Carbon monoxide in lung cell physiology and disease

Abstract

Carbon monoxide (CO) is an endogenously produced gas that has gained recognition as a biological signal transduction effector with properties similar, but not identical, to that of nitric oxide (NO). CO, which binds primarily to heme iron, may activate the hemoprotein guanylate cyclase, although with lower potency than NO. Furthermore, CO can modulate the activities of several cellular signaling molecules such as p38 MAPK, ERK1/2, JNK, Akt, NF-κB, and others. Emerging studies suggest that mitochondria, the energy-generating organelle of cells, represent a key target of CO action in eukaryotes. Dose-dependent modulation of mitochondrial function by CO can result in alteration of mitochondrial membrane potential, mitochondrial reactive oxygen species production, release of proapoptotic and proinflammatory mediators, as well as the inhibition of respiration at high concentration. CO, through modulation of signaling pathways, can impact key biological processes including autophagy, mitochondrial biogenesis, programmed cell death (apoptosis), cellular proliferation, inflammation, and innate immune responses. Inhaled CO is widely known as an inhalation hazard due to its rapid complexation with hemoglobin, resulting in impaired oxygen delivery to tissues and hypoxemia. Despite systemic and cellular toxicity at high concentrations, CO has demonstrated cyto- and tissue-protective effects at low concentration in animal models of organ injury and disease. These include models of acute lung injury (e.g., hyperoxia, hypoxia, ischemia-reperfusion, mechanical ventilation, bleomycin) and sepsis. The success of CO as a candidate therapeutic in preclinical models suggests potential clinical application in inflammatory and proliferative disorders, which is currently under evaluation in clinical trials.

INTRODUCTION

Carbon monoxide (CO) is a gaseous molecule that occurs ubiquitously in nature as the product of organic combustion processes (171). Since the middle of the twentieth century, the medical community has recognized that CO can evolve endogenously in living organisms as the by-product of metabolic activity (30, 148, 149). In both prokaryotes and eukaryotes, biological CO arises as the result of enzymatic heme degradation, catalyzed by the heme oxygenase enzymes (Fig. 1) (163, 164, 187). The characterization of the endogenous occurrence and enzymatic production of CO, and subsequent discoveries that CO at low concentration can influence cellular signal transduction pathways, leading to altered cellular functions, including adaptive responses, has led to the recent recognition of this gas as a potential physiological mediator (88, 134–138).¹

Fig. 1.

Interaction of carbon monoxide (CO) with biological systems. CO is endogenously produced in the body, primarily as the product of the erythrocyte-dependent turnover of hemoglobin-derived heme, and the cellular turnover of hemoproteins. Heme, a catalytic cofactor for hemoproteins, is degraded by the heme oxygenase (HO, EC: 1:14:99:3) enzymes. HO catalyzes the rate-limiting step in oxidative heme catabolism, to yield biliverdin-IXα, CO, and ferrous iron (Fe II), and requires 3 mol molecular oxygen (O₂) and reducing equivalents from NADPH. Biliverdin-IXα produced in the HO reaction is reduced to bilirubin-IXα by NAD(P)H biliverdin reductase (BVR). Non-heme sources may make a minor contribution to exogenous CO production. In the blood, CO binds hemoglobin to form carboxyhemoglobin (CO-Hb). Therapeutic CO may be applied at low concentration through inhalation or mechanical ventilation, or by administering CO-releasing molecules (CORMs).

The term “gasotransmitter” was introduced to describe a group of biologically relevant and related gases, including CO and other small gases such as nitric oxide (NO) and hydrogen sulfide (H₂S) (160, 176). NO was identified in 1986 as identical to the endothelial-derived relaxing factor responsible for regulation of blood vessel tone, laying the foundation for current acceptance of gaseous second messengers in biology (50). H₂S, a third gasotransmitter, has recently emerged as a modulator of vascular function, inflammation, and other diverse cellular processes, which have been reviewed extensively elsewhere (124, 160, 176). Like NO or H₂S, endogenously produced CO can exert a variety of biologic and physiologic functions (104, 134–136), which range from the regulation of vascular tone (156), mitochondrial homeostasis and biogenesis (82, 83, 157, 158), neurotransmission (169); to the modulation of inflammation (114), programmed cell death (16, 17), and cellular proliferation programs (99). Taken together, the evidence for physiological signaling roles of CO has displaced previous conceptions that endogenously derived CO represents solely a waste product of ordinary metabolism (138).

In addition to metabolic production, CO can also enter the body via the pulmonary inhalation portal (171). Environmental CO, which occurs as a common component of air pollution, wood smoke, and cigarette smoke (~5.9–17.4 mg CO/cigarette), is renowned for its systemic toxicity (21, 123, 171, 195). CO when presented at elevated concentrations can cause asphyxiation and death during accidental or occupational inhalation exposure (171). This property is directly attributed to the ability of CO to bind the heme-iron centers of hemoglobin (with an affinity ~250-fold that of oxygen), and generate carboxyhemoglobin (CO-Hb). The partial occupation of hemoglobin heme sites by CO impairs the release of O₂ from the remaining heme groups, resulting in reduced O₂ delivery to tissues (123, 171). The clinical toxicology of CO poisoning involves severe neurocognitive effects, cardiac dysfunction, and potential death, and has been reviewed extensively elsewhere (14, 53, 122, 123, 132).

One of the basic tenets of pharmacology is that the dosage of a compound will determine whether it potentially acts as a therapeutic or a poison (15). In this regard, research over the past two decades using animal models has uncovered a therapeutic window for this gas when applied at low concentrations, despite the well-publicized toxicity of CO at elevated concentrations. To date, an extensive collection of preclinical data in rodents and higher mammals has demonstrated salutary effects of CO in experimental models of organ injury and disease, in conditions involving aberrant inflammatory or proliferative states, and thereby has advanced the concept of CO as a gaseous therapy (104, 134, 135). These studies have collectively established a foundation for potential future clinical applications of this gas. As a result of these studies, a wealth of data has also emerged describing the physiological and cellular consequences of CO exposure in vitro and in vivo, and the underlying signaling mechanisms responsible for these effects.

Although the concept of gaseous molecule therapies, including CO (or its precedent, NO), was originally based on the proposition of direct application of the therapeutic gas by inhalation, recent years have also seen the development of chemical pro-drugs capable of delivering the gas in a controlled fashion. For example, like the native gas, CO-releasing molecules (CORMs) have also shown therapeutic potential in preclinical models of disease (103, 104). This review will focus on the cellular and molecular targets of CO in lung and vascular models, with an emphasis on cell signaling and functional outcomes. The therapeutic implications of CO will also be discussed in the context of lung and vascular diseases, as well as inflammatory disorders such as sepsis.

PHYSICOCHEMICAL PROPERTIES OF CO

Carbon monoxide (CO) is a diatomic molecule (molecular weight = 28.01), which exists as an invisible and odorless gas at ambient temperature and pressure (CO has a melting point of −205°C, and a boiling point of −191.5°C) (171). CO is water soluble (2.3 ml·100 ml⁻¹ at 20°C) and dissolves in some organic solvents such as chloroform (171). Unlike the free radical NO, which is highly reactive in biological systems and can engage in complex redox chemistry (81), CO is an inherently stable nonradical with little biological reactivity with respect to non-iron compounds. CO can ligate with cellular heme groups at the iron center, and has been demonstrated to form complexes with cellular hemoproteins, such as cytochrome-c oxidase (18, 23, 170), cytochrome p-450 (41, 55), soluble guanylate cyclase (sGC) (49, 154, 155), inducible nitric oxide synthase (153), and other cellular cytochromes (e.g., cytochrome b558) (31).

