The highly toxic gas carbon monoxide (CO) displays many physiological roles in several organs and tissues. Although many diseases, including cancer, hematological diseases, hypertension, heart failure, inflammation, sepsis, neurodegeneration, and sleep disorders, have been linked to abnormal endogenous CO metabolism and functions, CO administration has therapeutic potential in inflammation, sepsis, lung injury, cardiovascular diseases, transplantation, and cancer. Here, insights into the CO‐based therapy, characterized by the induction or gene transfer of heme oxygenase‐1 and either gas or CO‐releasing molecule administration, are reviewed. 2012 IUBMB IUBMB Life, 2012

Abbreviations CO, carbon monoxide; CO‐RM, CO‐releasing molecule; HO‐1, heme oxygenase‐1; IR, ischemia reperfusion; MAPK, mitogen‐activated protein kinase.

INTRODUCTION Carbon monoxide (CO) is a colorless, odorless, tasteless, and nonirritating but highly toxic gas generated by both natural and manufactured processes (1). However, in anaerobic bacteria and archaea, exogenous CO represents a source of carbon and energy (2, 3), whereas in eukaryotes endogenous CO acts as a physiological signaling molecule (4-8). CO displays many physiological roles in the neuronal, cardiovascular, and immune systems, as well as in the respiratory, reproductive, gastrointestinal, and urogenital apparatus, including anti‐apoptotic, anti‐inflammatory, anti‐oxidant, anti‐proliferative, and vasodilator effects (4, 5, 8, 9). Although many pathologies, including cancer, hematological diseases, hypertension, heart failure, inflammation, sepsis, neurodegeneration, and sleep disorders, have been linked to abnormal endogenous CO metabolism and functions (7-10), CO displays therapeutic actions (4, 11-14). An abundance of preclinical evidence shows the beneficial effects of CO in several pathological conditions, including inflammation, sepsis, lung injury, cardiovascular diseases, transplantation, and cancer (4, 7, 8, 10-12, 14). The therapeutic use of CO is based on (i) the induction or gene transfer of HO‐1 (15-17), (ii) the inhalation of gaseous CO, and (iii) the use of CO‐releasing molecules (CO‐RMs) (14). Here, recent insights into the CO‐based therapy are reviewed highlighting aspects relevant for human health.

INDUCTION OR GENE TRANSFER OF HO‐1 The induction of HO‐1 by various forms of oxidative stress stimuli has been implicated in a number of injuries and diseases such as ischemia‐reperfusion (IR) injury or inflammation (16-25). Since overexpression of the HO‐1 gene provides cytoprotection against oxidative stress, as demonstrated both in vitro and in vivo, the specific activation of HO‐1 gene expression by pharmacological modulation may represent a novel target for therapeutic intervention. Significant therapeutic potential of HO‐1 gene modulation has been implicated in various immune functions that are important in transplantation medicine. Pharmaceuticals, in particular those with low inherent toxicity, may be used to induce HO‐1 therapeutically. Notably, HO‐1 activation is induced by metabolites (e.g., heme, toxins, cytokines, and hormones) and diatomic gases (e.g., CO and NO) (18). Optimization and organ targeting of vectors for gene therapy approaches involving HO‐1 represents another avenue of active development. In this context, a preventive medicine approach may involve the screening of individuals for HMOX1 promoter polymorphisms to identify those individuals who may have an increased risk of disease (e.g., vascular diseases, atherosclerosis, diabetes mellitus, and cancer) (14, 23, 24, 26-28).

GASEOUS CO CO reduces the inflammation associated with allergen‐induced asthma in mice (29), protects against orthotopic lung transplantation (30) and lung injury caused by oxidants (31), and reverses established pulmonary hypertension (32). Moreover, the inhalation of CO, at concentrations ranging between 20 and 500 ppm, has been shown to exert cytoprotective effects in murine models of IR and organ transplantation (33). Furthermore, CO inhalation is effective against ischemic lung injury by attenuating deposition of fibrin in the microvasculature (34, 35) and by reducing neutrophil recruitment to alveoli in ventilator‐induced injury (36). However, the positive view emerging from the possible application of CO gas inhalation therapy in the treatment of lung disease has been challenged by several evidences indicating no significant benefit of CO gas treatment on hyperoxic acute lung injury and neurotoxicity at CO concentrations ranging between 200 and 500 ppm (9, 37).

