3.1. Role of CO in Vasoregulation
The putative vasoactive properties of CO depend on, in part, the stimulation of sGC and subsequent elevation of cGMP levels (10). CO-mediated activation of sGC leads to a several-fold increase in cGMP production, a potency approximately 30-100 times lower than that of its cognate gas NO (10). CO potentially serves as a substitute for NO, during NO-deficient states, but on the other hand may antagonize the activation of sGC by NO (66). CO released from heme by the action of HO activity regulates cGMP production in vascular tissues (16,17). Exposure of VSMC to exogenous CO elevated intracellular cGMP concentrations in these cells (17). Exposure of VSMC to hypoxia, an inducer of HO-1 in this cell type, also increased endogenous levels of cGMP, which required HO activity and HO-derived CO, but excluded the involvement of NO (16). CO released from VSMC acted in a paracrine fashion to stimulate the production of cGMP in co-cultured endothelial cells (57). Morita et al. have shown that endogenous VSMC-derived CO, as well as exogenously applied CO, inhibited the proliferation of cultured VSMC (16). The mechanism whereby exogenous or HO-de-rived CO attenuates cell growth in a cGMP-dependent fashion involved the downregulation of endot-helial-derived mitogens such as platelet-derived growth factor and endothelin-1 (57), and also the inhibition of E2F-1, a transcription factor involved in cell-cycle regulation (16).
In vivo models have also supported a role for CO in vasorelaxation. In an isolated perfused rat-liver model, Suematsu et al. detected the presence of CO in the effluent. Treatment with metallopor-phyrin HO inhibitors (i.e., ZnPPIX) diminished detectible CO levels, and increased perfusion pressure under constant flow conditions. The inhibitory effects of metalloporphyrins were reversed by the application of exogenous CO or cGMP analogs (i.e., 8-bromo-cGMP) in the perfusate (67).
In isolated porcine aortic rings, the HO inhibitor SnPPIX decreased endothelium-dependent ace-tylcholine-dependent vasorelaxation, in the presence of the NOS inhibitor ^-nitro-l-arginine-me-thyl-ester (l-NAME) (36). Conversely, the endothelium-dependent contractile response to phenylephrine in thoracic aortic rings was augmented in the presence of both ZnPPIX and ^-nitro-l-arginine (NNA), relative to treatment with NNA alone (68). In this system, exogenously applied CO relaxed the aortic rings in a cGMP-dependent fashion. Overexpression of HO-1 by AdHO-1 infection of the vessels inhibited phenylephrine-dependent vasoconstriction in isolated aortic rings. Furthermore, Ad-HO-1 infection induced cGMP production in VSMC, which presumably was due to the generation of CO. The effects of HO-1 expression on vasoconstriction and cGMP production were subject to inhibition by ZnPPIX and occurred in the presence of NOS inhibitors (i.e., l-NNA, l-NAME) (13). Thus, these effects are dependent on heme degradation and independent of NOS activity or NO generation. The effects of CO are not limited to the vascular smooth muscle, but also airway smooth muscle, whereby exogenously administered CO stimulated cGMP-dependent airway bronchodilation in guinea-pig trachea given histamine injections (14). On the other hand, cGMP-independent mechanisms of vasoregulation by CO have also been proposed. CO may dilate blood vessels by directly activating calcium-dependent potassium channels (KCa) (69-72). In interlobar arterial smooth muscle cells, the inhibition of HO activity by metalloporphyrins decreased endogenous CO production and decreased the number of open potassium channels (105 pS K). These effects increased vascular contractility in response to phenyephrine. The introduction of exogenous CO reversed the effects of metalloporphyrins on vascular contractility (71). CO dilated porcine cerebral arterioles by increasing the effective coupling of calcium sparks to KCa channels (72). The mechanism by which CO mediates these effects remains unclear.
The mitogen-activated protein kinase (MAPK) pathways, which transduce oxidative stress and inflammatory signaling, may represent an important target of CO action (20). The activation of MAPKs, a family of Ser/Thr protein kinases, responds to a variety of extracellular stimuli (73). Three major MAPK signaling pathways, which include extracellular signal-regulated protein kinase (ERK), p38 MAPK (p38), and c-Jun NH2-terminal protein kinase (JNK), have been identified in mammalian cells (73).