ENDOGENOUS PRODUCTION OF CO

The endogenous production of CO is largely attributed to the degradation of heme, with the majority (~85%) originating from erythrocyte-dependent hemoglobin turnover (30, 148, 149), as well as from the turnover of cellular hemoproteins such as mitochondrial cytochromes (80, 92). A minor component of endogenous CO production is believed to originate from non-heme sources that may include lipid peroxidation and cytochrome p-450-dependent drug metabolism (172). In discoveries beginning in 1968, heme-dependent CO production was shown to directly occur as the by-product of enzyme-dependent heme degradation catalyzed by heme oxygenases (HO, EC: 1:14:99:3) (163, 164). HO activity catalyzes the oxidative cleavage of heme to biliverdin-IXα, in a reaction that liberates ferrous iron and CO. The HO reaction requires molecular oxygen and an electron donor (i.e., NADPH cytochrome p-450 reductase) (163). Bilirubin-IXα is further enzymatically reduced to biliverdin-IXα by biliverdin reductase (BVR) (163, 165). Two genetically distinct isozymes of HO have been identified and characterized, an inducible isozyme (HO-1) that is highly transcriptionally responsive to cellular stress stimuli, and a constitutively expressed isozyme (HO-2) (71, 91). The molecular regulation and function of HO-1 and related enzymes have been extensively reviewed elsewhere (2, 92, 134, 135, 137).

THERAPEUTIC CO (INHALATION AND PHARMACOLOGICAL APPLICATION)

The potential for medical application of CO originally assumed inhalation as the primary route of administration. In this case, dosimetry is controlled by adjusting the concentration of the inspired gas mixture, and can be monitored by measurement of CO-Hb in the blood. As an alternative to inhalation, metal carbonyl-based chemical CO donor compounds (CORMs) have been developed as experimental therapeutics over the past two decades (46, 65, 103, 104). CORMs can be used to deliver therapeutic CO to biological systems by injection to the systemic circulation or application to the target site. An advantage of CORMs is that they deliver CO to tissues while limiting CO-Hb buildup associated with inhaled CO (104). A key consideration of this approach is that the safety and metabolic fate of the donor molecule or backbone, in addition to that of CO, must also be evaluated (65). Further considerations for the development of new CORMs include improved water solubility, tissue-specific targeting, and the ability to induce, control, or time the release of CO (65). Similar to gaseous CO, CORMs have demonstrated vasoactive effects (47), as well as anti-inflammatory effects in various injury and disease models (22, 82, 83, 140, 159).

The first CORM to be described was Mn₂CO₁₀ (CORM-1). Subsequently, ruthenium-based compounds were introduced: tricarbonyldichlororuthenium-(II)-dimer (CORM-2) and tricarbonylchoro(glycinato)-ruthenium (II) (CORM-3) (65, 103, 104). CORM-1 and CORM-2 are hydrophobic (103), while CORM-3 is water soluble and rapidly releases CO in physiological fluids (47). CORM-2 has been utilized primarily for in vitro studies, while CORM-3 is preferred for in vivo studies. A number of new generation water-soluble CORMs have been introduced in recent years, including CORM-A1 (sodium boranocarbonate), which releases CO in a pH and temperature-dependent fashion and has demonstrated vasodilatory and hypotensive effects (105); and CORM-S1 [dicarbonyl bis(aminoethylthiolato)iron(II)], which releases CO in response to visible light activation (79). Furthermore, a novel series of hydrophobic and hydrophilic CORMs (iron-allyl carbonyls) were shown to release CO at different rates (106). More recently, a novel manganese-based CORM (CORM-401), which releases CO in response to oxidant exposure, has been shown to exert potent proangiogenic and vasodilatory properties (44). The current frontier of CORMs development has introduced compounds that facilitate controlled release of CO, and these include micellar preparations (57), enzyme-activated CORMs (ET-CORMs) (152), and new generation radiation-sensitive (photo-activated) CORMs (PHOTO-CORMs) (95, 107, 118). Additional advances have yielded bifunctional hybrid molecules (189), or CORMs conjugated to nanoparticles and other biological scaffolds (carrier conjugates) for potential improvement of drug delivery (52, 70, 142).

EFFECTS OF CO ON CELL SIGNALING

CO has been shown to impact the regulation of multiple signaling pathways leading to altered cellular functions. While the proximal target of CO action has long been hypothesized to involve a heme-binding event, such as the heme of sGC, a heme-dependent mechanism has not been unequivocally identified across all studies. Mitogen-activated protein kinases (MAPKs) and other signal transduction molecules have been identified as secondary targets, though their integration with sGC and other proximal hemoprotein targets has also not been universally established across all studies. Emerging research also identifies mitochondrial cytochromes as proximal targets of CO action, though the hierarchy of mitochondrial regulation with other cytosolic or cellular signaling events remains incompletely elucidated. Furthermore, the effects of CO on cellular signaling events can vary in cell-type and/or model-specific fashion, adding further ambiguity. Thus, no universal pathway or unifying mechanism has emerged. Nevertheless, the following sections describe the key targets of CO that have been identified in recent years in a context-specific fashion.

Guanylate Cyclase

The soluble form of guanylate cyclase was among the first molecules proposed as a direct molecular target of CO, activating downstream signaling pathways (49). Binding of sGC by CO, akin to that of NO, results in the activation of the enzyme and subsequent production of the second messenger molecule guanosine 3′,5′-monophosphate (cGMP), which activates protein kinase G. CO binds the heme group of sGC in a hexa-coordinate fashion, whereas the binding of NO occurs in a penta-coordinate configuration. The activation potential of CO is much lower than that of NO (approximately 80-fold less potent) (154), and may not occur in the presence of NO. Nevertheless, the activation of sGC by CO has been implicated in neurotransmission (169), regulation of vascular tone (156), and the inhibition of vascular smooth muscle (SMC) proliferation (99, 100), and platelet aggregation (19). However, sGC as the primary proximal target of CO action has not been unequivocally demonstrated in all studies, and this can also vary in a cell-type and model-specific fashion.

NADPH Oxidase

The family of NADPH oxidases (NOX1-4, DUOX1-2), which include the phagocytic NOX (NOX2, gp91^phox) and a mitochondria-specific isozyme (NOX4), are cellular cytochrome-containing enzyme complexes responsible for the univalent reduction of molecular oxygen to superoxide anion (O₂⁻) (12). The formation of O₂⁻ by NOXs is utilized for diverse processes including host defense, vascular regulation, and cellular signaling (12). NOXs have been implicated as targets of CO action (109, 130, 161, 178). Inhibition of NOX-dependent reactive oxygen species (ROS) generation has been attributed to anti-inflammatory (109), antiproliferative (161), and antiapoptotic (178) effects of CO. For example, CO was shown to inhibit LPS induced ROS-dependent trafficking of Toll-like receptor-4 (TLR4) to lipid rafts, a proximal event to activation of the NF-κB-dependent inflammatory pathway, in cultured macrophages (109). TLR4 trafficking and activation, as well as the capacity for CO to inhibit the response, were sensitive to chemical inhibition of NOX, and abolished in gp91^phox-deficient macrophages (109). Experiments using CORM-2 demonstrated inhibition of cytochrome b558 (p21^phox) activity in vitro, the flavocytochrome component of NOX2, thereby implicating NOX2 as a regulatory target of CO action (161). Interestingly, NOX1 has also been implicated as a target in the inhibition of vascular SMC migration by endogenous CO (130). Recent studies have also proposed that endogenous HO-derived CO can downregulate mitochondrial NOX4, which in turn inhibits matrix metalloproteinase-1 expression and apoptosis in IL-1β-activated chondrocytes (133).

