NADPH oxidase-derived ROS and the regulation of pulmonary vessel tone

Abstract

Pulmonary vessel constriction results from an imbalance between vasodilator and vasoconstrictor factors released by the endothelium including nitric oxide, endothelin, prostanoids, and reactive oxygen species (ROS). ROS, generated by a variety of enzymatic sources (such as mitochondria and NADPH oxidases, a.k.a. Nox), appear to play a pivotal role in vascular homeostasis, whereas elevated levels effect vascular disease. The pulmonary circulation is very sensitive to changes in the partial pressure of oxygen and differs from the systemic circulation in its response to this change. In fact, the pulmonary vessels contract in response to low oxygen tension, whereas systemic vessels dilate. Growing evidence suggests that ROS production and ROS-related pathways may be key factors that underlie this differential response to oxygen tension. A major emphasis of our laboratory is the role of Nox isozymes in cardiovascular disease. In this review, we will focus our attention on the role of Nox-derived ROS in the control of pulmonary vascular tone.

this article is part of a collection on Hypertension and Novel Modulators of Vascular Tone. Other articles appearing in this collection, as well as a full archive of all collections, can be found online at http://ajpheart.physiology.org/.

Introduction

Since their discovery, reactive oxygen species (ROS) have aroused increasing interest because of evidence that they regulate important physiological and pathophysiological conditions. Under physiological conditions, the redox environment of vascular tissue is maintained in an overall reduced state via an interplay and balance between oxidants (reactive oxygen and nitrogen species) and antioxidant systems. Major ROS produced in the pulmonary vasculature are, among others, superoxide (O₂^·−), hydrogen peroxide (H₂O₂), hydroxyl radical (HO^·), and hydroperoxyl radical (HO₂^·), and the most important reactive nitrogen species emerge as nitric oxide (NO), nitrogen dioxide (NO₂), dinitrogen trioxide (N₂O₃), and peroxynitrite (Fig. 1). In this review, we decided to focus our attention on the ROS effects on pulmonary vascular tone. For a complete overview about reactive nitrogen species and their pathophysiological role, we refer the reader to an excellent review on the topic (167). Endogenous antioxidant systems include enzymes such as superoxide dismutase [SOD, which dismutes superoxide anion (O₂^·−) into H₂O₂], catalase (which converts H₂O₂ to water), peroxiredoxins (which catabolize H₂O₂ to water), glutathione peroxidase, peroxiredoxin, heme oxygenase, glutaredoxin and thioredoxin (which reduce the disulfide bridges of various target proteins), and nonenzymatic agents such as glutathione, β-carotene, retinol, vitamin C, and vitamin E (109). When endogenous antioxidant systems are inadequate or overwhelmed by ROS generation, increased ROS steady-state levels initiate multiple pathologies including diabetes (53, 74, 146), hypertension (40, 96, 125, 169), atherosclerosis (44, 76, 159), and hypoxia/reperfusion injury (89, 95, 190). In the lungs and cardiovascular system, principal sources of ROS include mitochondrial electron transport, xanthine oxidase, cytochrome P-450, cyclooxygenases, uncoupled NO synthase, lipoxygenases, and the NADPH oxidase (Nox) family of proteins (5). It is known that these different sources of ROS modulate each other through mechanisms of negative feedback and feed-forward processes. Dysregulation in these processes contribute to the development of diseases. For example, ROS produced from Nox can stimulate ROS production by the other sources, and vice versa, increased levels of mitochondria-derived ROS lead to Nox activation. In this review, we will focus our attention in analyzing the effect of the Nox-derived ROS. In the pulmonary system, Nox-derived ROS production is involved in diseases such as pulmonary hypertension (57, 103, 120), fibrosis (4, 41, 110), chronic granulomatous disease (144, 154), acute lung injury and acute respiratory distress syndrome (81, 175, 178), emphysema and asthma (1, 88, 126, 141, 156, 165, 174), chronic obstructive pulmonary disease (45, 129, 130), and lung cancer (72, 107) (Table 1). On the other hand, in the pulmonary vasculature, Nox-derived ROS contribute to the maintenance of vascular tone and regulate important processes such as cytoskeletal organization, cell migration, cell growth, proliferation, differentiation, and apoptosis (71). These seemingly contradictory phenotypes can be explained by the current theory that gradation and spatial orientation of ROS levels elicit distinct outcomes; that is, when levels of ROS exceed basal, distinct physiological and pathophysiological outcomes ensue, depending on the degree and delivery of ROS to effector pathways.

Fig. 1.

