Thiol-Redox Regulation in Lung Development and Vascular Remodeling

Abstract

Significance:

Redox homeostasis is finely tuned and governed by distinct intracellular mechanisms. The dysregulation of this either by external or internal events is a fundamental pathophysiologic base for many pulmonary diseases.

Recent Advances:

Based on recent discoveries, it is increasingly clear that cellular redox state and oxidation of signaling molecules are critical modulators of lung disease and represent a final common pathway that leads to poor respiratory outcomes.

Critical Issues:

Based on the wide variety of stimuli that alter specific redox signaling pathways, improved understanding of the disease and patient-specific alterations are needed for the development of therapeutic targets.

Further Directions:

For the full comprehension of redox signaling in pulmonary disease, it is essential to recognize the role of reactive oxygen intermediates in modulating biological responses. This review summarizes current knowledge of redox signaling in pulmonary development and pulmonary vascular disease.

Introduction

The evolutional transition from anaerobic to complex multicellular aerobic organisms required the ability to metabolize oxygen. Adenosine-5-triphosphate production via oxidative phosphorylation resulted in exposure of DNA, lipids, and proteins to reactive oxygen intermediates (ROIs), which include reactive oxygen and nitrogen species. Consequently, organisms developed a complex system of antioxidant defenses to bring about cellular homeostasis (Fig. 1).

FIG. 1.

Thiol regulation of alveolar development and vascular remodeling. Environmental and external factors stimulate the production of ROIs. ROIs activate several metabolic pathways that ultimately affect alveolar development and vascular remodeling. Homeostasis is mediated by transcription factors and post-translational modifications that regulate the final enzymatic responders. HIF, hypoxia-inducible factor; NFκB, nuclear factor κB; Nrf2, nuclear factor erythroid-derived 2-like 2; ROIs, reactive oxygen intermediates.

Oxidative stress (OS) refers to the perturbation of the balance between oxidants and antioxidants and is the pathophysiologic basis of many disease processes (47, 65, 133, 135, 170). Central to the concept of OS is an appreciation of the factors that modulate antioxidant defenses and the events that overwhelm these systems. Although ROIs are often viewed as harmful byproducts of aerobic metabolism, data support their important roles as secondary messengers in many intracellular signaling pathways (58, 143). Therapeutically, utilization of compounds intended to restore balance between oxidants and antioxidants has been tested with variable degrees of success [reviewed in Berkelhamer and Farrow (18)]. Recent approaches to prevent or treat disease have instead targeted master regulators of endogenous antioxidant responses (66, 190).

Lung development is guided by complex signaling pathways that regulate alveolar and vascular development to prepare the lung for executing gas exchange after birth. This fetal-to-neonatal transition offers a unique opportunity to understand the molecular mechanisms that govern redox balance. In this review, we examine the impact of OS on alveolar and pulmonary vascular development, review key pathways that regulate homeostasis and adaptation to environmental challenges with particular focus on the perinatal period, and consider emerging therapeutic approaches to ameliorate pulmonary vascular disease, including potential limitations of their implementation.

OS Defined

ROIs are byproducts of aerobic oxygen metabolism. Enhanced formation, especially in the setting of limited antioxidant defenses, results in damage to proteins, enzymes, lipids, and DNA (57). ROIs include superoxide (O₂ ^•−), hydrogen peroxide (H₂O₂), hydroxyl radical (^•OH), peroxynitrite (ONOO⁻), nitric oxide (NO), and nitrogen dioxide (NO₂). The chemical properties of each ROI directly correspond with reactivity and biological targets.

ROIs are predominantly produced by NADPH oxidases (NOXs) and mitochondria (143). Complexes I, II, and III generate O₂ ^•− as a byproduct of oxygen metabolism. Enzymes known as superoxide dismutases (SODs) reduce O₂ ^•− to H₂O₂. H₂O₂ regulates cellular proliferation and differentiation [see review Marinho et al. (108)]. Endoplasmic reticulum-derived ROIs contribute to pathology in the setting of mitochondrial dysfunction (99, 123).

Mitochondrial DNA is particularly susceptible to damage from ROIs. As a consequence, enzymatic function is negatively impacted, further amplifying mitochondrial ROI production (21). In turn, this leads to deleterious cellular consequences, including apoptosis through the cytochrome C pathway (67). Importantly, these effects are also dependent on the developmental and maturational stage of the redox system. Available evidence from extensive studies (See section Effects of altered oxygen tension on alveolar and vascular development) comparing baseline enzymatic capacity and their ability to response to external stimuli in preterm versus term lungs highlights these differences (6, 8, 10).

Effects of altered oxygen tension on alveolar and vascular development

There are few events in life that represent such drastic changes in oxygen exposure at the time of birth. Upon delivery, newborns experience a dramatic increase in oxygen exposure. In utero, the partial pressure of oxygen pressure is ∼20–25 mmHg. The extrauterine environment, in contrast, is ∼100 mmHg. This abrupt change means that infants are highly susceptible to ROI-mediated damage.

In premature infants, this scenario is further amplified. The immature lung of preterm infants is uniquely susceptible to oxidant injury. Both baseline antioxidant capacity and ability to upregulate endogenous defenses in response to OS are compromised in the developing lung (10, 60, 159). ROI levels are increased in prematurely born infants as reflected by elevations in plasma F2-isoprostane (38), cord blood 8-isoprostane (115), and plasma lipid peroxidation products (130). Compared with full-term infants, preterm infants have higher circulating levels of plasma unbound iron (30), which can further enhance oxidative damage through the Fenton reaction.

