-motive force nant sources of ROS within the mitochondrion, it is impor- across the inner mitochondrial membrane (FIGURE 13).A tant to note that mitochondrial enzymes beyond the ETC FMN center within the complex, which accepts electrons from have been reported to generate ROS. Pyruvate dehydroge- NADH, serves as the entry point to the ETC. In conditions in nase (887), ␣-ketoglutarate dehydrogenase (130, 887, 952), which electron transport through the ETC is slow, this FMN and monoamine oxygenase A and B (290) have all been can become reduced and react with oxygen, catalyzing super- demonstrated to serve as ROS sources. These sources, al- oxide production. This occurs in conditions of high NADH/ though lower in their ROS-generating capacity, potentially NADϩ ratios, such as when cellular ATP demand is low or the play a role in specific mitochondrial-dependent redox sig- ETC has sustained damage. In contrast, when electron supply naling pathways. Notably, other reactive species–produc- is high through the ETC, coenzyme Q downstream of complex ing enzymes have also been demonstrated to translocate I becomes reduced. A high proton-motive force present in this into the mitochondrion in specific conditions. For example, condition can then push electrons in the reverse direction from eNOS has been shown to enter the mitochondrion of pul- coenzyme Q to the complex I FMN, generating superoxide by monary smooth muscle cells upon stimulation with asym- reverse electron transport. This type of ROS generation occurs metric dimethyl arginine or endothelin-1 (768, 916). Addi- when succinate, the substrate for complex II, is present in high tionally, although controversial, NOX4 has been reported concentration and is responsible in vivo for the ROS generated to be localized within the mitochondrion either constitu- after ischemia upon reperfusion (171, 172). tively or in specific pathological conditions (152, 306, 540).

Complex III also represents a site of superoxide production 2. Physiological role of mitochondrial ROS within the ETC, although the rate of generation at this site is signiﬁcantly lower than by reverse electron transport Although it has been recognized since the 1960s that mito- through complex I. Complex III catalyzes the Q cycle in chondria produce ROS, the physiological role of this phe- which electrons are accepted from ubiquinone to be used to nomenon remained unclear until decades later. Accumulat- reduce cytochrome c for subsequent transfer to complex IV ing data now demonstrate that mitochondrial ROS both

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mediate essential vascular signaling pathways and can also 997). A similar pathway exists in the pulmonary vascula- contribute to the pathogenesis of cardiovascular disease. ture in which mitochondria-generated oxidants signal the This section will provide an overview of what is known conserved phenomenon of hypoxic vasoconstriction (995). about the role of mitochondrial ROS in cardiovascular On a cellular level, mitochondrial ROS have also been health and disease. shown to be required for the differentiation of some hematopoietic stem cells as well as human mesenchymal stem Early studies viewed mitochondrial ROS production as cells (707, 948). Additionally, mitochondrial ROS have pathological and focused on the deleterious effects of mito- been implicated in pathways leading to cell proliferation chondrial superoxide production. Mice completely lacking and senescence (233, 1046) as well as in the activation of MnSOD, leading to greater mitochondrial superoxide con- other major cell signaling molecules such as the MAP kinase centrations, died within weeks of birth (584), confirming JNK and NF-␬B (330, 689). the essentiality of mitochondrial superoxide scavenging. Further, mice partially or conditionally lacking MnSOD Notably, mitochondrial ROS modulate mitochondrial showed dilated cardiomyopathy characterized by left ven- function, number, and cellular metabolic balance through a tricle dilation, decreased wall thickness, and hypertrophy number of mechanisms. Mitochondrial hydrogen peroxide (584, 696). Notably, tissue from the left ventricle of heart can oxidize the catalytic subunit of AMP kinase, leading to failure patients showed an increase in superoxide produc- its autophosphorylation and subsequent activation to mod- tion and a decrease in MnSOD protein levels and activity, ulate metabolism within the cell (481, 1067). Although ac- consistent with a role for increased mitochondrial superox- tivation of AMP kinase can lead to mitochondrial biogene- ide in the pathogenesis of heart failure (814). It is now sis and increased mitochondrial number, this pathway can evident that mitochondrial ROS production also plays a also directly be stimulated by mitochondrial hydrogen per- major role in the pathogenesis of MI. During myocardial oxide-dependent activation of the MAP kinase Akt (911, ischemia, the ETC as well as the NADH and quinone pools 919). As a counterbalance, mitochondrial ROS have also become maximally reduced. Additionally, there is a signif- been shown to induce organellar degradation (e.g., mi- icant buildup of succinate, the substrate for complex II, tophagy) under specific conditions (810). Through these which further supports the reduction of the quinone pool. mechanisms, mitochondrial ROS potentially play a role as a Together, these conditions promote reverse electron trans- mediator in elaborate feedback pathways to regulate mito- fer upon reperfusion, which results in significant superoxide chondrial energetics and redox signaling. production from complex I (171). Pharmacological inhibitors of complex I, including classical inhibitors as well as The role of mitochondrial oxidants in the pathogenesis of NO donors and nitrite, decrease this reverse electron trans- some disease processes remains controversial. For example, fer–mediated ROS production, leading to attenuated in- two competing hypotheses have been proposed for the role farct size and protection of myocardial function after isch- of mitochondrial ROS in the pathogenesis of pulmonary emia/reperfusion in a number of animal models (discussed arterial hypertension. Studies by Schumacker and col- in the next section) (136, 165, 170, 864). leagues suggest that increases in pulmonary mitochondrial superoxide production leads to the release of intracellular 2ϩ Beyond the heart, changes in mitochondrial redox status Ca stores and subsequent vasoconstriction (996). To the can also have detrimental effects in the vascular wall and contrary, Archer and colleagues demonstrate in animal circulating cells. Decreased MnSOD activity or increased models of pulmonary arterial hypertension that mitochon- mitochondrial superoxide generation causes endothelial drial ROS production in the pulmonary artery smooth mus- dysfunction and impairs acetylcholine-dependent vasodila- cle cells is decreased, leading to inhibition of the KV1.5 channel, which ultimately results in membrane depolarization, particularly with age (1005). This dysfunction occurs 2ϩ not only through the direct reaction of superoxide with NO tion, intracellular Ca increases, and vasoconstriction but is also associated with the oxidative damage of mito- (645). Although the role of mitochondrial ROS in patho- chondrial DNA and proteins. Notably, specific polymor- genesis remains controversial and a hot area of research, phisms in the gene for MnSOD have been established as an there is no doubt that changes in mitochondrial redox status independent risk factor for coronary artery disease (317). play a role in disease progression.