Simplified schematic illustration of common forms of reactive oxygen and nitrogen species. O₂^·− derived from different sources such as XO, Nox enzymes, mithocondria, uncoupled endothelial NO synthase (eNOS) and cyclooxygenase (COX) is dismuted to H₂O₂ by SOD. H₂O₂ can be converted to H₂O by catalase or GPx. O₂^·− can also react with NO to generate the reactive nitrogen species ONOO⁻ that could be converted into NO₂^·, which reacts with protein tyrosine residues to generate NO₂-Tyr. This reaction can lead to a decrease in the bioavailability of NO, leading to endothelial dysfunction.

Table 1.

Involvement of Nox isoforms in lung disease

Nox Isoform	Localization	Pulmonary Disease Associated	References
Nox2, Nox4	Pulmonary artery endothelial cells	PAH, ALI/ARDS	(51, 56, 70, 90, 105, 156)
Nox4	Pulmonary artery smooth muscle cells	PAH	(51, 105)
Nox4	Myofibroblasts	Pulmonary fibrosis	(3, 36, 95)
Nox2	Macrophages/neutrophils	Chronic granulomatous disease	(125, 135)
Nox3	Endothelial cells of airways	Emphysema	(1, 122, 143)
DUOX1 and -2	Airway epithelial cells	COPD and asthma	(66, 166)
DUOX1 and -2	Lung carcinoma cells	Lung cancer	(93)

Multiple studies demonstrate the involvement of various NADPH oxidase (Nox) isoforms in the development and progression of different pulmonary diseases, such as pulmonary arterial hypertension (PAH), acute lung injury (ALI), pulmonary fibrosis, chronic granulomatous disease, emphysema, obstructive lung disorders [chronic obstructive pulmonary disease (COPD)], asthma, and lung cancer. ARDS, acute respiratory distress syndrome; DUOX, dual oxidase.

Nox Enzymes

The structure and function of Nox was first described in neutrophils. The phagocytic Nox is a multisubunit complex comprised of the flavocytochrome b₅₅₈, which is composed of two subunits, p22^phox and Nox2 (previously known as gp91^phox), as well as four cytoplasmic subunits, namely, p47^phox (organizer subunit), p67^phox (activator subunit), p40^phox,, and the small G protein, Rac. In resting phagocytes, this multienzyme complex is unassembled and inactive. Activation involves the phosphorylation and translocation of the cytoplasmic subunits of Nox to the membrane-integrated catalytic subunits, leading to the production of O₂^·−. Phosphorylation and activation of essential organizing subunit p47^phox initiates this process. To date, seven isoforms of the catalytic Nox subunit have been identified in humans and classified into three groups based on their basic structure (20) (Fig. 2). The first group is comprised of Nox1, Nox3, and Nox4, which share similar structural organization with Nox2. The second group includes dual oxidases 1 and 2 (DUOX1 and DUOX2), which possess an extracellular peroxidase-like domain and intracellular EF hand-type Ca²⁺-binding pockets involved in exquisite activation of these oxidases. The main product detected by DUOX1 and -2 is H₂O₂, but it remains controversial whether these enzymes produce O₂^·−, which rapidly dismutes to H₂O₂ or whether H₂O₂ is the main product. Third, Nox5 possesses an extra amino-terminal calmodulin-like domain that contains four calcium binding EF hands, in addition to the Nox2-like catalytic core. To date, four splice variants of Nox5 (Nox5α, Nox5β, Nox5γ, and Nox5δ) and a truncated variant (Nox5ε) have been identified in humans (16, 35). The different isoforms of the Nox family vary in their tissue expression, subcellular distribution, and mechanisms of activation and appear to be involved in distinct physiological functions in most cases (19).

Fig. 2.

Schematic structures of distinct identifying domains in Nox isoform catalytic subunits. A: Nox2 and Nox2-like isoforms (Nox1, 3, and 4) present a basic structure of 6 transmembrane domains containing two heme groups and COOH-terminal FAD and NADPH domains. B: in addition to those domains, Nox5 possesses Ca²⁺-binding EF motifs in the NH₂-terminal region. C: dual oxidasess, in addition to Ca²⁺-binding EF motifs, possess an NH₂-terminal peroxidase-like domain.

Nox1 is highly expressed in the colon epithelium, and to date two splicing isoforms have been described as NOH-1S and NOH-1L (15), also named Nox1-L and Nox1-S (78). Nox1 requires the presence of two cytosolic subunits: the p47^phox homolog, Nox organizer subunit 1 (NOXO1) and the p67^phox homolog, Nox activator subunit 1 (NOXA1). Four isoforms of NOXO1 have been described in humans (NOXO1α, NOXO1β, NOXO1γ, NOXO1δ) (36, 172). Despite the structural similarities among regulatory subunits of Nox2 and Nox1, different functional aspects are involved in the regulation of enzyme activity. For example, NOXO1 is localized to caveolae with Nox1 and p22^phox, whereas p47^phox localizes in the cytosol of resting cells. A hallmark characteristic of the Nox1 system is the lack of an autoinhibitory domain in NOXO1 (compared with p47^phox in the Nox2 system), which is invoked to explain the unique preassembled state of Nox1.