Premature infants often require high levels of supplemental oxygen to compensate for inefficient pulmonary gas exchange. They also experience frequent episodes of hypoxemia that also results in generation of ROIs (78). Animal studies elegantly demonstrate the importance of fetal hypoxia for lung branching morphogenesis, angiogenesis, and extracellular matrix deposition during the pseudoglandular and canalicular stages of lung development (64). In contrast, hyperoxic exposure during lung development leads to alveolar simplification and decreased gas exchange in animal models (19, 87). Hyperoxia inhibits endothelial proliferation by inactivation of fibroblast growth factors and vascular endothelial growth factor (VEGF) (68). These impairments contribute to the development of bronchopulmonary dysplasia (BPD), a leading cause of mortality and long-term morbidity in prematurity.

Oxygen tension influences organogenesis through the modulation of angiogenesis. Approximately 25% of extremely premature infants (<28 weeks' gestation) develop pulmonary hypertension (PH). Impaired pulmonary angiogenesis is a key factor in the development of PH in patients with BPD.

Members of the hypoxia-inducible factor (HIF) family of transcription factors are tightly regulated by O₂ tension. Disruption of HIF-mediated angiogenesis during lung development contributes to the pathophysiology of BPD. Under conditions of low O₂ tension, HIF is stabilized permitting translocation into the nucleus and binding to hypoxia-responsive elements in the promoter region of target genes (73, 169). The transition of the pulmonary circulation from the hypoxic fetal environment to the hyperoxic postnatal environment results in rapid molecular changes mediated via O₂-sensitive HIF signaling pathways. HIF-regulated proteins mediate the initial pulmonary adaptation and subsequent lung maturation by regulating the expression of VEGF. Redox epigenetic modifications also regulate HIF activity. S-glutathionylation, for example, regulates HIF stability during hypoxia, promoting revascularization after ischemia (175).

A key feature of BPD and associated PH is the vascular remodeling that occurs over time. Increased pulmonary arterial muscularization contributes to the pathogenesis of PH by enhancing vasoreactivity (51). Narrowing and stiffening of both the proximal and distal pulmonary arterioles reduce the size of the vessel lumen and decreases compliance resulting in increased right ventricular afterload (174). ROIs, specifically O₂ ^•− and H₂O₂, stimulate growth of fetal pulmonary artery smooth muscle cells (PASMCs) (178). This process is further exacerbated by ROIs generated in response to growth factors mitogenic for vascular smooth muscle proliferation (20). Conversely, antioxidants such as SOD and catalase attenuate cell proliferation and, at high doses, induce PASMC apoptosis (177).

Effects of inflammation on alveolar and vascular development and function

Inflammation and OS are closely related pathophysiological events that are tightly linked with one another (Fig. 2). Inflammatory cells produce ROIs at the site of inflammation leading to exaggerated OS. A variety of ROIs can initiate intracellular signaling pathways that result in enhanced proinflammatory gene expression. Hyperoxia alone induces proinflammatory cytokine expression leading to pulmonary endothelial cell (EC) and epithelial cell death (88). Levels of several proinflammatory cytokines have been detected in tracheal aspirates obtained from infants with BPD. Cytokine concentrations are increased as a function of duration of assisted mechanical ventilation and level of oxygen supplementation (24). Therapeutic anti-inflammatory interventions have yielded mixed results (167). Conversely, enhanced expression of antiapoptotic factors attenuates hyperoxic lung injury (176).

FIG. 2.

ROIs vicious cycle of inflammation. ROIs regulate the inflammatory cells influx after exposure to exogenous stimulus such as hyperoxia, volutrauma, or infection resulting in an additional release/production of proinflammatory cytokines that generates more ROIs. This provokes DNA damage, lipid peroxidation, and protein oxidation and ultimately destruction of the alveolar–capillary barrier, vascular leak, influx of inflammatory mediators, pulmonary edema, and cell death. ET-1, Endothelin-1; H₂O₂, hydrogen peroxide; IL, interleukin; O₂ ^•−, superoxide; TNFα, tumor necrosis factor alpha.

Damaged lung tissue releases chemotactic factors and inflammatory cytokines including interleukin (IL)-1, IL-8 (CXCL-8), and tumor necrosis factor alpha (TNF-α). These factors lead to an influx of neutrophils and other inflammatory cells resulting in an additional release/production of proinflammatory cytokines. Endothelin-1 (ET-1) is proinflammatory and is a potent vasoconstrictor expressed in high concentrations in the lung. Tracheal aspirates from infants that develop BPD show levels of ET-1, IL-6, and IL-8 that gradually increase from days of life 1 through 7. Leukocyte counts were also higher in tracheal aspirates from infants with BPD (128).

Hyperoxia-induced acute lung injury occurs secondary to inflammatory responses, leading to destruction of the alveolar–capillary barrier, vascular leak, influx of inflammatory mediators, pulmonary edema, and cell death. At the cellular level, both alveolar and interstitial macrophages express early response cytokines when exposed to hyperoxia, which, in turn, attract inflammatory cells to the lungs (25). The specific contribution of individual inflammatory mediators in the pathogenesis of BPD has been defined by utilizing lung-targeted overexpressing transgenic models that develop pulmonary phenotypes similar to infants with BPD despite lung development occurring at ambient oxygen tension (11, 28).