The studies of Otterbein et al. in this laboratory showed that the anti-inflammatory properties of CO are mediated by p38 MAPK and its upstream MAPK kinase (MKK3) (20). CO inhibited the expression of lipopolysaccharide (LPS)-induced pro-inflammatory cytokines in RAW 264.7 macrophages, including tumor necrosis factor (TNF)-a, interleukin (IL)-10, and macrophage inflammatory protein-1a, while simultaneously increasing expression of the anti-inflammatory cytokine IL-10. Similar observations were made in a mouse model of lung inflammation. Using mice genetically deficient for MKK3, the upstream kinase of p38 MAPK, Otterbein et al. demonstrated the critical role of the p38 MAPK pathway in the anti-inflammatory effect of CO in vivo. In this model, the inhibitory effect of CO on TNF-a production did not require cGMP or NO production, or depend on the ERK1/2 or JNK MAPK pathways (20). Sethi et al. demonstrated the inhibition of TNFa-induc-ible ERK1/2 activation by CO treatment in PAEC, indicating that CO can also downregulate signaling cascades initiated by pro-inflammatory cytokines (74).
The mechanisms by which CO modulates the MAPKs are not clear. We hypothesize that a proximal effector, most likely a heme-containing protein, initiates the signal upon binding of the CO to the heme moiety, possibly by modulation of reactive oxygen species (ROS) production and the redox state of the cell. With respect to CO and MAPK signaling in macrophages, the identity of the upstream CO target remains elusive.
Apoptosis, or programmed cell death, consists of a regulated cascade of events that results in the death of a cell in response to environmental cues. Distinct from necrosis, which involves membrane disruption, apoptosis requires the action of proteases and nucleases within an intact plasma membrane, and participates in tissue development and homeostasis (75). The biological significance of apoptosis varies in a tissue-specific manner. Exposure to CO has been shown to exert potent anti-apoptotic effects in vitro and in vivo in the context of I/R injury and organ transplantation (see following sections). The exogenous administration of CO or the overexpression of HO-1 prevented TNFa-induced apoptosis in murine fibroblasts (76). The inhibitory effect of CO on TNFa-induced apoptosis in endothelial cells depended on the modulation of the p38 MAPK pathway, since it could be abolished with the selective chemical inhibitor SB203580, or a p38 dominant-negative mutant (21). HO-1 or CO cooperated with nuclear factor (NF)-KB-dependent anti-apoptotic genes (c-IAP2 and A1) to protect against TNFa-mediated endothelial cell apoptosis (77).
The anti-apoptotic effect of CO on cytokine-treated rat aortic smooth-muscle cells was partially dependent on the activation of sGC and was associated with suppression of p53 and inhibition of mitochondrial cytochrome-c release (75). In this model, however, the investigators excluded a role for p38 MAPK in the anti-apoptotic effect of CO (78).
3.4. CO Protects Against Ischemia/Reperfusion Injury
I/R injury has long been associated with oxidative stress resulting from the reperfusion and reoxygenation of previously ischemic tissue. HO-1 may participate in the manifestation of ischemic preconditioning, a process of acquired cellular protection against I/R injury, as observed in guinea pig transplanted lungs (79). HO-1 overexpression provided potent protection against cold I/R injury in rat hearts and livers through an anti-apoptotic pathway (80,81).
Exogenously applied CO at low concentrations inhibited I/R-induced apoptosis in pulmonary artery endothelial cell (PAEC) cultures, associated with the CO-dependent activation specifically of the p38a isoform with parallel suppression of ERK and JNK activation (24). In addition to activation of p38a MAPK and its upstream MAPK kinase (MKK3), the anti-apoptotic effect of CO involved inhibition of Fas/FasL expression, and other apoptosis-related factors including caspases (-3, -8, -9) mitochondrial cytochrome-c release, Bcl-2 proteins, and poly (ADP-ribose) polymerase (PARP) cleavage (82). CO exposure also protected against I/R-induced lung injury in vivo. Chemical inhibition of p38 MAPK activity, or the use of the mkk3-/- null mouse abolished the anti-apoptotic effects of CO during I/R, likely by preventing the modulation of caspase-3 activity (24,82).