Importantly, although mitochondrial ROS can propagate 3. Pharmacological interventions at the level of cellular damage, low physiological concentrations of mito- mitochondrial function chondrial oxidants have been shown to mediate a number of essential cellular signaling pathways (290). For example, With the recognition that mitochondrial ROS play a role in mitochondria have been proposed to serve as a cellular disease pathogenesis, the development of antioxidants tar- oxygen sensor, such that in hypoxic conditions, mitochon- geted to the mitochondrion is an active area of research. drial ROS production by complex III leads to the stabiliza- Several compounds have been developed with encouraging tion of HIF1␣ and downstream hypoxic adaptation (84, results in vascular disease and other pathologies.

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A) MITOQ. The most extensively tested mitochondrial antiox- cumulates within the mitochondrion and binds to cardiolipin idant is probably MitoQ (FIGURE 14). This small molecule in the inner mitochondrial membrane where it scavenges ROS consists of the antioxidant ubiquinol linked to the lipophilic and prevents lipid peroxidation (921) (FIGURE 14). These pep- cation triphenylphosphonium (500). The positive charge of tides have been shown to mediate protection in animal models the triphenylphosphonium cation is delocalized over its of ischemia/reperfusion (922) and insulin resistance (27). large hydrophobic area, which allows it to freely be taken Phase I clinical trials established that the molecule was safe and into the mitochondria based on the mitochondrial mem- well tolerated (360). Multiple phase II trials are underway, brane potential (873). MitoQ has been used extensively in examining the effects of the drug in conditions such as skeletal animal models of disease and been shown to be protective muscle disorders (NCT02245620), mitochondrial disorders against ischemia/reperfusion injury, cardiac hypertrophy, (NCT02976038, NCT02805790, NCT02367014), and heart and hypertension (210, 211) among other pathologies. failure (NCT02814097, NCT02788747, NCT02914665).

MitoQ has also been tested in phase II clinical trials for C) MITOSNO AND SODIUM NITRITE. In addition to pure mitochondri- both Parkinson’s disease and hepatitis C (NCT00329056; al-targeted oxidant scavengers, potential therapeutics that NCT00433108). Although Parkinson’s patients taking Mi- modulate ETC activity to decrease mitochondrial ROS pro- toQ showed no difference in motor function compared with duction are being investigated. For example, MitoSNO those on placebo treatment, this trial did establish that ad- (FIGURE 14), a mitochondrially targeted S-nitrosothiol, was ministration of MitoQ to humans for 1 yr is safe (874). In shown in rodent models to signiﬁcantly attenuate infarct contrast, chronic hepatitis C patients treated with MitoQ size after MI (170). Mechanistically, this protection is de- for 28 days showed signiﬁcant lowering of liver injury pendent on MitoSNO transnitrosating a critical cysteine in markers such as circulating alanine transaminase (333). the active site of complex I. This S-nitrosation of Cys39 in These positive results have led to further phase IIB trials in the ND3 subunit of complex I inhibits its catalytic activity hepatitis as well as the initiation of clinical trials to test and, hence, attenuates reverse electron transport and ROS MitoQ in other vascular pathologies. production (170). Notably, other nitrogen-centered molecules, such as nitrite, have been shown to promote S-nitro- B) SZETO–SCHILLER-31. Szeto–Schiller peptides are another class sation of complex I and attenuate ischemia/reperfusion in- of molecules that target antioxidant activity to the mito- jury (864). Nitrite administered before or during cardiac chondrion. Szeto–Schiller-31 (SS-31) (Bendavia/elamipretide; ischemia has been shown to protect cardiac function and Stealth Peptides) is a small water-soluble tetrapeptide that ac- attenuate infarct size in a number of small and large animal models (226, 374, 864). These preclinical studies have led to ongoing clinical testing of nitrite as a therapeutic for MI as well as cardiac arrest.

