Heme Oxygenase-1
The "Emerging Molecule" Has Arrived

Danielle Morse and Augustine M. K. Choi

Department of Medicine, Division of Pulmonary, Allergy and Critical Care Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania

	Abstract

Top Abstract Introduction Induction and Regulation of... HO-1 and Signal Transduction HO-1 and Apoptosis HO-1 and Nitric Oxide HO-1 and Diseases of... Diagnostic and Therapeutic Uses... Conclusion References

Organisms on our planet have evolved in an oxidizing environment that is intrinsically inimical to life, and cells have been forced to devise means of protecting themselves. One of the defenses used most widely in nature is the enzyme heme oxygenase-1 (HO-1). This enzyme performs the seemingly lackluster function of catabolizing heme to generate bilirubin, carbon monoxide, and free iron. Remarkably, however, the activity of this enzyme results in profound changes in cells' abilities to protect themselves against oxidative injury. HO-1 has been shown to have anti-inflammatory, antiapoptotic, and antiproliferative effects, and it is now known to have salutary effects in diseases as diverse as atherosclerosis and sepsis. The mechanism by which HO-1 confers its protective effect is as yet poorly understood, but this area of invetsigation is active and rapidly evolving. This review highlights current information on the function of HO-1 and its relevance to specific pulmonary and cardiovascular diseases.

	Introduction

Top Abstract Introduction Induction and Regulation of... HO-1 and Signal Transduction HO-1 and Apoptosis HO-1 and Nitric Oxide HO-1 and Diseases of... Diagnostic and Therapeutic Uses... Conclusion References

Although life on this planet almost certainly began under anaerobic conditions, the environment changed ~ 2.5 billion years ago with the addition of oxygen to the atmosphere by early oxygenic photoautotrophic cells. Unwittingly, these cyanobacteria created an environment hostile to their very survival. The subsequent evolution of aerobic respiration made the best of a bad situation and allowed for the evolution of higher life forms, but our cells continue to contend with the toxic effects of oxygen. Over the millennia, cellular defense mechanisms have evolved as protection against oxidative stress. Prototypical antioxidants include superoxide dismutases, catalases, peroxidase, and nonenzymatic antioxidants such as vitamin C and vitamin E. One of the less well known but critical defenders of cellular homeostasis is the microsomal enzyme heme oxygenase.

Heme oxygenase (HO) made its debut onto the scientific stage in 1964, when Wise and coworkers first demonstrated the in vitro degradation of heme to biliverdin (1). This finding was confirmed by Tenhunen and colleagues (2, 3), who identified HO as the enzyme responsible for the catabolism of heme and went on to characterize the enzyme and its cellular localization. The enzyme settled into relative obscurity until the late 1980s, when an inducible form of HO was discovered (4). This inducible form, called heme oxygenase-1 (HO-1), proved to be identical to heat shock protein 32 (5); this observation sparked new interest in HO as a stress-responsive protein.

Cells respond to metabolic perturbations by producing specific stress proteins. Noting that HO-1 is induced by exposure to various forms of oxidative stress, Stocker proposed in 1990 that this enzyme might provide cellular protection (6). This view is supported by the observation that both HO and its substrate, heme, are highly conserved molecules across almost all forms of life, from algae to mammals. Molecules so evolutionarily conserved and ubiquitous generally serve a necessary and fundamental purpose. The first report of human HO-1 deficiency served to underscore this point: The boy in question suffered from growth failure, anemia, tissue iron deposition, lymphadenopathy, leukocytosis, and increased sensitivity to oxidant injury. He ultimately succumbed to a premature death (7). In an animal model of HO-1 deficiency, mice lacking the gene frequently die in utero, and those surviving to term display a phenotype similar to the HO-1-deficient boy (8). Clearly, HO-1 is necessary to the survival of organisms, consistent with Stocker's hypothesis that the enzyme protects against oxidative stress.

Beneficial effects of HO-1 have now been described in diseases as diverse as atherosclerosis and pre-eclampsia (Table 1). The mechanism by which this enzyme confers protection is only beginning to be unraveled, and this puzzle currently engages the attention of researchers worldwide. If the interest of the scientific community can be measured by the volume of publications dealing with a particular subject, then interest in heme oxygenase is increasing exponentially (Figure 1). The appeal is readily apparent: if we can understand how cells are able to protect themselves from oxidative stress, then our understanding and ability to intervene in disease processes will be immeasurably advanced.

View this table: [in this window] [in a new window]   TABLE 1 Diseases and physiologic functions associated with changes in heme oxygenase-1

View larger version (18K): [in this window] [in a new window]   Figure 1.   Graph illustrating the number of publications concerning HO-1 cataloged ny Medline from 1990 to the present.

At least part of the antioxidant function of HO-1 may depend upon the prevention of free heme from participating in pro-oxidant reactions (5). A major focus of interest, however, is in the products of enzymatic heme breakdown as mediators of cytoprotection. HO accomplishes the first rate-limiting step of heme degradation as it cleaves the -meso carbon bridge of heme molecules to yield equimolar quantities of biliverdin IXa, free iron, and carbon monoxide (CO) (Figure 2). Biliverdin is subsequently converted to bilirubin through the action of biliverdin reductase, and free iron is sequestered by ferritin. The three products of this reactionbilirubin, CO, and ferritin induced by free iron releaseall have antioxidant function (9). These molecules are candidate effectors of the anti-inflammatory, antiapoptotic, and antiproliferative functions of HO-1, and they represent targets of active research.

View larger version (6K): [in this window] [in a new window]   Figure 2.   Catalytic reaction of HO.

As previously noted, there exist several isoforms of HO, all products of separate genes. Two of these isoforms have been extensively characterized: HO-1, an inducible isoform, and HO-2, which is constitutively synthesized. A third isoform, HO-3, was more recently identified; this enzyme is structurally similar to HO-2, but acts as a less efficient heme catalyst. HO-1 is the principle focus of this review. Since this subject was last reviewed in a 1996 issue of AJRCMB (12), a great deal of progress has been made in the study of HO-1, particularly with regard to function. This article is not exhaustive but seeks to summarize what is known about the induction, regulation, and function of HO-1, ending with a discussion of HO-1 in specific human diseases.

	Induction and Regulation of HO-1

Top Abstract Introduction Induction and Regulation of... HO-1 and Signal Transduction HO-1 and Apoptosis HO-1 and Nitric Oxide HO-1 and Diseases of... Diagnostic and Therapeutic Uses... Conclusion References

HO-1 is inducible by more diverse stimuli than any other enzyme described to date (13). The common characteristic of these many inducers is their ability to cause oxidative stress. These include, but are not limited to: heme, hyperoxia, hypoxia, heat shock, endotoxin, hydrogen peroxide, cytokines, UV light, heavy metals, and nitric oxide (14). HO-1 expression is primarily regulated at the transcriptional level (12, 23), although there are interspecies variations in the regulation of HO-1. For example, the rat HO-1 promoter has a heat shock-responsive element; during heat shock increased binding of heat shock nuclear factor to the promoter region of the gene causes HO-1 message stabilization (27). By contrast, the heat shock element is not functional in the human HO-1 gene (28).

Various regulatory elements have been identified in the promoter region of HO-1, including binding sites for oxidative stress-responsive transcription factors such as nuclear factor (NF)-B, which is present in the human HO-1 gene, and activator protein-1 (AP-1) (12, 29, 30). The role of the AP-1 family of transcription factors in the regulation of HO-1 is currently being investigated, but it would appear that NF-E2-related factor 2 (Nrf2) is of particular importance (31). Work is ongoing to demonstrate which HO-1 gene elements mediate activation in response to particular stimuli. The mouse gene, which is the best characterized with regard to inducer-dependent transcriptional regulation, contains one proximal and two distal enhancers. The first distal enhancer is located 4 kb upstream from the transcription initiation site (32), and the other is located 10 kb upstream (33). Some stimuli, such as lipopolysaccharide (LPS), require the distal enhancers for gene activation (34), whereas others such as hypoxia do not (16). The mouse promoter does not function alone in response to a number of stimuli tested, including cadmium, heme, and 12-O-tetradecanoylphorbol-13-acetate (32, 35). The human HO-1 gene is not regulated in an identical fashion; the promoter has been shown to be functionally active in response to various inducers such as UV irradiation, cadmium, and heme (36). Iron- and hyperoxia-responsive regions appear to reside outside the promoter region, however, implying that the human HO-1 gene requires distal enhancer elements for induction by some agents (25).

Within the enhancer regions of the mouse HO-1 gene, there is a 10-bp sequence that is necessary for induction of the gene by all agents except hypoxia. This sequence is referred to as the stress response element (StRE). The mouse StRE contains the binding site for the activator protein-1 (AP-1) family of transcription factors. Mutation of the AP-1 binding site within the StREs abolishes gene activation by heavy metals, hydrogen peroxide, arsenate, and LPS in vitro (14, 33), providing evidence of the role of AP-1 proteins in HO-1 gene regulation. Lee and coworkers have presented both in vitro and in vivo data demonstrating that that AP-1 activation represents one mechanism mediating hyperoxia-induced HO-1 gene transcription in the lung (37), and that AP-1 acts in concert with signal transducer and activator of transcription (STAT) (38). More recently, Rensing and colleagues have demonstrated that the induction of HO-1 after hemorrhagic shock and resuscitation is mainly regulated by AP-1 in a reactive oxygen species (ROS)-dependent manner (39).