Nox3 is principally expressed in the inner ear and less notably in fetal brain and kidney tissues (14) and seems to generate low levels of O₂^·− in a cytosolic subunit-independent manner and high levels of O₂^·− in the presence of activator and organizer subunits (p47^phox and p67^phox, NOXO1 and NOXA1) (14).

In contrast to other Nox isoforms, Nox4, which is highly expressed in the kidney, endothelial cells, and pulmonary artery smooth muscle cells (PASMCs), does not require cytosolic subunits p47^phox and p67^phox or their homologues for activation and thus appears to be constitutively active or regulated by heretofore undefined cytosolic subunits. Intriguingly, Lyle and colleagues (108) showed that the polymerase δ-interacting protein-2 associates with p22^phox to activate Nox4.

Nox5 is highly abundant in the spleen and testis (16) and is less abundant in vascular tissue, cells of the gastrointestinal tract, reproductive system, and fetal organs (16, 35, 97). The regulation of Nox5 activity appears not to require the presence of cytosolic subunits that are recognized as necessary for other Nox isoforms (16). Nox5 has also been shown to bind p22^phox, but the significance of this is unclear because changes in the expression level of p22^phox, or dominant negative constructs of p22^phox, do not affect Nox5 activity (21, 91). The elevation of intracellular calcium promotes the occupation of the calcium-binding EF-hand domains of Nox5. This leads to conformational changes among the domains that trigger O₂^·− production (16).

DUOX1 and DUOX2 were originally identified and cloned from the epithelium of the thyroid gland (31, 43, 49). DUOXs are widely expressed in epithelial tissues, including epithelia of the airways (61, 66, 155), alveoli, intestinal tract (52, 65), salivary glands (65), and prostate (177). When DUOX2 is partially glycosylated, it is located to the endoplasmic reticulum (ER) and generates O₂^·−, whereas when it is fully glycosylated, it is transported to the plasma membrane and generates H₂O₂, (7, 69, 196). DUOX proteins require the presence of two maturation factors, DUOX activator 1 and 2 (DUOXA1 and DUOXA2), which allow proper DUOX processing and trafficking to the membrane (69). Only the combination of the DUOXs with their corresponding DUOXAs renders the complex active and allows exit from the ER (68). In experiments with transfection of DUOX2 into PCCl3 cells and Cos7 cells, an enhanced generation of H₂O₂ in response to Ca²⁺ has been shown (42, 152). Moreover, site-directed mutation of the predicted Ca²⁺-binding glutamate residues to glutamine in the EF hands of DUOX1 (E839Q and E875Q) or DUOX2 (E843Q and E879Q) resulted in an almost complete loss of function (152). In addition, NOXA1 was recently shown to interact with and inhibit DUOX1 in a Ca²⁺-dependent fashion, possibly involving the COOH-terminal region of DUOX1 (133). The levels of expression of DUOX1 and DUOX2 are selectively upregulated by inflammatory cytokines (IL-4 and IL-13) and interferon-γ, respectively (77).

Expression of Nox Isoforms in the Lung

In total lung tissue, mRNA for Nox1, Nox2, Nox4, DUOX1, DUOX2, p22^phox as well as the cytosolic subunits NOXA1, NOXO1, p47^phox, p40^phox, and p67^phox are expressed (51, 65, 120) (Table 2). In this section, we will discuss Nox isoform expression across the different sections of the lung tissue and in the different layers of the pulmonary vasculature.

Table 2.

Expression and function of Nox isoforms in lung tissue

Nox Isoform	Distribution	Function	References
DUOX1 and -2	Epithelial airway	Host defense, mucin production, cellular migration and differentiation	(60, 139, 176)
Nox2, Nox4	Pulmonary endothelial cells	Control of vascular tone, vascular cell growth and remodeling, angiogenesis, proinflammatory cytokine production	(24, 104, 118)
Nox4	Pulmonary smooth muscle cells	Differentiation and hypoxia-induced proliferation	(32,144)
Nox4	Myofibroblasts	Differentiation	(42)

Distinct isoforms of Nox (especially Nox2, Nox4, DUOX1, and DUOX2) are expressed in lung and participate in multiple physiological and pathological processes such as control of vascular tone, cell growth, and differentiation.

Airway epithelium and alveolar cells.