D. XO

Xanthine oxidoreductase (XOR) is expressed as a xanthine dehydrogenase (XDH) but can become XO by posttranslational modiﬁcations (FIGURE 15). XOR is a dimeric protein containing one molybdopterin cofactor, two iron/sulfur clusters (2Fe/2S), and one molecule of FAD per 145-kDa subunit. The N-terminal domain contains the two Fe/S clusters, a central domain contains the FAD cofactor, and a NADϩ/H binding site and the C-terminal domain contains the molybdopterin cofactor and a purine binding site (268). The enzyme has an important role in the catabolism of purines, catalyzing the stepwise oxidations of hypoxanthine to xanthine and xanthine to uric acid. Purine oxidation takes place in the molybdopterin site where the reaction leads to the reduction of the Mo6ϩ to Mo4ϩ. The electrons are then transported within the enzyme, ﬁrst to the

Fe/S clusters and then to the FAD. The fully reduced FADH2 can reduce NADϩ to form NADH.

1. ROS generation by XOR

FIGURE 14. Structures of selected mitochondria-targeted During normal operation, XDH shows a limited produc- compounds. tion of superoxide/hydrogen peroxide via electron leak

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A C Xanthine Hypoxanthine

MPT e–

Fe2S2 e–

Fe2S2 e– FIGURE 15. Architecture of xanthine dehydrogenase FAD (XDH)/zanthine oxidase. A and B indicate the arrangement of the domains in the XDH/xanthine oxidoreductase (XOR) monomer. The middle, FAD-containing do- NAD+ NADH main (yellow), is connected to the N-terminal domain

containing two iron/sulfur clusters (Fe2S2) (green) and the molybdopterin domain (pink) by two ﬂexible linkers. B Posttranslational changes modify the protein activity from a XDH (A), NADH-producing enzyme, to the xan- Xanthine Hypoxanthine thine oxidase (XO) (B), hydrogen peroxide/superoxide- generating enzyme. C: three-dimensional structure of the XDH monomer (PDB:1FO4) (268). MPT e–

Fe2S2 e–

Fe2S2 e– FAD

• – O2 H2O2 / O2

through the reduced FAD cofactor to O2. This reaction only 2. Pharmacological interventions targeting XO produces significant levels of ROS if NADϩ levels are low; however, these conditions can happen in the setting of vas- Extensive evidence supports a role of XO in the develop- cular hypoxia (406, 562). ment of cardiovascular disease (137, 148, 157, 282, 430, 466, 561, 633). As XO has become an interesting pharma- Oxidation of cysteine residues 535 and 992 in rat or bovine cological target in the treatment of heart and cardiovascular XDH or limited proteolysis turns XDH into XO (23, 72, diseases, numerous studies have used different strategies for 693, 773, 803, 994). XO shows a decreased affinity for the inhibition of XO. ϩ NAD and increased O2 affinity. The comparison of the A) TUNGSTEN. Tungsten can be used as a global inhibitor of crystal structures for XDH and XO indicates the movement ϩ molybdenum-containing enzymes as it replaces the Mo of a loop that partially blocks the NAD binding site and atom in the molybdopterin, rendering the enzymes that use alters the electrostatics of the FAD environment (268, 542). this cofactor inactive. Because of this mechanism of action, Altogether, these changes in substrate affinity lead to an it should be noted that effects independent of XO will also increased electron flow from the molybdopterin domain to ϩ be observed because of inhibition of AO, sulfite oxidase, oxygen instead of NAD . Thus, XO catalyzes the oxidation and mARC. Tungsten is a powerful tool for the study of of hypoxanthine to xanthine and subsequently xanthine to molybdenum enzymes in cell and animal studies and has uric acid, generating hydrogen peroxide and superoxide in a been found to alleviate atherosclerosis in ApoEϪ/Ϫ mice reaction amplified under ischemia and hypoxia (147, 307, (833). However, because of its broad effects, the clinical use 496, 497, 499). of tungsten is not feasible.

XO is expressed in ECs (393), and its activity is increased B) ALLOPURINOL. Allopurinol is structurally similar to the XO by angiotensin II (551), TNF-␣, LPS (117, 311), IL-6, and substrate hypoxanthine and is metabolized to the active hypoxia (410, 748). There are low-circulating levels of compound oxypurinol (FIGURE 16). Both allopurinol and plasma XO in baseline conditions that can increase in oxypurinol have been used for the treatment of gout for disease conditions by the release from damaged cells in decades (305, 710). Beyond its use for hyperuricemia and liver or other tissues (1010). The binding of plasma XO gout, more recent studies have used allopurinol and oxy- to ECs can lead to vascular dysfunction (45, 71, 562, purinol as XO inhibitors for the treatment of cardiovascu- 612, 1010). lar disease in human studies. In small trials, the pharmaco-

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covalent modiﬁcation in the enzyme that inactivates XO after complex dissociation (629, 703, 821). Topiroxostat is currently under study for the treatment of hyperuricemia in clinical trials (NCT02327754; NCT02837198) (441, 442).

E) NITRITE. Nitrite has also been used to inhibit XO-catalyzed ROS generation and to take advantage of its nitrite reductase activity to generate NO (reviewed in sect. IIB3) while decreasing superoxide production (1071). Recent work indicates that this treatment can be effective in the reduction of blood pressure in hypertensive patients (357).

