Heme Oxygenase-1: From Bench to Bedside

CONTENTS

A Brief History of Heine Oxygenase

Products of Heme Oxygenase Activity

Carbon Monoxide

Bilirubin

Iron

Heme Oxygenase and Nitric Oxide

Heme Oxygenase and the Lung

Acute Lung Injury

Asthma

Chronic Obstructive Pulmonary Disease

Pulmonary Fibrosis

Pulmonary Vascular Disease

Lung Transplantation

Exhaled Breath Analysis of Carbon Monoxide

Clinical Application

Summary and Future Directions

And from the standpoint of medicine as an art for the prevention and cure of disease, the man who translates the hieroglyphics of science into the plain language of healing is certainly the more useful.

-Sir William Osler (1849-1919)

Increasingly in clinical medicine we are asked to look beyond perturbations of physiology or anatomic abnormalities to the molecular basis of disease and health. Tools to detect global changes in gene and protein expression in response to stress are now at our disposal, allowing insights into disease in ways that once seemed science fiction. With these new technologies, however, comes a bewildering array of new information for the medical practitioner to digest. Sometimes the molecule of the moment, like nitric oxide, finds a central place in medical school curricula everywhere. Other discoveries remain lost-at least for a time-in the noise. One of several molecules emerging from the background as an important regulator and mediator of diseases of the lung and intensive care unit is the enzyme heme oxygenase-1 (HO-1). The seemingly pedestrian function of this enzyme is to break down heme to release bilirubin, carbon monoxide, and iron. The products released in the dismantling of heme could be viewed as mere waste, perhaps toxic in their own rights, but the parallels between carbon monoxide and nitric oxide generation by the body hint at something more. We now know nitric oxide to be a highly biologically active and pluripotent molecule. Could it be that the products of heme catabolism function in similar fashions? As we describe in this article, the consequences of the simple act of heme catabolism reach beyond what the original discoverers of heme oxygenase first imagined.

A BRIEF HISTORY OF HEME OXYGENASE

The story of heme oxygenase takes us back to our beginnings. As oxygen gathered in the primordial nitrogen-containing atmosphere, our distant evolutionary ancestors were confronted simultaneously with a new opportunity and a new danger. As pulmonologists are acutely aware, the oxygen necessary to sustain multicellular life is inherently toxic. To make use of oxygen, cells had to evolve mechanisms for surviving oxidative stress. One of the most ubiquitous such mechanisms is the enzyme heme oxygenase. From algae to humans, this enzyme is remarkably preserved; this preservation is an indication of its success as a strategy for protection and its importance for survival. As a measure of the critical nature of this enzyme, there has been one reported case of heme oxygenase deficiency in a human, and the afflicted boy did not survive childhood (1). It is likely that mutations of heme oxygenase are generally lethal in utero, and indeed the mother of this boy had experienced two intrauterine fetal deaths.

Heme oxygenase was discovered in 1968 when Tenhunen and colleagues described the mechanism for catabolism of heme (2). Subsequently, three distinct isoforms of the enzyme were described. Two of these are constitutively activated (HO-2 and HO-3) and one is an inducible form (HO-1) (3, 4). It was not immediately evident that heme oxygenase might have a function beyond recycling heme, in itself an important function. On the other hand, it was not abundantly evident why so many organisms would devote energy to the degradation and resynthesis of hemoproteins. The spleen is required to remove a harmful compound such as heme from the circulation and to facilitate the recovery of iron from senescent red blood cells; hence the spleen expresses a high level of HO-1. However, it is not obvious why heme oxygenase should be present in other organs or in organisms without circulating blood-such as algae-where the need for hemoprotein turnover is less clear. Moreover, the ability of HO-1 to be induced by stimuli other than free heme makes little sense if the sole object of activation is heme catabolism. Indeed, HO-1 is induced by a remarkable array of cellular insults and this is a key to understanding its function.