Mitochondria as Targets for CO Action

Mitochondria have been strongly implicated as a primary target of CO action. Many of the mitochondrial cytochromes responsible for respiration have heme functional groups that potentially serve as binding targets for CO. The cellular toxicity of CO has long been known, such that CO at elevated concentrations can impair mitochondrial function leading to inhibition of mitochondrial respiration (23). In this regard, CO at high concentration can inhibit the terminal enzymatic step in the electron transport chain (cytochrome-c oxidase) by binding to the cytochrome a3-subunit (23). Several recent studies have demonstrated the modulation of mitochondrial (mt)ROS production and modulation of mitochondrial membrane potential by CO (13, 25, 85, 197). In macrophages, upregulation of mtROS by low concentration CO was attributed to inhibition of cytochrome-c oxidase (197), which promoted anti-inflammatory effects via p38 MAPK. These studies reported maintenance of cellular ATP and increase of mitochondrial membrane potential in the presence of CO at the concentrations used (197). Furthermore, upregulation of mtROS in macrophages was attributed to adaptive cellular signaling through upregulation of peroxisome proliferator-activated receptor-γ (PPARγ), downregulation of the proinflammatory mediator Egr-1 (13), and stabilization of the hypoxia-inducible factor-1-α (HIF-1α) (25), leading to adaptive gene expression and anti-inflammatory effects. Gaseous CO was shown to upregulate mtROS in epithelial cells, in the context of upregulating the cytoprotective autophagy program (85).

In isolated cardiac mitochondria, CO released from CORM-3 was demonstrated to exert a mitochondrial uncoupling effect. At low concentrations, CORM-3 increased mitochondrial oxygen consumption, but it limited respiration at higher concentrations via inhibition of cytochrome-c oxidase. CORM-3 also caused time-dependent decline of mitochondrial membrane potential and increase of complex I-dependent mtROS production. In contrast, CORM-3 inhibited succinate-inducible (complex II)-dependent mtROS production in isolated mitochondria (90). CO released from CORM-401 was also shown to cause mitochondrial uncoupling, accompanied by increased oxygen consumption, reduction of ATP production, and impairment of cellular glycolysis. The mitochondrial uncoupling effect of CORM-401, but not the effect on glycolysis, was further shown to be dependent on mitochondrial large-conductance calcium-regulated potassium ion channels (mitoBK_Ca) (68).

The administration of CO was shown to downregulate mtROS production in lung epithelial cells under conditions of hyperoxia, where ROS production was elevated due to high oxygen tension (83). CO was also observed to downregulate mtROS in macrophages in the presence of proinflammatory stimuli such as LPS and ATP (67). Treatment with LPS and ATP caused mitochondrial depolarization and cytosolic translocation of mtDNA in macrophages, whereas CO exposure abolished these effects (67).

In conclusion, the reported roles of CO effects on mitochondrial respiration, mitochondrial membrane potential, and mtROS generation are clearly dose-dependent. The detection of mtROS can be complicated by differential effects of CO on other enzymatic sources of ROS, such as NOX1-4, and can also vary in the absence or presence of inducing stimuli. The contribution of mitochondria-specific NOX (NOX4) in CO-dependent ROS flux currently remains unclear. Nevertheless, the regulation of mtROS may represent a common mechanism by which CO exerts effects on downstream signaling pathways, yet this has not been unequivocally demonstrated across all studies. Further research will be required to clarify the relative roles of mitochondrial versus cytosolic ROS production as transducing mechanisms of CO effects, both in response to CO alone or in the presence of proinflammatory stimuli.

In addition to modulation of respiratory chain activity and basic mitochondrial functions, CO has been shown to stimulate mitochondrial biogenesis, a cellular program for the de novo generation of mitochondria (82, 157, 158). CO-dependent stimulation of mitochondrial biogenesis, as demonstrated in the heart and in skeletal mitochondria, involves increased proliferator-activated receptor-γ coactivator-1α (PGC-1α), nuclear respiratory factor 1 (NRF-1), and its downstream regulator, mitochondrial transcription factor A (TFAM), factors that initiate the biogenesis program. Furthermore, the stimulation of mitochondrial biogenesis by CO is dependent on sGC, activation of the prosurvival Ser/Thr kinase Akt, and evolution of mtROS (i.e., H₂O₂) but independent of NO production (158). CO stimulation of cardiac mitochondrial biogenesis was shown to be protective in the context of doxorubicin-induced cardiomyopathy (157). Similarly, application of CORM-3 prevented mitochondrial function decline and stimulated mtROS-dependent activation of mitochondrial biogenesis in septic mice (82). These studies describing stimulation of mitochondrial biogenesis by CO suggest a potential novel mechanism by which CO treatment can lead to adaptive responses to systemic stress.

Ion Channels as Targets of CO Action

CO has been implicated as a regulator of ion channel activity, which may contribute to the observed signaling effects of CO towards various biological functions, including vasoregulation and neurotransmission (120, 185). One the most widely studied are high-conductance, Ca²⁺-dependent K⁺ channels (BK_Ca) (120, 185), which are implicated in smooth muscle relaxation and vasodilation (175). Several explanations have been proposed for the stimulatory action of CO on the BK_Ca channel but the precise nature of the activating mechanism remains unclear (120). There is controversial evidence for direct interactions of CO with hydrophobic protein domains, heme-iron (38, 60, 63, 120, 128, 175, 185, 188), and more recently, with a cyanide-sensitive non-heme metal binding site (162). Additional reported ion channels activated by CO include the ATP-gated purinogenic P2X2 receptor (184), and the tandem P domain channel TREK1 (K2P2.1) (33), which have been implicated in sensory neurotransmission and neuronal excitability, respectively. Variable effects of CO were reported towards the activity of voltage gated L-type Ca²⁺ channels, which include demonstrated activation in intestinal cells (through a mechanism involving mobilization of NO and cGMP) (89), and channel inhibition in cardiomyocytes (through a mechanism dependent on redox modification subsequent to enhanced mtROS production) (144, 166). CO has also been shown to exert variable effects on epithelial Na⁺ channels (ENaC) (5, 177). For example, one study has shown that CO inhibits epithelial Na⁺ uptake, which was associated with impaired alveolar fluid clearance in the rabbit lung (5). In contrast, in vitro studies describe activation of ENaC in normoxic mouse epithelial cells by CO (177). The authors proposed a regulatory role for endogenous HO activity as an oxygen sensor for the regulation of channel activity, based on oxygen tension-dependent variable effects of heme on channel activity (177). CO has been shown to inhibit the activities of the voltage-gated delayed rectifier K⁺ channel, Kv2.1 (32) and the cardiac Na⁺ channel Nav1.5 (40) through redox-dependent mechanisms. Curiously, CORM-2 also inhibited the purinergic P2X4 receptor, but in a mechanism independent of CO release (186). Taken together, these intriguing observations suggest a regulatory role for CO, as well as pharmaceutical effects of CO-donor compounds by modulating ion channel activity in diverse model systems. To date, few studies have related the modulation of ion channel activity by CO with its cytoprotective effects. For example, inhibition of Kv2.1 channels by CO was related to antiapoptotic effects in neurons (4, 32). Downregulation of L-type channels by CO was associated with protection from ischemic necrosis in cardiomyocytes (166). In contrast, potentiation of L-type channels by CO in astrocytes was reported to stimulate VEGF-dependent angiogenesis through a mechanism dependent on AMP-activated protein kinase (AMPK)-α, NAD-dependent deacetylase sirtuin-1, and stabilization of PGC-1α (27). Further research is needed to determine how ion-channel dependent responses are integrated with other signaling targets of CO to coordinate cytoprotective responses. The potential relevance of these systems to lung physiology and pathophysiology remains incompletely understood.