CO‐RELEASING MOLECULES The development of compounds that carry and release CO into biological systems, for experimental administration of CO, has been shown to be a valid alternative to the use of gaseous CO. Indeed, CO‐RMs display anti‐inflammatory effects, anti‐apoptotic properties, anti‐microbial activity, therapeutic potential in vascular diseases, and application in organ preservation. Remarkably, the rate of CO release from CO‐RMs can be finely modulated allowing the modification of the ensuing biological effects (12, 14, 38, 39). Transition metal carbonyls with structures simpler than HbCO are the best examples of how CO covalently bound to a metal can be carried and released under appropriate conditions (12, 14, 39, 40). CORM‐1 (Mn 2 CO 10 ) and CORM‐2 ([Ru(CO) 3 Cl 2 ‐dimer]) promote vasodilatation, hypotension, and reno‐protection in vivo (41-43). CORM‐2 also has anti‐inflammatory, anti‐carcinogenic, anti‐apoptotic, anti‐proliferative, and pro‐angiogenic effects (41, 44-51). CORM‐3 ([Ru(CO) 3 Cl(glycinate)]) displays cardio‐protective, reno‐protective, anti‐inflammatory, anti‐ischemic, and vasodilator effects; furthermore, CORM‐3 acts as an inhibitor of platelet aggregation (45, 52-60) (Table 1). CORM‐2, CORM‐3, and CORM‐371 ([Me 4 N][Mn(CO) 4 (thioacetate) 2 ]) exert a significant bactericidal effect decreasing O 2 consumption and growth of Pseudomonas aeruginosa (61). Table 1. Chemical structure and pharmacological actions of the most relevant CO‐releasing molecules (CO‐RMs) showing therapeutic potential Molecule Chemical structure Pharmacological action References CORM‐1 Vasodilator 41-43 Reno‐protective CORM‐2 Vasodilator 41 44-51 61 Reno‐protective Anti‐inflammatory Anticarcinogenic Proangiogenic Antiapoptotic Antiproliferative Bactericidal CORM‐3 Vasodilator 45 52-61 Reno‐protective Cardio‐protective Anti‐inflammatory Anti‐ischemic Antiplatelet aggregation Bactericidal CORM‐A1 Vasodilator 57 62 67 Reno‐protective Anti‐ischemic Antiapoptotic CORM‐F3 Vasodilator 62 63 Anti‐inflammatory Anti‐ischemic Antioxidant CORM‐F3 (η‐4‐(4‐bromo‐6‐methyl‐2‐pyrone)tricarbonyl iron (0)) causes vasorelaxation in isolated aortic rings and inhibits the inflammatory response of a murine monocyte macrophage cell line stimulated with endotoxin. When the bromide at the 4‐position of the 2‐pyrone CORM‐F3 is substituted with a chloride group (η‐4‐(4‐chloro‐6‐methyl‐2‐pyrone)tricarbonyl iron (0)) (CORM‐F8), the rate of CO release is significantly decreased (4.5‐fold). A further decrease is observed when the 4‐ and 6‐positions are substituted with a methyl group (η‐4‐(4‐methyl‐6‐methyl‐2‐pyrone)tricarbonyl iron (0)) (CORM‐F11) or a hydrogen group (η‐4‐(4‐chloro‐2‐pyrone)tricarbonyl iron (0)) (CORM‐F7), respectively. Interestingly, CORM‐F3 and CORM‐F8, containing halogens at the 4‐position and the methyl group at the 6‐position of the 2‐pyrone ring, have been found to be less cytotoxic when compared with other CO‐RMs (62, 63) (Table 1). CO‐RM ALF492 (tricarbonyldichloro(thiogalactopyranoside)ruthenium(II)) controls CO delivery in vivo without affecting O 2 transport by hemoglobin, fully protecting mice against experimental cerebral malaria and acute lung injury. The protective effect is CO dependent and mediated by the expression of HO‐1, which contributes for the observed protection. CO‐RM ALF492, in combination with the antimalarial drug artesunate, is an effective adjunctive and adjuvant treatment for experimental cerebral malaria conferring protection after the onset of severe disease (64). CORM‐S1 ((dicarbonyl‐bis(cysteamine)Fe(II))) releases CO under irradiation with visible light activating CO‐sensitive ion channels, thus arguing to contribute to CO‐mediated vasodilatation (65). [Mn(CO) 4 {S 2 CNMe(CH 2 CO 2 H)}] is a CO‐RM providing at least three moles of CO per mole of compound. CO is rapidly released in the presence of either a CO receptor or a ligand that prevents CO re‐binding (66). CORM‐A1 (Na 2 [H 3 BCO 2 ]), which does not contain a transition metal carbonyl but a boron atom to which a carboxyl group is covalently bound and converted to CO through hydrolysis, causes vasodilator, hypotensive, anti‐ischemic, and anti‐apoptotic effects (57, 62, 67). Moreover, CORM‐A1 is bacteriostatic rather than bactericidal in vitro eliciting only a moderate and transient decrease in O 2 consumption by Pseudomonas aeruginosa (61) (Table 1). CO‐RM actions are partially inhibited by the soluble guanylate cyclase inhibitor 1H‐[1,2,4]oxadiazolo[4,3‐a]quinoxalin‐1‐one and intensified by the (3‐(5′‐hydroxymethyl‐2′‐furyl)‐1‐benzylindazole), an activator of the soluble guanylate cyclase/cGMP pathway. To date, it is controversial whether CO‐RMs are affected or not by blockers of K ATP potassium channels (e.g., iberiotoxin) (14, 67-70). As a whole, CO‐RMs, administered either intraperitoneally or by intravenous infusion, show vasodilator and reno‐protective effects, with CORM‐2, CORM‐3, and CORM‐A1 also showing anti‐inflammatory, anti‐apoptotic, anti‐ischemic, anti‐carcinogenic, anti‐proliferative, pro‐angiogenic, anti‐aggregatory, and cardio‐protective activities, as well as regulate mitochondrial respiration. In addition to the pharmaceutical use, CO‐RMs may represent a valid expedient to explore the interaction of CO with various cellular targets and to identify and elucidate CO action mechanisms (12, 14, 39).