Stress-induced molecules are proteins generated or activated by cells in response to stressors such as fever, heat, infection, toxins, or radiation. They are able to not only protect cells against danger posed by an immediate stress but also fortify cells to withstand future stresses, even from a different source. For example, an animal exposed experimentally to a sublethal dose of lipopolysaccharide, simulating a bacterial infection, will later be better equipped to survive toxic levels of oxygen than an animal that was not exposed to lipopolysaccharide (5). In the 1980s, reports emerged of a 32-kD mammalian stress protein induced by a wide range of stresses (6-8). The protein became known as heat shock protein-32 (HSP-32) by virtue of its induction by hyperthermia in the rat (9). It soon became clear that HSP-32 was identical to the previously described HO-1 (10, 11). Although the human HO-1 gene is not inducible by hyperthermia (10) (except in the hepatoma cell line Hep3B [12]), the HSP-32 nomenclature is still occasionally used to refer to human HO-1.

PRODUCTS OF HEME OXYGENASE ACTIVITY

To understand the function of HO-1, it is necessary to examine the products of its activity. In breaking down heme, HO-1 releases carbon monoxide, iron, and biliverdin, which is subsequently converted to bilirubin (Figure 1). Each of these products of enzymatic activity plays unique and often protective roles in the human body.

Carbon Monoxide

Carbon monoxide, a colorless, odorless diatomic gas, is best known in the medical community for its lethal properties. In 1963, however, the physiologist R. Coburn and colleagues at the University of Pennsylvania demonstrated that CO is produced by the bodies of humans and other animals (13). Interestingly, this discovery was made two decades before Ignarro and Moncada famously proved that another diatomic gas, nitric oxide, is also produced in our bodies (14, 15). Unlike the gas championed by Ignarro, Moncada, and others, the functional physiologic role of carbon monoxide languished in relative obscurity until recently. However, since the 1990s, a number of investigators have shown that low concentrations of CO can exert biological functions as diverse as neurotransmission (16, 17), protection against cell death (18), antiinflammation (19), protection against oxidative injury (20), inhibition of cell proliferation (21, 22), and tolerance of organ transplantation (23, 24). Analogous to NO, some activities of CO have been shown to be cyclic guanosine monophosphate (cGMP) dependent, such as inhibition of platelet aggregation and smooth muscle relaxation (25-27). Other CO-mediated effects are independent of cGMP, involving the mitogen-activated protein kinase (MAPK) pathways or other as yet undescribed pathways (Figure 2). The MAP kinases constitute hierarchic phosphorylation cascades responsible for transducing inflammatory signals from the cell surface through to the nucleus, which may result in cellular activation and production of cytokines that amplify inflammation (Figure 2B). The antiinflammatory action of CO has been shown to involve the downregulation of proinflammatory cytokines, such as tumor necrosis factor a, interleukin (IL)-1ß, and IL-6 together with augmentation of the antiinflammatory cytokine IL-10 via the p38 and c-Jun N-terminal kinase-MAPK pathways (19, 28). The antiapoptotic action of CO has also been shown to involve the p38 MAPK system (18). The biochemical mechanism by which nonreactive CO activates MAPKs is not clear at this time. One can speculate that CO affects phosphorylation of MAPKs either indirectly by modulating the redox state of cells or by directly activating hemecontaining moieties, which in turn could promote MAPK phosphorylation. A report by Maines and colleagues demonstrating that biliverdin reductase can be phosphorylated suggests that byproducts of heme catalysis such as CO or bilirubin could affect the phosphorylation state of signaling molecules (29).

In addition to being an effector of the actions of HO-1, carbon monoxide may also serve as a marker for enzyme activity. Changes in the rate of production of CO can be measured by breath excretion, and the gas could theoretically be used to monitor the course of diseases. This method for monitoring disease is appealing as it is fast, noninvasive, and could be used serially to gauge responses to interventions. Investigators in this field envision using levels of CO and other molecules in the breath to monitor the course of disease in the same way pulmonary function testing is used today.

A further clinical application for CO could involve therapeutic administration of the gas to grafts before transplantation or to individuals by inhalation in the way NO is used in some clinical situations now. The concentration of CO currently being used in animal studies is in the range of 250-500 ppm (Table 1) (30-48), and beneficial effects have been observed at doses as low as 10 ppm. For reference, the concentration of single-breath CO used to measure diffusing capacity during pulmonary function testing is 3,000 ppm, and the current guideline for workplace limits of CO exposure over an 8-hour work day is 35-50 ppm (49, 50). The carboxyhemoglobin concentrations at equilibrium associated with these levels of inhaled carbon monoxide are outlined in Table 2 (51, 52). The exposure time required to achieve equilibrium carboxyhemoglobin levels varies from 8 hours for the highest concentration of 1,000 ppm to 24 hours for a concentration of 25 ppm (52).