The primary sources of ROS production in airway epithelium are DUOX1 (66, 77, 98, 155) and DUOX2 (61, 77, 155). However, the expression of other Nox isoforms, Nox1, Nox2, and their regulatory proteins, has been reported (98). mRNA levels of DUOX1 and DUOX2 are regulated by cytokines. Reportedly, IL-4 and IL-13 (produced by Th2) induce a moderate increase of DUOX1 mRNA, whereas interferon-γ (from Th1) markedly increases mRNA for DUOX2, perhaps suggesting a constitutive role of DUOX1 in noninflamed airways and an inducible role of DUOX2 in infection and inflammation (77). Other Nox enzyme functions in airway epithelia include host defense (65), response to mechanical stress (34), regulation of matrix metalloproteinase-12 (MMP-12), MMP gene expression (98, 135), production of epithelial mucins (158), induction of cell death (199), regulation of lung development, epithelial cell differentiation (59, 77), and epithelial integrity (93, 188).

In type I pneumocytes, Nox2, p22^phox, p67^phox, p47^phox, and p40^phox subunits, as well as Rac1, are present at substantially higher levels than in type II cells (168) in which ROS production is reportedly derived primarily from mitochondria (143). In type II cells DUOX1 is expressed (there is no significant expression of DUOX2), but the functions of this Nox isoform in alveolar cells remains unknown (58).

Pulmonary vasculature.

In the pulmonary vasculature, Nox isozymes are activated by a wide range of stimuli including G protein-coupled receptor agonists, angiotensin II, thrombin, endothelin, serotonin, thromboxane A₂ (38, 105), cytokines [tumor necrosis factor-α (TNF-α) and transforming growth factor-β (TGF-β)] (166), mechanical forces, and shear stress. Proposed functions of Nox-derived ROS include NF-κβ activation (25), MAPK activation (136), cell proliferation (119), and potassium channel regulation in response to changes in the oxygen concentration (9, 186).

Pulmonary endothelium.

Generally speaking, endothelial cells express Nox1, Nox2, and Nox4 (and Nox5 in human cells) and their cofactors (70, 173). In human pulmonary artery endothelial cells and human lung microvascular endothelial cells, mRNA levels for Nox4 are much higher compared with mRNA for Nox2 (27, 139). Interestingly, in these cells, Nox4 silencing induces Nox2 mRNA upregulation, and in Nox2^−/− mice mRNA Nox4 upregulation was described, suggesting that a compensatory regulation induced by their respective deficiency exists for Nox2 and Nox4 mRNA expression (138). Nox endothelial activity is increased in response to pulmonary ischemia (6) and hyperoxia (136), is associated with activation of ATP-sensitive K⁺ channels, and results in stimulated endothelial cell proliferation and NO production (119, 198). The roles of endothelial Noxs include control of vascular tone, vascular cell growth, angiogenesis, and inflammation. Studies suggest a role for both H₂O₂ (119) and O₂^·− (79) in response to general pulmonary endothelial injury.

PASMCs.

In PASMCs, both Nox1 and Nox4 are expressed, whereas Nox4 seems to be the most abundant isoform (24, 120, 139). Nox4 expression in PASMCs can be induced by several stimuli including hypoxia/ischemia, shear stress, and ER stress (19, 97, 120). Nox4 seems to be required for the expression of smooth muscle differentiation markers and maintenance of smooth muscle actin-based stress fibers as suggested by the correlation between the decrease of Nox4 expression and the loss of smooth muscle differentiation markers, such as smooth α-actin and myosin heavy chain, in multiple passages of freshly isolated PASMCs (37, 166). Both Nox1 and Nox4 induce proliferation of PASMCs (120). In particular, it has been shown, that Nox4 is involved in this process through induction by TGF-β1 (166).

Fibroblasts.

In lung fibroblasts, p47^phox, p67^phox, p22^phox, Nox1, and Nox4 are expressed but not Nox2 (47), and Nox4 expression dominates Nox1. Oxidase-derived H₂O₂ generation is observed in response to TGF-β (170), irradiation with α-particles (127), rhinovirus infection (47), and hypoxia (99). Nox4 is upregulated by hypoxia, inflammatory mediators, and fibrotic stimuli, such as TNF-α and TGF-β, contributing to fibroblast activation and transdifferentiation. The consequences of Nox4-induced ROS by TGF-β in pulmonary fibroblasts include IL-8 upregulation (47) and induction of epithelial cell death through a paracrine mechanism (176). The TGF-β-induced Nox4-derived ROS production in human fibroblasts seems to be extracellular, in contrast to what is observed in human PASMCs in which the activation of Nox4 by TGF-β leads to intracellular ROS production (166, 176). Experiments using siRNA-mediated knockdown of Nox4 demonstrated that Nox4 contributed to the increase in ROS generation under hypoxic conditions, stimulated proliferation, and inhibited apoptosis in pulmonary fibroblasts (99).