Nrf2-Dependent Responses

CO can activate a feedback mechanism to further stimulate its endogenous production through the upregulation of HO-1 gene expression via activation of the transcription factor NF-E2-related factor-2 (Nrf2). Nrf2, a master regulator of the antioxidant response, is anchored in the cytoplasm by Kelch-like ECH-associated protein 1 (Keap1). Cellular exposure to electrophiles and other inducing stimuli promote the dissociation of Keap1, thus permitting activation and translocation of Nrf2 to the nucleus (194). By binding to antioxidant-responsive elements/stress-responsive elements (ARE/StRE) in the 5′-regulatory region of HO-1 (HMOX1, Hmox1) genes, Nrf2 acts as a major transcriptional regulator of HO-1 gene expression (2, 3). Furthermore, Nrf2 orchestrates the cellular antioxidant response by regulating the expression of other genes involved in detoxification and antioxidant responses: including those encoding NAD(P)H quinone oxidoreductase, glutathione reductase, glutathione peroxidase-2, glutamate–cysteine ligase complex, glutathione-S-transferases, ferritin heavy and light chains, and others (54, 102). Nrf2 has been implicated as an important mediator of antioxidant defenses in the lung (194). For example, mice genetically deficient in Nrf2 (Nrf2^−/−) were sensitized to inflammatory responses and emphysema induced by chronic cigarette smoke exposure (62, 126), and were also sensitized to asthma and airway inflammation (127).

Endogenous CO, generated from HO-1, can activate Nrf2 nuclear translocation through upregulation of phosphoinositide 3-kinase (PI3K)/Akt and consequent inhibition of glycogen synthase kinase-3β. This pathway results in further stimulation of HO-1, and activation of the mitochondrial biogenesis program through the stimulation of NRF-1 (121). A pathway dependent on Src, epidermal growth factor receptor, and downstream PI3K/Akt-signaling was shown to mediate the activation of Nrf2 and subsequent HO-1 gene expression in human tracheal smooth muscle cells in response to CORM-2 treatment (190). The neuroprotective effects conferred by inhaled CO (250 ppm) in a murine ischemic shock model were associated with HO-1 induction and were abolished in Nrf2^−/− mice (173). Furthermore, the anti-inflammatory effects of CORM-2 in mice, with respect to reduction of proinflammatory cytokine production and protection from LPS-induced mortality, were abrogated in Nrf2^−/− mice (125). Taken together, these studies indicate that the therapeutic potential of CO is coupled to Nrf2-dependent antioxidant responses, including the stimulation of endogenous CO production via HO-1, and these mechanisms may be significant in adaptive responses of the lung and other organs.

EFFECTS OF CO ON CELLULAR INFLAMMATION RESPONSES

Cytokine Regulation

CO has been demonstrated to exert anti-inflammatory effects when applied to cultured macrophages at low concentration, including downregulation of proinflammatory cytokines (e.g., IL-1β, TNF-α) and upregulation of the anti-inflammatory cytokine IL-10 (114). CO confers these effects through MAPKs, which are critical mediators of inflammatory and stress responses (114) (Fig. 2). The anti-inflammatory effect of CO, as demonstrated in LPS-stimulated macrophages, required activation of the MKK3/p38β MAPK pathway (114). Additional studies have implicated downregulation of JNK or ERK1/2 in the downregulation of IL-6 by CO in macrophages and pulmonary epithelial cells, respectively (101, 112). In addition to modulation of cytokines, CO was shown to inhibit TLR4 trafficking and activation through mechanisms involving inhibition of NOX-dependent ROS generation (109) and the modulation of caveolin-1-TLR4 interactions at the plasma membrane (180). CO was also proposed to confer anti-inflammatory effects in macrophages via the mtROS-dependent induction of PPARγ, which led to downregulation of the proinflammatory mediator Egr-1 (13). The anti-inflammatory effects of CO have potential therapeutic value in diseases where aberrant inflammation is implicated in the pathogenic response, including acute lung injury, transplant rejection, and sepsis.

Fig. 2.

Effect of CO on cellular inflammation pathways. CO has been implicated in the downregulation of inflammatory pathways. CO can inhibit the production of the proinflammatory cytokines (IL-1β, IL-6, TNF-α) and upregulate anti-inflammatory cytokine IL-10, through a mechanism dependent on activation of the MKK3/p38 MAPK pathway. Additionally, CO has been shown to modulate the trafficking and activation of Toll-like receptor-4 (TLR4) at the plasma membrane. CO has been proposed as an inhibitor of NOD-, leucine rich region- and pyrin domain-containing-3 (NLRP3) inflammasome activation, through the stabilization of mitochondria, leading to modulation of mitochondrial reactive oxygen species (mtROS) and inhibition of mtDNA release, key triggers for inflammasome activation under proinflammatory conditions.

Inflammasome Regulation

Inflammasomes are cytosolic protein complexes responsible for the regulation of caspase-1. Activation of caspase-1 by proteolytic cleavage governs the maturation and secretion of proinflammatory cytokines, including IL-1β (IL-1β) and IL-18. Among the known inflammasomes, the NOD-, leucine rich region- and pyrin domain-containing-3 (NLRP3) inflammasome has been recently implicated in the pathogenesis of several acute or chronic inflammatory diseases (35, 84, 143). Current research suggests that CO can regulate the activation of the NLRP3 inflammasome. Our recent studies demonstrate that application of CO inhibited caspase-1 activation and secretion of IL-1β and IL-18 in bone marrow-derived macrophages (BMDMs) in response to LPS and ATP treatment, a NLRP3 inflammasome activation model (67). CO also inhibited IL-18 secretion in BMDMs in response to LPS and nigericin, an alternate NLRP3 inflammasome activation model. LPS and ATP stimulation induced the formation of molecular complexes between NLRP3 and the adaptor ASC, or NLRP3 and caspase-1, whereas CO inhibited the formation of inflammasome complexes. In this model, the presence of CO inhibited mitochondrial depolarization induced by LPS and ATP in macrophages (67). These results suggest that low concentration CO negatively regulated NLRP3 inflammasome activation by preserving mitochondrial function (67). Application of CORM-2 downregulated caspase-1 activation and IL-1β maturation and secretion during endoplasmic reticulum stress-induced inflammation (76). In contrast to these observations, another report demonstrates that CO can promote ATP secretion from bacteria, which in turn can upregulate NLRP3 inflammasome activation by stimulating the purinergic receptor (P2X7R) in bacteria-exposed macrophages (183). The reasons for these contrasting findings remain unclear at present, but they may be related to differences between bacterial sepsis and inflammation induced by cell-free LPS. Although further studies will be needed to clarify the precise mechanisms, CO shows potential as a regulator of the NLRP3 inflammasome and secretion of IL-1β and IL-18, which may contribute to its modulatory effects on inflammation and innate immune responses.