Bilirubin

Long considered a waste product of heme catabolism, bilirubin has also enjoyed a rehabilitation of its reputation. Biliverdin, the precursor to bilirubin, has been found to have antiviral activity, and bilirubin is now recognized as being a potent antioxidant manufactured by the body. In neural cell culture, for example, a minute amount of bilirubin is capable of protecting cells from 10,000-fold higher concentrations of the oxidant hydrogen peroxide (53). Normal human serum bilirubin concentrations are high enough to provide a substantial portion of the total known antioxidant capacity of serum (54). Clinical implications of this antioxidant role for bilirubin are beginning to be realized. For instance, higher baseline serum bilirubin levels have been shown to correlate with a lower incidence of myocardial infarction in men, probably because of the inhibitory effect of bilirubin on the oxidation of low-density lipoproteins (55). It has been proposed that the common hyperbilirubinemia of newborns, although in some circumstances resulting in neurologic toxicity, may represent a protective adaptation to counter the oxidative effect of breathing oxygen for the first time (56, 57).

Iron

Free iron is capable of participating in deleterious oxidation reactions, and for this reason it is carefully sequestered in biological systems. By releasing iron from heme, HO-1 is potentially contributing to a prooxidant state within the cell. However, the iron released by HO activity also increases the synthesis of ferritin, which stores free iron and has well-known cytoprotective properties. It has been argued that some of the protective effect of HO-1 induction is attributable to the increase in cellular ferritin that rapidly follows (58). In addition, HO-1 can repress the expression of a highly active ferrous iron-ATPase transporter involved in iron efflux from cells (59). In the absence of HO-1, iron efflux from cells can be decreased and can potentially contribute to cell death. Mice with a deletion of the HO-1 gene display increased tissue and intracellular accumulation of iron (60), supporting the thesis that coregulation of ferritin and the iron transporter may contribute to the cellular protection conferred by HO-1.

HEME OXYGENASE AND NITRIC OXIDE

The parallels between heme oxygenase and nitric oxide synthase, the enzyme that gives us nitric oxide, are difficult to ignore. Both enzymes have constitutively expressed and inducible forms, both generate diatomic gases, both inducible forms are responsive to a similar array of stimuli, and both are present in multiple organs and tissues. The gases produced by these two systems share similar molecular masses, solubilities in water, and basal rates of production (61). Some of the functional similarities between these gases are outlined in Table 3. Important differences exist as well. There are differences in the specific inducers and regulators of the enzymes that lead to the production of CO or NO. For instance, low oxygen tension has been shown to induce HO-1 activity but not nitric oxide synthase activity in smooth muscle cells, suggesting that CO may be responsible for regulating smooth muscle cell tone under conditions of hypoxia (62). Significant differences also exist between the two gaseous enzymatic products. NO, with its unpaired electron, is a free radical capable of undergoing various oxidative and reductive reactions, whereas CO is relatively inert. The similarities and differences between these enzymes are not mere curiosities but point to the functions and possible interactions between these key gas-generating systems.

Nitric oxide has well established functions as a signaling molecule in the vascular system and neuronal system. In addition, NO can affect airway smooth muscle, inhibit platelet aggregation, and participate in host defense in the lung and elsewhere. The effects of NO on pulmonary vascular tone and matching of ventilation with perfusion have been studied extensively, in part with the hope that inhaled NO could be a viable therapy for diseases involving pulmonary hypertension and ventilation-perfusion mismatch. Administration of inhaled NO has been attempted with variable success to treat chronic obstructive pulmonary disease (COPD) (63, 64), persistent pulmonary hypertension of newborns (65, 66), and severe hypoxemia in both infants (67) and adults with adult respiratory distress syndrome (68, 69) and in those who have undergone lung transplantation (70, 71). If inhaled NO has failed so far to achieve its full promise as a therapy for lung disease, this may be due to properties intrinsic to the molecule. NO has an affinity for hemoglobin that is about 1,500 times that of CO (72). This means that NO binds or reacts with hemoglobin extremely quickly, leading to an effective half-life in the lung on the order of a few seconds, and probably limits its effect to the immediate lung tissue with which it comes in contact (73). Furthermore, although NO is quite stable under anaerobic conditions, it will form more potent oxidizing agents such as nitrogen dioxide when exposed to oxygen (74). NO reacts even more efficiently with superoxide anion, to form the potent oxidant peroxynitrite, than does superoxide dismutase with superoxide anion (75). Peroxynitrite has been implicated in airway or alveolar inflammation and edema (76) and inhibits the ability of surfactant protein A to aggregate phospholipids (77). Although it is not known how much inhaled NO might contribute to lung injury, there is some evidence of deterioration in lung function during inhalation of NO in healthy volunteers. In a study of 191 normal subjects inhaling NO, the study subjects experienced a mean fall in arterial oxygen tension of 7 mm Hg at low doses (15-20 ppm); at higher concentrations airway resistance increased (78).