Epigenetic effects of mechanical ventilation, oxygen toxicity, and inflammation on alveolar and vascular responses

The term “epigenetics” refers to heritable changes in gene expression not involving changes to the underlying DNA sequence. Epigenetics modulates the regulation of protein expression by switching genes on or off and can be inherited stably during cell differentiation and development (2) The three most studied epigenetic mechanisms are DNA methylation, noncoding RNA, and histone modifications (95, 124, 153).

Hyperoxia-related alterations in cell signaling and gene expression have been thoroughly reviewed (182, 191). Hyperoxia has the potential to alter the gene activity in lung cells by inducing DNA modifications. ROIs modify cytosine with the oxidative conversion of 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine. Peroxides can also modify cytosine to 5-chlorocytosine, which mimics 5-mC (124). These changes can induce DNA methylation by inhibiting the binding of DNA methyltransferase 1 (Dnmt1) to DNA (107). Subsequent alterations in methylation patterns of CpG sequences can result in gene silencing (40).

MicroRNAs (miRs) are a class of noncoding RNAs involved in the regulation of gene expression. miRs play important roles in alveolar and endothelial development. For example, data obtained from 50 preterm infants identified 4 miRs (miR-133b, miR-7, miR-152, and miR-30a-3p) as significantly associated with the development of BPD (184). miR-206 expression is lower in lungs from a mouse model of BPD, whereas blood miR-206 levels are lower in a cohort of BPD patients than in non-BPD controls. Overexpression of miR-206 in lung cells resulted in increased apoptosis, and decreased proliferation, migration, and adhesion (194). Individual members of the miR-17 ∼ 92 cluster regulate many molecular pathways implicated in BPD development (116), including HIF (157), matrix metalloproteinases, collagens, and p53 (23). Expression of this cluster is decreased in experimental and human BPD and expression is inversely correlated with Dnmt expression and promoter methylation (140).

The noxious effects of ROIs condition the developing lung toward aberrant pulmonary responses. Additional negative effects are seen on cardiopulmonary function across the lifespan and are driven by altered pulmonary immune and OS responses (189). These effects appear to be mediated, at least, in part, by permanent changes in histone deacetylase activity (104).

Antioxidant Systems: Effects on Alveolar and Vascular Signaling and Implications for Lung Diseases

A complex system of enzymatic and nonenzymatic antioxidant systems modulates oxidant balance within the lung. Key antioxidant defenses include the SODs; the glutathione (GSH) and thioredoxin (Trx) systems with their respective peroxidases and reductases; hemeoxygenases, catalases, and small molecular weight antioxidants including vitamin C and E (170). Antioxidant defenses are themselves regulated by the presence of ROIs, although the ability to respond to OS is influenced by species, gender, and age (55, 93).

The GSH redox system

The tripeptide glutathione (l-γ-glutamyl-l-cysteinyl-glycine, GSH) is present in the millimolar range in nearly all mammalian cells, making it the most abundant intracellular antioxidant. The GSH redox system is crucial in maintaining intracellular homeostasis with subsequent effects on normal cellular physiological processes.

The GSH system is one of the most critical antioxidant defense systems in the lung (113). Almost 90% of cellular GSH is cytosolic with 10% in the mitochondria and a small percentage in the endoplasmic reticulum (81, 114). GSH scavenges electrophilic and oxidizing compounds either directly or via reactions catalyzed by glutathione-S-transferases (GSTs). GSH detoxifies H₂O₂ and lipid peroxides in reactions involving glutathione peroxidases. This reaction generates GSH disulfide (GSSG) which is subsequently reduced to GSH in an nicotinamide adenine dinucleotide phosphate (NADPH)-dependent reaction catalyzed by GSH reductase (Fig. 3). GSH is also utilized by glutaredoxins (Grxs) by serving as disulfide reductases and de-glutathionylating enzymes. GSH can also undergo thiol–disulfide exchange reactions (protein − SSG + GSH → protein − SH + GSSG).

FIG. 3.

Glutathione redox cycle. GSH is synthesized from the amino acids l-glutamate, l-cysteine, and glycine in a two-step pathway requiring energy from ATP. Glu and Cys are combined via the action of GCL. This dipeptide then combines with Gly via a reaction from GS. GSH undergoes a redox reaction using glutathione peroxidase GPx to detoxify ROIs such as H₂O₂. GSH is converted to an oxidized form (GSSG) and is recycled back to GSH by the enzymatic reaction of GR, which requires the cofactor NADPH to form a redox cycle. ATP, adenosine-5-triphosphate; Cys, cysteine; GCL, glutamate cysteine ligase; GR, glutathione reductase; GS, glutathione synthetase; GSH, glutathione; NADPH, nicotinamide adenine dinucleotide phosphate.

Maintenance of a high intracellular GSH/GSSG ratio (>90%) minimizes accumulation of disulfides and promotes an intracellular reducing environment. When oxidants or other environmental stressors alter the GSH/GSSG ratio, this shift influences a variety of cellular signaling processes including transcription factor and cellular activation (4, 79). The oxidize balance of GSH also appears to depend on the subcellular compartment. For example, the GSH/GSSG ratio is lower in the endoplasmic reticulum (81).

Regulation and antioxidant functions

GSH exerts a key role in regulating OS in the pathogenesis of respiratory diseases. Depletion of blood GSH has been documented in premature infants after a few days of life (85), and blood GSH correlates with gestational age (97). The major determinants of de novo GSH synthesis are the availability of the sulfur amino acid precursor cysteine (Cys) and glutamate cysteine ligase (GCL) activity, the rate limiting step in de novo GSH synthesis. Cys is oxidized to cystine, which is readily transported into mammalian cells as a naturally occurring analogue of Cys (110). Inside cells, cystine is reduced back to Cys.