ROS-Induced Regulation of Pulmonary Vascular Tone

Nox-derived ROS (both O₂^·− and H₂O₂) play an important role as mediators and signaling molecules capable of activating multiple pathways involved in the control of pulmonary vascular tone, cell proliferation and apoptosis, inflammation, and fibrosis. One of the potential mechanisms by which ROS are able to regulate pulmonary vessel tone is via the control of the cellular redox potential (87). The pulmonary vascular responses to exogenously added ROS are heterogeneous because O₂^·− or H₂O₂ can lead to contractile or relaxant responses. This heterogeneity might be explained by the nature (H₂O₂ or O₂^·−) and dose of ROS applied on the existence of a previous contractile tone and on the nature of the preconstrictor used.

O₂^·− induces constriction of pulmonary artery, triggering Rho-kinase-mediated Ca²⁺ sensitization (92), and is involved in hypoxia-induced vasoconstriction, as suggested by the fact that inhibitors of SOD, such as diethyldithiocarbamate or triethylenetetramine, produce larger potentiation of the hypoxic contraction (2, 101) and that EC-SOD overexpression in the lung ameliorates monocrotaline-induced pulmonary hypertension (PH) in rats (90).

More controversial is the effect of H₂O₂ on the regulation of pulmonary vascular tone. Studies provide evidence that H₂O₂ constricts both isolated perfused rat lung (28) and isolated, perfused rabbit lung (157), and also isolated rat and rabbit pulmonary arteries (86, 132, 137, 145, 160). These findings are substantiated by our finding in systemic vessels (11, 12). The possible mechanisms involved in H₂O₂-induced vasoconstriction are stimulation of serine esterase with subsequent activation of phospholipase A₂ and production of arachidonic acid products (33, 87, 160), activation of PKC, and activation of tyrosine kinases in smooth muscle and endothelium (32, 128, 195, 197, 200). H₂O₂ is also responsible for the elevation of intracellular calcium concentration ([Ca²⁺]_i) in PASMC during hypoxia (150, 183), and multiple similarities have been drawn between H₂O₂-induced vasoconstriction and hypoxic pulmonary vasoconstriction. In contrast, it has been shown that H₂O₂ produces concentration-dependent relaxation of precontracted isolated bovine arteries by a mechanism independent of the endothelium or prostaglandin mediators but related to soluble guanylate cyclase and cGMP (30). A similar H₂O₂-induced vasodilatation is described in isolated perfused rabbit lungs (29). Finally, in arteries stimulated with PGF₂α or phenylephrine, a biphasic response is observed (64, 106, 113). Taken together, the variable effects of H₂O₂ could be tone dependent and related to the dose of H₂O₂ applied; it seems that low doses of H₂O₂ are involved in physiological vasodilatory control of pulmonary tone, whereas high or supraphysiological doses of H₂O₂ are implicated in pathophysiological response leading to vasoconstriction and pulmonary hypertension (87).

In addition, it has been shown that ROS controls vascular tone via the release of or interaction with other vasoactive factors. For example, ROS interacts with cyclooxygenase (COX) (140). The relationship between ROS and COX is complex because COX is a potential source of O₂^·− in the pulmonary circulation (140), and it has been shown that thromboxane A₂ stimulates Nox-derived ROS (38); conversely, ROS can stimulate COX activity. Another way for ROS to control vascular tone is its reaction with NO (the most powerful vasodilator in endothelial cells). O₂^·− can react with NO in a diffusion-limited manner to form peroxynitrite, which could also be converted into nitrogen dioxide and nitrotyrosine. These reactions cause a decrease in NO bioavailability that leads to impaired vascular relaxation, upregulation of proinflammatory and prothrombotic molecules and endothelial dysfunction in pulmonary arteries.