Similar anti-inflammatory effects of CO have now been demonstrated in models of I/R injury of the heart, kidney, and small bowel (83). CO protected against liver I/R injury via activation of the p38 MAPK (84). Homozygous ho-1 null mice (hmox-1-/-) displayed increased mortality in a model of lung I/R injury. Inhalation of CO (1000 ppm) partially compensated for the HO-1 deficiency in hmox-1-/- mice, and improved survival following I/R (15). In this model, the authors propose that the protection provided by CO involved the enhancement of fibrinolysis via the cGMP-dependent inhibition of plasminogen activator inhibitor-1 (PAI-1) expression (15). Mice treated with a guanylate cyclase inhibitor, ODQ, were not rescued by CO from I/R-induced lethality.
3.5. Role of HO-1/CO in Atherosclerosis
HO-1 can be induced in both endothelial and vascular smooth muscle cells by pro-atherogenic stimuli, including oxidized low-density lipoprotein (LDL), lipid metabolites, shear stress, and angiotension II (85). HO-1 also confers protection in animal models of arteriosclerosis, where it may be found in atherosclerotic lesions (86). HO-1 is highly upregulated in the endothelium, and in the foam cells of intimal lesions from humans and apolipoprotein E-deficient mice (86). Induction of endogenous HO-1 by chemical treatment (hemin) reduced the formation of atherosclerotic lesions in LDL-receptor knockout mice fed high-fat diets, relative to untreated or SnPPIX-treated controls (87). The adenovirally mediated transduction of HO-1 into ApoE-deficient mice inhibited the formation of arteriosclerotic plaques relative to control mice (88). The mechanism by which HO-1 protects against arterioslcerosis may involve, in part, the inhibition of platelet aggregation by HO-derived CO.
3.6. CO Inhibits Cellular Proliferation and Vascular Stenosis Associated With Balloon Injury
Under homeostatic conditions, a tightly regulated balance exists between apoptosis and cellular proliferation. Loss of growth control is typically associated with neoplasia. In the case of vascular stenosis, hyperproliferation of VSMC results in occlusion of the vascular lumen.
CO has been shown to block vascular cell proliferation in a number of models. Exogenous application of HO-1, by gene transfer or exogenous CO application, inhibited VSMC proliferation in vitro by G0/G1 arrest, which required cGMP production, the G1 cyclin-dependent protein kinase inhibitor p21cip1, and activation of p38 MAPK (12,13,17). Adenoviral-mediated overexpression of HO-1 (AdHO-1) in pigs inhibited vascular cell proliferation and lesion formation in a model of arterial injury. Conversely, HO-1-/- mice subjected to arterial injury displayed increased vascular cell proliferation, and developed hyperplastic lesions in comparison to HO-1+ + controls (13). Employing a model of intimal hyperplasia where smooth muscle cells proliferate uncontrollably following balloon angioplasty of the carotid artery or chronic rejection of a transplanted aorta, exposure to CO completely prevented stenosis of the vessel (12). Pretreatment of a rat with CO (250 ppm) for just 1 h significantly reduced the neointimal proliferation seen at 14 d after balloon angioplasty relative to control animals that did not receive CO treatment. The mechanisms involved in this effect required activation of p38 MAPK and cGMP production (12). The application of HO-1 by adenovirally mediated gene transfer also protected against intimal hyperplasia following vascular balloon injury (89).
3.7. Protective Roles of HO-1/CO in Organ Transplantation
Expression of the stress protein HO-1 in rodent allografts and xenografts correlates with long-term graft survival in several models of transplantation (23,30,90). A higher expression of several protective genes has been observed in acute renal allograft rejection episodes in a rodent model of renal transplantation, where HO-1 expression increased in the allograft in response to immune injury (90). The reduced expression of HO-1 in chronic rejection as compared with acute rejection represents either an inadequate response to injury or a consequence of prior injury that jeopardizes further tissue response to immune attack (90).