Other cis-acting DNA elements that have been studied include hypoxia-inducible factor-1 (HIF-1) and interleukin (IL)-6 response element. HIF-1 is a transcriptional activator of many oxygen-sensitive genes, and has been shown to bind to hypoxia response elements in the mouse HO-1 gene (16). Mutation of the hypoxia response elements abolishes HIF-1 binding and hypoxia-dependent gene activation (16), confirming that this activator mediates HO-1 expression in response to hypoxia. Yang and coworkers have corroborated this finding with their studies using rat renal medullary interstitial cells (40). IL-6 is one of several cytokines known to activate HO-1 gene transcription (23). A sequence motif in the HO-1 5' flanking region that conforms to the consensus IL-6 response element has been identified (41).

AP-1 is a well-known redox-sensitive transcription factor activating a number of genes involved in adaptive responses to oxidative stress, including HO-1. AP-1 belongs to the family of basic region/leucine zipper (bZIP) transcription factors which includes Jun-Jun and Jun-Fos dimers, and the more recently described NF-E2, Nrf1, and Nrf2. There is good evidence that Nrf2 modulates HO-1 gene expression by binding to a recognition site for Cap'n'Collar/basic leucine zipper (CNC-bZIP) in one of the inducible enhancers (E1) (42, 43). Results obtained using a yeast 2 hybrid system further suggest that Nrf2 may heterodimerize with activating transcription factor 4 to exert its effect (44).

	HO-1 and Signal Transduction

Top Abstract Introduction Induction and Regulation of... HO-1 and Signal Transduction HO-1 and Apoptosis HO-1 and Nitric Oxide HO-1 and Diseases of... Diagnostic and Therapeutic Uses... Conclusion References

The MAP kinases (mitogen-activated protein kinases) are components of signaling cascades that respond to extracellular stimuli by targeting transcription factors, resulting in the modulation of gene expression. Three major MAP kinase subfamilies have been described: extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 (Figure 3). ERK was the first cascade to be elucidated; it is activated by mitogens and is primarily involved in the regulation of growth and proliferation. The remaining two cascades respond to cellular stress signals and are hence sometimes referred to as stress-activated protein kinases. There is a good deal of overlap among the cascades both in their targets and upstream activators. The specifics of these interactions have been extensively reviewed elsewhere (45).

View larger version (22K): [in this window] [in a new window]   Figure 3.   Simplified diagram illustrating signal transduction by MAP kinases.

Given that both HO-1 and the MAP kinases are activated by stressful stimuli, and that tyrosine phosphorylation is involved in the induction of HO-1 (48), it is reasonable to postulate a relationship between the MAP kinases and HO-1. Lu and coworkers have reported that constitutive activation of ERK and p38 pathway components results in increased HO-1 reporter gene activity, and that dominant negative components abrogate arsenite-induced reporter gene activity (49, 50). These findings were confirmed using chemical inhibitors of the ERK and p38 pathways (51). The JNK pathway does not appear to be involved in the response of HO-1 to arsenite (50).

There is evidence of MAP kinase pathway involvement downstream of HO-1 activation as well. CO, one of the products of heme catabolism by HO-1, has cytoprotective and antiapoptotic effects that appear to be mediated through MAP kinase pathways, specifically the p38 pathway (11, 52, 86). Sethi and colleagues confirmed that CO can upregulate the p38 MAP kinase pathway while simultaneously downregulating ERK (53).

	HO-1 and Apoptosis

Top Abstract Introduction Induction and Regulation of... HO-1 and Signal Transduction HO-1 and Apoptosis HO-1 and Nitric Oxide HO-1 and Diseases of... Diagnostic and Therapeutic Uses... Conclusion References

One common cellular response to oxidative injury is apoptosis, and the involvement of reactive oxygen species in programmed cell death makes HO-1 a good candidate as an antiapoptotic molecule. Fibroblasts (54) and neurons (55) overexpressing HO-1 are resistant to stress-mediated cell death, and fibroblasts deficient in the HO-1 gene are particularly susceptible to stressful or toxic insults (55). Similarly, human renal epithelial cells resist cisplatin-induced apoptosis and necrosis when HO-1 activity is enhanced by chemical induction or gene overexpression, and mice lacking the gene for HO-1 exhibit increased susceptibility to apoptosis and necrosis after treatment with cisplatin (56). The authors of these studies have postulated that HO-1 inhibits apoptosis by regulating cellular pro-oxidant iron. Ferris and coworkers correlated protection of cells by HO-1 with a decrease in intracellular iron amounts and reproduced the protective effect by iron chelation (54).

Using mouse fibroblasts, Petrache and colleagues have demonstrated that conditional overexpression of HO-1 prevents tumor necrosis factor (TNF)- induced apoptosis. The antiapoptotic effect is lost in the presence of tin protoporphyrin, a specific inhibitor of HO activity, and in cells overexpressing antisense HO-1. These authors reported that the exogenous administration of CO also prevents TNF-- induced apoptosis, suggesting that the antiapoptotic effect of HO-1 may be mediated via CO (57). Corroborating a role for CO in the antiapoptotic function of HO-1, Brouard and coworkers have shown that exposure of endothelial cells to exogenous CO prevents TNF--induced apoptosis. They have further demonstrated that this effect is mediated via the p38 MAP kinase pathway (52).

Whether HO-1 exerts its antiapoptotic effect principally through the action of CO, the regulation of cellular iron, or even the generation of bilirubin (58) is a matter for further study. It is possible that two or more of the byproducts of HO-1 are required for full protection, or that the requirements differ depending on cell type and apoptotic stimulus. Given the relevance of apoptosis to so many disease processes, this will doubtless be an area of active investigation in the future.

	HO-1 and Nitric Oxide

Top Abstract Introduction Induction and Regulation of... HO-1 and Signal Transduction HO-1 and Apoptosis HO-1 and Nitric Oxide HO-1 and Diseases of... Diagnostic and Therapeutic Uses... Conclusion References

When nitric oxide (NO) was unveiled as the long-sought endothelium-derived relaxing factor in the 1980s (59), the concept of a gaseous molecule acting as a second messenger was entirely novel. It is now clear that NO has numerous vital biologic roles, including neurotransmission, host defense, inhibition of platelet aggregation, and regulation of blood flow (60). The generation of NO by nitric oxide synthase (NOS) bears an uncanny resemblance to the production of CO by HO. Both diatomic gasses are produced endogenously by enzymes that have constitutive and inducible forms. Both NO and CO share affinity for the heme molecule, and both are capable of activating guanylyl cyclase (63). One important difference between NO and CO is that NO is a highly reactive gas by virtue of its unpaired electron, whereas CO is inert. These similarities (and this difference) between the two molecules make interactions between NO and HO a natural subject for investigation.

There is some evidence that NO and CO have parallel signaling actions via soluble guanylyl cyclase (sGC) in select tissues. sGC acts as a receptor for NO, mediating many of its biologic effects. NO stimulates sGC by binding to the prosthetic heme of the enzyme leading to as much as a 400-fold activation of the purified enzyme (63, 66). CO, like NO, binds to the heme group of sGC with high affinity but leads to a far lower level of activation of the purified enzyme (67). CO has been shown to stimulate cyclic 3'5'-guanosine monophosphate (cGMP) production and to promote cGMP-dependent activities such as inhibition of platelet aggregation and smooth muscle relaxation (64, 65, 68). In the nervous system, HO-2 colocalizes with cGMP and/or sGC in cells with little or no NOS expression, supporting a link between HO and sGC. Moreover, in isolated neural cell preparations, a direct relationship between HO activity and cGMP production has been observed (69).

The inducible form of NOS (iNOS) and HO-1 are upregulated by common stimuli such as ROS and cytokines (70). NO itself has also been shown to induce HO-1 expression in various cell types by a cGMP-independent pathway (26, 71). This induction may depend upon changes in mRNA stability (71, 72), or may be transcriptionally regulated (26); the mechanism appears to vary with the cell type and the NO exposure conditions. Given the overlap in the biologic functions of CO and NO, it is possible that some actions of NO are exerted by the induction of HO-1 and elaboration of CO.

It has also been proposed that HO activity may serve under some circumstances to modulate NO production. Chemical inhibition of HO activity by zinc protoporphyrin-IX (ZnPP) in isolated smooth muscle strips (74) and macrophages (75) results in increased NO production, suggesting that HO may exert an inhibitory effect on NOS. This suggestion is further supported by the observation that induction of HO-1 in macrophages suppresses NO generation (75). As NOS is a hemoprotein, it is reasonable to postulate that CO generated by HO activity could bind to NOS, causing inactivation. It has been demonstrated that CO is in fact capable of binding to NOS (76), and that exogenously administered CO inhibits NOS activity (77). Inhibition of NOS by HO could be important in view of the fact that NO is a free radical, and as such is able to react nonspecifically with many cellular components, causing unwanted effects (78, 79). Maines has proposed that in the case of NO interaction with sGC heme, HO may promote a reversal of the reaction (13). This could prevent tonic activation of sGC by NO, which dissociates from heme at an exceedingly slow rate (80).

	HO-1 and Diseases of the Lung and Vascular System

Top Abstract Introduction Induction and Regulation of... HO-1 and Signal Transduction HO-1 and Apoptosis HO-1 and Nitric Oxide HO-1 and Diseases of... Diagnostic and Therapeutic Uses... Conclusion References

The salutary effects of HO-1 activation have been studied in nearly every organ system and a growing number of diseases (Table 1). What follows is a brief summary of some of the work that has been done to date relating to diseases of the lung and cardiovascular system.