Adaptive Immune Responses

In addition to modulation of inflammasome-dependent and independent cytokine responses, CO has been implicated in the modulation of adaptive immune functions. CO has been implicated in tolerogenic responses to endotoxin, decreasing the surface expression of TLR4/myeloid differentiation factor-2 (MD2) in dendritic cells (DC) and neutrophils. CO preconditioning conferred protection in endotoxin induced mice and was associated with reduced recruitment of DCs and neutrophils to peripheral blood (129). Recently, CO has been shown to induce immunoresponsive gene-1 in macrophages, which promotes LPS tolerance by increasing A20 expression via modulation of ROS production (64). Treatment with exogenous CO inhibited LPS-induced maturation of DCs and protected against in vivo and in vitro antigen-specific inflammation (24). Application of CO also blocked antigen presentation by DCs at the step of endosome-lysosome fusion (147). The ex vivo treatment of DCs with CO was shown to augment dendritic-cell based therapy in a type 1 diabetes model. Application of CO-conditioned DCs inhibited the accumulation and pathogenic activity of autoreactive CD8⁺ T cells in the pancreas (147). Taken together, these examples illustrate the complex and incompletely understood effects of CO in adaptive immune response, and warrant further investigation.

Resolution of Inflammation

Recent findings suggest that therapeutic CO can also modulate the resolution of inflammation, which represents a relatively new area of study. Lipid mediators (LMs), which include eicosanoids and recently described omega-3-derived “specialized pro-resolution mediators” (SPMs), represent the key signaling molecules in resolution of inflammation (20). The therapeutic potential of CO was tested against Streptococcus pneumoniae pneumonia in baboons (34). Plasma obtained from S. pneumoniae-infected baboons displayed significantly reduced levels of LMs/SPMs, including eicosapentaenoic acid-derived E-series resolvins (RvE) and lipoxins, suggesting that pneumonia can deregulate pro-resolution programs in baboons. CO inhalation increased the levels of plasma RvE and lipoxins relative to control levels in infected baboons. These results suggest that low-dose CO can restore altered SPM profiles during pneumonia (34). CO inhalation therapy was also shown to reduce neutrophil infiltration in the lung in a mouse model of ischemia-reperfusion (I/R)-induced lung injury and conferred additive protection when combined with intravenous application of the pro-resolving mediator resolvin D1 (RvD1). Both CO and RvD1 application were shown to reduce neutrophil-platelet aggregates in mouse blood, and reduce leukotrienes and thromboxane B2 production in I/R lungs. CO also reduced neutrophil-platelet aggregation in human whole blood (146). These intriguing studies suggest that the activation of pro-resolving programs may add an additional underlying mechanism to the anti-inflammatory protection afforded by CO.

EFFECTS OF CO ON CELLULAR PROLIFERATION

Carbon monoxide (CO) is endogenously produced by vascular smooth muscle cells (SMCs) under conditions of hypoxia and can modulate cGMP levels in both endothelial cells and SMCs (98–100). Further studies by this group demonstrated that endogenous or exogenous CO can inhibit the proliferation of SMCs via upregulation of cGMP and downregulation of the cell cycle regulatory factor E2F-1 (99). In addition, SMC-derived CO was shown to have paracrine antiproliferative effects on endothelial cells by downregulating endothelial cell expression of endothelin-1 and platelet-derived growth factor-B (98). Further studies have identified cGMP-dependent activation of p38 MAPK as a signaling pathway leading to the upregulation of p21^Waf1/Cip1, a regulator of cell cycle progression, in the antiproliferative effects of CO in vascular SMCs (73, 117). The p38 MAPK-dependent upregulation of the lipid raft-associated tumor suppressor protein caveolin-1 was also implicated in the antiproliferative response (73). Experiments in human airway SMCs demonstrated a role for downregulation of ERK1/2 and cyclin D and for upregulation of p21^Waf1/Cip1 and mtROS in the antiproliferative effects of CO (151, 161). In experiments using CORM-2, both NOX2 and mitochondria were implicated as regulatory targets of CO action, resulting in downregulation of NOX-derived ROS and simultaneous upregulation of mtROS. In support of this hypothesis, the NOX inhibitors diphenylene iodonium or apocynin and the respiration inhibitor rotenone (which increases mtROS) were shown to exert antiproliferative effects in this model (161). CO exposure also exerted antiproliferative and antifibrotic effects in pulmonary fibroblasts through the downregulation of cyclins A/D expression, upregulation of p21^Waf1/Cip1, and inhibition of extracellular matrix formation (196). Studies in cancer models demonstrate that CO can also inhibit tumor growth by inducing growth arrest in cancer cells (181). The observed antiproliferative effects of CO have potential therapeutic value in diseases where aberrant cell proliferation and/or tissue remodeling has been implicated, including vascular injury (73, 117), pulmonary fibrosis (196), pulmonary hypertension (198), and cancer (181).

EFFECTS OF CO ON ENDOTHELIAL CELL GROWTH, VASCULAR REPAIR, AND ANGIOGENESIS

In contrast to the antiproliferative effects observed in fibroblasts and SMCs, both exogenous CO and HO-1-derived CO can act as potent regulators of endothelial cell growth and angiogenesis. Exposure to CO gas or CORM treatment promoted endothelial cell growth and motility in vitro (61, 182). CO induced endothelial cell growth through NO-dependent phosphorylation of retinoblastoma protein. In vivo, CO application as gas or CORM-2 promoted endothelial repair after vascular injury in mice and rats (61, 182). Furthermore, CO was shown to drive reendothelization via endothelial nitric oxide synthase (eNOS)-dependent NO production and subsequent NO-dependent mobilization of endothelial progenitor cells from bone marrow (182). Both CORM-2 and chemical activators of HO-1 (i.e., hemin, Δ-12 prostaglandin-J2) stimulated VEGF production in endothelial cells (66). Furthermore, proangiogenic activity in endothelial cells was upregulated by CORM-2 treatment and HO-1 expression and downregulated by inhibition of HO activity (66). HO-1 was demonstrated as an essential intermediate in the activity of the angiogenic factor stromal cell-derived factor 1 (SDF-1). Exogenous CO was shown to compensate for HO-1 deficiency, in maintaining the proangiogenic effect of SDF-1 in isolated aorta from Hmox1^−/− mice (36). In contrast, antiangiogenic properties of CO have also been reported. For example, CO was shown to inhibit VEGF-induced proliferation and proangiogenic activity in human umbilical vein endothelial cells. In this model, CO suppressed VEGF-induced phosphorylation of Akt and VEGF receptor-2. CO inhibited basic fibroblast growth factor-2-dependent angiogenesis in vivo (1). Taken together, these studies suggest that CO can exert complex effects on endothelial cell growth and angiogenesis, which require additional resolution.