Adenoviral-HO-1 gene therapy resulted in remarkable protection against rejection in rat liver transplants (91). The upregulation of HO-1 protected pancreatic islet cells from Fas-mediated apoptosis in a dose-dependent fashion, supporting an anti-apoptotic function of HO-1 (92,93). HO-1 may confer protection in the early phase after transplantation by inducing Th2-dependent cytokines such as IL-4 and IL-10, while suppressing interferon-gamma and IL-2 production, as demonstrated in a rat-liver allograft model (94). The induction of HO-1 in rats undergoing liver transplantation with adenoviral-HO-1 gene therapy resulted in protection against I/R injury and improved survival after transplantation, possibly by suppression of Th1-cytokine production and decreased apoptosis after reperfusion (95).
Beneficial effects of HO-1 modulation have been described in xeno-transplantation models, where HO-1 gene expression appears functionally associated with xenograft survival (23,30). In a mouse to rat heart transplant model, the effects of HO-1 upregulation could be mimicked by CO administration, suggesting that HO-derived CO suppressed the graft rejection (23). The authors proposed that CO suppressed graft rejection by inhibition of platelet aggregation, a process that facilitates vascular thrombosis and myocardial infarction. The ability of CO to suppress inflammation is likely involved in xenograft transplant models in which 400 ppm CO for 2 d prevented rejection for up to 50 d (23). The modulatory effects of CO on platelet aggregation, vasodilation, and pro-inflammatory cytokines all potentially contribute to the favorable outcome in xenograft transplantation (12).
Lung transplantation has become an accepted treatment modality for end-stage lung disease. After lung transplantation, there remains a persistent risk of acute and chronic graft failure, as well as of complications of the toxic immunosuppressive regimen used (96). Compared to other solid organ transplants, the success of lung transplantation has been severely limited by the high incidence of acute and chronic graft rejection. The frequency and severity of episodes of acute rejection are the predominant risk factors for chronic airway rejection, manifested as obliterative bronchiolitis (OB) (97,98). Data from rodent allograft studies as well as from clinical lung transplantation show that the lung, in comparison to other solid organs, is highly immunogenic. Despite advances in immunosuppression, the incidence of acute rejection in lung graft patients can be as high as 60% in the first postoperative month (99,100). OB, which may develop during the first months after transplantation, is the main cause of morbidity and death following the first half-year after transplantation, despite therapeutic intervention. Once OB has developed, re-transplantation remains the only therapeutic option available (101). Little is known about the pathophysiological background of OB. The possible determinants of developing OB include ongoing immunological allograft response, HLADR mismatch, cytomegalovirus infection, acute rejection episodes, organ-ischemia time, and recipient age (101).
Until recently, only very limited research data were available on the possible role for HO-1 in allograft rejection after lung transplantation. Increased HO-1 expression has been detected in alveolar macrophages from lung tissue in lung-transplant recipients with either acute or chronic graft failure, when compared to stable recipients (102).
In recent studies from this laboratory, Song et al. demonstrate that the level of HO-1 expression correlated to the acute rejection grade level in lung fibroblasts from a lung-transplant patient (22). The effects of CO were examined in a rat model of lung transplantation. Orthotopic left lung transplantation was performed in LEW rat recipients from BN rat donors. HO-1 mRNA and protein expression were markedly elevated in transplanted rat lungs compared to sham-operated lungs. Animals were exposed to continuous inhalation CO (500 ppm) or air. Transplanted lungs developed severe intra-alveolar hemorrhage and intravascular coagulation. In the presence of continuous CO exposure (500 ppm), however, the gross anatomy and histology of transplanted lungs showed dramatic preservation relative to air-treated controls. Furthermore, transplanted lungs displayed increased apoptotic cell death compared with the transplanted lungs of CO-treated recipients, as assessed by TUNEL and caspase-3 immunostaining. CO exposure inhibited the induction of IL-6 mRNA expression in lung and serum caused by the transplantation. Gene array analysis revealed that CO also downregulated other pro-inflammatory genes, including macrophage inflammatory protein (MIP)-1a and macrophage migration inhibitory factor (MIF), and growth factors such as platelet-derived growth factor (PDGF), which were upregulated by transplantation (22).
In organ transplantation, the I/R injury that occurs leads to rapid endothelial cell apoptosis. The loss of endothelial cells in the vessels serving the organ results in a rapid cascade of events including thrombosis that can ultimately result in the rejection of the organ. These data suggest CO limits lung graft injury by maintaining cell viability and suppressing inflammation.
Was this article helpful?