HO-1 and the Lung

As an organ that provides an interface between the environment and the circulation, the lung affords an ideal environment for studying the effects of oxidative stress. HO-1 is readily induced in the lung by numerous stresses and has been implicated in the maintenance of homeostasis in the face of oxidative injury.

One common model of pulmonary oxidative stress is exposure to hyperoxia. Increased HO-1 activity has been observed in the lungs of rodents exposed to hyperoxic conditions (81), and overexpression of HO-1 in isolated pulmonary epithelial cells (81) and rat fetal lung cells (82) has been shown to confer protection against hyperoxic injury. This same protective phenomenon has been demonstrated in vivo with adenoviral transfer of HO-1 into rat lungs (83): rats overexpressing HO-1 demonstrate marked resistance to oxygen-induced lung injury and survive longer in a hyperoxic environment. However, there may be a threshold effect for cytoprotection by HO-1. Suttner and coworkers observed that although moderate overexpression of HO-1 in fibroblasts conferred protection against oxidative injury, higher levels of HO-1 were detrimental (82).

There is evidence that the CO elaborated during heme catabolism by HO-1 accounts for at least some of the protective effects of HO-1 in the lung. Otterbein and colleagues reported that rats exposed to hyperoxia in the presence of a low concentration of CO (250 ppm) exhibit less lung injury than control rats exposed to oxygen alone (84). A similar study by Clayton and associates demonstrated a statistically significant reduction in pulmonary edema with exposure to CO and hyperoxia, but no difference in other markers of lung injury (85). Interestingly, the latter study did find that inhaled CO abrogated a hyperoxia-induced increase in HO-1 protein expression. The mechanism by which CO might provide pulmonary cytoprotection has not been entirely elucidated, but downregulation of proinflammatory cytokines such as TNF- and IL-1 along with augmentation of the anti-inflammatory cytokine IL-10 appear to play a role (86). The potential role for IL-10 has been further supported by a recent murine study demonstrating resistance to LPS-induced lung injury and enhanced IL-10 production by alveolar macrophages after transfer of HO-1 cDNA (87).

As anti-inflammatory actions of HO-1 continue to be described, there is growing interest in the role of HO-1 in asthma, which is now understood to be an inflammatory disease involving activated T lymphocytes and their cytokine products (88, 89). Exhaled CO, a marker of HO activity, is elevated in humans with asthma (90). The inflammatory cells of patients with asthma have increased HO-1 protein content (91), and enhanced airway HO-1 immunostaining has also been described in a murine model of asthma (92). Given the evidence for HO-1 involvement in asthma and the proposed anti-inflammatory properties of CO (86), Chapman and coworkers sought an immunoregulatory role for CO in aeroallergen-induced inflammation in mice (93). The authors demonstrated a reduction in bronchoalveolar lavage levels of IL-5, prostaglandin E2, leukotriene B4, and eosinophils in mice treated with inhaled CO. Although these findings do not definitively indicate that symptoms of asthma will respond to exogenous or endogenous CO, they do suggest that HO and CO play a role in the modulation of allergic inflammation.

In the past year, HO-1 has been implicated in a number of other pulmonary diseases. Smokers are now known to have increased airway expression of HO-1 (94), and microsatellite polymorphisms in the HO-1 gene promoter have been linked with increased susceptibility to emphysema (95). Hypoxia-induced pulmonary hypertension, vascular remodeling, and inflammation have been attenuated through enhanced HO-1 expression in rodent models (96, 97). Influenza virus-induced lung injury in mice has also been prevented through transfer of HO-1 cDNA (98). Oxidative injury contributes to all of these disease processes, and it stands to reason that HO-1 would be of central importance in the maintenance of homeostasis under these circumstances. HO-1 will likely prove to play a role in the full range of pulmonary disease processes and, perhaps one day, in their prevention or cure.

HO-1 and the Cardiovascular System

A potential role for HO in the regulation of vascular tone was first surmised in 1984, when McGrath and Smith (99) and McFaul and McGrath (100) exposed isolated perfused rat hearts to exogenous CO and noted an increase in coronary blood flow. They concluded that CO caused dilation of the coronary arteries and that the effect was reversible because coronary flow returned to control level when the CO was removed. Several years later, other investigators reported that heme, a potent inducer of HO-1, lowers blood pressure in spontaneously hypertensive rats (101) and that inhibition of HO activity increases mean arterial blood pressure and total peripheral resistance in rats (102). It has since been demonstrated that CO promotes arterial vasodilation through activation of guanylyl cyclase in a manner similar to but independent of NO (103). The physiologic significance of a stress-induced mediator of vasodilation separate from the NOS/NO system remains to be fully elucidated, but one potential teleologic explanation is that HO-1 serves to maintain perfusion after vascular injury.

This thesis is supported by several findings related to HO-1 in the cardiovascular system. First, HO-1 expression has been shown to inhibit the growth of vascular smooth muscle cells in vitro and in vivo (107). Vascular smooth muscle cell-derived CO has been shown to inhibit endothelial cell platelet derived growth factor and endothelin-1 expression resulting in inhibited smooth muscle cell growth (108). CO can also inhibit hypoxia-induced vascular endothelial growth factor induction in smooth muscle cells (109). These observations have obvious implications with regard to vascular remodeling after injury. Next, ischemia-reperfusion injury, shear stress, and hypoxia are potent inducers of HO-1 (103, 110). Third, HO-1 induction has been shown to inhibit leukocyte adhesion through the action of bilirubin (113) and platelet aggregation via the production of CO (112). Fourth, and most convincingly, HO-1 provides protection against ischemia-reperfusion tissue injury and vascular balloon injury in a number of models (114).

The studies demonstrating a protective effect of HO-1 in vascular injury have generated a great deal of excitement because of their relevance to so many human disease processes. Yet and coworkers recently showed that HO-1 overexpression confers protection against ischemia-reperfusion-induced myocardial tissue injury (114). A role for bilirubin has been implicated in the amelioration of postischemic myocardial dysfunction conferred by HO-1 (115). Interestingly, several studies in humans have correlated low serum bilirubin levels with ischemic heart disease (116- 118). In a model of ischemic lung injury, it is CO that appears to protect against lethality through modulation of the fibrinolytic axis (119). CO has also been implicated in the prolonged survival of mouse-to-rat cardiac transplants expressing HO-1. Exposure of graft donor and recipient animals to exogenous CO is associated with suppression of graft rejection; this effect is associated with inhibition of platelet aggregation, thrombosis, myocardial infarction, and apoptosis in the transplanted organ (120). Balloon injury has been used as a model of vascular injury to demonstrate that HO-1 is induced locally in the vessel (121), that preinduction of HO-1 can prevent the injury (121, 122), that inhibition of HO-1 can worsen the injury (121), and that overexpression of HO-1 through adenovirus-mediated delivery can inhibit injury-induced vascular neointima formation (123). These findings have been further supported by a human study demonstrating that HO-1 gene promoter microsatellite polymorphisms are associated with restenosis after percutaneous transluminal angioplasty (124).

	Diagnostic and Therapeutic Uses of HO-1 and Its Byproducts

Top Abstract Introduction Induction and Regulation of... HO-1 and Signal Transduction HO-1 and Apoptosis HO-1 and Nitric Oxide HO-1 and Diseases of... Diagnostic and Therapeutic Uses... Conclusion References

What practical applications can we foresee in the near future for HO-1 and its byproducts? Based on our current knowledge of the function, regulation, and properties of HO-1, several approaches have been proposed for diagnostic and therapeutic purposes.

From a diagnostic point of view, there is growing interest in monitoring HO-1 activity in disease states. It is clear from the work cited above that HO-1 is activated in many pathologic situations, and it may be possible to use this association to predict disease flares or monitor therapy. Because CO generated by HO-1 is excreted by the lungs, quantification of exhaled CO can be used to track enzymatic activity. Elevated levels of CO in the breath have been described in asthma (125, 126), cystic fibrosis (127), diabetes (128), and critical illness (129). Increases in the amount of exhaled CO have also been correlated with disease exacerbations (130, 131). Standardization of equipment and reference values for the measurement of exhaled CO is being undertaken by researchers in this field; their efforts will lead to a better appreciation for how CO monitoring may fit into clinical practice.

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 (83, 98, 123), but 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. As noted above, high serum bilirubin levels correlate with less cardiovascular disease in humans; whether this is cause and effect, and whether bilirubin levels could safely be manipulated in humans, is unknown. Administration of exogenous CO has been shown to protect against lung injury and transplant rejection (84, 120), among other ills, but whether this poisonous gas could be given harmlessly to humans is an unanswered question. 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. Motterlini and coworkers have described the use of chemical CO donors in the form of transition metal carbonyls (132), which represents a novel approach to administering this gas. Treatment of organ grafts with CO before transplant is another use of the gas which does not involve direct inhalation. The future will show which (if any) of these approaches will ultimately be of benefit to humans.

