Abstract-Carbon monoxide, which is generated in mammals during the degradation of heme by the enzyme heme oxygenase, is an important signaling mediator. Transition metal carbonyls have been recently shown to function as carbon monoxide-releasing molecules (CO-RMs) and to elicit distinct pharmacological activities in biological systems.In the present study, we report that a water-soluble form of CO-RM promotes cardioprotection in vitro and in vivo. Specifically, we found that tricarbonylchloro(glycinato)ruthenium(II) (CORM-3) is stable in water at acidic pH but in physiological buffers rapidly liberates CO in solution. Cardiac cells pretreated with CORM-3 (10 to 50 mol/L) become more resistant to the damage caused by hypoxia-reoxygenation and oxidative stress. In addition, isolated hearts reperfused in the presence of CORM-3 (10 mol/L) after an ischemic event displayed a significant recovery in myocardial performance and a marked and significant reduction in cardiac muscle damage and infarct size. The cardioprotective effects mediated by CORM-3 in cardiac cells and isolated hearts were totally abolished by 5-hydroxydecanoic acid, an inhibitor of mitochondrial ATP-dependent potassium channels. Predictably, cardioprotection is lost when CORM-3 is replaced by an inactive form (iCORM-3) that is incapable of liberating CO. Using a model of cardiac allograft rejection in mice, we also found that treatment of recipients with CORM-3 but not iCORM-3 considerably prolonged the survival rate of transplanted hearts. These data corroborate the notion that transition metal carbonyls could be used as carriers to deliver CO and highlight the bioactivity and potential therapeutic features of CO-RMs in the mitigation of cardiac dysfunction. Key Words: transition metal carbonyls Ⅲ carbon monoxide-releasing molecules Ⅲ myocardial ischemia Ⅲ heart transplantation Ⅲ reperfusion injury M ammalian cells constantly generate carbon monoxide (CO) gas via the endogenous degradation of heme by a family of constitutive (HO-2) and inducible (HO-1) heme oxygenase enzymes. 1,2 Firstly described as a putative neural messenger, 3 CO is now regarded as a versatile signaling molecule having essential regulatory roles in a variety of physiological and pathophysiological processes that take place within the cardiovascular, nervous, and immune systems. Indeed, CO produced in the vessel wall by heme oxygenase enzymes possesses vasorelaxing properties and has been shown to prevent vasoconstriction and both acute and chronic hypertension through stimulation of soluble guanylate cyclase. 4 -10 Endogenous CO appears to modulate sinusoidal tone in the hepatic circulation, 11 control the proliferation of vascular smooth muscle cells 12 and suppress the rejection of transplanted hearts. 13 The biological action of heme oxygenase-derived CO is substantiated by the pharmacological effects observed when this gas is applied exogenously to in vitro and in vivo systems. At concentrations ranging from 10 to 500 ppm, CO gas has been reported to mediate potent antiinfl...

Abstract-Carbon monoxide (CO) is generated in living organisms during the degradation of heme by the enzyme heme oxygenase, which exists in constitutive (HO-2 and HO-3) and inducible (HO-1) isoforms. Carbon monoxide gas is known to dilate blood vessels in a manner similar to nitric oxide and has been recently shown to possess antiinflammatory and antiapoptotic properties. We report that a series of transition metal carbonyls, termed here carbon monoxide-releasing molecules (CO-RMs), liberate CO to elicit direct biological activities. Specifically, spectrophotometric and NMR analysis revealed that dimanganese decacarbonyl and tricarbonyldichlororuthenium (II) dimer release CO in a concentration-dependent manner. Moreover, CO-RMs caused sustained vasodilation in precontracted rat aortic rings, attenuated coronary vasoconstriction in hearts ex vivo, and significantly reduced acute hypertension in vivo. These vascular effects were mimicked by induction of HO-1 after treatment of animals with hemin, which increases endogenously generated CO. Thus, we have identified a novel class of compounds that are useful as prototypes for studying the bioactivity of CO. In the long term, transition metal carbonyls could be utilized for the therapeutic delivery of CO to alleviate vascular-and immuno-related dysfunctions. A lthough it has been known for a long time that carbon monoxide (CO) is generated in the human body, 1 only in recent years have scientists begun to explore the possible biological activities of this gaseous molecule. The main endogenous source of CO is heme oxygenase, which exists in constitutive (HO-2 and HO-3) and inducible (HO-1) isoforms; heme serves as substrate for HO-1 and HO-2 in the formation of CO, free ferrous iron, and biliverdin, the latter being rapidly converted to bilirubin by biliverdin reductase. 2 There is general consensus, supported by extensive published reports, that HO-1 represents a pivotal inducible defensive system against stressful stimuli, including UVA radiation, carcinogens, ischemia-reperfusion damage, endotoxic shock, and several other conditions characterized by production of oxygen-derived free radicals. [2][3][4] As part of its physiological and cytoprotective actions, heme oxygenase-derived CO appears to play a major role as neurotransmitter, 5-7 regulator of sinusoidal tone, 8 inhibitor of platelet aggregation, 9 and suppressor of acute hypertensive responses. 10,11 In addition, exogenously applied CO has been shown to protect against lung injury in vivo, 12,13 prevent both production of proinflammatory cytokines 14 and endothelial cell apoptosis, 15 and suppress graft rejection in mouse-to-rat cardiac transplants 16 ; all these effects are simulated by transfection of the HO-1 gene. Thus, consistent findings reveal a series of important cellular functions that support a versatile and previously unidentified role for CO. It is interesting that many of the novel properties pertaining to CO have strong analogies with the well-established biological activities elicited by nitri...