Hypoxic Pulmonary Vasoconstriction

The physiological response to short-term hypoxia is a reversible contraction of the PASMC, a compensatory physiological response that serves to redirect blood to better ventilated areas of the lung and preserve proper ventilation of the systemic circulation (54). The response of the pulmonary circulation to a decrease in partial pressure of oxygen is discordant with the response of the systemic circulation in which arterial smooth muscle cells (SMCs) relax in response to hypoxia maintaining a fairly constant blood flow to important organs and tissues. This suggests that oxygen-sensing mechanisms in vascular smooth muscle in each circulatory system are different. Despite many recent studies, the mechanism involved in hypoxic pulmonary vasoconstriction (HPV) is, to date, not fully understood. The redox theory of HPV involves a redox-based O₂ sensor regulating the activity of effector proteins, and there is strong evidence indicating a role of ROS as signaling intermediates in this response (8, 118). The redox theory, initially proposed by Weir and colleagues (118), describe a hypoxia-induced decrease in ROS production leading to potassium channel inhibition, membrane depolarization, and cell contraction. Hypoxia has also been reported to decrease intracellular concentration of ROS ([ROS]_i) in isolated rat lungs and PASMCs (9, 116, 117), as well as in microsome-enriched fractions of calf pulmonary arteries (121, 124). Michelakis et al. (116) has shown that hypoxia decreases H₂O₂ in rat PASMCs but not in renal (systemic) artery SMCs. In addition, hypoxia has been found to decrease H₂O₂ levels in both cultured human pulmonary and coronary (systemic) artery SMCs (114). Conversely, Waypa et al. (182) showed that hypoxia produces an increase in ROS production in cultured rat pulmonary and renal artery SMCs, and other studies (149, 150) indicate that hypoxia causes a large increase in ROS and Nox activity in freshly isolated mouse PASMCs but not in mesenteric SMCs. Although still a matter of debate, the concept that a paradoxical increase in ROS arises during hypoxia is gaining traction (179). Many studies have revealed that hypoxia increases intracellular ROS concentrations ([ROS]_i) in isolated rabbit and goat lungs, rat and dog pulmonary arteries, and cultured calf, dog, and rat PASMCs (26, 62, 84, 102, 112, 134, 180–182). In accordance with these findings, studies using a novel, redox-sensitive, ratiometric fluorescent protein sensor (RoGFP) (181, 182) and a developed, genetically encoded, specific ROS biosensor HyPer (22) reveal that hypoxia augments ROS signals in PASMCs. The explanation for these potentially odd results could be the use of different experimental conditions, but also recent studies suggest that these contrary hypotheses may hold true. That is, hypoxia causes a decrease in ROS generation in the mitochondrial matrix compartment, whereas it increases ROS production in the mitochondrial intermembrane space, which diffuses to the cytosol (182). This can lead to the activation of Nox proteins and a further increase in ROS levels (150).

One of the mechanisms by which hypoxia-derived ROS may lead to pulmonary artery vasoconstriction involves inhibition of K_V channels (principally K_v 1.5 and K_v 2.1), depolarization, activation of Ca²⁺ voltage-dependent L-type channels, and an increase [Ca²⁺]_i in PASMCs (8). In support of this notion, many research groups have reported that exogenous H₂O₂, similar to hypoxia, inhibits K_V (38), induces an increase in [Ca²⁺]_i in PASMCs (183) (100), and vasoconstriction in pulmonary arteries (28, 86, 151, 157, 160, 189, 194).

Role of Nox-derived ROS in HPV.

Many studies show that HPV is biphasic with an acute phase occurring in seconds/minutes and a sustained phase developing over several minutes to hours. Nox and mitochondria are the major sources of intracellular ROS generation mediating hypoxic responses in PASMCs (185, 191), and they are interdependent at least in acute HPV. The first evidence of a role for Nox in HPV came from experiments in which iodonium compounds or apocynin (both nonspecific Nox inhibitors) attenuates the hypoxic increase in [ROS]_i (112, 123), inhibits K_V currents (39, 184), increases [Ca²⁺]_i, and induces contraction of PASMCs (112) and HPV in isolated lungs and pulmonary arteries (63, 73, 122, 123, 185, 186). Experiments using other Nox inhibitors, such as 4-(2-aminoethyl)bensenesulfonyl fluoride in isolated rabbit lung (186) and cadmium sulfate in isolated rat pulmonary artery further support the role of Nox in HPV. Although, Nox2^−/− mice show normal or reduced hypoxic responses (10, 103); in p47^phox−/− mice, acute but not sustained HPV is inhibited (185), suggesting that a Nox isoform other than Nox2 using p47^phox contributes to the acute phase of HPV. Recently, a role for Nox4 in HPV has been shown by Ahmad et al. (3). They showed that Nox4-derived H₂O₂ maintains a basal relaxation under normoxic condition, which is removed by hypoxia leading to acute HPV. Increasing Nox4 expression with TGF-β was also observed to enhance HPV (192). Wolin et al. (75, 192) suggest that the mechanism of HPV in isolated bovine pulmonary arteries originates from the ability of pulmonary arteries to maintain increased smooth muscle levels of cytosolic NADPH, which sustain Nox4-derived H₂O₂ production. Moreover, Diebold et al. (48) showed that hypoxia induces the upregulation of Nox4 mRNA though hypoxia-inducible factor-1α (HIF-1α), which stimulates the synthesis of matrix metalloproteinases (MMPs), activation of MAPKs, transactivation of growth factor receptors, and signaling molecules and transcription factors involved in the proliferation of PASMCs and angiogenesis (109). In human lung adenocarcinoma A549 cells, an upregulation of Nox1 mRNA and protein occurred during hypoxia, accompanied by the activation of HIF-1-dependent target gene expression (heme oxygenase-1 mRNA, hypoxia-responsive element reporter gene activity), followed by enhanced ROS generation. HIF-1 induction is inhibited by diphenylene iodonium (DPI) and catalase, suggesting that hypoxic upregulation of Nox1 and subsequently augmented ROS generation may activate HIF-1-dependent pathways (67). Rathore et al. (150) recently revealed that hypoxia markedly increases Nox activity in PASMCs through activation of protein kinase C ε (PKCε) (150). Previous findings from the same group showed that the hypoxic activation of PKCε is secondary to increasing mitochondrial ROS (149), suggesting that hypoxia may enhance mitochondrial ROS generation, which activates PKCε and then augments Nox activity to cause further generation of intracellular ROS. Furthermore, several inhibitors of PKC isoforms inhibit HPV (17), and studies show the role for PKCζ in the activation of Nox induced by hypoxia (62). ROS also can activate a number of PKCs, and PKCα is involved in H₂O₂-induced contraction in pulmonary arteries (145). Whether various PKCs are simultaneously required remains unknown.