CO‐BASED THERAPY Inflammation and Sepsis The anti‐inflammatory action of CO has been demonstrated in several animal models, suggesting a possible therapeutic application for inflammatory diseases (7, 12, 14). CO‐RMs have been shown to attenuate acute inflammatory response both in vitro and in vivo and to ameliorate neuroinflammatory response in microglia (71, 72). The efficacy of CO administration has also been observed in models of carrageenan‐induced inflammation (47). Moreover, positive effects of gaseous CO have been observed in a mouse model of cerebral malaria in which the brain inflammation is responsible for much of the morbidity and mortality, with symptoms closely resembling those observed in parasite‐infected patients (64, 73). Very recently, the use of CORM‐A1 has been hypothesized to represent a novel therapeutic strategy for the treatment of multiple sclerosis (74). CO and CO‐RMs administration appears to be useful also in acute hepatitis (75, 76) and sepsis (77, 78). CO inhalation after injection of a lethal dose of endotoxins has been demonstrated to rescue 20–90% of mice from fulminant hepatitis (79). In contrast, no anti‐inflammatory effects of CO inhalation on systemic cytokine production during experimental endotoxemia have been reported in humans (80). Systemic administration of CORM‐2 in septic mice attenuates the accumulation of neutrophils, the expression of intracellular adhesion molecule 1, and the activation of nuclear factor κ‐light‐chain enhancer of activated B cells; moreover, CORM‐2 administration decreases both the NO and the reactive oxygen species production (81). The administration of either CORM‐2 or CORM‐3 in rodents with cecal ligation and puncture‐induced sepsis results in the reduced migration of polymorphonuclear leukocytes and in the increased survival rate (78, 82). CORM‐3 also shows bactericidal effects in both immunocompetent and immunosuppressed mice (83); however, preliminary studies do not show direct effects of CO on bacterial survival (14). In post‐operative ileus, intestinal contractility and transit were restored in mice after treatment with either CO or CORM‐3 or CORM‐A1 (33, 84, 85). Phase I and II clinical trials have been planned to determine whether inhalation of very low doses of CO before and after colon surgery may shorten the duration of normal post‐operative ileus and/or prevent the development of post‐operative ileus complications in patients undergoing colon surgery (ClinicalTrials.gov Identifier: NCT01050933; ClinicalTrials.gov Identifier: NCT01050712). Lung Injury The beneficial effect of CO on lung diseases is openly debated. Indeed, although in vivo models of lung injury induced by acid aspiration (86), aeroallergens (29, 87), bleomycin (88), hyperoxia (31, 89), IR (34, 90-92), and mechanical stretch (36) highlighted efficacy of inhaled CO, other studies did not evidence any beneficial impact of CO (9, 37, 93–95). Lung protection by induction or gene transfer of HO‐1 and CO has been demonstrated in vitro and in vivo in several models of experimental acute lung injury and sepsis (96). The administration of low doses of CO after cardiopulmonary bypass attenuates lung injury in a pig model (97). Cytoprotective effects elicited by CO in the lung appear to be mediated by mitogen‐activated protein kinases (MAPK; e.g., p38 and MAPK kinase 3), peroxysome proliferator‐activated receptor‐γ, caspase‐3, caveolin‐1, and cGMP‐dependent mechanisms (31, 34-36, 98, 99). Interestingly, the feasibility and anti‐inflammatory effects of low‐dose CO inhalation in patients with chronic obstructive pulmonary disease have been demonstrated (ref. 100; ClinicalTrials.gov, Identifier: NCT00122694). Moreover, the effects of inhaled CO on local pulmonary inflammatory responses following endotoxin administration have been recently demonstrated (ClinicalTrials.gov, Identifier: NCT00094406). Finally, a phase II clinical trial