1 Carbon monoxide (CO), one of the end products of heme catabolism by heme oxygenase, possesses antihypertensive and vasodilatory characteristics. We have recently discovered that certain transition metal carbonyls are capable of releasing CO in biological fluids and modulate physiological functions via the delivery of CO. Because the initial compounds identified were not water soluble, we have synthesized new CO-releasing molecules that are chemically modified to allow solubility in water. The aim of this study was to assess the vasoactive properties of tricarbonylchloro(glycinato)ruthenium(II) (CORM-3) in vitro and in vivo. 2 CORM-3 produced a concentration-dependent relaxation in vessels precontracted with phenylephrine, exerting significant vasodilatation starting at concentrations of 25-50 mM. Inactive CORM-3, which does not release CO, did not affect vascular tone. 3 Blockers of ATP-dependent potassium channels (glibenclamide) or guanylate cyclase activity (ODQ) considerably reduced CORM-3-dependent relaxation, confirming that potassium channels activation and cGMP partly mediate the vasoactive properties of CO. In fact, increased levels of cGMP were detected in aortas following CORM-3 stimulation. 4 The in vitro and in vivo vasorelaxant activities of CORM-3 were further enhanced in the presence of YC-1, a benzylindazole derivative which is known to sensitize guanylate cyclase to activation by CO. Interestingly, inhibiting nitric oxide production or removing the endothelium significantly decreased vasodilatation by CORM-3, suggesting that factors produced by the endothelium influence CORM-3 vascular activities. 5 These results, together with our previous findings on the cardioprotective functions of CORM-3, indicate that this molecule is an excellent prototype of water-soluble CO carriers for studying the pharmacological and biological features of CO.

Bilirubin is a potent antioxidant generated intracellularly during the degradation of heme by the enzyme heme oxygenase. The purpose of this study was to determine the role of increased cardiac bilirubin in protection against postischemic myocardial dysfunction. Rat hearts were isolated and perfused according to the Langendorff technique to evaluate the recovery of myocardial function after 30 min of global ischemia and 60 min of reperfusion. We found that upregulation of the inducible isoform of heme oxygenase (HO-1) by treatment of animals with hemin 24 h before ischemia ameliorated myocardial function and reduced infarct size (tetrazolium staining) on reperfusion of isolated hearts. Tin protoporphyrin IX, an inhibitor of heme oxygenase activity, completely abolished the improved postischemic myocardial performance observed after hemin-mediated HO-1 induction. Likewise, cardiac tissue injury was exacerbated by treatment with tin protoporphyrin IX. Increased cardiac HO-1 expression and heme oxygenase activity were associated with enhanced tissue bilirubin content and an increased rate of bilirubin release into the perfusion buffer. Furthermore, exogenously administered bilirubin at concentrations as low as 100 nanomolar significantly restored myocardial function and minimized both infarct size and mitochondrial damage on reperfusion. Our data provide strong evidence for a primary role of HO-1-derived bilirubin in cardioprotection against reperfusion injury.

Carbon monoxide (CO) is a resourceful gas as recent advances in the area of cell signaling are revealing an unexpected physiological role for CO in the cardiovascular, immune and nervous systems. Transition metal carbonyls have been lately discovered to function as CO-releasing molecules (CO-RMs) and elicit distinct pharmacological activities in biological systems. Studies currently ongoing in our laboratories are investigating both the chemical and bioactive features of a series of water-soluble CO-RMs and their specific utilization as vasoactive mediators, anti-inflammatory agents and inhibitors of cellular and tissue damage. The data presented in this review corroborate the notion that transition metal carbonyls could be used as carriers to deliver CO in mammals and highlight the bioactivity and potential therapeutic features of CO-RMs in the mitigation of cellular and organ dysfunction.