Limits to the therapeutic value and potential toxicities of NO have not caused it to be abandoned as a possible treatment for lung disease. Rather, new avenues for achieving the same ends are now being explored, such as manipulation of the cGMP signaling pathway or use of NO donors. Another approach that has been suggested is to exploit the connection between NO and heme oxygenase. In 1995, investigators at the University of Pittsburgh demonstrated that increasing cellular nitric oxide levels lead to increased expression of heme oxygenase (79). This has since been confirmed by others (80-82) and has led to speculation that HO-1 is in fact responsible for cytoprotective effects attributed to NO. It could be theorized that a switch has evolved for circumstances of great stress in which NO, as a highly reactive molecule best at mediating local events, turns on HO-1 to release bilirubin and CO with their antiinflammatory, antiapoptotic, and cytoprotective effects. This theory is attractive in that NO might be bypassed as a therapy in favor of more "downstream" molecules.

The truth of the interactions among heme oxygenase, nitric oxide, and carbon monoxide is probably much more complicated than HO-1 acting as a downstream effector for NO. Although NO and CO share many biological functions such as neurotransmission, vasoregulation, and inhibition of platelet aggregation, they are not interchangeable molecules. There are circumstances in which NO and CO have been shown to have discrete, synergistic, or even antagonistic effects (83-85). It has also been proposed that HO activity may serve under some circumstances to modulate NO production; in fact, both increased HO-1 activity and exogenously administered CO have been shown to inhibit NOS and thereby suppress NO generation (86, 87). More recently, CO was shown to induce NO production in a model of liver injury (88). Although clearly both NO- and CO-generating systems participate in a wide variety of critical biological events, there is still much to be learned about the interactions between these two systems.

HEME OXYGENASE AND THE LUNG

The lung, as the interface between the atmosphere and the rest of the body, is particularly vulnerability to oxidative injury. In addition, the lung shares with other organs the risk of injury due to infections, inflammation, ischemia-reperfusion, and other insults. Lungs therefore require potent defense mechanisms, and in fact have higher levels of antioxidant enzymes than almost any other organ. Many of these enzymes, such as catalase and glutathione peroxidase, are constitutively active and protect the lungs against everyday insults. HO-1 is one of the few inducible molecules that can protect the lungs from an increased oxidant burden under more stressful circumstances. One illustration of this role for HO-1 comes from an experiment conducted in the intensive care unit, where investigators drew arterial and central venous blood samples to compare the levels of carboxyhemoglobin. It had previously been assumed that carbon monoxide was predominantly produced in the spleen and liver and subsequently excreted by the lungs. If this were the case, one would expect to find higher venous than arterial levels of carboxyhemoglobin. Surprisingly, these investigators found not only that arterial and central venous carboxyhemoglobin levels were higher in critically ill patients than in healthy humans, as one might expect, but that arterial carboxyhemoglobin was significantly higher than central venous carboxyhemoglobin, suggesting that a significant amount of CO is being generated by the lungs (89). CO is also excreted by the lungs, and this opens new possibilities for monitoring heme oxygenase activity in the body.