FIGURE 16. Structures of xanthine oxidase substrates and inhibitors. E. Other Sources of ROS

Other proteins have been related to the direct or indirect logical inhibition of XO with allopurinol/oxypurinol im- production of ROS in the cardiovascular system, including, proved cardiovascular conditions in several pathologies, but not limited to, peroxidases, cyclooxygenases (COX), including heart failure (58, 278), dilated cardiomyopathy lipoxygenases, monoamine oxidase, heme oxygenase (HO), (148), and diabetes (137, 224). Amelioration of endothelial function in smokers was also observed (387). Despite these glucose oxidase, cytochrome b5 reductase, and CYPOR initial results, subsequent trials with larger number of pa- (209, 505, 790, 908). In this section, we will briefly describe tients have failed to report substantial positive effects as some of these ROS sources. compared with the placebo groups (361, 405), although the meta-analysis of available studies indicates some improve- 1. Peroxidases and COX ment of cardiovascular function (430). Altogether, the effect of allopurinol in cardiovascular disease is still contro- Mammalian peroxidases are heme proteins that catalyze versial. It has also been pointed out that allopurinol and the oxidation of a wide range of substrates. These proteins oxypurinol have limited capacity to inhibit XO bound to are activated by their reaction with hydrogen peroxide to glycosaminoglycans (620). It is very possible that the use of generate an oxidizing heme species that can hydroxylate or XO inhibitors needs to be complemented with other thera- oxygenate a substrate. Mammalian peroxidases include pies targeting other sources of endothelial dysfunction thyroid peroxidase, myeloperoxidase (MPO), lactoperoxi- (928). dase, eosinophil peroxidase, and COX. MPO, lactoperoxi- dase, and eosinophil peroxidase are integral components of C) FEBUXOSTAT. Febuxostat (2-(3-cyano-4-isobutoxyphenyl)- the immune defense (516). 4-methyl-5-thiazole carboxylic acid) (TEI-6720, TMX-67) (FIGURE 16) is a XO inhibitor developed by Teijin, with A) MPO. MPO is found predominantly in neutrophils and higher selectivity and potency than allopurinol and oxy- monocytes and catalyzes the reaction of hydrogen peroxide purinol (702). The selectivity is partly due to a lack of to produce several reactive species, mainly hypochlorous structural resemblance with purines; this also reduces side acid (HOCl) (discussed in sect. IVB) (516, 1021). Macro- effects of purine and pyrimidine metabolism (1041). The higher affinity and specificity of febuxostat can have advan- phage localization in the atherosclerotic plaque leads to tages over allopurinol and oxypurinol. Notably, the activity detectable MPO activity and products of HOCl oxidation of febuxostat is not impaired by XO immobilization on the (213, 414). Several studies have correlated low circulating surface of ECs (620). Febuxostat was approved in 2008 in MPO levels with better cardiovascular prognosis (239, 260, Europe and by the Food and Drug Administration in 2009 644, 690, 1059). for the treatment of gout (commercial name Uloric in United States, Adenuric in Europe) (76, 77, 837). Studies in B) COX. COX are proteins from the peroxidase family, evo- rodents have shown improvement in atherosclerosis and lutionarily related to the peroxidase enzymes of the immune hypertension after treatment with febuxostat (697, 859). system (1052). They utilize lipid substrates, generally ara- The safety of febuxostat for the treatment of cardiovascular chidonic acid, and produce a variety of prostaglandins, lipid disease in humans is still under investigation (331, 610). oxidation products with important physiological effects (156). Two isoforms are expressed in mammals: COX-1 D) TOPIROXOSTAT. Topiroxostat (FYX-051, 4-[5-pyridin-4-yl- and COX-2, and both of them have been related to endo- 1H-[1, 2, 4] triazol-3-yl]pyridine-2-carbonitrile) (FIGURE thelial dysfunction (981, 982). Interestingly, some interplay 16) is a novel XO inhibitor developed by Fuji Yahuhin Co. between COX-2 and other ROS/RNS-generating proteins Ltd. Topiroxostat is a potent inhibitor of XO and induces a such as iNOS (52) and XO (701) have been described.

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2. Lipoxygenases sistent with an important role in the processing of heme from red blood cell turnover (713, 805). The interplay be- Lipoxygenases catalyze the insertion of oxygen in polyun- tween HO-1, CO production, and NOX inactivation is a saturated fatty acids (119, 535, 1040) and are also biolog- focus of current interest in cardiovascular research (685, ical sources of superoxide (537). Several lipoxygenases have 793, 805, 924). been related to inﬂammation and cardiovascular disease, in particular, 5-lipoxygenase and 12/15-lipoxygenase (203, IV. OTHER ROS 597, 636).

