Carbon Monoxide Suppresses Neointima Formation in Transplant Arteriosclerosis by Inhibiting Vascular Progenitor Cell Differentiation.

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Carbon monoxide suppresses arteriosclerotic lesions associated with chronic graft rejection and with balloon injury.

Carbon monoxide (CO), one of the products of heme oxygenase action on heme, prevents arteriosclerotic lesions that occur following aorta transplantation; pre-exposure to 250 parts per million of CO for 1 hour before injury suppresses stenosis after carotid balloon injury in rats as well as in mice. The protective effect of CO is associated with a profound inhibition of graft leukocyte infiltration/activation as well as with inhibition of smooth muscle cell proliferation. The anti-proliferative effect of CO in vitro requires the activation of guanylate cyclase, the generation of cGMP, the activation of p38 mitogen-activated protein kinases and the expression of the cell cycle inhibitor p21Cip1. These findings demonstrate a protective role for CO in vascular injury and support its use as a therapeutic agent.

Carbon monoxide protects against liver failure through nitric oxide-induced heme oxygenase 1.

Carbon monoxide (CO) and nitric oxide (NO) each have mechanistically unique roles in various inflammatory disorders. Although it is known that CO can induce production of NO and that NO can induce expression of the cytoprotective enzyme heme oxygenase 1 (HO-1), there is no information whether the protective effect of CO ever requires NO production or whether either gas must induce expression of HO-1 to exert its functional effects. Using in vitro and in vivo models of tumor necrosis factor alpha-induced hepatocyte cell death in mice, we find that activation of nuclear factor kappaB and increased expression of inducible NO are required for the protective effects of CO, whereas the protective effects of NO require up-regulation of HO-1 expression. When protection from cell death is initiated by CO, NO production and HO-1 activity are each required for the protective effect showing for the first time an essential synergy between these two molecules in tandem providing potent cytoprotection.

Heme oxygenase-1: from biology to therapeutic potential.

Heme oxygenase-1 (HO-1) is a stress-responsive enzyme that catabolizes free heme into carbon monoxide, iron (which induces the expression of heavy-chain ferritin, an iron-sequestering protein) and biliverdin (which is converted to bilirubin by biliverdin reductase). Over the past few years it has become apparent that these 'arms' of the HO-1 system can act protectively in a variety of experimental models of disease; there is also evidence that HO-1 and bilirubin have protective actions in humans. Here, we present a model for the beneficial actions of the products of heme degradation, and we discuss the potential clinical applications of enhancing the HO-1 system.

Blockade of p38 MAPK inhibits chronic allograft vasculopathy.

Long-term survival after solid-organ transplantation is hampered by chronic changes in the arteries of the grafts, called chronic allograft vasculopathy (CAV). The lesions consist mainly of proliferating vascular smooth muscle cells that cause narrowing of the vessels; these lesions can develop within a few months. There is no effective treatment to prevent CAV. We previously noted that the pharmacological inhibition of p38 mitogen-activated protein kinase (MAPK) suppresses the proliferation of vascular smooth muscle cells. We hypothesized that in vivo inhibition of p38 MAPK in mice bearing allogeneic aortic allografts would prevent CAV. We here report that blockade of p38 MAPK, a signaling molecule involved in cell division, apoptosis, and cell death, markedly suppresses CAV. Given recent data indicating that inhibition of p38 MAPK is a promising approach for the treatment of autoimmune diseases plus our present findings, p38 MAPK blockade for CAV seems a reasonable approach to consider for clinical application.

Toll-like receptor 4 suppression leads to islet allograft survival.

Carbon monoxide (CO) exposure of an islet donor frequently leads to islet allograft long-term survival and tolerance in recipients. We show here that CO confers its protective effects at least in part by suppressing Toll-like receptor 4 (TLR4) up-regulation in pancreatic beta cells. TLR4 is normally up-regulated in islets during the isolation procedure; donor treatment with CO suppresses TLR4 expression in isolated islets as well as in transplanted grafts. TLR4 up-regulation allows initiation of inflammation, which leads to islet allograft rejection; islet grafts from TLR4-deficient mice survive indefinitely in BALB/c recipients and show significantly less inflammation at various days after transplantation compared with grafts from a control donor. Isolated islets preinfected with a TLR4 dominant negative virus before transplantation demonstrated prolonged survival in recipients. Despite the salutary effects of TLR4 suppression, HO-1 expression is still needed in the recipient for islet survival: TLR4-deficient islets were rejected promptly after being transplanted into recipients in which HO-1 activity was blocked. In addition, incubation of an insulinoma cell line, betaTC3, with an anti-TLR4 antibody protects those cells from cytokine-induced apoptosis. Our data suggest that TLR4 induction in beta cells is involved in beta cell death and graft rejection after transplantation. CO exposure protects islets from rejection by blocking TLR4 up-regulation.