Acute Lung Injury

In the lungs, HO-1 is highly expressed in alveolar macrophages, but is also found in epithelial cells, fibroblasts, and endothelial cells (90-92). Many of the initial studies done to characterize the location and function of HO-1 in the lung used rodent models of acute lung injury, such as hyperoxia. The delivery of high concentrations of oxygen to the lung is likely harmful to humans, and certainly causes lung damage in other animals. Mice and rats exposed to more than 95% oxygen develop lung edema and die within 70 to 100 hours of continuous exposure. In this model of lung injury, increased immunohistochemical staining for HO-1 protein is seen diffusely throughout the alveolar and bronchiolar epithelium and the infiltrating cells, indicating that the concentration of the enzyme is increased (93). When the lungs are homogenized and assayed for function of HO-1, the enzyme activity is also found to be increased (93). This provides circumstantial evidence of the importance of HO-1 in protection against lung injury, but further proof comes from experiments showing changes in lung response to injury based on increased or decreased activity of HO-1. Thus, chemical inhibition of HO-1 activity will increase the susceptibility of rats to lung injury from endotoxin (94). Conversely, when the expression of HO-1 is increased in the bronchiolar epithelium by adenoviral gene transfer, the rats are protected against injury when subsequently exposed to hyperoxia (95). Exposure of mice to a product of HO-1 activity, carbon monoxide, also results in protection against hyperoxic lung injury (96). These experiments show that HO-1 is increased in the setting of lung injury, and that its increased activity serves to protect the lung against damaging influences.

In attempting to translate findings from rodent studies to humans, consideration must be given to the differences in expression of the HO-1 gene among species. The induction of HO-1 is regulated at the level of transcription, and some differences exist in the molecular mechanisms regulating basal and inducible transcription of the rat, mouse, and human HO-1 genes. The level of expression of HO-1 is influenced by the proximal promoter region, the proximal enhancer, and distal enhancers. As was previously mentioned, the rat HO-1 protein is classified as a heat shock protein because of its responsiveness to thermal stress. The 5' regulatory region of the rat gene contains consensus heat shock elements similar to those found in the promoter regions of other heat shock genes (9). The mouse HO-1 promoter also contains three putative heat shock elements, but in vivo HO-1 transcription is not induced by thermal stress (97). Instead, most transcriptional activity in the mouse is mediated by multiple copies of the stress response element located within two upstream enhancer regions (98). The stress response element sequences are essentially conserved between human and mouse HO-1 genes, suggesting that these play a dominant role in humans as well. A new regulatory locus was described in the human gene, however, and ongoing work may uncover substantive differences between mouse and human HO-1 gene regulation (98). This new regulatory locus may represent a novel enhancer internal to the human HO-1 gene that, in conjunction with the HO-1 promoter, regulates induction of the endogenous HO-1 gene.

In addition to animal studies demonstrating increased resistance to hyperoxia after gene transfer of HO-1 or treatment with CO, in vitro studies have shown that the overexpression of HO-1 in lung epithelial cells or rat fetal lung cells causes growth arrest and prevents apoptosis in response to increased oxygen tension (99). This suggests that HO-1 may protect against hyperoxia-induced lung injury by conferring resistance to cell death. This scheme is complicated, however, by studies from Dennery and colleagues. Dennery's group showed that mice lacking the gene for HO-2 were more sensitive to the lethal effects of hyperoxia than were wild-type mice (60), but that HO-1 gene-deleted mice were actually more resistant to hyperoxia than the corresponding wild-type mice (100). Work by Piantadosi and colleagues further showed that although oxygen tolerance could be induced in rats by increasing HO-1 activity, this tolerance also occurred in the presence of HO inhibitors (101), bringing into question the mechanism of protection. Although the preponderance of evidence points to a protective role for HO-1 and CO in hyperoxic lung injury, much remains unknown about the mechanism of this protection.

Asthma

If asthma is considered the result of two main pathogenic mechanisms, bronchospasm and inflammation, there are theoretic grounds to seek the involvement of heme oxygenase in both. Two products of heme oxygenase activity, carbon monoxide and bilirubin, have well described antiinflammatory properties. In addition, carbon monoxide, by increasing levels of cGMP, could theoretically lead to relaxation of bronchial smooth muscle in a manner analogous to nitric oxide. There is now reasonably good evidence that HO-1 activity is affected by the course of asthma, and it appears probable that HO-1 activity also affects the course of disease. Work by several groups of investigators has shown that levels of HO-1 expression and exhaled CO are higher in subjects with asthma than in normal subjects (102-104) and that exhaled CO levels increase with allergen challenge (105) and decrease with steroid treatment (106, 107). Carboxyhemoglobin levels have also been shown to increase during exacerbations and to decrease after treatment (108). Not all studies have shared similar findings, however; some investigators have reported no change in exhaled CO with allergen challenge or steroid treatment (109-111).