	Conclusion

Top Abstract Introduction Induction and Regulation of... HO-1 and Signal Transduction HO-1 and Apoptosis HO-1 and Nitric Oxide HO-1 and Diseases of... Diagnostic and Therapeutic Uses... Conclusion References

Oxidative injury is now understood to be the common denominator in manyindeed, mostpathologic processes. An antidote to oxidative damage has become a holy grail of medical research. The discovery of a molecule which is ubiquitous in nature, cytoprotective, antiapoptotic, and anti-inflammatory is cause for great excitement. It is to be hoped that investigators will one day understand the mechanisms by which HO-1 exerts its remarkable effects, and that this understanding will lead to cures for the many infirmities caused by oxidative stress. Until that time, the quest to delineate the complex interplay of HO-1 and its enzymatic products with biologic systems will continue to be pursued by a growing number of researchers in every medical discipline.

	Footnotes

Address correspondence to:

(Received in original form March 14, 2002).

	References

Top Abstract Introduction Induction and Regulation of... HO-1 and Signal Transduction HO-1 and Apoptosis HO-1 and Nitric Oxide HO-1 and Diseases of... Diagnostic and Therapeutic Uses... Conclusion References

1. Wise, C.D., and D.L. Drabkin. 1964. Degradation of haemoglobin and hemin to biliverdin by a new cell-free system obtained from the hemophagous organ of dog placenta. Fed. Proc. 23: 323 .

2. Tenhunen, R., H. S. Marver, and R. Schmid. 1968. The enzymatic conversion of heme to bilirubin by microsomal heme oxygenase. Proc. Natl. Acad. Sci. USA 61: 748-755 [Free Full Text].

3. Tenhunen, R., H. S. Marver, and R. Schmid. 1969. Microsomal heme oxygenase: characterization of the enzyme. J. Biol. Chem. 244: 6388-6394 [Abstract/Free Full Text].

4. Maines, M. D., G. M. Trakshel, and R. K. Kutty. 1986. Characterization of two constitutive forms of rat liver microsomal heme oxygenase. J. Biol. Chem. 261: 411-419 [Abstract/Free Full Text].

5. Keyse, S. M., and R. M. Tyrrell. 1989. Heme oxygenase is the major 32-kDa stress protein induced in human skin fibroblasts by UVA radiation, hydrogen peroxide, and sodium arsenite. Proc. Natl. Acad. Sci. USA 86: 99-103 [Abstract/Free Full Text].

6. Stocker, R.. 1990. Induction of heme oxygenase as a defense against oxidative stress. Free Rad. Res. Commun. 9: 101-112 [Medline].

7. Yachie, A., Y. Niida, T. Wada, N. Igarashi, H. Kaneda, T. Toma, K. Ohta, Y. Kasahara, and S. Koizumi. 1999. Oxidative stress causes enhanced endothelial cell injury in human heme oxygenase-1 deficiency. J. Clin. Invest. 103: 129-135 [Medline].

8. Poss, K. D., and S. Tonegawa. 1997. Heme oxygenase 1 is required for mammalian iron reutilization. Proc. Natl. Acad. Sci. USA 94: 10919-10924 [Abstract/Free Full Text].

9. Oberle, S., P. Schwartz, A. Abate, and H. Schroder. 1999. The antioxidant defense protein ferritin is a novel and specific target for pentaerithrityl tetranitrate in endothelial cells. Biochem. Biophys. Res. Commun. 261: 28-34 [Medline].

10. Yesilkaya, A., R. Altinayak, and D. K. Korgun. 2000. The antioxidant effect of free bilirubin on cumene-hydroperoxide treated human leukocytes. Gen. Pharmacol. 35: 17-20 [Medline].

11. Otterbein, L. E., and A. M. K. Choi. 2000. Heme oxygenase: colors of defense against cellular stress. Am. J. Physiol. Lung Cell. Mol. Physiol. 279: L1029-L1037 [Abstract/Free Full Text].

12. Choi, A. M. K., and J. Alam. 1996. Heme oxygenase-1: function, regulation, and implication of a novel stress-inducible protein in oxidant-induced lung injury. Am. J. Respir. Cell Mol. Biol. 15: 9-19 [Abstract].

13. Maines, M. D.. 1997. The heme oxygenase system: a regulator of second messenger gases. Annu. Rev. Pharmacol. Toxicol. 37: 517-554 [Medline].

14. Camhi, S. L., J. Alam, L. Otterbein, S. L Sylvester, and A. M. Choi. 1995. Induction of heme oxygenase-1 gene expression by lipopolysaccharide is mediated by AP-1 activation. Am. J. Respir. Cell Mol. Biol. 130: 387-398 .

15. Carraway, M. S., A. J. Ghio, J. D. Carter, and C. A. Piantadosi. 2000. Expression of heme oxygenase-1 in the lung in chronic hypoxia. Am. J. Respir. Cell Mol. Biol. 278: L806-L812 .

16. Lee, P. J., B. H. Jiang, B. Y. Chin, N. V. Iyer, J. Alam, G. L. Semenza, and A. M. Choi. 1997. Hypoxia-inducible factor-1 mediates transcriptional activation of the heme oxygenase-1 gene in response to hypoxia. J. Biol. Chem. 272: 5375-5381 [Abstract/Free Full Text].

17. Lee, P. J., J. Alam, G. W. Wiegand, and A. M. K. Choi. 1996. Overexpression of heme oxygenase-1 expression in human pulmonary epithelial cells results in cell growth arrest and increased resistance to hyperoxia. Proc. Natl. Acad. Sci. USA 93: 10393-10398 [Abstract/Free Full Text].

18. Keyse, S. M., and R. M. Tyrrell. 1987. Both near ultraviolet radiation and the oxidizing agent hydrogen peroxide induce a 32-kDa stress protein in normal human skin fibroblasts. J. Biol. Chem. 262: 14821-14825 [Abstract/Free Full Text].

19. Lautier, D., P. Luscher, and R. M. Tyrrell. 1992. Endogenous glutathione levels modulate both constitutive and UVA radiation/hydrogen peroxide inducible expression of the human heme oxygenase gene. Carcinogenesis 13: 227-232 [Abstract/Free Full Text].

20. Ewing, J. F., and M. D. Maines. 1991. Rapid induction of heme oxygenase 1 mRNA and protein by hyperthermia in rat brain: heme oxygenase 2 is not a heat shock protein. Proc. Natl. Acad. Sci. USA 88: 5364-5368 [Abstract/Free Full Text].

21. Eyssen-Hernandez, R., A. Ladoux, and C. Frelin. 1996. Differential regulation of cardiac heme oxygenase-1 and vascular endothelial growth factor mRNA expressions by hemin, heavy metals, heat shock and anoxia. FEBS Lett. 382: 229-233 [Medline].

22. Carraway, M. S., A. J. Ghio, J. L. Taylor, and C. A. Piantadosi. 1998. Induction of ferritin and heme oxygenase-1 by endotoxin in the lung. Am. J. Physiol. Lung Cell Mol. Physiol. 275: L583-L592 [Abstract/Free Full Text].

23. Rizzardini, M., M. Terao, F. Falciani, and L. Cantoni. 1993. Cytokine induction of haem oxygenase mRNA in mouse liver: Interleukin 1 transcriptionally activates the haem oxygenase gene. Biochem. J. 290: 343-347 .

24. Agarwal, A., F. Shiraishi, G. A. Visner, and H. S. Nick. 1998. Linoleyl hydroperoxide transcriptionally upregulates heme oxygenase-1 gene expression in human renal epithelial and aortic endothelial cells. J. Am. Soc. Nephrol. 9: 1990-1997 [Abstract].

25. Fogg, S., A. Agarwal, H. S. Nick, and G. A. Visner. 1999. Iron regulates hyperoxia-dependent human heme oxygenase-1 gene expression in pulmonary endothelial cells. Am. J. Respir. Cell Mol. Biol. 20: 797-804 [Abstract/Free Full Text].

26. Durante, W., M. H. Kroll, N. Christodoulides, K. J. Peyton, and A. I. Schafer. 1997. Nitric oxide induces heme oxygenase-1 gene expression and carbon monoxide production in vascular smooth muscle cells. Circ. Res. 80: 557-564 [Abstract/Free Full Text].

27. Raju, V. S., and M. D. Maines. 1993. Coordinated expression and mechanisms of induction of HSP32 (heme oxygenase-1) mRNA by hyperthermia in rat organs. Biochem. Biophys. Acta 1217: 273-280 .

28. Okinaga, S., K. Takahashi, K. Takeda, M. Yoshizawa, H. Fujita, H. Sasaki, and S. Shibahara. 1996. Regulation of human heme oxygenase-1 gene expression under thermal stress. Blood 87: 5074-5084 [Abstract/Free Full Text].

29. Lavrosky, Y., M. L. Schwartzman, R. D. Levere, A. Kappas, and N. G. Abraham. 1994. Identification of binding sites for transcription factors NF-kappa B and AP-2 in the promoter region of the human heme oxygenase-1 gene. Proc. Natl. Acad. Sci. USA 91: 5987-5991 [Abstract/Free Full Text].

30. Takeda, K., S. Ishizawa, M. Sato, T. Yoshida, and S. Shibahara. 1994. Identification of a cis-acting element that is responsible for cadmium-mediated induction of the human heme oxygenase gene. J. Biol. Chem. 269: 22858-22867 [Abstract/Free Full Text].

31. Otterbein, L. E., and A. M. K. Choi. 2002. The saga of leucine zippers continues in response to oxidative stress. Am. J. Respir. Cell Mol. Biol. 26: 161-163 [Free Full Text].