Early in life, Cys may be an essential amino acid, as the transsulfuration pathway producing Cys from methionine and serine via homocysteine is not fully active. Consistent with this likelihood is the fact that cystathionine-γ-lyase activity is barely detectable in human fetal tissues (139, 154, 172). Provision of Cys stimulates de novo GSH synthesis and restores GSH stores in pathologic situations (12, 183). Preterm infants appear to have a proportionally maturational capacity for intrahepatic de novo Cys synthesis (147). Studies comparing [GSH] and [Cys] in umbilical cord blood from very low birth weight infants (gestational age <32 weeks and/or birth weight <1500 g) with those of healthy full-term newborns demonstrated that GSH depletion is present at the time of birth in premature infants (97). GSH depletion does not appear to be a result of defective synthesis as GSH synthetic rates in isolated erythrocytes are indistinguishable between preterm and term infants. The depletion is likely driven by a dramatic reduction in Cys delivery to the preterm fetus after birth. This premise is supported by evidence of a 40% reduction in umbilical cord vein [Cys] in preterm infants compared with that in term infants and a 43% reduction in maternal blood [Cys] in mothers delivering preterm babies.

Collectively, low GSH in premature neonates may result from the combination of increased fetal GSH utilization and insufficient maternal–fetal Cys delivery. Unfortunately, Cys supplementation alone to preterm infants after birth neither increases [GSH] nor enhances GSH synthesis (162). Finally, other sources and potential therapeutic targets for GSH synthesis are the amino acid glutamine, by conversion to glutamate (142) and serine, which affects the biosynthesis of selenium (Se) and, consequently, selenoprotein activity and expression (173).

OS induces de novo GSH synthesis via activation of specific transcription factors. These factors include nuclear factor erythroid-derived 2-like 2 (Nrf2) via the antioxidant response element (ARE), activator protein-1, and nuclear factor κB (NFκB). Dysregulation of GSH synthesis is increasingly recognized as a key factor in the pathogenesis of lung disease.

Therapeutic augmentation to prevent/treat lung and vascular diseases

Given the importance of the GSH system in lung diseases, various approaches have been attempted to modify its regulation. N-acetylcysteine (NAC) can act as a thiol donor and a Cys precursor. In contrast to GSH, NAC does not have to be broken down or transported intact when given orally. The efficacy of oral NAC has been examined in individuals with chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF), and fibrosing alveolitis with varying degrees of success (16, 134, 156). In premature infants, a multicenter double-blind placebo-controlled trial of intravenously administered NAC did not decrease the incidence or severity of BPD or result in improved lung function (1, 141).

These therapeutic challenges are likely driven by poor bioavailability and the transient nature of the increases in Cys or GSH. A systematic review examined six trials to determine the effects of supplementing parenteral nutrition with Cys, cystine, or precursor NAC on neonatal growth and short- and long-term outcomes (152). Growth was not significantly impacted by Cys (one trial) or NAC supplementation (one trial). Plasma Cys levels were significantly increased by Cys but not NAC supplementation. There were no effects on death by 36 weeks' postmenstrual age, BPD, composite death or BPD, retinopathy of prematurity (ROP), severe ROP, necrotizing enterocolitis requiring surgery, periventricular leukomalacia, intraventricular hemorrhage (IVH), or severe IVH.

The role of NAC in the management of other disease processes has been studied in depth. For instance, ROIs are an important pathogenetic factor in the lungs of patients with COPD (137). Administration of NAC together with cigarette smoke prevented the loss of pulmonary GSH and abolished the effects of cigarette smoke in a murine model of COPD (39). Results from clinical trials are conflicting with unresolved questions related to timing and patient selection for therapeutic administration (22, 46). A similar scenario has also evolved for the use of NAC in treatment of IPF. Although clinical trials did not show an overall benefit on pulmonary function, a subgroup of patients with specific genotypes did benefit (131). These studies indicate that perhaps antioxidant therapies should be studied in the context of personalized medicine.

The Trx system

The Trx system is a key player in redox homoeostasis in mammals. The Trx system is composed of Trx reductase (TXNRD) and Trx. Mammalian TXNRDs are NADPH-dependent FAD-containing enzymes (70, 168) belonging to a family of pyridine nucleotide-disulfide oxidoreductases, which also includes lipoamide dehydrogenase, NADH peroxidase, GSH reductase, and several other enzymes. Mammalian TXNRDs are characterized by the presence of the C-terminal redox-active motif Gly-Cys-Sec-Gly, in which selenocysteine (Sec) is essential for optimal catalytic function (195). Both Trx and TXNRD are present in mammals in distinct isoforms and are either predominantly cytosolic (Trx1 and TXNRD1) or mitochondrial (Trx2 and TXNRD2) (80, 100). Trx1, TXNRD1, Trx2, and TXNRD2 are essential for development since deletion leads to embryonic lethality (109).

Trx acts as a direct antioxidant via reduction of H₂O₂ by the Trx peroxidases, also called peroxiredoxins (126). Several proteins contain –SH groups that are susceptible to oxidative inactivation, typically through disulfide bond formation. Trx(SH)₂ regulates protein activity via thiol–disulfide exchange reactions with protein mixed disulfides (PSSX) that, coupled with reduction by TXNRD, results in net reduction of PSSX by NADPH (Fig. 4).

FIG. 4.