Role of Nox-derived ROS in pulmonary vascular remodeling.

Chronic hypoxia induces irreversible change in vascular remodeling characterized by medial and adventitial thickening of the muscular and elastic vessels and muscularization of previously nonmuscular more distal small vessels. This leads to a thickening of the vessel wall and reduction of the lumen area, increased vascular resistance, and thus development of pulmonary hypertension and right ventricular hypertrophy. Distinct segments of the pulmonary artery appear to contribute to this process. Endothelial cells from the intima exhibit atypical proliferation and participate in the formation of plexiform lesions. Nox2, Nox4, and p22^phox are pivotal for proliferation of pulmonary endothelial cells (18, 142); SMCs from the medial layer exhibit hyperplasia and enhanced contractility (50). Both Nox1 and Nox4 are involved in the proliferation of SMCs (115, 120, 166). In particular, Nox4 is known to be induced by TGF-β and has been related to the TGF-β-dependent proliferation of SMCs in the pulmonary artery (82). In contrast to the mechanisms of TGF-β1 signaling observed in normoxic human PASMCs (166), the hypoxic release of TGF-β1 increases insulin-like growth factor binding protein (IGFBP-3) expression through phosphatidylinositol 3-kinase (PI3K) signaling with subsequent serine/threonine kinase (Akt) phosphorylation and further Nox4 activation. Moreover, IGFBP-3 increases Nox4 gene expression, resulting in PASMC proliferation (82).

The adventitial layer is thickened via hypoxia-induced proliferation of adventitial fibroblasts (153, 164). Furthermore, transdifferentiation of adventitial fibroblasts into myofibroblasts or SMCs appears to be critically involved in this process (162). Several studies demonstrate a role of Nox4 in the pathogenesis of hypoxia-induced pulmonary arterial remodeling and pulmonary hypertension, as well as idiopathic pulmonary arterial hypertension (IPAH) (120). In adventitial fibroblasts, a higher level of Nox4 mRNA expression than Nox1 is observed under normal conditions and this expression is significantly upregulated under hypoxic conditions (99). Consistent with this, a significant increase of Nox4 mRNA expression was observed under hypoxic conditions in adventitial fibroblasts from the lungs of patients with IPAH compared with healthy donors (99). Silencing of Nox4 causes reduction of H₂O₂ levels under normoxic and hypoxic conditions, suppression of the hypoxia-induced ROS increase, a decrease in adventitial fibroblast proliferation, and enhanced apoptosis (99).

Pulmonary Arterial Hypertension

Pulmonary arterial hypertension (PAH) is characterized by vascular remodeling and enhanced constrictor vasoreactivity. In PAH, there is a progressive decrease in arterial lumen and an increase of pulmonary arterial pressure that ultimately leads to right ventricular failure and death (148). Three mechanisms principally contribute to the decrease of the arterial lumen: the increase in contractility of the pulmonary resistance arteries, the proliferation and remodeling of pulmonary endothelial SMCs and fibroblasts, and the thrombosis. Accumulating evidence indicates that ROS contributes to all three mechanisms mentioned above, which are involved in the pathogenesis of PAH. Liu et al. (103) show that a disruption of the murine Nox2 gene completely prevents chronic hypoxia-induced PAH and vascular remodeling. In addition to studies supporting a role for Nox2 in PAH in response to hypoxia (103), it has been demonstrated that PAH is also characterized by the induction of Nox4, perhaps in response to an initial activation of Nox2 within the pulmonary endothelium, which is responsible for increased ROS generation and SMC proliferation (120). Hypoxia-dependent development of PAH in mice is linked to increased Nox4 expression in PASMCs (120), suggesting a role for Nox4 in the vascular remodeling associated with hypoxia-induced PAH. As mentioned above, hypoxia increases the expression of TGF-β (85) and Nox4 expression (166); Nox4 has also been shown to be critical for hypoxia-inducible factor 2α (HIF-2α) expression and transcriptional activation in renal carcinoma cells (111). This suggests that in hypoxic vascular remodeling, a mechanism involving HIF-2α, TGF-β, and Nox4 is operant.