EFFECTS OF CO ON PROGRAMMED CELL DEATH

CO has been shown to impact cell death and survival in a cell- and stimulant-dependent manner in experimental cell death models including TNF-α stimulation, I/R and/or anoxia-reoxygenation (A/R)-induced injury, and hyperoxia exposure. CO has been shown to protect against inducers of apoptosis, including TNF-α exposure and I/R injury through the activation of the MKK3/p38 MAPK and NF-κB pathways (16, 17, 75, 191–193) (Fig. 3). For example, the antiapoptotic effect of CO in endothelial cells against TNF-α required p38 MAPK- and NF-κB-dependent expression of antiapoptotic genes (16, 17). CO exerts its antiapoptotic effects through MKK3/p38 MAPK-mediated downregulation of Fas/Fas ligand interaction and by maintaining levels of antiapoptotic proteins Bcl-2 and Bcl-X_L (17, 191). CO exposure also protected rat hepatocytes from TNF-α-induced apoptosis modulation of the NF-κB pathway (75). CO exposure stimulated mtROS production, which activated PI3K and subsequent Akt phosphorylation, leading to degradation of I-κB and activation of NF-κB (75). CO-induced activation of the PI3K/Akt pathway also protected against TNF-α-induced apoptosis in cerebral microvascular endothelial cells (10). The PI3K/Akt pathway attenuated endothelial cell apoptosis during A/R injury through activation of signal transducer and activator of transcription-3 (STAT3) and attenuation of Fas expression and of caspase-3 activity (192). CO also protected pancreatic β-cells against TNF-α-induced apoptosis in a cGMP-dependent manner (56).

Fig. 3.

Effect of CO on cell death pathways. CO can modulate apoptotic signaling and prosurvival pathways leading to cytoprotection. CO-dependent antiapoptotic effects have been related to the upregulation of the MKK3/p38 MAPK pathway, resulting in modulation of the expression and activation of apoptotic effector molecules. CO may target mitochondria, leading to modulation of mtROS flux and downstream adaptive signaling. Furthermore, the stimulation of phosphoinositide 3-kinase (PI3K)/Akt and NF-κB-dependent prosurvival pathways has been associated with the protective effects of CO.

While CO has consistently been shown to protect against cell death from multiple injuries, the precise pathways have been conflicting and have exhibited cell type-specific variations. During hypoxia, unlike TNF-α-induced apoptosis, CO exposure protected against cell death by decreasing hyperoxia-induced NOX-dependent ROS formation, leading to inhibition of Fas-dependent apoptosis and modulation of Bcl-2-related proteins (178). CO exposure was protective against apoptosis in retinal I/R injury, but variable effects on p38 phosphorylation were reported dependent on experimental conditions (141, 167).

CO suppressed Fas-activating antibody (Jo2)-induced apoptosis of murine lung endothelial cells through both inhibition of Fas-associated protein with death domain (FADD) phosphorylation via JNK and inhibition of caspase-8 activation via cellular FLICE (FADD-like IL-1β-converting enzyme)-like inhibitory protein (c-FLIP) (179). CO also conferred cytoprotection against apoptosis through activation of ERK1/2 and NF-κB prosurvival pathways in this model (179). In BMDMs, CO also protects against A/R injury through mtROS-mediated HIF-1α activation and increased expression of anti-inflammatory cytokine transforming growth factor-β (25). CO has also been shown to protect against hypoxia-induced necrosis in cardiac myocytes by inhibiting L-type Ca²⁺ channel-mediated influx of Ca²⁺ (166). To date, few studies have examined the effect of CO on cellular necroptosis, a programmed form of necrotic cell death. Treatment with CORM-A1 reduced hepatocyte necroptotic cell death after ethanol exposure (9). The inhibitory potential of CO on cell death has been recapitulated using in vivo models of I/R and organ transplantation as discussed in subsequent sections.

EFFECTS OF CO ON CELLULAR AUTOPHAGY

Autophagy is a genetically regulated cellular homeostatic program for the lysosome-dependent turnover of cellular organelles and proteins that can be regulated by cellular stress and nutrient starvation (26). The processing of autophagy substrates is dependent on their recognition and sequestration in double-membraned compartments called autophagosomes, which subsequently fuse to lysosomes where the cargoes are digested by lysosomal enzymes. Recent studies from this laboratory have demonstrated that CO can stimulate the autophagy program in cultured epithelial cells. CO exposure increased the expression and activation of the autophagy protein microtubule-associated protein-1 light chain 3B (LC3B) in mouse lung and in cultured human alveolar or bronchial epithelial cells. Moreover, CO was shown to increase autophagosome formation in pulmonary epithelial cells (85). In this context, CO was shown to stimulate the production of mtROS in the absence of other inducing stimuli typically required for activation of autophagy. The enhanced autophagy response to CO exposure was inhibited by both cellular antioxidants (e.g., N-acetyl-l-cysteine) and mitochondria-specific antioxidants (e.g., mito-TEMPO) (85). Recent studies have also explored protective effects of CO application to mesenchymal stromal cells (MSCs) with the goal of enhancing the therapeutic potential of MSC-based therapies (51). CO enhanced autophagy in MSCs and protected the cells from oxidative stress and apoptosis, whereas CO preconditioning failed to confer protection in MSCs derived from autophagy-deficient mice (51). These observations, taken together, support a role for autophagy as an underlying mechanism for the cytoprotective effects of CO.

PROTECTIVE EFFECTS OF CO IN ORGAN INJURY MODELS

CO has been shown to confer protection in preclinical animal models of lung injury and disease. Although the mode of protection and the underlying mechanisms have been reported to vary in a model-specific faction, the observed protection phenotypes have been primarily associated with anti-inflammatory effects, and with the modulation of apoptotic and proliferation programs. Additional mechanisms have also been implicated including systemic effects on vasoregulation, autophagy, and innate immune responses. The following sections highlight major findings in this area.

Oxidative Lung Injury

Inhalation of supplemental oxygen (hyperoxia) is commonly used as supportive therapy for patients with respiratory failure. However, hyperoxia has also been shown to be a consistent experimental model of lung injury. Hyperoxia (>95% fraction of inspired O₂) in rodents causes acute lung injury associated with an excessive inflammatory response. Inhalation of CO at low concentrations (e.g., 250 ppm) has been shown to extend survival and protect the lung against the effects of hyperoxia in both rats and mice (115, 116). Compared with hyperoxia-only controls, the application of CO ameliorated lung injury markers including neutrophil influx in the airways, fibrin deposition, edema, protein accumulation in the bronchioalveolar lavage (BAL) fluid, and lung tissue apoptosis after hyperoxia exposure (115, 116). The protection afforded by CO treatment against the lethal effects of hyperoxia was associated with the inhibition of proinflammatory cytokine (e.g., TNFα, IL-1β, IL-6) production and was dependent on the upregulation of the MKK3/p38β MAPK pathway (116). Similarly, CO was also shown to confer anti-inflammatory and tissue-protective effects in a model of hyperoxia-induced bronchopulmonary dysplasia in neonatal mice (8, 45). CO ameliorated alveolar simplification in this model and was associated with the reduction of inflammatory cell influx including monocytes (F4/80⁺, CD11c⁺, and CD11b⁺) and neutrophils (Gr-1⁺), and inhibition of proinflammatory cytokines, including C-C motif chemokine ligand 2 (8).