The Trx system. Trx is maintained in a reduced state by the enzyme TXNRD. TrxR reduces the active site of oxidized Trx (TrxS₂) from a disulfide to a dithiol [Trx(SH)₂] with NADPH as a cofactor. Trx intervenes in reduction of disulfide in proteins and as a direct antioxidant via reduction of H₂O₂ by Prx. Prx, peroxiredoxins; Trx, thioredoxin; TXNRD, Trx reductase.

Regulation and direct antioxidant functions

Trx activity depends on the redox state of its active site. If the Trx-active site is inactivated by oxidation or by endogenous inhibitors, then catalytic activity is impaired. Hyperoxia potently upregulates Trx system expression. Greater than 95% hyperoxia enhances cellular ROI generation, increases apoptosis, induces Trx1 and TXNRD1 expression, but decreases TXNRD1 activity (59). In ECs, Trx1 regulates a broad array of cellular functions that are important for angiogenesis. These functions include regulation of transcriptional activation (NFκB, HIF-1α, and Ref-1), post-translational protein modifications (PSSX and PSNO), cellular migration and proliferation (NO), apoptosis (ASK-1), and intracellular signaling (20). Trx1 also exhibits classical antioxidant properties in ECs by protecting against H₂O₂-mediated toxicity (53, 181).

The airways and alveoli are covered with a thin layer of epithelial lining fluid (ELF). The ELF provides the first line of defense from the environment and the composition of the ELF is most reliably detected by bronchoalveolar lavage. Approximately 10% of the total proteome found in bronchoalveolar lavage contains antioxidant proteins including Trx1 (32). The precise role of Trx1 in the ELF in lung diseases is unclear.

Therapeutic augmentation to prevent/treat lung and vascular diseases

Several studies have linked impairment of the Trx1 system with hyperoxic injury and altered lung development (165). In alveolar epithelial cells, overexpression of human TRX-1 significantly decreased hyperoxia-induced alveolar damage (185) In lungs from hyperoxia-exposed newborn mice, total Trx1 and TXNRD1 protein levels are not different over the first 7 days of life (164); however, Trx1 is persistently oxidized. It is unclear whether hyperoxia induces a change in Trx1 subcellular localization. Preterm baboons that were either continuously exposed to 100% O₂ and mechanical ventilation or received O₂ as needed to normalize arterial oxygenation for 6 or 10 days had increase in lung Trx1 and TrxR1 gene expression (45). The importance of Trx in lung diseases was tested in newborn mouse models targeting TXNRD, which catalyzes the reduction of oxidized thioredoxin-1 (Trx1) (192). Paradoxically, TXNRD inhibition attenuates lung injury in newborn and adult murine models of acute respiratory distress syndrome and BPD in an Nrf2-dependent manner (Fig. 5) (26, 52, 102).

FIG. 5.

Regulation of the Trx system and effects on hyperoxic lung injury. Lung histology at 14 days of life of newborn C3H/HeN mice treated with a single intraperitoneal dose of 25 mg/kg ATG, a TXNRD inhibitor, or saline and exposed to room air or hyperoxia (85% O₂) for 14 days. Scale bars: 100 μm. Reprinted with permission of the American Thoracic Society. Copyright © 2018 American Thoracic Society. Li et al. (102) The American Journal of Respiratory Cell and Molecular Biology is an official journal of the American Thoracic Society.

Regulation and consequence of glutathionylation and nitrosation

Proteins can be post-translationally regulated via reversible redox modifications of amino acid side chains or cofactors. Cysteinyl thiol groups represent the major targets of reversible redox modifications; however, methionine and Sec residues undergo reversible redox modifications as well. Cys residues are often present in the active site of proteins and are necessary for tertiary and quaternary structure of proteins. The number of homologous proteins containing at least one Cys expanded along with evolution, highlighting the importance of their signaling and regulatory functions in increasingly complex organisms (117).

Thiol groups can be oxidized in various ways. Two protein thiols can be oxidized to a disulfide, forming a strong inter- or intramolecular bridge. A single protein thiol may also form a disulfide with GSH, termed glutathionylation, or free Cys, termed cysteinylation or thiolation. Cysteinyl thiols may also react with H₂S to form persulfides, ROIs to form sulfenic acids, or NO resulting in nitroso-thiols in a process termed S-nitrosation. Not every surface-exposed cysteinyl residue can undergo any or all of these oxidative modifications. The susceptibility of cysteinyl side chains to undergo S-nitrosation, for example, is determined by the electrostatic and hydrophobic environment of the thiol. Thus, the microenvironment of the cysteinyl side chains determines their reactivity toward different compounds and, therefore, the specificity of redox signaling in general (41, 61).

As a redox signaling switch, glutathionylation can be efficiently and specifically removed by glutaredoxin-1 via a thiol–disulfide exchange reaction in the presence of GSH, NADPH, and GSH reductase (72). In recent studies, the role of glutathionylation in lung disease has been demonstrated by modulation of NFκB by GST, which, in turn, activated proinflammatory cytokine production in lipopolysaccharide (LPS)-exposed epithelial cells (89).

In mammalian cells, l-Arg-dependent NO synthases, neuronal NOS (NOS1), inducible NOS (NOS2), and endothelial NOS (NOS3) are the major sources of endogenous NO. Stimulus-coupled activation or induction of NO synthases mediates or modulates a broad range of cellular signaling pathways. The physiological influence of NO is exerted predominantly through post-translational modification and functional regulation of proteins. Nitrosation of Cys residues represents a large part of the ubiquitous influence of NO on the cellular function of Trx and TXNRD and have been implied in transnitrosation reactions (146).