32. Alam, J., J. Cai, and A. Smith. 1994. Isolation and characterization of the mouse heme oxygenase-1 gene: distal 5' sequences are required for induction by heme and heavy metals. J. Biol. Chem. 269: 1001-1009 [Abstract/Free Full Text].

33. Alam, J., S. Camhi, and A. M. K. Choi. 1995. Identification of a second 5' distal region that functions as a basal and inducer-dependent transcriptional enhancer of the mouse heme oxygenase-1 gene. J. Biol. Chem. 270: 11977-11984 [Abstract/Free Full Text].

34. Camhi, S. L., J. Alam, G. W. Wiegand, B. Y. Chin, and A. M. Choi. 1998. Transcriptional activation of the HO-1 gene by lipopolysaccharide is mediated by 5' distal enhancers: role of reactive oxygen intermediates and AP-1. Am. J. Respir. Cell Mol. Biol. 18: 226-234 [Abstract/Free Full Text].

35. Alam, J., and D. Zhining. 1992. Distal AP-1 binding sites mediate basal level enhancement and TPA induction by heavy metals. J. Biol. Chem. 267: 21894-21900 [Abstract/Free Full Text].

36. Tyrell, R. M., L. A. Applegate, and Y. Tromvoukis. 1993. The proximal promoter region of the human heme oxygenase gene contains elements involved in stimulation of transcriptional activity by a variety of agents including oxidants. Carcinogenesis 14: 761-765 [Abstract/Free Full Text].

37. Lee, P. J., J. Alam, S. L. Sylvester, N. Inamdar, L. Otterbein, and A. M. K. Choi. 1996. Regulation of heme oxygenase-1 expression in vivo and in vitro in hyperoxic lung injury. Am. J. Respir. Cell Mol. Biol. 14: 556-568 [Abstract].

38. Lee, P. J., S. L. Camhi, B. Y. Chin, J. Alam, and A. M. Choi. 2000. AP-1 and STAT mediate hyperoxia-induced gene transcription of heme oxygenase-1. Am. J. Physiol. Lung Cell. Mol. Physiol. 279: L175-L182 [Abstract/Free Full Text].

39. Rensing, H., H. Jaeschke, I. Bauer, C. Patau, V. Datene, B. H. Pannen, and M. Bauer. 2001. Differential activation pattern of redox-sensitive transcription factors and stress-inducible dilator systems heme oxygenase-1 and inducible nitric oxide synthase in hemorrhagic and endotoxic shock. Crit. Care Med. 29: 1962-1971 [Medline].

40. Yang, Z., and A. Zou. 2001. Transcriptional regulation of heme oxygenase by HIF-1 in renal medullary interstitial cells. Am. J. Physiol. Renal Physiol. 281: F900-F908 . [Abstract/Free Full Text]

41. Yuan, J., U. M. Wgenka, C. Lutticken, J. Buschmann, T. Decker, C. Schindler, P. C. Heinrich, and F. Horn. 1994. The signaling pathways of interleukin-6 and gamma interferon converge by the activation of different transcription factors which bind to common responsive DNA elements. Mol. Cell Biol. 14: 1657-1668 [Abstract/Free Full Text].

42. Alam, J., D. Stewart, C. Touchard, S. Bionapally, A. M. Choi, and J. L. Cook. 1999. Nrf2, a Cap'n'Collar transcription factor, regulates induction of the heme oxygenase-1 gene. J. Biol. Chem. 274: 26071-26078 [Abstract/Free Full Text].

43. Alam, J., C. Wicks, D. Stewart, P. Gong, C. Touchard, S. Otterbein, A. M. K. Choi, M. E. Burrow, and J. Tou. 2000. Mechanism of heme oxygenase-1 gene activation by cadmium in MCF-7 mammary epithelial cells: role of p38 kinase and Nrf2 transcription factor. J. Biol. Chem. 275: 27694-27702 [Abstract/Free Full Text].

44. He, C. H., P. Gong, B. Hu, D. Stewart, M. E. Choi, A. M. K. Choi, and J. Alam. 2001. Identification of activating transcription factor 4 (ATF4) as an Nrf2-interacting protein. J. Biol. Chem. 276: 20858-20865 [Abstract/Free Full Text].

45. Graves, J. D., J. S. Campbell, and E. G. Krebs. 1995. Protein serine/threonine kinases of the MAPK cascade. Ann. N.Y. Acad. Sci. 766: 320-343 [Medline].

46. Bokemeyer, D., A. Sorokin, and M. J. Dunn. 1996. Multiple intracellular MAP kinase signaling cascades. Kidney Int. 49: 1187-1198 [Medline].

47. Kyriakis, J. M., and J. Avruch. 1996. Protein kinase cascades activated by stress and inflammatory cytokines. Bio Essays 18: 567-577 [Medline].

48. Masuya, Y., K. Hioki, R. Tokunaga, and S. Taketani. 1998. Involvement of the tyrosine phosphorylation pathway in induction of human heme oxygenase-1 by hemin, sodium arsenite and cadmium chloride. J. Biochem. 124: 628-633 [Abstract/Free Full Text].

49. Lu, T. H., R. W. Lambrecht, J. Pepe, Y. Shan, T. Kim, and H. L. Bonkovsky. 1998. Molecular cloning, characterization, and expression of the chicken heme oxygenase-1 gene in transfected primary cultures of chick embryo liver cells. Gene 207: 177-186 [Medline].

50. Elbirt, K. K., and H. L. Bonkovsky. 1999. Heme oxygenase: recent advances in understanding its regulation and role. Proc. Assoc. Am. Phys. 111: 438-447 . [Medline]

51. Elbirt, K. K., A. J. Whitmarsh, R. J. Davis, and H. L. Bonkovsky. 1998. Mechanism of sodium arsenite-mediated induction of heme oxygenase-1 in hepatoma cells: role of mitogen activated protein kinases. J. Biol. Chem. 273: 8922-8931 [Abstract/Free Full Text].

52. Brouard, S., L. E. Otterbein, J. Anrather, E. Tobiasch, F. H. Bach, A. M. Choi, and M. P. Soares. 2000. Carbon monoxide generated by heme oxygenase-1 suppresses endothelial cell apoptosis. J. Exp. Med. 192: 1015-1026 [Abstract/Free Full Text].

53. Sethi, J. M., and A. M. K. Choi. 2002. Differential modulation by exogenous carbon monoxide of TNF--stimulated mitogen activated protein kinase signaling in rat pulmonary artery endothelial cells. Antioxidant & Redox Signaling (In press)

54. Ferris, C. D., S. R. Jaffrey, A. Sawa, M. Takahashi, S. D. Brady, R. K. Barrow, S. A. Tysoe, H. Wolosker, D. E. Baranano, S. Dore, K. D. Poss, and S. H. Snyder. 1999. Haem oxygenase-1 prevents cell death by regulating cellular iron. Nat. Cell Biol. 1: 152-157 . [Medline]

55. Chen, K., K. Gunter, and M. D. Maines. 2000. Neurons overexpressing heme oxygenase-1 resist oxidative stress-mediated cell death. J. Neurochem 75: 304-313 [Medline].

56. Shiraishi, F., L. M. Curtis, L. Truong, K. Poss, G. A. Visner, K. Madsen, H. S. Nick, and A. Agarwal. 2000. Heme oxygenase-1 gene ablation or expression modulates cisplatin-induced renal tubular apoptosis. Am. J. Physiol. Renal Physiol 278: F726-F736 . [Abstract/Free Full Text]

57. Petrache, I., L. E. Otterbein, J. Alam, G. W. Wiegand, and A. M. Choi. 2000. Heme oxygenase-1 inhibits TNF--induced apoptosis in cultured fibroblasts. Am. J. Physiol. Lung Cell Mol. Physiol. 278: L312-L319 [Abstract/Free Full Text].

58. Dore, S., M. Takahashi, C. D. Ferris, R. Zakhary, L. D. Hester, D. Guastella, and S. H. Snyder. 1999. Bilirubin, formed by activation of heme oxygenase-2, protects neurons against oxidative stress injury. Proc. Natl. Acad. Sci. USA 96: 2445-2450 [Abstract/Free Full Text].

59. Palmer, R. M., A. G. Ferrige, and S. Moncada. 1987. Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor. Nature 327: 524-526 [Medline].

60. Bredt, D. S., P. M. Hwang, and S. H. Snyder. 1990. Localization of nitric oxide synthase indicating a neural role for nitric oxide. Nature 347: 768-770 [Medline].

61. Stuehr, D. J., S. S. Gross, I. Sakuma, R. Levi, and C. F. Nathan. 1989. Activated murine macrophages secrete a metabolite of arginine with the bioactivity of endothelium-derived relaxing factor and the chemical reactivity of nitric oxide. J. Exp. Med 169: 1011-1020 [Abstract/Free Full Text].

62. Engelman, D. T., M. Watanabe, N. Maulik, G. A. Cordis, R. M. Engelman, J. A. Rousou, J. E. Flack, D. W. Deaton, and D. K. Das. L-Argininine reduces endothelial inflammation and myocardial stunning during ischemia/reperfusion. Ann. Thorac. Surg. 60:1275-1281.

63. Humbert, P., F. Niroomand, G. Fischer, B. Mayer, D. Koesling, K. H. Hinsch, H. Gausepohl, R. Frank, G. Schultz, and E. Bohme. 1990. Purification of soluble guanylyl cyclase from bovine lung by a new immunoaffinity chromatographic method. Eur. J. Biochem 190: 273-278 [Medline].