Heme oxygenase-1, carbon monoxide, and bilirubin induce tolerance in recipients toward islet allografts by modulating T regulatory cells.

Heme oxygenase-1 (HO-1) induction in, or carbon monoxide (CO), or bilirubin administration to, donors and/or recipients frequently lead to long-term survival (>100 days) of DBA/2 islets into B6AF1 recipients. We tested here whether similar treatments show value in a stronger immunogenetic combination, i.e., BALB/c to C57BL/6, and attempted to elucidate the mechanism accounting for tolerance. Induction of HO-1, administering CO or bilirubin to the donor, the islets or the recipient, prolonged islet allograft survival to different extents. Combining all the above treatments (the "combined" protocol) led to survival for >100 days and antigen-specific tolerance to 60% of the transplanted grafts. A high level of forkhead box P3 (Foxp3) and transforming growth factor beta (TGF-beta) expression was detected in the long-term surviving grafts. With the combined protocol, significantly more T regulatory cells (Tregs) were observed surrounding islets 7 days following transplantation. No prolongation of graft survival was observed using the combined protocol when CD4+ CD25+ T cells were predepleted from the recipients before transplantation. In conclusion, our combined protocol led to long-term survival and tolerance to islets in the BALB/c to C57BL/6 combination by promoting Foxp3+ Tregs; these cells played a critical role in the induction and maintenance of tolerance in the recipient.

Therapeutic applications of bilirubin and biliverdin in transplantation.

Bilirubin is the end product of heme catabolism by heme oxygenases. The inducible form of these enzymes is heme oxygenase-1 (HO-1), which is the rate-limiting enzyme that can degrade heme into equimolar quantities of carbon monoxide (CO), biliverdin, and free iron. Biliverdin is very rapidly converted to bilirubin by the enzyme biliverdin reductase, and free iron upregulates the expression of ferritin. HO-1 is a ubiquitous stress protein and is induced in many cell types by various stimuli. Induced HO-1 exerts antiinflammatory effects and modulates apoptosis. Expression of HO-1 in vivo suppresses the inflammatory responses in endotoxic shock, hyperoxia, acute pleurisy, and organ transplantation, as well as ischemia-reperfusion injury, and thereby provides salutary effects in these conditions. Accumulating evidence indicates that biliverdin/bilirubin can mediate the protective effects of HO-1 in many disease models, such as IRI and organ transplantation, via its antiinflammatory, antiapoptotic, antiproliferative, and antioxidant properties, as well as its effects on the immune response. This review attempts to summarize these protective roles as well as the molecular mechanisms by which biliverdin/bilirubin benefit IRI and solid-organ transplantation, including chronic rejection, and islet transplantation.

Heme oxygenase-1 in organ transplantation.

Cells have a plethora of defense mechanisms that are activated upon exposure to oxidative stress. These aim at limiting the deleterious effects of oxidative stress and re-establishing homeostasis. In the particular context of organ transplantation, these defense mechanisms contribute to sustain graft survival via at least two interrelated mechanisms. First, cytoprotection per se should support survival and function of cells within a transplanted organ. Second, cytoprotection could reduce immunogenicity of the graft and modulate the activation of the recipient's immune system to promote regulatory (suppressive) responses that sustain graft survival. Others and we have gathered evidence, to suggest that the stress-responsive enzyme Heme Oxygenase-1 (HO-1 encoded by the gene Hmox1) acts in such a manner. Upon organ transplantation, HO-1 is ubiquitously expressed in a transplanted organ, becoming the rate-limiting enzyme in the catabolism of heme into carbon monoxide (CO), iron (Fe) and biliverdin (1). There is accumulating evidence to support the notion that HO-1 expression in a graft and in the recipient can prevent rejection and promote immune tolerance. We will argue that these effects are mediated to a large extent by limiting the deleterious effects of free heme as well as by the inherent cytoprotective and/or anti-inflammatory effects of the end-products generated via heme catabolism.