Deficient NO signaling plays a critical role in the pathogenesis of PH (129), and nitrite anion (NO₂ ⁻) contributes to local NO bioavailability and NOS formation by acting as a stable nonreactive NO pool (3). Inhaled ethyl (alkyl) nitrite, a volatile gas that acts as a NO₂ donor, was more effective than inhaled NO (iNO) in preventing hyperoxic lung injury in neonatal rats (9) and was highly efficacious as an acute pulmonary vasodilator in human neonates (122). These observations support NO₂ as a promising therapeutic strategy. Systemic or inhaled sodium nitrite (NaNO₂) is an alternative NO₂-based therapy that is strongly protective in adult experimental models of chronic PH (13, 54). A recent study in hypoxia-exposed neonatal/juvenile mice reported that systemically administered NaNO₂ prevented and reversed PH and increased lung NO contents (86). Unlike iNO, NaNO₂ did not increase overall lung nitrosation.

Lastly, recent studies conducted in hydrogen sulfide (H₂S) signaling have revealed the importance of persulfides (RSSH) in redox biology and have recognized protein polysulfidation as a signaling regulatory mechanism (82, 127, 132). Cystathionine β-synthase and cystathionine γ-lyase produced cysteine persulfide (Cys-SSH) from Cys (127). An endogenous sulfur transfer system involving Cys-SSH generates glutathione persulfide (GSSH) that exists at concentrations greater than 100 μM in vivo (94). Because reactive persulfide species such as Cys-SSH and GSSH have higher nucleophilicity than parental Cys and GSH do, these reactive species exhibit strong scavenging activities against oxidants, which contributes to redox signaling regulation (94).

Redox Regulation of Transcription Factors

Nuclear factor erythroid-derived 2-like 2

Nrf2 is a ubiquitously expressed basic leucine zipper (bZIP) transcription factor that contains a Cap‘n'Collar structure. Nrf2 induces antioxidant and xenobiotic response genes via ARE activation in the promoter/enhancer regions of target genes and enhances survival from oxidative insults (138). ARE activation leads to increased transcription of genes that provide direct antioxidants, including Trx and TXNRD, increased GSH levels via de novo synthesis, and stimulation of NADPH synthesis (31, 35, 160, 186, 193).

The Nrf2 system is one of the most important regulators of detoxification and OS responses in mammalian cells. Nrf2 is activated in response to numerous exogenous and endogenous stressors, including electrophilic agents and ROIs. Upon activation, Nrf2 accumulates in the nucleus where it heterodimerizes with one of several other ubiquitous bZIP family members, and binds to ARE(s) in the promoter region of target genes, resulting in promotion of cell survival and enhancement of detoxifying enzymes, antioxidant enzymes, metabolic enzymes, transcription factors, and proteases (76, 105, 112). Under normal conditions, Nrf2 is bound to its endogenous inhibitor Keap1, an ubiquitin E3 ligase that constantly targets Nrf2 for ubiqutination and proteasomal degradation. Keap1 is a sensor for Nrf2-activating compounds. Oxidation of or electrophilic binding to key Cys residues on Keap1 causes a conformational change (33, 96), resulting in blockage of Nrf2 ubiquitination and degradation. This permits nuclear accumulation of Nrf2.

Hyperoxia-exposed Nrf2^−/− mice demonstrated decreased survival upon exposure to hyperoxia when compared with wild-type mice (36). In response to hyperoxia, wild-type mice displayed marked increases in the expression of Nrf2-regulated genes such as glutathione peroxidase 2 (Gpx2) and NQO1. Whereas Nrf2^−/− showed minimal or undetectable levels of Gpx2 and NQO1 in the lung, lung development was characterized by a marked reduction in alveolar and vascular development (111).

Inflammation is also modulated by Nrf2. Hyperoxia-exposed newborn Nrf2^−/− mice showed evidence of enhanced necrosis and apoptosis in cells from bronchoalveolar lavage. Protein exudation in air spaces, alveolar inflammation and developmental disruption, and perivascular–peribronchiolar edema were more severe and frequent in Nrf2 ^−/− neonates than in wild-type mice after exposure to 100% oxygen for 3 days (36). Epigenetic modifications also regulate Nrf2 expression. In extrapulmonary organs, miRs have reportedly been shown to regulate Nrf2 either directly (miR-153, miR-27a, miR-142-5p, and miR-144) or by targeting Keap1 (miR-200a and miR-7) (91, 125). The effects in the pulmonary parenchyma remain mostly unexplored.

Given its key role in modulating endogenous antioxidant responses, Nrf2 has become an attractive therapeutic target to promote positive outcomes in diseases characterized by excessive oxidative and/or inflammatory stress. Keap1 is primary target of Nrf2 activators, most of which are electrophilic in nature and react with key thiol groups on Keap1 (29). Many of the compounds being studied as Nrf2 activators are from natural sources or have structures derived from natural compounds. Unfortunately, gaps in understanding of downstream effector pathways governed by Nrf2 activation have hindered successful translation of preclinical studies to the bedside.

Nuclear factor κB

The NFκB family of transcription factors regulates the expression of hundreds of genes that are involved in regulating cell growth, differentiation, development, and apoptosis. There are five evolutionarily conserved members that bind as homodimers or heterodimers and possess a Rel-homology (RHD) domain that is essential for DNA binding and dimerization (75, 121). Under quiescent conditions, NFκB dimers are inactive via interactions with IκB proteins in the cytoplasm. Upon stimulation or activation, phosphorylation by IκB kinase (IKK) promotes nuclear translocation of NFκB, resulting in transcriptional activation of associated genes.