Thiols are very important antioxidants that protect cells against oxidative insults. Recently, a different and new physiological role has been defined for these compounds because of their involvement in nitric oxide (NO) binding and transport in biological systems. In view of these characteristics, we examined the effect of thiols and NO on the expression of the inducible form of heme oxygenase (HO-1), a stress protein that degrades heme to carbon monoxide and biliverdin. Cultured bovine aortic endothelial cells exposed to the NO donors sodium nitroprusside (SNP) and S-nitroso-N-acetylpenicillamine (SNAP) resulted in increased heme oxygenase activity and HO-1 expression. Co-incubation with N-acetylcysteine, a precursor of glutathione synthesis, significantly attenuated heme oxygenase induction by SNP and SNAP, and a reduction in heme oxygenase activity was also observed when cells were preincubated with N-acetylcysteine for 16 h prior to exposure to NO donors. This effect appears to be associated with NO stabilization by thiols through the formation of S-nitrosothiols. Hydroxocobalamin, a specific NO scavenger, significantly decreased endothelial heme oxygenase activity, indicating a direct involvement of NO released by NO donors to regulate the expression of this stress protein. Moreover, superoxide anion (O 2 . ) and its reaction product with NO, peroxynitrite (ONOO ؊ ), were found to partially contribute to the observed NO-mediated activation of endothelial heme oxygenase. Thus, we suggest the existence of a dynamic equilibrium among free NO, O 2 . , and endogenous glutathione, which might constitute an interactive signaling mechanism modulating stress and adaptive responses in tissues.Heme oxygenase is a widely distributed enzyme in mammalian tissues, and its main function is associated with the degradation of heme to biliverdin, iron, and carbon monoxide (CO).1 Two distinct isoforms of the protein have been characterized revealing that one of the isozymes is constitutive (HO-2), whereas the other is inducible (HO-1) (1). Thus, if the first enzyme is constitutively expressed and is part of the normal cellular metabolism, the second is regarded as a heat shock protein, and its expression is elicited by many conditions and factors that produce an imbalance in the cellular functions. Various agents, including heavy metal ions (2), oxidative stress (3), endotoxins (4), and hemoglobin (5) are capable of inducing HO-1 in different tissues, and recent findings showed that nitric oxide (NO) donors increase HO-1 mRNA in the brain (6) and in cultured hepatocytes (7). Accordingly, we have reported the ability of diverse NO releasing agents to modulate heme oxygenase activity in aortic endothelial cells (8).The physiological importance of HO-1 induction following stress situations is not fully understood, although it has been hypothesized that the expression of this gene is part of the defensive mechanism that cells and tissues are capable of mounting against different stress stimuli. To sustain this idea are the findin...

Complement research experienced a renaissance with the discovery of a third activation route, the lectin pathway. We developed a unique model of total lectin pathway deficiency, a mouse strain lacking mannan-binding lectin-associated serine protease-2 (MASP-2), and analyzed the role of MASP-2 in two models of postischemic reperfusion injury (IRI). In a model of transient myocardial IRI, MASP-2-deficient mice had significantly smaller infarct volumes than their wild-type littermates. Mice deficient in the downstream complement component C4 were not protected, suggesting the existence of a previously undescribed lectin pathway-dependent C4-bypass. Lectin pathway-mediated activation of C3 in the absence of C4 was demonstrated in vitro and shown to require MASP-2, C2, and MASP-1/3. MASP-2 deficiency also protects mice from gastrointestinal IRI, as do mAb-based inhibitors of MASP-2. The therapeutic effects of MASP-2 inhibition in this experimental model suggest the utility of anti-MASP-2 antibody therapy in reperfusion injury and other lectin pathway-mediated disorders.

The enzyme heme oxygenase, which exists in inducible (HO-1) and constitutive (HO-2) isoforms, catalyzes the degradation of heme to biliverdin and CO in mammalian tissues. CO has been implicated in the control of vascular tone in a manner similar to that for NO. In the present study, we investigated the contribution of the heme oxygenase/CO pathway to the modulation of acute hypertensive responses in vivo induced by (1) alphaalphaHb, a chemically modified hemoglobin known to scavenge NO, and (2) NG-nitro-L-arginine methyl ester (L-NAME), a competitive NOS inhibitor. Experiments were carried out in conscious rats in which femoral arteries and veins were surgically catheterized 1 or 5 days before treatment with the vasoconstrictor agents. Intravenous infusion of alphaalphaHb (8% solution) or L-NAME (30 micromol/kg) [corrected] produced an acute and significant increase in mean arterial pressure (P<0.05) in rats at 5 days after catheter implantation. In contrast, no change in blood pressure was observed when alphaalphaHb or L-NAME was infused 1 day after the surgical intervention. The suppression of the hypertensive response observed at 1 day after surgery correlated with a significant (P<0.05) HO-1 expression in aorta, heart, and liver as well as increased aortic CO production and cGMP levels. At 1 day after surgery, pretreatment of animals with the heme oxygenase inhibitor zinc protoporphyrin IX (50 micromol/kg IP) markedly decreased aortic CO and cGMP levels and completely restored the vasoconstrictor effects of both alphaalphaHb and L-NAME. These results provide evidence for a crucial role of the heme oxygenase/CO pathway in the regulation of blood pressure under stress conditions in vivo.