3. AO A. Peroxynitrite

Ϫ The Mo-containing protein AO has also been described as a Peroxynitrite (ONOO ) is a strong oxidizing and nitrating potential source of superoxide in vivo (538, 539). Although agent formed by the reaction of NO with superoxide (Eq. the function of AO in mammals is yet to be established 23). This reaction occurs at a diffusion-limited rate and is (434, 935), AO appears to play an important role in drug governed by the flux of NO and superoxide production metabolism and other metabolic pathways (335, 512, 513, (78). Another physiological route by which peroxynitrite 756, 920). Studies with rat AO have shown a significant can be formed is through the reaction of hydrogen peroxide production of superoxide when the enzyme uses NADH as with nitrite in acidic conditions (Eq. 24) (808). a substrate (539). Recent work on the human enzyme shows Ϫ Ϫ NO· ϩ O · → ONOO (23) that the production of superoxide by the human AOX1 2 ϩ → ϩ isoform is lower than that of the rat enzymes, and the en- H2O2 HNO2 ONOOH H2O(24a) zyme is not reactive toward NADH (304). However, a Ϫ Ϫ ONOOH ϩ OH → ONOO ϩ H O(24b) known single nucleotide polymorphism in the AOX1 gene 2 was shown to produce a mutant enzyme with increased Despite its rapid rate of formation, peroxynitrite reacts rel- superoxide formation rates in vitro (304). The possible im- atively slowly in comparison with other RNS, with many pact of these single nucleotide polymorphisms in vivo re- biomolecules, including metal centers, proteins, DNA, and mains to be determined. lipids (78, 526, 709). Additionally, studies show peroxynitrite can traverse cell membranes by both diffusion and 4. CYPOR and CYP through ion channels, suggesting that peroxynitrite can mediate oxidation/nitration distant from its site of formation CYPOR is a diflavin protein, structurally and evolutionarily (221, 611). related to the reductase domain of the NOS enzymes (377) that functions as a general reductase of the many CYP pro- The reaction of peroxynitrite with biomolecules is generally teins. The P450 system is conspicuous for its role in detox- considered as a detrimental event. Peroxynitrite can directly ification and hormone synthesis (675, 1013). CYPOR, ei- react with a variety of metal centers, resulting in their oxi- ther directly or in combination with the CYP proteins, has dation. In the cardiovascular system, peroxynitrite can ox- been described as a source of superoxide (758, 851). The idize heme proteins, such as Hb and Mb, resulting in the presence of these proteins in endothelial cells makes them a oxidation of their heme oxygen–carrying site from ferrous significant source of superoxide in the vasculature (295, iron to ferric heme (101, 942). In the mitochondrion, a 298). Because of the functional similarities with the dehy- similar reaction oxidizes the electron transport protein cy- drogenase domain of NOX, some NADPH-dependent su- tochrome c (942). Reaction of peroxynitrite with Zn/S clus- peroxide measurements can be unable to differentiate ters such as that present in eNOS, causes dysfunction of the CYPOR from NOX as the source of superoxide (783). enzyme as outlined above (sect. IIIB3) (78, 526). Addition- ally, peroxynitrite reacts rapidly with Fe/S clusters leading 5. HO to the inactivation of critical enzymes such as mitochondrial aconitase (155, 401). HO mediates the degradation of heme in mammals. This enzyme catalyzes the degradation of the protoporphyrin IX Peroxynitrite is perhaps most well recognized for its reac- (heme b) to biliverdin (805, 806). This process generates tion with cysteine and tyrosine residues in proteins. Per- ferrous iron (Fe2ϩ) and CO, which are well-known toxic oxynitrite-dependent cysteine oxidation results in the for- species (122, 739, 795, 959), but CO can also be, in low mation of a sulfenic acid intermediate that can further be concentrations, a signaling molecule (713, 739, 805, 807). oxidized to a disulfide. This type of oxidation modulates the Three HO isoforms exist; the inducible isoform HO-1 is, to activity of a number of enzymes. For example, peroxyni- date, the most relevant to vascular physiology. HO-1 ex- trite-mediated oxidation of ETC components such as com- pression in basal conditions is low or undetectable in most plex I and complex V results in inhibition of mitochondrial tissues. High levels of HO-1 are present in the spleen, con- function (767). Oxidation of tyrosine phosphatases results