ROIs and NFκB interact in various cellular pathways. Crosstalk between NFκB and c-Jun N-terminal kinase (JNK) prevents sustained JNK activation and prevents cell death via apoptosis and necrosis (119, 120). NFκB activation modulates a cascade of antioxidant mechanisms including manganese superoxide dismutase (MnSOD, or SOD2) (48, 90). MnSOD is a mitochondrial enzyme that protects cells from OS by converting O₂ ^•− to H₂O₂. Ferritin heavy chain, heme oxygenase 1 (HO-1), Trx1, and Trx2 are also upregulated by NFκB activation (48, 92, 98).

Genetic models have revealed novel roles for NFκB in the lung. IKKβ overexpression increased proinflammatory gene expression, neutrophilic infiltration, and pulmonary edema in mice (34). In airway epithelia, dominant negative overexpression of IκB-α (IκBαSR) limited intranasal LPS-induced neutrophilic infiltration and TNF-α expression (136). Alveolar macrophages are key drivers of pulmonary inflammation due to enhanced production of cytokines. These cytokines, in turn, activate NFκB in other intrapulmonary cells. In rats, alveolar macrophage depletion inhibited immunoglobulin-induced pulmonary NFκB activation. Restoration of NFκB activation by TNF-α administration implies that TNF-α is a key mediator of NFκB activation in the lung (101).

NFκB contributes to lung morphogenesis by modulating EC proliferation and by regulating angiogenesis (83). In contrast to adult lungs, NFκB is constitutively activated in neonatal mouse lungs. NFκB inhibition blocks alveolarization through effects on pulmonary vascular development (83). Similarly, genetic deletion of IKKβ expression impairs alveolar development (103). Additional data implicate NFκB in the pathogenesis of cystic fibrosis, asthma, and lung cancer (77, 118, 158).

Emerging evidence indicates the crosstalk between Nox-produced ROIs, NOX isoforms, and NFκB, especially in the case of PH. In lungs and pulmonary arteries of lambs with PH, enhanced NFκB activity is attenuated upon Nox4 inhibition (179). This and other Nox subunits are, in turn, regulated by HIF (180). NFκB upregulates HIF-1α transcription in response to hypoxia in PASMCs (17). These pathways are intriguing as potential therapeutic targets.

Hypoxia-inducible factor

Mitochondria-derived ROIs control activation of HIF-1α (144, 150). HIF activation regulates a variety of genes under hypoxia. Transcriptional activation of HIF-regulated genes is driven by both oxygen-sensitive HIF-1α and oxygen-insensitive HIF-1β (145). In normoxia, HIF-1α undergoes ubiquitin-mediated degradation regulated by hydroxylation of conserved proline residues under control of prolyl hydroxylases (PHDs) (84). PHD activity is suppressed by hypoxia, thereby permitting the accumulation, heterodimerization, and translocation of HIF-1α. HIFs are key drivers of embryogenesis that occurs under hypoxic conditions. Fetal lung HIF mRNA and protein levels are comparatively greater with HIF-1α localized predominantly in the branching epithelia (69). Although also expressed by epithelia, HIF-2α is also expressed in mesenchyme and vascular endothelia (69).

Mechanical ventilation reduces HIF-1α and HIF-2α protein expression, thus contributing to abnormal pulmonary development, respiratory distress syndrome, and BPD (71). Thus, PHD inhibitors have been shown to preserve HIF expression, improve pulmonary development, and enhance lung function in a baboon model of BPD (5). Expression of HIF-2α is also decreased in the lungs of neonatal rats exposed to chronic hypoxia, another stimulus that impairs alveolar development and decreases pulmonary vascular growth in mice (166). Enhancement of HIF signaling by either selective or nonselective inhibition of PHD-mediated HIF degradation increased angiogenesis in vitro, and increased PECAM-1, VEGF, and FLT-1 levels (7).

Relevant to the pathophysiology of PH, hypoxia causes mitochondrial production of ROIs that trigger the activation of hypoxic transcriptional responses through the inhibition of prolyl hydroxylase (PHD2), a negative regulator of HIF-α stability (151, 171). In mice exposed to chronic hypoxia (10% O₂), partial HIF-1α or HIF-2α deficiency improved attenuated increases in pulmonary arterial pressure, right ventricular hypertrophy, and pulmonary vascular remodeling (27). Selective deletion of HIF-1α leads to attenuated pulmonary vascular remodeling and PH in chronically hypoxia-exposed mice; however, right ventricular morphology was unchanged despite attenuated pulmonary pressures. These findings suggest that right ventricular hypertrophy in the absence of PH may be caused by direct cardiac HIF induction, independent of elevated pulmonary artery pressure (14). Therapies aimed at regulating HIF levels may be beneficial for the treatment of lung diseases.

Selenium: Effects on Oxidant and Antioxidant Systems

As an essential trace element, the importance of Se in humans is well established. Se deficiency causes serious health effects in humans. Selenoproteins are so named due to the presence of Sec residues while selenoenzymes contain Sec in the catalytic active site (148). Thus, Se is important for the proper function of antioxidant enzymes such as GPx and TXNRD (161). Selenoprotein P and GPx are the most abundant selenoproteins in blood accounting for up to 50%–80% of all selenoproteins (49). Se deficiency exacerbates hyperoxic lung injury in adult rats (74).