64. Ramos, K. S., H. Lin, and J. J. McGrath. 1989. Modulation of cyclic guanosine monophosphate levels in cultured aortic smooth muscle cells by carbon monoxide. Biochem. Pharmacol 38: 1368-1370 [Medline].

65. Brune, B., and V. Ullrich. 1987. Inhibition of platelet aggregation by carbon monoxide is mediated by activation of guanylate cyclase. Mol. Pharmacol 32: 497-504 [Abstract].

66. Stone, J. R., and M. A. Marletta. 1995. Heme stoichiometry of hetero- dimeric soluble guanylate cyclase. Biochemistry 34: 14668-14674 [Medline].

67. Stone, J. R., and M. A. Marletta. 1994. Soluble guanylate cyclase from bovine lung: activation with nitric oxide and carbon monoxide and spectral characterization of the ferrous states. Biochemistry 33: 5636-5640 [Medline].

68. Utz, J., and V. Ullrich. 1991. Carbon monoxide relaxes ilial smooth muscle through activation of guanylate cyclase. Biochem. Pharmacol 41: 1195-2001 [Medline].

69. Ingi, T., J. Cheng, and G. V. Ronnett. 1996. Carbon monoxide: an endogenous modulator of the nitric oxide-cyclic GMP signaling system. Neuron 16: 835-842 [Medline].

70. Foresti, R., and R. Motterlini. 1999. The heme oxygenase pathway and its interaction with nitric oxide in the control of cellular homeostasis. Free Rad. Res 31: 459-475 [Medline].

71. Hartsfield, C. L., J. Alam, J. L. Cook, and A. M. K. Choi. 1997. Regulation of heme oxygenase-1 gene expression in vascular smooth muscle cells by nitric oxide. Am. J. Physiol. 273: L980-L988 [Abstract/Free Full Text].

72. Bouton, C., and B. Demple. 2000. Nitric oxide-inducible expression of heme oxygenase-1 in human cells. J. Biol. Chem. 275: 32688-32693 [Abstract/Free Full Text].

73. Motterlini, R., R. Foresti, M. Intaglietta, and R. M. Winslow. 1996. NO-mediated activation of heme oxygenase: endogenous cytoprotection against oxidative stress to endothelium. Am. J. Physiol. 270: H107-H114 [Abstract/Free Full Text].

74. Chakder, S., S. Rathi, X. L. Ma, and S. Rattan. 1996. Heme oxygenase inhibitor zinc protoporphyrin-IX causes activation of nitric-oxide synthase in the rabbit internal anal sphincter. J. Pharmacol. Exp. Ther 277: 1376-1382 [Abstract/Free Full Text].

75. Turcanu, V., M. Dhouib, and P. Poindron. 1998. Nitric oxide synthase inhibition by haem oxygenase decreases macrophage nitric oxide dependent cytotoxicity: a negative feedback mechanism for the regulation of nitric oxide production. Res. Immunol. 149: 741-744 [Medline].

76. McMillan, K., D. S. Bredt, D. J. Hirsch, S. H. Snyder, J. E Clark, and B. S. S. Masters. 1992. Cloned, expressed rat cerebellar NOS containing stoichiometric amounts of heme which binds CO. Proc. Natl. Acad. Sci. USA 89: 11141-11145 [Abstract/Free Full Text].

77. White, K. A., and M. A. Marletta. 1992. Nitric oxide synthase is a cytochrome P-450 type hemoprotein Biochemistry 31: 6627-6631 [Medline].

78. Stamler, J. S., D. J. Singel, and J. Loscalzo. 1992. Biochemistry of nitric oxide and its redox-activated forms. Science 258: 1898-1902 [Abstract/Free Full Text].

79. Hibbs, J. B., R. R. Taintor, Z. Vavrin, and E. M. Rachlin. 1988. Nitric oxide: a cytotoxic activated macrophage effector molecule. Biochem. Biophys. Res. Commun. 157: 87-94 [Medline].

80. Gibson, Q. H., and F. J. W. Roughlon. 1955. The reactions of sheep haemoglobin with nitric oxide. J. Physiol. 128: 69-70 .

81. Lee, P. J., J. Alam, S. L. Sylvester, N. Inamdar, L. Otterbein, and A. M. Choi. 1996. Regulation of heme oxygenase-1 expression in vivo and in vitro in hyperoxic lung injury. Am. J. Respir. Cell Mol. Biol. 14: 556-568 .

82. Suttner, D. M., K. Sridhar, C. S. Lee, T. Tomura, T. N. Hansen, and P. A. Dennery. 1999. Protective effects of transient HO-1 overexpression on susceptibility to oxygen toxicity in lung cells. Am. J. Physiol. Lung. Cell Mol. Physiol. 276: L443-L451 [Abstract/Free Full Text].

83. Otterbein, L. E., J. K. Kolls, L. L. Mantell, J. L. Cook, J. Alam, and A. M. Choi. 1999. Exogenous administration of heme oxygenase-1 by gene transfer provides protection against hyperoxia-induced lung injury. J. Clin. Invest. 103: 1047-1054 [Medline].

84. Otterbein, L. E., L. L. Mantell, and A. M. Choi. 1999. Carbon monoxide provides protection against hyperoxic lung injury Am. J. Physiol. Lung Cell Mol. Physiol. 276: L688-L694 [Abstract/Free Full Text].

85. Clayton, C. E., M. S. Carraway, H. B. Suliman, E. D. Thalmann, K. N. Thalmann, D. E. Schmechel, and C. A. Piantadosi. 2001. Inhaled carbon monoxide and hyperoxic lung injury in rats. Am. J. Physiol. Lung Cell Mol. Physiol. 281: L949-L957 [Abstract/Free Full Text].

86. Otterbein, L. E., F. H. Bach, J. Alam, M. Soares, H. Tao, Lu, M. Wysk, R. J. Davis, R. A. Flavell, and A. M. Choi. 2000. Carbon monoxide has anti-inflammatory effects involving the mitogen-activated protein kinase pathway. Nat. Med. 6: 422-428 [Medline].

87. Inoue, S., M. Suzuki, Y. Nagashima, S. Suzuki, T. Hashiba, T. Tsuburai, K. Ikehara, T. Matsuse, and Y. Ishigatsubo. 2001. Transfer of heme oxygenase 1 cDNA by a replication-deficient adenovirus enhances interleukin 10 production from alveolar macrophages that attenuates lipopolysaccharide-induced acute lung injury in mice. Hum. Gene Ther 12: 967-979 [Medline].

88. Azzawi, M., B. Bradley, P. K. Jeffery, A. J. Frew, A. J. Wardlaw, G. Knowles, B. Assoufi, J. V. Collins, S. Durham, and A. B. Kay. 1990. Identification of activated T lymphocytes and eosinophils in bronchial biopsies in stable atopic asthma. Am. Rev. Respir. Dis. 142: 1407-1413 [Medline].

89. Bousquet, J., P. Chanez, J. Y. Lacoste, G. Barneon, N. Ghavanian, I. Enander, P. Venge, S. Ahlstedt, J. Simony-Lafontaine, and P. Godard. 1990. Eosinophilic inflammation in asthma. N.. Engl. J. Med. 323: 1033-1039 [Abstract].

90. Zayasu, K., K. Sekizawa, S. Okinaga, M. Yamaya, T. Ohrui, and H. Sasaki. 1997. Increased carbon monoxide in exhaled air of asthmatic patients. Am. J. Respir. Crit. Care Med. 156: 1140-1143 [Abstract/Free Full Text].

91. Horvath, I., L. E. Donnelly, A. Kiss, P. Paredi, S. A. Kharitonov, and P. J. Barnes. 1998. Raised levels of exhaled carbon monoxide are associated with an increased expression of heme oxygenase-1 in airway macrophages in asthma: a new marker of oxidative stress. Thorax 53: 668-672 [Abstract/Free Full Text].

92. Kitada, O., T. Kodama, K. Kuribayasi, D. Ihaku, M. Fujita, T. Matsuyama, and M. Sugita. 2001. Heme oxygenase-1 (HO-1) protein induction in a mouse model of asthma. Clin. Exp. Allergy 31: 1470-1477 [Medline].

93. Chapman, J. T., L. E. Otterbein, J. A. Elias, and A. M. K. Choi. 2001. Carbon monoxide attenuates aeroallergen-induced inflammation in mice. Am. J. Physiol. Lung Cell Mol. Physiol 281: L209-L216 [Abstract/Free Full Text].

94. Maestrelli, P., A. H. El, Messlemani, O. De Fina, Y. Nowicki, M. Saetta, C. Mapp, and L. M. Fabbri. 2001. Increased expression of heme oxygenase (HO)-1 in alveolar spaces and HO-2 in alveolar walls of smokers. Am. J. Respir. Crit. Care Med 164: 1508-1513 [Abstract/Free Full Text].

95. Yamada, N., M. Yamaya, S. Okinaga, K. Nakayama, K. Sekizawa, S. Shibahara, and H. Sasaki. 2000. Microsatellite polymorphism in the heme oxygenase-1 gene promoter is associated with susceptibility to emphysema. Am. J. Hum. Genet 66: 187-195 [Medline].

96. Cristou, H., T. Morita, C. M. Hsieh, H. Koike, B. Arkonac, M. Perrella, and S. Kourembanas. 2001. Prevention of hypoxia-induced pulmonary hypertension by enhancement of endogenous heme oxygenase-1 in the rat. Circ. Res. 86: 1224-1229 [Abstract/Free Full Text].