New experimental data point to a possible therapeutic effect for heme oxygenase in asthma. Heme oxygenase and bilirubin have been shown to decrease airway smooth muscle contractility in an ex vivo model (112). Similarly, low-concentration inhaled carbon monoxide both attenuates aeroallergen-induced inflammation (47) and reduces airway hyperresponsiveness in mice (41). Heme oxygenase may also have an inhibitory effect on the process of lung remodeling that is seen in asthma. In a guinea pig model of aeroallergen-induced airway remodeling, activation of heme oxygenase prevented the increase in bronchial smooth muscle area, whereas inhibition of HO had the opposite effect. The investigators attributed this finding to the antiproliferative effect of bilirubin, which they showed was capable of inhibiting human airway smooth muscle proliferation in vitro (113). Similarly, low concentrations of carbon monoxide have been shown to inhibit human airway smooth muscle cell proliferation (22).

Chronic Obstructive Pulmonary Disease

Exposure to reactive oxygen species and an imbalance in oxidant-antioxidant status are thought to be major contributors to the pathogenesis of COPD. Glutathione presents a first line of defense against oxidants in the lung; when glutathione is depleted, HO-1 expression is increased, suggesting a complementary defensive role against oxidative stress (114, 115). It is not surprising therefore that accumulating evidence points to a role for HO-1 in the pathogenesis of COPD. Smoking has been shown to increase airway expression of HO-1 (116); however, patients with COPD have lower HO-1 expression in alveolar macrophages than do control subjects (117). A microsatellite polymorphism in the promoter for HO-1 has been associated with the development of emphysema, and this polymorphism was shown to cause decreased promoter responsiveness to hydrogen peroxide in vitro (118). This suggests that an abnormal response of HO-1 to oxidative stress may be linked to the development of COPD. Further work needs to be done to better define the involvement of HO-1 in the pathogenesis of COPD.

Pulmonary Fibrosis

The mechanism by which pulmonary fibrosis is initiated and progresses remains undetermined. Most theoretic schemes focus on the fibroblast and its response to interactions with other cells in the lung because it is the final common mediator of fibrosis. Cell-cell interactions are complex, however, and leukocytes, fibroblasts, and epithelial cells are all capable of influencing one another in almost any combination. The part that HO-1 plays in this intricate process is poorly defined but represents an area of active research. Immunohistochemical analysis of lung tissue from patients with various forms of interstitial lung disease reveals increased expression of HO-1, primarily in alveolar macrophages (119). A salutary effect for HO-1 in fibrotic lung disease has been proposed, and there is now mounting evidence that this is the case. A report described suppression of lung fibrosis in a murine bleomycin model by adenoviral transfer of the HO-1 gene; the investigators attributed this effect to inhibition of apoptotic cell death (120). Both bilirubin (121) and inhaled carbon monoxide (30) have been shown to have similarly suppressive effects on fibrosis in bleomycin models of lung injury. Low concentrations of carbon monoxide slow fibroblast proliferation, inhibit matrix synthesis, and alter the cytokine milieu, which may in part account for a modulatory effect on fibrosis (30, 122). A report (123) demonstrating the protective effects of HO chemical inhibitor on bleomycin-induced fibrosis is surprising, considering the above-mentioned reports that HO-1 and its by-products CO and bilirubin attenuate bleomycin-induced pulmonary fibrosis (30, 121, 122). This differential effect may reflect either the nonselective effect of the chemical inhibitor of HO used in this study or the complex mechanism by which HO-1 may regulate differential responses of the lung to profibrotic effects of bleomycin.

Pulmonary Vascular Disease

Some of the earliest studies examining the biological actions of HO-1 in the body focused on vascular effects. Because CO was known to induce cGMP, it was hypothesized that CO might regulate vascular tone in a manner similar to nitric oxide. In 1987, increased coronary blood flow in response to CO was demonstrated in isolated perfused rat hearts (124). Subsequently, HO-1 and CO were shown to have effects on systemic blood pressure and hepatic blood flow in animal models (125, 126). CO has direct vasodilatory activity via activation of soluble guanylate cyclase in smooth muscle cells (127), and it can also affect vascular tone indirectly through inhibition of the vasoconstrictors endothelin-1 and platelet-derived growth factor-B (128). In addition to these vasodilatory effects, CO may also prevent vascular remodeling by inhibiting smooth muscle cell proliferation (21, 22). Both hypoxia (21) and ischemia-reperfusion injury (48) are potent inducers of HO-1, and this could represent the body's attempt to restore blood flow and oxygenation to tissues or to limit the oxidative injury associated with decreased blood flow.