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Hypochlorous acid (HOCl) is generated by the enzyme MPO, localized within leukocytes by the following reaction Beyond cysteine oxidation, peroxynitrite can mediate the (300) (Eq. 25) Ϫ covalent addition of a nitro group ( NO2) to the aromatic H O ϩ ClϪ → HOCl ϩ OHϪ (25) ring of tyrosine, resulting in tyrosine nitration. This modi- 2 2 fication is measured as the hallmark of peroxynitrite pres- This highly reactive oxidizing and chlorinating agent plays ence and is traditionally viewed as a sign of nitrative or a major role in the microbicidal actions of neutrophils (15, oxidative stress (78, 765). Importantly, protein nitration 1021). Although HOCl reacts with a variety of biomolecule can also occur through the reaction of nitrite with heme targets (755), in proteins, it predominantly oxidizes cys- proteins in the presence of hydrogen peroxide (153, 154). teine and methionine residues to disulfide and methionine Thus, methodology beyond nitrotyrosine measurement is sulfoxide, respectively (726). With regards to metal centers, required to confirm the presence of peroxynitrite in biolog- HOCl can react with heme and Fe/S centers to mediate ical systems. oxidative bleaching. Additionally, HOCl reacts more slowly with tyrosine residues to mediate the formation of Although a growing number of proteins have now been 3-chlorotyrosine (1019). Notably, 3-chlorotyrosine is uti- shown to be nitrated, multiple studies have shown that lized as a marker of the presence of both MPO (as this is the nitration is a selective process (47, 484). Tyrosines for only enzyme known to generate HOCl) and HOCl. Oxida- which the aromatic ring is closer to the surface of a protein, tion of amino acids and metal centers of enzymes results in in which there is a neighboring negative charge, and those in the HOCl-dependent impairment of a number of critical hydrophobic regions are more likely to be nitrated (66, enzymes. 877). Several studies provide comprehensive lists of proteins identified to be nitrated in physiology and disease (44, The role of HOCl in the modification of proteins and lipids 73, 381, 709). These proteins include, but are not limited is particularly well studied and pertinent to the pathogene- to, enzymes involved in antioxidant defenses such as Mn- sis of vascular disease. In the context of atherosclerosis, SOD (219, 220), detoxification enzymes such as CYP (587), HOCl has been shown to oxidize both high and low density nucleic acid regulatory proteins such as histone deacetylases lipoproteins (90, 470, 881, 1042). Furthermore, chlori- (457) and histones (453, 504), and cell structural proteins nated proteins and lipids are present in atherosclerotic such as tubulin (730, 732) and myosin heavy chain (648). plaques (90, 415, 621). Hypochlorous acid decreases eNOS Importantly, the nitration of these proteins decreases en- activity and NO bioavailability by a number of direct and zyme activity, disrupts metabolism and cellular detoxifica- indirect mechanisms, disrupting protective NO signaling tion, perturbs cytoskeletal organization, and ultimately (2, 464, 1054). The oxidation and chlorination of proteins, contributes to the cytotoxic effects of peroxynitrite. such as matrix metalloproteinases, leads to detrimental vascular remodeling (314). Although peroxynitrite is traditionally regarded as a harmful species, it should be noted that it may have some bene- C. Hydroxyl Radical ficial signaling properties. For example, lipids are also a significant biological target for peroxynitrite, and although The hydroxyl radical (OH·) is one of the most reactive spe- the species can abstract a hydrogen atom to initiate lipid cies formed in biological systems. In this context, OH· is peroxidation, a detrimental process that can lead to cyto- generally formed as a product of Fenton reactions involving toxicity in many cases (65, 766), peroxynitrite can also free iron or copper ions (Fe2ϩ;Cuϩ) and hydrogen peroxide mediate lipid nitration (54, 1027). Nitrated fatty acids are a (Eq. 26) (281, 986) or from the reaction of hydrogen per- class of electrophilic signaling molecules which have been oxide with superoxide (Haber–Weiss reaction) (Eq. 27) shown to react with a number of cellular targets to mediate (394). protective inflammatory and metabolic signaling (830). Pri- 2ϩ ϩ → 3ϩ ϩ · ϩ Ϫ Fe H2O2 Fe OH OH (26a) mary mechanisms of nitrated fatty acid signaling include ϩ ϩ → 2ϩ ϩ · ϩ Ϫ activation of PPAR␥ (55, 583) and Michael addition to Cu H2O2 Cu OH OH (26b) Ϫ· ϩ → · ϩ ϩ Ϫ thiol-containing proteins in a number of signaling pathways O2 H2O2 OH O2 OH (27) (53, 955). Through these signaling mechanisms, nitrated lipids mediate a number of physiological effects in animal The process is generally more complex than indicated in Eq. models including attenuation of inflammatory bowel dis- 26 and includes the formation of a transient metal/peroxide

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· ϩ · → complex that can react in a different fashion depending on NO2 NO N2O3 (29) the metal ligands and possible substrates. Therefore, in some cases, the reaction may not produce free hydroxyl As discussed previously in section IIB2, the formation of radical but other metal-based oxidizing species (759, 1020). N2O3 in the red blood cell via an anhydrase reaction involving Hb, NO, and nitrite (69) constitutes a possible pathway · Although the endogenous production of OH in healthy to preserve NO bioactivity (367). conditions is apparently negligible (474), the excess of hydrogen peroxide and superoxide in oxidative stress condi- 3. Carbonate radical tions can yield an increased amount of this toxic species (1072). The carbonate radical can be formed in vivo by the reaction of the hydroxyl radical with carbonate or bicarbonate (138) In physiology, perhaps the most common source of Fenton or via the reaction of peroxynitrite with carbon dioxide chemistry is the presence of increased concentrations of free (Eq. 30) (112, 608, 640). iron and/or copper (122, 458, 959). In the cardiovascular · ϩ 2Ϫ → Ϫ ϩ ·Ϫ system, this can be related to aging (122) or hemolytic dis- OH CO3 OH CO3 (30a) ease (1026). The source of this iron is most likely plasma- · ϩ Ϫ → Ϫ ϩ ·Ϫ OH HCO3 H2O CO3 (30b) released Hb; whereas Hb or heme are not catalysts for the Fenton reaction, the reaction of plasma Hb with peroxides Several examples of biological oxidations mediated by the can cause the release of free iron (389, 759). bicarbonate anion, in particular through interactions with peroxynitrite, have been reported in the literature [e.g., (46, The hydroxyl radical reacts with multiple organic sub- 635, 918, 1056)]. In relation to cardiovascular biology, it is strates including, but not limited to, DNA, lipids, and pro- important to note that XO has also been characterized as a teins at rates near the diffusion limits. This lack of specific- target and source of carbonate radical (111). ity makes the use of antioxidants to prevent hydroxyl radical toxicity impractical. Ideally, the best strategies will be based on the chelation of the active metal ions in an envi- V. CROSS-TALK BETWEEN ROS AND RNS ronment that blocks their redox reactivity. Proteins like SOURCES ceruloplasmin, transferrin, or lactoferrin limit the reaction (388, 1020). The binding of NO to heme iron can also limit Our description of sources of NO and ROS may give the heme reactivity and prevent iron release from heme (482). impression of a rigorous separation of roles for each protein or enzymatic system. This is certainly not the situation in physiological systems. We have discussed how some en- D. Other Species zymes, in particular eNOS, can produce different species in response to posttranslational modifications or lack of co- The reactions of NO can generate other reactive species that factors or substrates (sects. IIA1 and IIIB). Current research have a poorly characterized role in vivo. Among these spe- has greatly advanced our knowledge on how different sys- · cies, we will mention the nitrogen dioxide radical (NO2 ), tems can influence the NO/ROS generation from other ·Ϫ dinitrogen trioxide (N2O3), and carbonate radical (CO3 ) sources in which it has been termed ROS-induced ROS (46, 692). release (1066, 1069).