to determine whether low concentration of inhaled CO is effective in treating idiopathic pulmonary fibrosis has been planned (ClinicalTrials.gov Identifier: NCT01214187). Cardiovascular Diseases The pharmacological induction or gene transfer of HO‐1, the direct administration of CO via inhalation, and the use of CO‐RMs have been suggested as therapeutic targets in the treatment of vascular diseases (13, 14, 101). The pharmacological induction and gene transfer of HO‐1 ameliorate vascular dysfunction in animal models of atherosclerosis, postangioplasty restenosis, vein graft stenosis, thrombosis, myocardial infarction, and hypertension, whereas inhibition of HO‐1 activity or gene deletion exacerbates these disorders (101). Products of HO‐1 induction (i.e., CO, biliverdin, and bilirubin) exert potent cyto‐ and vaso‐protective (i.e., anti‐inflammatory, anti‐oxidant, anti‐apoptotic, anti‐thrombotic, and vaso‐active) actions and preserve vascular homeostasis at sites of arterial injury by influencing the proliferation, migration, and adhesion of vascular smooth muscle cells, endothelial cells, endothelial progenitor cells, and leukocytes (101). CO inhibits smooth muscle cell proliferation and prevents vascular intimal hyperplasia in animal models of airway and vascular proliferative diseases (102-106). The effects of CO on vascular smooth muscle cells are NO independent and require cGMP and p38 MAPK, whereas the effects of CO on endothelial cells are NO‐ and NO synthase‐dependent, involving the modulation of RhoA and Akt signaling pathways (14). In rodent models of established pulmonary arterial hypertension, CO inhalation restores thickened pulmonary arteries and the right heart to normal size and pressure (32). CO has been shown to produce vasodilatation and to improve microvascular hemodynamics (e.g., vessel diameter, red blood cell velocity, and functional capillary density) in the hamster window chamber model (107). CO‐RMs have been shown to possess anti‐hypertensive and anti‐aggregatory effects (41, 56, 108). Moreover, CO‐RMs have vasodilator and anti‐apoptotic effects in the cerebral circulation (42, 48, 109). CORM‐2 treatment provides cardioprotection against acute doxorubicin‐induced cardiotoxicity in mice, and this effect may be attributed to CORM‐2‐mediated anti‐oxidant and anti‐apoptotic properties (70). Cancer CO‐RMs may possess chemoprotective properties (12, 14, 38, 39). In particular, CORM‐2 may modulate inflammation‐induced colon carcinogenesis by modulating nuclear factors involved in the transcription of genes implicated in the development of intestinal inflammation and cancer progression. Indeed, CORM‐2 reduces the expression of inducible NOS and interleukin‐6 and ‐8 caused by pro‐inflammatory cytokines. CORM‐2 inhibits metalloproteinase‐7 (also named matrilysin) expression via interleukin‐6, which is involved in carcinogenesis (71). Furthermore, topical CORM‐2 treatment of female albino Skh:hr‐1 hairless mice, chronically irradiated with daily minimally erythemogenic doses of solar‐simulated UV radiation (to induce photocarcinogenesis), reduces the acute and chronic inflammatory erythema reaction, as well as the chronic epidermal hyperplasia accompanying tumor outgrowth (50). Overall, the CORM‐2 treatment provides a significant moderate inhibition of early tumor appearance dose dependently, significantly reduces the average tumor multiplicity, increases the regression of established tumors dose dependently, and inhibits the formation of large locally invasive tumors. The CORM‐2 treatment also reduced the expression of immunosuppressive interleukin‐10 in the uninvolved epidermis and dermis of tumor‐bearing mice and enhances immunopotentiating epidermal interleukin‐12 expression (50). Lastly, CORM‐3 can be used as an effective therapeutic adjuvant in the treatment of nephrotoxicity caused by cisplatin, a widely used antineoplastic agent (58).