Evidence from animal studies supports a role for HO-1 in pulmonary vascular disease. Enhanced activity of HO-1 in the lung has been shown to prevent the development of hypoxic pulmonary hypertension and to inhibit structural remodeling of the pulmonary vessels in rats (129). Conversely, HO-1 null mice have been shown to have maladaptive responses to hypoxia, with severe right ventricular dilation, infarcts, and mural clot formation (130). Studies have also demonstrated protection against vascular injury by CO (131) and vascular constriction by HO-1 (132) involving an antiproliferative effect on vascular smooth muscle cells. The mechanism(s) by which HO-1 and CO mediate this effect on proliferation may involve the p21 pathway (131-134). Interestingly, overexpression of HO-1 can also promote apoptosis of vascular smooth muscle cells (135). These antiproliferative and proapoptotic effects of HO-1 in vascular cells may play critical roles in the remodeling and repair process in vascular injuries. In an animal model of hepatopulmonary syndrome, NO-dependent increases in HO-1 expression appeared to contribute to the blunted hypoxic pressor response seen in this condition (136). Evidence from human studies is still lacking, but given the interest in the role of NO in pulmonary vascular disease, studies of HO-1 and CO are likely to follow.

Lung Transplantation

Lung transplantation represents the last therapeutic option for advanced lung disease of many etiologies. Unfortunately, the success of lung transplantation has been limited when compared with that of other organ transplants, such as kidney or liver. Much of the disparity in outcomes may be attributed to the high rate of graft failure in lung transplantation due to bronchiolitis obliterans (BO). BO accounts for more than 30% of all deaths occurring after the third postoperative year ( 137). The pathologic progression of BO is thought to be due to ineffective epithelial regeneration and aberrant tissue repair in response to various insults, leading to excessive fibroproliferation (138).

HO-1 expression is increased in alveolar macrophages of lung transplant recipients with either acute or chronic rejection (139), implying a possible immunomodulatory effect akin to that shown in a rat liver allograft model, where HO-1 was shown to induce helper T cell type 2-dependent cytokines, such as IL-4 and IL-10, while suppressing interferon-? and IL-2 production (140). In a rat orthotopic lung transplantation model, low-concentration CO exposure was shown to protect against rejection (23). The investigators demonstrated both antiinflammatory and antiapoptotic effects of CO; both of these effects may be contributing factors to the graft protection afforded by CO. Another publication points to an immunomodulatory role for CO via suppression of lymphocyte proliferation; this would also be expected to improve graft survival in transplantation (141). Transplanted organs are vulnerable to ischemia-reperfusion injury as well as immunologic assaults, and both HO-1 and CO have been shown to protect lungs against this sort of injury (48, 142). In a lung model of ischemia-reperfusion injury, protection by CO was attributed to the suppression of plasminogen activator inhibitor-1 (48). Thus, theoretic considerations and animal studies point to possible beneficial effects of HO-1 in the setting of lung transplantation, but human studies are needed to finally define the role of HO-1.

EXHALED BREATH ANALYSIS OF CARBON MONOXIDE

There has been increasing interest in the analysis of breath constituents as a way of diagnosing and monitoring disease in the lungs. Carbon monoxide is one of several gases generated in the body that can be noninvasively measured in exhaled breath. Approximately 80% of the CO formed from heme oxygenase activity is exhaled (143), which makes breath measurement a potentially viable way to monitor changes in enzyme activity. It could thus theoretically be possible to monitor the degree of inflammation and oxidative stress in diseases such as asthma, chronic obstructive pulmonary disease, and cystic fibrosis. Indeed, increases in the level of exhaled CO have been reported in asthma and other lung diseases (102-104), and these levels have been shown to change with therapy or exacerbations (105-107). Because the techniques for monitoring breath are completely noninvasive, they could theoretically be used repeatedly to give information about the kinetics of disease, and they could be used in children or individuals who are too sick to perform lung function testing. In fact, one promising application for this technology is in monitoring hyperbilirubinemia of newborns, as CO and bilirubin are produced in equimolar amounts. A good correlation has been found between infant end-tidal CO concentration and serum bilirubin concentration (144).