Se deficiency and supplementation

Se accumulation during development is primarily via third trimester transplacental acquisition. In the fetal liver, Se accumulates between the 20th and 40th gestational week (15). This means that the majority of premature infants are born with Se deficiency. There exists a linear relationship between umbilical cord Se levels, gestational age, and birth weight (106). When compared with term infants, premature infants umbilical cord Se levels are significantly lower (62). Further exacerbating Se deficiency of prematurity, preterm infants receive minimal or no Se supplementation postnatally as a consequence of prolonged need for parenteral nutrition. In premature infants, parenteral nutrition and formula feeding are associated with impaired Se status when compared with infants receiving human milk during the first weeks to months of life (42, 50, 149).

Several studies have investigated the effects of Se supplementation. In a prospective randomized study of adult patients with systemic inflammatory response syndrome and multiple organ failure, sodium selenite supplementation correlated with deceased mortality (196). In premature infants in a large randomized double-blind study, Se supplementation did not improve neonatal respiratory outcome. However, lower maternal and infant Se concentrations before randomization were associated with an increased risk of oxygen dependency at 28 days and oxygen dependency at 28 days or death (44). A possible explanation for this is that the first few days of life are critical to prevent OS and protect the developing lung, and supplementation in this study was began on average at day 4 of life. A 2003 meta-analysis indicated that low plasma Se correlated with increased BPD incidence, oxygen need, and poor pulmonary outcomes in premature infants. Se supplementation decreased late onset sepsis, but not BPD, in extremely premature infants (43).

Perhaps a more useful way to study Se is as a possible therapeutic coadjutant in the endogenous antioxidant response. Nrf2 activation results in increased synthesis of Sec-containing enzymes whose enzymatic function is directly related to Se bioavailability. Studies in mouse club cells clearly showed that the effects of inhibiting TXNRD inhibition on Nrf2 activation are Se dependent (Fig. 6) (163). Ultimately, these studies showed that Se positively influences Nrf2 nuclear levels and AFN-mediated Nrf2 transcriptional activation. The implications of this lay in the need for optimization of conditions for the maximum effectiveness of drugs that target redox homeostasis.

FIG. 6.

Se enhances Nrf2 nuclear localization and ARE-luciferase activity in AFN-treated mtCCs. Nuclear Nrf2 expression and ARE-luciferase activity. mtCCs were cultured in 5% fetal bovine serum-containing media containing 0, 10, 25, or 100 nM Na₂SeO₃. (A) Cells were cultured in the presence or absence of 0.5 μM AFN for 1 h and Nrf2 expression was determined by Western blotting of nuclear fractions. (B, C) mtCCs were cultured as described, transfected with ARE-luciferase and Renilla luciferase plasmid DNA for 24 h, and incubated in the presence or absence of 0.5 μM AFN for 18 h. (B) Luciferase activity in 0, 10, 25, or 100 nM Na₂SeO₃-supplemented cells. (C) Fold-change luciferase activity in control and AFN-treated cells. Data (mean ± standard error of the mean, n = 3) were analyzed by one-way ANOVA followed by Tukey's post hoc analysis (A, B) or t-test (C). *p < 0.05 versus respective (Na₂SeO₃); ^$ p < 0.03 versus 0 nM Na₂SeO₃ + AFN; ^# p < 0.02 versus 10 mM Na₂SeO₃ + AFN. Reprinted from Redox Biology Tindell et al. (163), with permission from Elsevier. ARE, antioxidant response element; mtCC, mouse transformed clara cells.

Se toxicity

Se toxicity has negative consequences on the endocrine system, including enhanced type 2 diabetes risk and increased incidence of melanoma and lymphoid cancers. Several case reports and epidemiologic studies describe a wide range of symptoms from Se overexposure (37, 187, 188). The majority of these reports describe overexposure as a result of excessive daily Se intake from food, water, or nutritional supplements. Chronic exposure to environmental Se negatively correlated with health outcomes in human populations. Se overdose in humans causes selenosis. Although quite rare, selenosis can cause amyotrophic lateral sclerosis (56). Mechanistically, cellular exposure to high-dose Se can increase intracellular ROIs, which is considered as the main mediator of Se-induced cell toxicity (155). Although Se is regarded as an essential factor of antioxidant enzyme production, it is chemically able to form intramolecular disulfide bond (S-Se) with thiol groups or Cys residues indirectly generating ROIs (63). The issue of dose of intake shall be, therefore, taken into serious account when using Se as a nutritional or therapeutic agent.

Concluding Remarks

Redox signaling significantly contributes to pulmonary disease. ROIs are not only deleterious through their effects on lipids, proteins, and DNA, but are also key regulators of signaling process through processes such as glutathionylation and nitrosation. The GSH and the Trx systems are fundamental for redox homeostasis, regulation of vascular and alveolar development, and control of inflammation that make these systems attractive for investigation as novel therapeutic targets. Regulatory systems including Nrf2, NFκB, and HIF-1α, which are fundamental for cellular response in disease, are redox sensitive. Enhanced understanding of these mechanisms could lead to additional novel therapeutic approaches. Lastly, optimization of endogenous antioxidant responses through correction of trace mineral deficiencies, as evidenced by the role of Se in redox homeostasis, could promote successful translation of preclinical therapies to the bedside. Despite the large amount of experimental evidence of redox modulation in pulmonary disease, the vast majority of clinical trials in humans have been disappointing. This lack of success represents an opportunity to optimize therapeutic approaches for specific diseases and patient populations.

Footnotes

Abbreviations Used

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