97. Minamino, T., H. Christou, C. M. Hsieh, Y. Liu, V. Dhawan, N. G. Abraham, M. A. Perrella, S. A. Mitsialis, and S. Kourembanas. 2001. Targeted expression of heme oxygenase-1 prevents the pulmonary inflammatory and vascular responses to hypoxia. Proc. Natl. Acad. Sci. USA 98: 8798-8803 [Abstract/Free Full Text].

98. Hashiba, T., M. Suzuki, Y. Nagashima, S. Suzuki, S. Inoue, T. Tsuburai, T. Matsuse, and Y. Ishigatubo. 2001. Adenovirus-mediated transfer of heme oxygenase-1 cDNA attenuates severe lung injury induced by the influenza virus in mice. Gene Ther. 8: 1499-1507 [Medline].

99. McGrath, J. J., and D. L. Smith. 1984. Response of rat coronary circulation to carbon monoxide and nitrogen hypoxia. Proc. Soc. Exp. Biol. Med. 177: 132-136 [Medline].

100. McFaul, S. J., and J. J. McGrath. 1987. Studies on the mechanism of carbon monoxide-induced vasodilation in the isolated perfused rat heart. Toxicol. Appl. Pharmacol. 87: 464-473 [Medline].

101. Levere, R. D., P. Martasek, B. Escalante, M. L. Schwartzman, and N. G. Abraham. 1990. Effect of heme arginate administration on blood pressure in spontaneously hypertensive rats. J. Clin. Invest 86: 213-219 .

102. Johnson, R. A., M. Lavesa, B. Askari, N. G. Abraham, and A. Nasjletti. 1995. A heme oxygenase product, presumably carbon-monoxide, mediates a vasodepressor function in rats. Hypertension 25: 166-169 [Abstract/Free Full Text].

103. Morita, T., M. A. Perrella, M. E. Lee, and S. Kourembanas. 1995. Smooth muscle cell-derived carbon monoxide is a regulator of vascular cGMP. Proc. Natl. Acad. Sci. USA 92: 1475-1479 [Abstract/Free Full Text].

104. Morita, T., and S. Kourembanas. 1995. Endothelial cell expression of vasoconstrictors and growth factors is regulated by smooth muscle cell-derived carbon monoxide. J. Clin. Invest. 96: 2676-2682 .

105. Sammut, I. A., R. Foresti, J. E. Clark, D. J. Exon, M. J. Vesely, P. Sarathchandra, C. J. Green, and R. Motterlini. 1998. Carbon monoxide is a major contributor to the regulation of vascular tone in aortas expressing high levels of haeme oxygenase-1. Br. J. Pharmacol. 125: 1437-1444 [Medline].

106. Suematsu, M., N. Goda, T. Sano, S. Kashiwagi, T. Egawa, Y. Shinoda, and Y. Ishimura. 1995. Carbon monoxide: an endogenous modulator of sinusoidal tone in the perfused rat liver. J. Clin. Invest. 96: 2431-2437 .

107. Duckers, H. J., M. Boehm, A. L. True, S.-F. Yet, H. San, J. L. Park, R. C. Webb, M.-E. Lee, G. J. Nabel, and E. G. Nabel. 2001. Heme oxygenase-1 protects against vascular constriction and proliferation. Nat. Med. 7: 693-698 [Medline].

108. Kourembanas, S., T. Morita, Y. Liu, and H. Christou. 1997. Mechanisms by which oxygen regulates gene expression and cell-cell interaction in the vasculature. Kidney Int. 51: 438-443 [Medline].

109. Liu, Y., H. Christou, T. Morita, E. Laughner, G. L. Semenza, and S. Kourembanas. 1998. Carbon monoxide and nitric oxide suppress the hypoxic induction of vascular endothelial growth factor gene via the 5' enhancer. J. Biol. Chem 273: 15257-15262 [Abstract/Free Full Text].

110. Maines, M. D., R. D. Mayer, J. F. Ewing, and W. K. McCoubery. 1993. Induction of kidney heme oxygenase-1 (HSP32) mRNA and protein by ischemia/reperfusion: possible role of heme as both promoter of tissue damage and regulator of HSP32. J. Pharmacol. Exp. Ther 264: 457-462 [Abstract/Free Full Text].

111. Maulik, N., D. T. Engelman, M. Watanabe, R. M. Engelman, and D. K. Das. 1996. Nitric oxide: a retrograde messenger for carbon monoxide signalling in ischemic heart. Mol. Cell. Biochem. 157: 75-86 [Medline].

112. Wagner, C. T., W. Durante, N. Christodoulides, J. D. Hellums, and A. I. Schafer. 1997. Hemodynamic forces induce the expression of heme oxygenase in cultured vascular smooth muscle cells. J. Clin. Invest 100: 589-596 [Medline].

113. Hayashi, S., R. Takamiya, T. Yamaguchi, K. Matsumoto, S. J. Tojo, T. Tamatani, M. Kitajima, N. Makino, Y. Ishimura, and M. Suematsu. 1999. Induction of heme oxygenase-1 suppresses venular leukocyte adhesion elicited by oxidative stress: role of bilirubin generated by the enzyme. Circ. Res 85: 663-671 [Abstract/Free Full Text].

114. Yet, S.-F., R. Tian, M. D. Layne, Z. Y. Wang, K. Maemura, M. Solovyeva, B. Ith, L. G. Melo, L. Zhang, J. S. Ingwall, V. J. Dzau, M.-E. Lee, and M. A. Perrella. 2001. Cardiac-specific expression of heme oxygenase-1 protects against ischemia and reperfusion injury in transgenic mice. Circ. Res 89: 168-173 [Abstract/Free Full Text].

115. Clark, J. E., R. Foresti, P. Sarathchandra, H. Kaur, C. J. Green, and R. Motterlini. 2000. Heme oxygenase-1 derived bilirubin ameliorates postischemic myocardial dysfunction. Am. J. Physiol. 278: H643-H651 .

116. Greabu, M., R. Olinescu, F. A. Kummerow, and D. O. Crocnan. 2001. The levels of bilirubin may be related to an inflammatory condition in patients with coronary heart disease. Acta Pol. Pharm. 58: 225-231 [Medline].

117. Vítek, L., M. Jirsa, M. Brodanová, M. Kaláb, Z. Marecek, V. Danzig, L. Novotný, and P. Kotal. 2001. Gilbert syndrome and ischemic heart disease: a protective effect of elevated bilirubin levels (abstr). Jpn. J. Cardiovasc. Dis. Prev. 36: 40 .

118. Djoussé, L., D. Levy, A. Cupples, J. C. Evans, R. B. D'Agostino, and C. Ellison. 2001. Total serum bilirubin and risk of cardiovascular disease in the Framingham Offspring study. Am. J. Cardiol. 87: 1196-1200 [Medline].

119. Fujita, T., K. Toda, A. Karimova, S.-F. Yan, Y. Naka, S.-F. Yet, and D. J. Pinsky. 2001. Paradoxical rescue from ischemic lung injury by inhaled carbon monoxide driven by derepression of fibrinolysis. Nat. Med. 7: 598-604 [Medline].

120. Sato, K., J. Balla, L. Otterbein, R. N. Smith, S. Brouard, Y. Lin, E. Csizmadia, J. Sevigny, S. C. Robson, G. Vercellotti, A. M. Choi, F. H. Bach, and M. P. Soares. 2001. Carbon monoxide generated by heme oxygenase-1 suppresses the rejection of mouse-to-rat cardiac transplants. J. Immunol 166: 4185-4194 [Abstract/Free Full Text].

121. Togane, Y., T. Morita, M. Suematsu, Y. Ishimura, J. I. Yamazaki, and S. Katayama. 2000. Protective roles of endogenous carbon monoxide in neointimal development elicited by arterial injury. Am. J. Physiol. Heart Circ. Physiol. 278: H623-H632 [Abstract/Free Full Text].

122. Aizawa, T., N. Ishizaka, J. Taguchi, S. Kimura, K. Kurokawa, and M. Ohno. 1999. Balloon injury does not induce heme oxygenase-1 expression, but administration of hemin inhibits neointimal formation in balloon-injured rat carotid artery. Biochem. Biophys. Res. Commun. 261: 302-307 [Medline].

123. Tulis, D. A., W. Durante, X. Liu, A. J. Evans, K. J. Peyton, and A. I. Schafer. 2001. Adenovirus-mediated heme oxygenase-1 gene delivery inhibits injury-induced vascular neointima formation. Circulation 104: 2710-2715 [Abstract/Free Full Text].

124. Exner, M., M. Schillinger, E. Minar, W. Mlekusch, G. Schlerka, M. Haumer, C. Mannhalter, and O. Wagner. 2001. Heme oxygenase-1 gene promoter microsatellite polymorphism is associated with restenosis after percutaneous transluminal angioplasty. J. Endovasc. Ther. 8: 433-440 . [Medline]

125. Zayasu, K., K. Sekizawa, S. Okinaga, M. Yamaya, and H. Sasaki. 1997. Increased carbon monoxide in exhaled air of asthmatic patients. Am. J. Respir. Crit. Care Med. 156: 1140-1143 .