Although there has been growing interest in monitoring diseases by breath analysis, this technique is only just beginning to find a role outside the research laboratory. Problems with monitoring exhaled levels of CO include significant variations in the concentration of exhaled gas among individuals with the same disease and confounding variables such as smoking or environmental CO (145). Techniques for monitoring exhaled gases are still being refined; CO can be measured in real time or in collected breath, and there is some debate as to what procedures are most accurate and reproducible. Variations in ventilation will result in changes in the concentration of exhaled gases, which can also limit the accuracy of measurements. Although these technical difficulties need to be resolved, it is possible that with refined techniques for measuring exhaled breath and more universally accepted standards, these tests will eventually find their way into the offices of general pulmonologists.

Clinical Application

The abundant preclinical data described above, demonstrating the functional role of HO-1 in models of human lung diseases, have established a solid foundation for future application to human lung diseases. Undoubtedly, more rigorous investigations are needed to determine whether heme oxygenase and its byproducts, be it carbon monoxide, bilirubin, or ferritin, can be used either as diagnostic biomarkers or as therapies to treat human lung diseases. These rigorous investigations need to address critical questions regarding the optimal pharmacokinetics of HO-1 detection or delivery, mode of exogenous delivery, cost, and competing therapy. Furthermore, significant challenges lie ahead in the potential application of inhaled CO in human diseases. The known toxicity and lethality of CO poisoning from tissue hypoxia are well known when humans are accidentally exposed to high levels of CO. The experiences of the scientific community with inhaled NO can provide invaluable insights concerning the protocol needed for inhaled CO. However, because of the avid binding of CO to hemoglobin (Table 2) and its commensurate ability to cause tissue hypoxia, the major challenges facing investigators interested in delivering inhaled CO to patients lie in the "safe" dosing of inhaled CO. The algorithm reported by Peterson and Stewart represents a viable starting point in guiding us to the levels of carboxyhemoglobin achieved in the context of duration and concentration of inhaled CO delivery (146). A report by Motterlini and colleagues in using CO-releasing compounds (147) to achieve vascular effects similar to those of CO lends promise to perhaps viable alternative methods to administer CO. Although CO-releasing compounds, being chemicals, open new commercial opportunities and technical problems, they represent viable options to circumvent the toxicity of increased carboxyhemoglobin levels with inhaled CO.

SUMMARY AND FUTURE DIRECTIONS

If we knew what we were doing, it wouldn't be called research, would it?

-Albert Einstein (1879-1955)

So far, the basic research on heme oxygenase has only just begun to make an impact on clinical medicine. Some diseases may be associated with hypofunction of the constitutive HO-2 system. Examples of these could include vascular disease or defects in neurotransmission. Other disease states could be improved by increased activity of the inducible enzyme, HO-1, as outlined above. The obvious target and ultimate goal of most research in HO-1 is to find a therapeutic use, and there have been several approaches to this issue. Gene transfer has been attempted in animals with promising results, although current limitations to this approach in humans are widely appreciated. The antioxidant and cytoprotective effects of CO, bilirubin, and ferritin have been demonstrated experimentally, but given the potential toxicities of these products of HO-1 they have yet to be used therapeutically in human studies. Whether bilirubin levels could be safely or successfully manipulated in humans is unknown. Low concentrations of inhaled CO are currently used diagnostically to estimate lung-diffusing capacity in patients, so it is not unthinkable that CO could be used therapeutically. The concentration of gas used in some animal experiments would result in carboxyhemoglobin levels close to those found in smokers, and so may not critically affect oxygen delivery. CO-releasing molecules (transition metal carbonyls) developed by Motterlini and colleagues (146) represent a novel approach to administering CO without inhalation, which could be beneficial in some disease states. It is probable that we will see, in the not too distant future, human trials involving the heme oxygenase system. Only after human studies have been completed will we know for certain whether heme oxygenase research can complete the journey from bench to bedside.

Conflict of Interest Statement: D.M. has received a GEMI Fund research grant for study of biological gases. A.M.K.C. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

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