One of the early events contributing to the pathophysiology of all forms of PAH is endothelial dysfunction (148). A role for Nox2 in hypoxia-induced endothelial dysfunction involving intrapulmonary arteries has been established. In addition, ROS generation from Nox-independent sources, xanthine oxidase (83), or uncoupled endothelial NO synthase (eNOS) (94) may also contribute to hypoxia-induced vascular dysfunction and PAH.

Several strategies designed to diminish ROS levels, through scavenging ROS or through the inhibition of the sources of ROS, have been proposed as potential therapeutic treatments for PAH. For example, treatment with recombinant human SOD is able to enhance ROS clearance, which leads to enhanced eNOS expression and function in animal models (neonatal lambs) of persistent pulmonary hypertension of the newborn (PPHN) (55). Interestingly, Kamezaki et al. (90) recently reported that gene transfer of extracellular SOD (EC-SOD) protects rats against monocrotaline-induced PH, suggesting that extracellular O₂^·− is important in the disease process. Manipulation of EC-SOD in models of vascular and lung diseases confirm a role for extracellular O₂^·− in the pathogenesis of a number of disease models associated with fibrosis and tissue remodeling (13, 23, 56, 60, 147). Using wild-type (WT) and transgenic mice overexpressing human EC-SOD in the lung, Nozik-Grayck et al. (131) show a protective effect of lung EC-SOD overexpression on chronic hypoxic-induced PAH and vascular remodeling. This protective effect is suggested to involve the suppression of redox-sensitive transcription factor early growth response-1 (Egr-1) pathway (131). In addition, catalase and SOD mimetics have proven beneficial in PH (46). Moreover, inhibition of xanthine oxidase (83) or eNOS (193) has shown beneficial effects in rat neonates with PH and in caveolin-1 knockout mice with PH, respectively. Also, soluble guanylate cyclase activators have yielded beneficial effects in preclinical studies in different animal models of PAH (163). Finally, in the clinic, use of phosphodiesterase-5 inhibitors has also been reported to reduce ROS levels (80).

Conclusion

For nearly two decades, Nox-derived ROS have been described as critical mediators of systemic vascular hypertension. While ROS vasoconstrictor and vasodilator effects in pulmonary vessels have been studied for at least as long, interest in the role of Nox in the complex orchestration of events leading to pulmonary hypertension has burgeoned in recent years. Nox isoforms appear to subserve a pivotal role in lung signaling, leading to marked changes in pulmonary airway, pneumocyte, and vascular cell phenotypes, including proliferation, hypertrophy, and apoptosis, resulting in acute lung injury, emphysema, and pulmonary hypertension among other pathologies. Evidence has emerged for a highly influential role of Nox in pulmonary hypertension; however, much of these data are either associative or are gleaned from in vitro studies. Studies designed to modulate Nox isoforms in a pulmonary vascular cell-specific manner will be needed to draw more definitive conclusions. Even less well studied is the direct or indirect contribution Nox isoforms play in right ventricular failure, the major cause of death in patients with PH. As past drug therapy has been focused solely on various pulmonary vascular targets and has largely been disappointing, emphasis should be placed on targeting individual or combinations of Nox in both the pulmonary vasculature and the right ventricle. For this reason, major priority is now being given to the discovery of specific Nox small molecule and peptidic inhibitors and therapies targeting these drugs to the pulmonary vasculature and right ventricle.

GRANTS

This work was support by National Heart, Lung, and Blood Institute (NHLBI) Grant R01-HL-079207 and by the Ri.MED Foundation. H. C. Champion is supported by NHLBI Grants PO1-HL-103455 and UO1-HL-108642-01 and grants from the Vascular Medicine Institute, the Institute for Transfusion Medicine, and the Hemophilia Center of Western Pennsylvania.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

Author contributions: G.F. drafted manuscript; H.C.C. and P.J.P. edited and revised manuscript; P.J.P. approved final version of manuscript.