Ischemia-Reperfusion Injury

Tissue-protective effects of CO have been demonstrated in a model of pulmonary I/R injury in mice which can cause lung injury associated with activation of Fas-dependent apoptosis in situ (191, 193). In this model, CO conferred antiapoptotic effects through upregulation of the MKK3/p38α MAPK pathway, resulting in the inhibition of apoptotic tissue markers, including the expression of Fas/FasL and Bcl-2 family proteins, activation of caspases-3, -8, -9, cleavage of PARP, and release of cytochrome c (191, 193). Consistently, the therapeutic effect of CO was compromised in Mkk3^−/− mice or by chemical inhibition of p38 MAPK (191). Furthermore, inhaled CO was shown to rescue HO-1-deficient mice (hmox-1^−/−) from mortality induced by pulmonary ischemia (48). CO conferred protection in this model by promoting fibrinolysis (48). These effects of CO were dependent on the downregulation of cGMP and ERK1/2-dependent expression of the proinflammatory factor Egr-1, resulting in the reduced expression of the inhibition of plasminogen activator inhibitor-1 (PAI-1), an inhibitor of fibrinolysis (96). Consistently, PAI-1-deficient mice (Serpine1^−/−) were protected from ischemic insult and CO afforded no further protection to these mice (96). In hepatic I/R injury, CO has also been shown to attenuate apoptosis through activation of the miR-34a/SIRT1 pathway (72).

Transplant Rejection

Organ transplantation has been a frequently used model of I/R injury in preclinical studies. I/R injury may also represent an underlying pathogenic mechanism of graft failure during organ transplantation. CO, when applied at low concentration (e.g., 250 ppm), has been demonstrated to reduce apoptosis, inflammation, and transplant-associated I/R injury, thus reducing the incidence of graft rejection and preserving graft function. In a rat model of orthotopic left lung transplantation, exogenous application of CO significantly protected the graft and reduced hemorrhage, fibrosis, and thrombosis (150). Furthermore, CO inhibited lung cell apoptosis after transplantation and attenuated lung and systemic proinflammatory cytokine production (e.g., macrophage inflammatory protein-1α, macrophage migration inhibitory factor, and IL-6) (150). Furthermore, protection against I/R injury was conferred in syngeneic rat orthotopic lung transplantation by inhaled CO administered to either the donor or the recipient (77). Delivery of CO to lung grafts by saturation of the preservation media also reduced I/R injury and inflammation in syngeneic rat orthotopic lung transplantation (78). In a vascular transplantation model, aortic graft transplant recipients exposed to CO displayed reduced vascular remodeling and reduced leukocyte, macrophage, and T cell infiltration in the graft (117). Preexposure of murine pancreatic islet cells to CO improved islet cell survival and function after transplant (56). The mechanisms underlying the protective effects of CO in organ transplantation are currently believed to involve primarily anti-inflammatory, immunomodulatory, and antiapoptotic effects, though other mechanisms such as inhibition of cell proliferation and improved perfusion may contribute to the observed protection (6, 7, 110, 117, 119). In addition to lung and vascular transplantation, the protective effects of CO are now substantiated in transplantation models of the pancreas, heart, intestine, kidney, and liver (7, 42, 56, 69, 87, 110, 111, 139, 174).

Mechanical Ventilation-Induced Lung Injury

Mechanical ventilation (MV) in rodent lungs causes cyclical stretch and activation of sterile inflammatory pathways, resulting in ventilator-induced lung injury (VILI). This experimental model is used as an animal model of acute respiratory distress syndrome (ARDS) in humans. The therapeutic potential of CO has been demonstrated in rodent models of MV-induced VILI (37, 58, 59). In mice subjected to MV at moderate tidal volume, CO conferred tissue protection when administered through the ventilator circuit (37, 58, 59). CO reduced MV-induced proinflammatory cytokine and chemokine production and reduced markers of lung injury such as alveolar protein and cellular influx, lung neutrophil recruitment, and pulmonary edema (58, 59). The protective effects of CO were shown independently to require caveolin-1 (59) and the activation of PPARγ, leading to the inhibition of Egr-1 (58). These studies, taken together, suggest that MV in the presence of CO may provide protection in VILI. However, more studies are required to elucidate the precise mechanisms underlying the therapeutic potential of CO in VILI models.

Bleomycin-Induced Lung Injury (Fibrosis)

Idiopathic pulmonary fibrosis (IPF) is a fatal disease with no known etiology characterized by scarring or thickening of lung tissues associated with fibroblast hyperproliferation and extracellular matrix remodeling (145). Bleomycin, a redox cycling compound that generates O₂⁻ and H₂O₂, is a frequently used chemotherapeutic agent that is a known inducer of pulmonary fibrosis in humans. Intratracheal administration of bleomycin in mice is used to model IPF in animals. Exogenous CO treatment can provide protection against bleomycin-induced fibrotic lung injury in mice (196). In mice treated with bleomycin intratracheally and then exposed to CO or ambient air, the lungs from CO-treated animals displayed reduced lung hydroxyproline, collagen, and fibronectin levels relative to air-treated bleomycin-injured controls. The protective effect of CO in this model was associated with an antiproliferative effect of CO on fibroblast proliferation associated with the increased expression of p21^Waf1/Cip1 and inhibition of cyclins A/D expression (196).

Pulmonary Hypertension

Pulmonary arterial hypertension (PAH) is characterized by progressive pulmonary arteriole smooth muscle remodeling, resulting in increases in pulmonary vascular resistance and, ultimately, right ventricular failure (43). Administration of CO was shown to provide protection in rodent models of monocrotaline-induced and hypoxia-induced PAH. Compared with controls, intermittent exposure to CO reversed established PAH and right ventricular hypertrophy, restored right ventricular and pulmonary arterial pressures, as well as pulmonary vascular morphology. The ability of CO to reverse PAH was dependent on endothelial nitric oxide synthase (eNOS)-dependent NO generation. The protective effect of CO was endothelial cell-dependent and was associated with increased apoptosis and decreased cellular proliferation of vascular SMCs (198). Further studies have shown that CO decreased pulmonary artery vascular resistance and inhibited hypoxic vasoconstriction, through activation of the sGC/cGMP, and the hyperpolarization of potassium channels (39).

Endotoxemia and Sepsis

CO has demonstrated anti-inflammatory effects in animal models of endotoxin shock and sepsis (114). CO preconditioning reduced the production of serum TNF-α, IL-1β, IL-6, and increased the production of the anti-inflammatory cytokine IL-10. Furthermore, CO preconditioning also reduced organ injury and prolonged survival following LPS challenge (101, 114). Anti-inflammatory effects of CO through modulation of pro- or anti-inflammatory cytokine production were diminished in heat shock factor-1 knockout (hsf1^−/−) mice, suggesting a role for the heat shock response in vivo (74). Anti-inflammatory effects of CO have been recently documented in higher mammals including swine and non-human primates (NHPs) (94, 97). CO pretreatment improved organ function and reduced lung inflammation in LPS-challenged swine. CO inhibited serum levels of the proinflammatory IL-1β in response to LPS, while inducing the anti-inflammatory cytokine IL-10 after LPS challenge in this model (94). Anti-inflammatory effects were also reported in NHPs subjected to LPS inhalation (97). CO exposure following LPS inhalation decreased TNF-α release in BAL fluid and reduced pulmonary neutrophilia without affecting IL-6 and IL-8. The anti-inflammatory effects of CO in this model required elevated doses (500 ppm) that were associated with excessive CO-Hb buildup (97). These studies highlight interspecies variation in the dosimetry and efficacy of CO therapy with respect to modulation of inflammation.