1. Nitrogen dioxide Mitochondria and NOX are the main effectors of downstream signaling to other sources and to each other. Mito- · Nitrogen dioxide (NO2 ) is formed in the reaction of NO chondrial uncoupling and increased ROS generation with oxygen (Eq. 28) (509, 1018). A more reactive species activates the mitochondrial permeability transition pore than NO, it is often used as a surrogate for the detection of (mPTP), allowing the release of ROS to the cytosol (1069). NO. Other formation routes include the reactions of nitrite In response to the increased levels of ROS, the PKC is acti- with oxygenated Hb (501) and peroxynitrite decomposi- vated, which in turn, can activate NOXs (319, 776). Other tion (46). routes of mitochondrial triggered activation of NOX in- NO· ϩ O ↔ O NO· (28a) volve the activation of NOX1 via activation of PI3K and 2 2 Rac1. The blockade of mitochondrial ROS by rotenone was · ϩ · → · O2NO NO 2NO2 (28b) shown to prevent NOX1 activation (FIGURE 17) (564).

2. Dinitrogen trioxide Indeed, activated NOX can in turn, increase ROS production by the mitochondria (11, 116). This process appears to

Dinitrogen trioxide (N2O3) is a strong nitrosating species require the function of mitochondrial ATP-dependent po- also formed in the oxidation of NO (Eq. 29) (691, 692, tassium channels (KATP), as shown by the ability of KATP 1018). inhibitors to block the increase of mitochondrial ROS pro-

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Mitochondria

mPTP PI3K K ATP PKC FIGURE 17. Cross-talk between mitochondria, NADPH oxidase (NOX), and other reactive oxygen species NOS XO (ROS) sources. Mitochondrial ROS are released to the cytosol via the mitochondrial permeability transition pore (mPTP). These can cause activation of ROSROS ROSROS PKC and phosphatidylinositol 3-kinase (PI3K) kinases and Rac1 GTPase with subsequent activation of NOX enzymes and further ROS production. Alterna- tively, NOX can release ROS that acti- PKC vate protein kinase C, isoform ␧ (PKC␧) PI3K and in turn activate the mitochondrial Rac1 ATP-dependent potassium channels

(KATP), increasing mitochondrial ROS. Cytosolic ROS can increase ROS-generating activity of endothelial nitric oxide synthase (eNOS) and xanthine oxidase (XO).

NOX

duction after angiotensin II–induced activation of NOX the direct inhibition of a single source of oxidative stress. (242, 507). Of note, the activation of NOX via angiotensin Recent work indicates that this can be a feasible approach II and the angiotensin II type 1 receptor is mediated by PKC; (206, 207, 234, 836). The development of novel, speciﬁc ␧ however, the isoform PKC is also an activator of the KATP mitochondrial inhibitors (discussed in sect. IIIC3)isa channel, which, as indicated, also stimulates mitochondrial promising step in this direction (8, 679, 923, 1062). ROS production (FIGURE 17) (187, 242).

Another case of signiﬁcant cross-talk between NOX and VI. CONCLUDING REMARKS: ADVANCES, mitochondria has been provided by studies of nitroglycerin CHALLENGES, AND THERAPEUTIC resistance. In this case, the development of tolerance was OPPORTUNITIES mainly due to mitochondrial ROS release and was independent of NOX, whereas the activation of NOX, triggered by Over the last decade, our knowledge about NO and ROS mitochondrial ROS, was the effector of the endothelial dys- generation has increased substantially. From the initial function associated to nitroglycerin tolerance (1004). ideas considering that oxidative species need to be sup- pressed and that oxidative stress could be combated with Secondary to the main role of mitochondria and NOX, but adequate antioxidant treatment, we now can appreciate the not to be ignored, XO and eNOS can also amplify the critical signaling properties of NO, hydrogen peroxide, or formation of ROS; for instance, XO is indirectly activated by angiotensin II, increasing cellular ROS production (551). superoxide. A direct role of mitochondrial ROS on XO activation has also been described (362). As discussed in previous section, The initial approach to the treatment of oxidative stress was increased ROS eventually causes eNOS uncoupling via BH based on the supplementation with different antioxidants, 4 ␤ depletion, Zn/S cluster breakdown, and other routes, fur- in particular vitamins C and E and -carotene. The use of ther perpetuating the generation of superoxide and other antioxidants has been markedly ineffective when not dam- ROS (FIGURE 17) (488, 570). aging (97, 390, 400, 785). In fact, the situation has been termed the “antioxidant paradox” (399, 400). The failure The existence of many different routes interconnecting of these studies, along with their limitations, has been dis- ROS/RNS sources may suggest that the normalization of cussed at length in this review, and a number of explana- dysfunctional conditions is a herculean task; however, it tions for this phenomenon have been proposed (67, 158, also presents the promise of normalizing a disease state by 252, 400, 672, 949).