126. Horvath, I., L. E. Donelly, P. Paredi, S. A. Kharitonov, and P. J. Barnes. 1998. Elevated levels of exhaled carbon monoxide are associated with an incresased expression of heme oxygenase-1 in airway macrophages in asthma: a new marker of chronic inflammation. Thorax 53: 668-672 .

127. Paredi, P., S. A. Kharitonov, D. Leak, P. L. Shah, D. Cramer, M. E. Hodson, and P. J. Barnes. 2000. Exhaled ethane is elevated in cystic fibrosis and correlates with carbon monoxide levels and airway obstruction. Am. J. Respir. Crit. Care Med. 161: 1247-1251 [Abstract/Free Full Text].

128. Paredi, P., W. Biernacki, G. Invernizzi, S. A. Kharitonov, and P. J. Barnes. 1999. Exhaled carbon monoxide levels elevated in diabetes and correlated with glucose concentration in blood: a new test for monitoring the disease? Chest 116: 1007-1111 [Abstract/Free Full Text].

129. Scharte, M., H. G. Bone, H. Van Aken, and J. Meyer. 2000. Increased carbon monoxide in exhaled air of critically ill patients. Biochem. Biophys. Res. Commun. 267: 423-426 [Medline].

130. Yamara, M., K. Sekizawa, S. Ishizuka, M. Monma, and H. Sasaki. 1999. Exhaled carbon monoxide levels during treatment of acute asthma. Eur. Resp. J. 13: 757-760 [Abstract].

131. Antuni, J. D., S. A. Kharitonov, D. Hughes, M. E. Hodson, and P. J. Barnes. 2000. Increase in exhaled carbon monoxide during exacerbations of cystic fibrosis. Thorax 55: 138-142 [Abstract/Free Full Text].

132. Motterlini, R., J. E. Clark, R. Foresti, P. Sarathchandra, B. E. Mann, and C. J. Green. 2002. Carbon monoxide-releasing molecules: characterization of biochemical and vascular activities. Circ. Res. 90: e17-e24 [Abstract/Free Full Text].

133. Siow, R. C. M., H. Sata, and G. E. Mann. 1999. Heme oxygenase-carbon monoxide signaling pathway in atherosclerosis: anti-atherogenic actions of bilirubin and carbon monoxide? Cardiovasc. Res. 41: 385-394 [Abstract/Free Full Text].

134. Ohta, K., and A.Yachie, K. Rujimoto, H. Kaneda, T. Wada, T. Toma, A. Seno, Y. Kasahara, H. Yokoyama, H. Seki, S. Koizumi. 2000. Tubular injury as a cardinal pathologic feature in human heme oxygenase-1 deficiency. Am. J. Kid. Dis. 35: 863-870 [Medline].

135. Nath, K. A., G. Balla, G. M. Vercellotti, J. Balla, H. S. Jacob, M. D. Levitt, and M. E. Rosenberg. 1992. Induction of heme oxygenase is a rapid, protective response in rhabdomyolysis in the rat. J. Clin. Invest. 90: 267-270 .

136. Smith, M. A., R. K. Kutty, P. L. Richey, S. D. Yan, D. Stern, G. J. Hader, B. Wiggert, R. B. Petersen, and G. Perry. 1994. Heme oxygenase-1 is associated with the neurofibrillary pathology of Alzheimer's disease. Am. J. Pathol. 145: 42-47 [Abstract].

137. Mautes, A. E., D. H. Kim, F. R. Sharp, S. Panter, M. Sata, N. Maida, M. Bergeron, K. Guenther, and L. J. Noble. 1998. Induction of heme oxygenase-1 (HO-1) in the contused spinal cord of the rat. Brain Res. 795: 17-24 [Medline].

138. Chen, W., D. M. Hunt, H. Lu, and R. C. Hunt. 1999. Expression of antioxidant protective proteins in the rat retina during prenatal and postnatal development. Invest. Opthalmol. Vis. Sci 40: 744-751 [Abstract/Free Full Text].

139. Matz, P. G., P. R. Winstein, and F. R. Sharp. 1997. Heme oxygenase-1 and heat shock protein 70 induction in glia and neurons throughout rat brain after experimental intracerebral hemorrhage. Neurosurgery 40: 152-160 [Medline].

140. Montecot, C., J. Seylaz, and E. Pinard. 1998. Carbon monoxide regulates cerebral blood flow in epileptic seizures but not in hypercapnia. Neuroreport 9: 2341-2346 [Medline].

141. Verma, A., D. J. Hirsch, C. E. Glatt, G. W. Ronnett, and S. H. Snyder. 1993. Carbon monoxide: a putative neural messenger. Science 259: 381-384 [Abstract/Free Full Text].

142. Sato, H., R. C. Siow, S. Bartlett, S. Taketani, T. Ishi, S. Bannai, and G. E. Mann. 1997. Expression of stress proteins heme oxygenase-1 and -2 in acute pancreatitis and pancreatic islet betaTC3 and acinar AR42J cells. FEBS Lett. 405: 219-223 [Medline].

143. Donat, M. E., K. Wong, W. A. Staines, and A. Krantis. 1999. Heme oxygenase immunoreactive neurons in the rat intestine and their relationship to nitrergic neurons. J. Auton. Nerv. Syst. 77: 4-12 [Medline].

144. Suematsu, M., and Y. Ishimura. 2000. The heme oxygenase-carbon monoxide system: a regulator of hepatobiliary function. Hepatology 31: 3-6 [Medline].

145. Kyokane, T., S. Norimizu, H. Taniai, T. Yamaguchi, S. Takeoka, E. Tsu, and - chida, M. Naito, Y. Nimura, Y. Ishimura, and M. Suematsu. 2001. Carbon monoxide from heme catabolism protects against hepatobiliary dysfunction in endotoxin-treated rat liver. Gastroenterology. 120: 1227-1240 [Medline].

146. Rensing, H., I. Bauer, V. Datene, C. Patau, B. H. Pannen, and M. Bauer. 1999. Differential expression pattern of heme oxygenase-1/heat shock protein 32 and nitric oxide synthase-II and their impact on liver injury in a rat model of hemorrhage and resuscitation. Crit. Care Med. 27: 2766-2775 [Medline].

147. Levere, R. D., R. Staudinger, G. Loewy, A. Kappas, S. Shibahara, and N. G. Abraham. 1993. Elevated levels of heme oxygenase activity and mRNA in peripheral blood adherent cells of acquired immunodeficiency syndrome patients. Am. J. Hematol. 43: 19-23 [Medline].

148. Otterbein, L., B. Y. Chin, S. L. Otterbein, V. C. Lowe, H. E. Fessler, and A. M. K. Choi. 1997. Mechanism of hemoglobin-induced protection against endotoxemia in rats: a ferritin-independent pathway. Am. J. Physiol. Lung Cell. Mol. Physiol. 272: L268-L275 [Abstract/Free Full Text].

149. Downard, P. J., M. A. Wilson, D. A. Spain, P. J. Matheson, Y. Siow, and R. N. Garrison. 1997. Heme oxygenase-dependent carbon monoxide production is a hepatic adaptive response to sepsis. J. Surg. Res. 71: 7-12 [Medline].

150. Van Uffelen, B. E., B. M. de Koster, J. Van Steveninck, and J. G. Elferink. 1996. Carbon monoxide enhances human neutrophil migration in a cyclic GMP-dependent way. Biochem. Biophys. Res. Commun. 226: 21-26 [Medline].

151. Mancuso, C., P. Preziosi, A. B. Grossman, and P. Navarra. 1997. The role of carbon monoxide in the regulation of neuroendocrine function. Neuroimmunomodulation 4: 225-229 [Medline].

152. Kim, C. K., and C. L. Rivier. 2000. Nitric oxide and carbon monoxide have a stimulatory role in the hypothalamic-pituitary-adrenal response to physico-emotional stressors in rats. Endocrinology 141: 2244-2253 [Abstract/Free Full Text].

153. Henningsson, R., P. Alm, and I. Lundquiest. 1997. Occurrence and putative hormone regulatory function of a constitutive heme oxygenase in rat pancreatic islets. Am. J. Physiol. Cell Physiol. 273: C703-C709 [Abstract/Free Full Text].

154. Baum, M., E. Schiff, D. Kreiser, P. A. Dennery, D. K. Stevenson, T. Rosenthal, and D. S. Seidman. 2000. End-tidal carbon monoxide measurements in women with pregnancy-induced hypertension and preeclampsia. Am. J. Obstet. Gynecol. 183: 900-903 [Medline].

155. Acevedo, C. H., and A. Ahmed. 1998. Hemeoxygenase-1 inhibits human myometrial contractility via carbon monoxide and is upregulated by progesterone during pregnancy. J. Clin. Invest. 101: 949-955 [Medline].

156. Lyall, F., A. Barber, L. Myatt, J. N. Bulmer, and S. C. Robson. 2000. Hemeoxygenase expression in human placenta and placental bed implies a role in regulation of trophoblast invasion and placental function. FASEB J. 14: 208-219 [Abstract/Free Full Text].

157. Poss, K. D., and S. Tonegawa. 1997. Heme oxygenase-1 is required for mammarian iron reutilization. Proc. Natl. Acad. Sci. USA 94: 10919-10924 .

158. Hedlund, P., L. Ny, P. Alm, and K. E. Andersson. 2000. Cholinergic nerves in human corpus cavernosum and spongiosum contain nitric oxide synthase and heme oxygenase. J. Urol. 164: 868-875 [Medline].