In addition to mechanisms related to downregulation of cytokine and inflammatory mediator production, CO has been shown to improve bacterial clearance in sepsis models, though the mechanisms remain incompletely understood (29, 113, 183). The pharmacological application of CORM-2 enhanced bacterial phagocytosis in vivo and rescued HO-1-deficient mice from sepsis-induced mortality after cecal-ligation and puncture (CLP) surgery (29). Furthermore, CORM treatment using the compound ALF-186 improved intestinal bacterial clearance in a model of chronic colitis in Il10^−/− mice, and enhanced bactericidal activity in wild-type and HO-1-deficient macrophages (113). In an Escherichia coli infection model, endogenous HO-derived CO was associated with enhanced macrophage phagocytosis and this was shown to require NLRP3 and caspase-1-dependent immune responses (183). Our laboratory has recently demonstrated that CO inhalation (250 ppm) either as pretreatment or as posttreatment improved mouse survival in the CLP model (86). The protective effects of CO in CLP were dependent on the induction of Beclin 1-dependent autophagy and phagocytosis, the reduction of inflammation, and enhanced bacterial clearance from lung, liver, and blood. The prosurvival effects of CO in CLP were dependent on the autophagy program, as they were reduced in autophagy-compromised (Becn1^+/−) mice (86).

POTENTIAL FOR INHALATION CO AS A THERAPY FOR HUMAN DISEASE

CO has been demonstrated as an effective anti-inflammatory modulator in preclinical models of organ injury and disease, both in rodents and in higher animals (e.g., swine and primates). On the basis of these observations, continuing efforts are underway to develop inhaled CO (iCO) as a therapy for clinical application. Previous clinical trials have demonstrated the safety of iCO application in humans. In a randomized, double-blinded, placebo-controlled, two-way crossover trial, experimental endotoxemia was induced in healthy volunteers by injection of 2 ng/kg LPS, and inhalation CO (iCO) was applied at 500 ppm CO for 1 hour (which increased CO-Hb to 7%), with air inhalation as the placebo control. Under these conditions, iCO did not modulate the inflammatory response as measured by systemic cytokine production, with no adverse effects of iCO reported (93). In a different human clinical trial, iCO was applied to patients with chronic obstructive pulmonary disease (COPD) (11). In this study, ex-smoking patients with stable COPD were subjected to iCO (100–125 ppm, 2 hours/day for 4 days), which increased CO-Hb levels to 4.5%. Application of iCO to patients with stable COPD did not cause adverse effects, and patients exhibited a trend in reduction of sputum eosinophils and improvement of methacholine responsiveness (11). We have recently completed a multicenter phase IIa, double-blinded, sham-controlled, clinical trial, in which IPF patients were randomized to treatment with inhaled CO at 100–200 ppm or to inhaled 21% oxygen for 2 hours/day, twice/week, for 12 weeks (131). Application of iCO did not affect serum concentration of matrix metalloproteinase-7 (MMP7), the primary study end point, following 12 weeks of treatment, relative to controls. No differences were observed in physiologic measures, incidence of acute exacerbations, hospitalization, death or patient-reported outcomes, with no differences in the distribution of adverse events between the groups. We concluded that inhaled CO is well tolerated and can be safely administered to IPF patients, albeit with no significant changes in study end points (131). Our findings warrant further testing the efficacy of iCO in future IPF clinical trials.

CONCLUSIONS

Much has been learned about the cellular signaling networks that are modulated by exposure to CO, including major signal transduction pathways associated with the regulation of inflammation, cell death, and cellular proliferation programs. An emerging concept is that mitochondria may represent a common target of CO action, such that the resulting flux in mitochondria-derived mediators and mtROS generation may be associated with the regulation of downstream signaling effects of CO. Although the heme moieties of cellular and mitochondrial cytochromes have been identified as candidate proximal targets of CO, not all studies have unequivocally identified a heme-dependent mechanism. Furthermore, model and tissue-specific variation in molecular pathways have left some ambiguity and unanswered questions such that no unifying mechanism has emerged. CO has now been shown to confer cyto- and tissue protection in a number of animal models of organ injury and diseases. Further research is needed to identify how discrete signaling targets of CO are integrated in a context-specific fashion to coordinate cytoprotection. In addition to the lung and vascular models covered in this review, salutary effects of CO therapy have been upheld in other diverse models of disease, including metabolic syndrome (83), traumatic brain injury (28), preeclampsia (168), and others. The successful demonstration of cyto- and tissue-protective effects of inhaled CO in preclinical animal models using rodents and higher mammals has led to continued efforts toward the clinical application of CO, both as iCO, or through pharmacological delivery of CORMs. Human clinical trials now in progress attempt to harness the therapeutic effects of CO observed in animal models and aim to characterize the safety and efficacy of iCO as a therapeutic drug. Barriers to progress include differences in pulmonary physiology between humans and experimental animals, as well as regulatory issues. Nevertheless, several studies have now been completed that lend support for the safety and feasibility of iCO therapy (11, 93, 131).

Further progress in therapeutic use of iCO is dependent on the completion and analysis of several ongoing clinical trials in organ transplantation, pulmonary fibrosis, and critical illness (sepsis/ARDS) (104, 108). At present the therapeutic potential of CO remains to be realized in the clinical setting.

Glossary

AMPK

AMP-activated protein kinase

ARDS

Acute respiratory distress syndrome

BK_Ca

Ca²⁺-dependent K⁺ channels

BMDM

Bone-marrow-derived macrophages

BVR

Biliverdin reductase

CLP

Cecal ligation and puncture

CO

Carbon monoxide

CO-Hb

Carboxy-hemoglobin

CORM

Carbon monoxide releasing molecule

cGMP

Guanosine 3′,5′-monophosphate

Egr-1

Early growth response-1

EnaC

Epithelial Na⁺ channels

HO-1

Heme oxygenase-1

HIF-1α

Hypoxia-inducible factor-1-α

H₂S

Hydrogen sulfide

iCO

Inhaled CO

IL

Interleukin

I/R

Ischemia-reperfusion

JNK

c-Jun NH₂-terminal kinase

MAPK

Mitogen-activated protein kinase

MSCs

Mesenchymal stromal cells

mtROS

Mitochondrial reactive oxygen species

MV

Mechanical ventilation

NHP

Non-human primate

NO

Nitric oxide

NOX

NADPH oxidase

NF-κB

Nuclear factor-κB

NLRP3

NOD, leucine rich region- and pyrin domain-containing-3

NRF-1

Nuclear respiratory factor-1

Nrf2

NF-E2-related factor-2

sGC

Soluble guanylate cyclase

p21^Waf1/Cip1

Cyclin-dependent kinase inhibitor-1

PGC-1α

Proliferator-activated receptor-γ coactivator-1α

PI3K/Akt

Phosphoinositide 3-kinase/Akt

PPARγ

Peroxisome proliferator-activated receptor-γ

ROS

Reactive oxygen species

RvD1

Resolvin-D1

SMC

Smooth muscle cell

TLR4

Toll-like receptor-4

VILI

Ventilator-induced lung injury

GRANTS

This work was supported by National Institutes of Health-National Heart, Lung, and Blood Institute Grants P01 HL-108801, R01 HL-079904, and R01 HL-060234 (A. M. K. Choi).

DISCLOSURES

A.M.K. Choi is a co-founder of Proterris, Inc., and is on the Scientific Advisory Board at Proterris. No other conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

K.C.M. prepared figures; S.W.R. and K.C.M. drafted manuscript; S.W.R., K.C.M., and A.M.K.C. edited and revised manuscript; S.W.R. and A.M.K.C. approved final version of manuscript.