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At the same time, we are increasingly appreciative of the sGC. Advances on NOX research have led to the develop- cross-talk between different radical species, generation ment of novel inhibitors on their way to widespread use for pathways, and cellular compartments. Different examples the treatment of cardiovascular disease and other patholo- of species interaction have been discussed throughout this gies. New highly speciﬁc inhibitors of other ROS sources

review; for instance, the oxidation of BH4 by superoxide like the XO-targeted febuxostat and mitochondrial-tar- will trigger eNOS uncoupling with decreased NO produc- geted drugs provide additional pathways to modulate cel- tion and increased superoxide production. In another ex- lular ROS production. We foresee that the availability of ample, NO, through its high affinity for heme groups or via these specific drugs and a better biomarker-based diagnosis S-nitrosation, can act as an important modulator of NOX of the dysregulated systems will allow for more personal- (176, 315, 846) or can also modulate the insertion of heme ized treatments. in the receptor sGC. The generation of NO through NOS enzymes and oxidative pathways in normoxic conditions is Finally, antioxidants targeted to specific cells or organelles supplemented by the actions of reductive, nitrite-dependent can still become relevant in the management of oxidative pathways in hypoxia (603, 968). We also note how some stress pathologies, in particular, but not limited to, mito- proteins are able to switch, in an oxygen-dependent man- chondria-targeted antioxidants (873). The development of ner, from NO scavengers into nitrite-reducing, NO-produc- these treatments directed toward specific organs, tissue, and ing systems. The ability to interact with different oxidation cell types can be one of the next big advances in the field. states of sGC and use pharmacological tools to recover ACKNOWLEDGMENTS sGC-dependent signaling provides a new tool for the treatment of conditions in which NO signaling is compromised. We thank Patrick Pagano (University of Pittsburgh), Maria Further research aimed to the understanding of these dy- Eugenia Cifuentes-Pagano (University of Pittsburgh), Eric namics will be critical for the treatment of endothelial dys- E. Kelley (West Virginia University), Mauro Siragusa function and other redox imbalance-mediated pathologies. (Goethe University, Frankfurt am Main, Germany), and members of the Gladwin laboratory for critical reading of The interactions between mitochondria and ROS-produc- our manuscript. Coordinates for the sGC model were ing proteins, especially NOXs, are of particular current in- kindly provided by William Montfort (University of Ari- terest (116, 206, 234, 836). Mitochondria are a physiolog- zona). Because of the immense volume of literature in the ically significant source of ROS with important implica- field, we are sure that many important works are not in- tions in physiology, not only in cardiovascular disease but cluded in the reference list. We sincerely apologize to the in many other conditions like aging (57, 84, 136, 165, 170, authors for these unintentional omissions. 290, 864, 997). GRANTS It is also significant that many of our advances have evolved not only from the clinical field but also from basic science. This work was supported by National Institutes of Health Chemical knowledge of NO and ROS reactivity has been Grants R01 HL098032, R01 HL125886, P01 HL103455, available well before its biological relevance was estab- T32 HL110849, and T32 HL007563 (to M.T. Gladwin), lished. This knowledge is critical for the understanding of R21 ES027390 (to J. Tejero), and funding from the Institute the biology of ROS and can provide new tools and strate- for Transfusion Medicine and the Hemophilia Center of gies for the study of ROS biology (232). The studies on Western Pennsylvania (to M.T. Gladwin and S. Shiva). purified protein systems, although necessarily simplistic, have provided insights on the reactivity of most proteins DISCLOSURES involved in NO and ROS biology that have been widely validated in vivo. Conversely, the study of genetic patholo- M.T. Gladwin is a coinventor on a National Institutes of gies such as chronic granulomatous disease has helped to Health government patent application for the use of nitrite establish the relevance of NOX proteins even before these salts in the treatment of cardiovascular diseases. J. Tejero were thoroughly characterized. and S. Shiva do not declare any conflicts of interest, finan- cial or otherwise. In summary, we have progressed from a simpler approach leveraging antioxidant vitamins and ROS scavengers, REFERENCES which failed, to a more fundamental discovery of the enzymatic sources and reaction pathways of ROS. The future 1. Aamand R, Dalsgaard T, Jensen FB, Simonsen U, Roepstorff A, Fago A. Generation of treatments need to address these issues. It is clearer now nitric oxide from nitrite by carbonic anhydrase: a possible link between metabolic activity and vasodilation. 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