CO AND TRANSPLANTATION CO and CO‐RMs display beneficial effects on various organ and cell transplantation systems, including heart (33, 110, 111), lung (33, 112, 113), intestine (33, 114), kidney (33, 115-119), liver (120, 121), pancreas (33), and vascular diseases (33, 122). Overall, CO administration results particularly useful in the preservation of organs for transplantation (33, 57, 59, 123), preventing IR injury, as well as allograft and xenograft rejection (33, 116). In particular, CO improves cardiac energetics by protecting the heart during reperfusion after cardiopulmonary bypass in pigs (124). Moreover, CO and CO‐RMs administration diminishes IR injury associated with cardiac rejection after transplantation in a model of cardiac allograft rejection in rodents (52, 125). Notably, intravenous infusion of CORM‐3 before reperfusion reduces infarct size, fibrillation, and tachycardia in a mouse myocardial infarct model of coronary artery occlusion (52, 53, 110). Treatment with CORM‐2 improves survival after aortic transplantation in HO‐1‐deficient mice, reducing platelet aggregation in the graft (111) and confirming antiaggregatory properties of CO and CORM‐3 (56). These cardio‐protective mechanisms mediated by CO‐RMs probably involve potassium channels (14). The addition of CO to organ preservation solution can improve the function of the transplanted kidney and the survival of the recipient. Indeed, it has been demonstrated that the use of a CO‐containing preservation solution (i) diminishes oxidative injury and the upregulation of inflammatory participants in the kidney, as assessed 3 h after transplantation, (ii) improves renal function and reduces histological injury at 1 month after transplantation, and (iii) enhances survival of the recipients of a kidney over 100 days after transplantation (33, 126). Moreover, it has been demonstrated that ex vivo application of CO in preservation solution prevents transplant‐induced renal IR injury in pigs (118). Using a pig kidney allograft model of delayed graft function, it has been demonstrated that intraoperative administration of inhaled CO at low nontoxic doses reduces IR injury and allows earlier return of function in kidney with delayed graft function (117). In a rat transplant model, CORM‐2‐derived CO protects renal transplants from IR injury by modulating inflammation. All recipients receiving CORM‐2‐treated isografts survived the transplant process and had near‐normal serum creatinine levels, whereas all control animals died of uremia by the third post‐operative day. This beneficial effect was also observed in isografted Lewis recipients receiving kidneys perfused with CORM‐3, indicating that CO‐RMs have direct effects on the kidney (119). Furthermore, pre‐treatment of human umbilical vein endothelial cells in culture with CORM‐2 for 1 h significantly reduces (i) cytokine‐induced NADPH‐dependent production of superoxide and activation of the nuclear factor‐κB and (ii) the upregulated expression of the adhesion proteins E‐selectin and intercellular adhesion molecule‐1, as well as leukocyte adhesion to the endothelial cells (119). Low dose of CORM‐3 has been shown to significantly ameliorate the effects of IR in a porcine model of controlled non heart‐beating donor kidney transplantation (115). As proposed for the heart and kidney transplantation, CO‐RMs could be used as adjuvant therapeutics in preservation solutions to limit the injury sustained by donor livers during cold storage prior to transplantation. CORM‐3 addition to preservation solution prevents the injury caused by cold storage in a rat model of an isolated normothermic perfused liver system. Indeed, CORM‐3 acts by significantly improving the perfusion flow during reperfusion (by almost 90%) and by decreasing the intrahepatic resistance (by 88%) when compared with livers cold‐preserved in preservation solution alone. CORM‐3 supplementation also preserves good metabolic capacity as indicated by hepatic O 2 consumption, glycogen content, and release of lactate dehydrogenase. Liver histology is also partially preserved by CORM‐3 treatment (123).

CONCLUSION AND PERSPECTIVES The well‐known adverse effects of CO intoxication are counterbalanced by beneficial effects of CO administration (either as induction or gene transfer of HO‐1 or as a gas or as a CO‐RM), opening new scenarios in the treatment of IR injury, acute and chronic inflammation and sepsis, lung injury, cardiovascular diseases, cancer, and organ transplantation. In particular, gaseous CO has been already evaluated in phase I testing in healthy humans with dose‐escalation studies of the inhaled gas; actually, phase II testing are ongoing. However, CO gas is not tissue specific, this representing an important characteristic of CO‐RMs. Indeed, CO‐RMs have the potential to reduce excessive exposure through tissue‐specific delivery as well as through a range of CO‐released kinetics. As a whole, CO should be considered as a Janus molecule showing both toxic and beneficial effects.

Acknowledgements This work was partially supported by grants from the Italian Ministry of Education, University and Research (CLAR 2011 to A. di Masi and to P. Ascenzi). The authors apologize to many authors of the outstanding studies that were not cited here due to space limitation.