Carbon monoxide: from the origin of life to molecular medicine.

Carbon monoxide, long considered only as a toxic gas, has recently been shown to mediate potent anti-inflammatory and other salutary effects in rodents when it is used at low doses. Carbon monoxide is one of the products of the degradation of heme by heme oxygenase 1. Until recently, these beneficial effects of carbon monoxide were shown only when it was given before a stress stimulus. Hagazi and colleagues have recently shown that this substance is effective even when it is given after a disease process has started. The effects of low doses of carbon monoxide are complemented by the production of biliverdin and probably also by ferritin, which are additional products of heme degradation.

Heme oxygenase-1 is essential for and promotes tolerance to transplanted organs.

This investigation focused on obtaining a further understanding of the role of heme oxygenase-1 (HO-1) in tolerance induction. Hearts from C57BL/6 (H-2b) mice survived long-term when transplanted into BALB/c (H-2d) recipients treated with the tolerance-inducing regimen of anti-CD40L antibody (MR-1) plus donor-specific transfusion (DST). Grafts did not, however, survive long-term in (HO-1-/-) recipients given the same treatment. Similarly, long-term survival induced by DST was ablated when HO-1 activity was blocked by zinc protoporphyrin IX (ZnPPIX). We further asked whether modulation of HO-1 expression/activity could be used to promote the induction of graft tolerance. DST alone (day 0) failed to promote any prolongation of survival of DBA/2 (H-2d) hearts transplanted into B6AF1 (H-2(b,k/d)) recipients. However, long-term survival and (dominant peripheral) tolerance were readily induced when DST was combined with induction of HO-1 expression by cobalt protoporphyrin IX (CoPPIX). HO-1 induction plus DST led to a significant up-regulation of Foxp3, TGF-beta, IL-10, and CTLA4, which suggests a prominent role for CD4+CD25+ regulatory T cells (Tregs). In fact, the tolerogenic effect of HO-1 plus DST was dependent on CD4+CD25+ Tregs as suggested by adoptively transferring these cells into irradiated recipients under various regimens. Taken together, these findings show that expression of HO-1 in a graft recipient can be essential for long-term graft survival and for induction of tolerance and that modulation of HO-1 expression/activity can be used therapeutically to synergize in the generation of graft tolerance.

Bilirubin: a natural inhibitor of vascular smooth muscle cell proliferation.

BACKGROUND: Bilirubin, a natural product of heme catabolism by heme oxygenases, was considered a toxic waste product until 1987, when its antioxidant potential was recognized. On the basis of observations that oxidative stress is a potent trigger in vascular proliferative responses, that heme oxygenase-1 is antiatherogenic, and that several studies now show that individuals with high-normal or supranormal levels of plasma bilirubin have a lesser incidence of atherosclerosis-related diseases, we hypothesized that bilirubin would have salutary effects on preventing intimal hyperplasia after balloon injury. METHODS AND RESULTS: We found less balloon injury-induced neointima formation in hyperbilirubinemic Gunn rats and in wild-type rats treated with biliverdin, the precursor of bilirubin, than in controls. In vitro, bilirubin and biliverdin inhibited serum-driven smooth muscle cell cycle progression at the G1 phase via inhibition of the mitogen-activated protein kinase signal transduction pathways and inhibition of phosphorylation of the retinoblastoma tumor suppressor protein. CONCLUSIONS: Bilirubin and biliverdin might be potential therapeutics in vascular proliferative disorders.

Bilirubin can induce tolerance to islet allografts.

Induction of heme oxygenase-1 (HO-1) expression in recipients of allogeneic islets can lead to long-term survival (>100 d) of those islets. We tested whether administration of bilirubin would substitute for the beneficial effects of HO-1 expression in islet transplantation. Administering bilirubin to the recipient (B6AF1) or incubating islets in a bilirubin-containing solution ex vivo led to long-term survival of allogeneic islets in a significant percentage of cases. In addition, administering bilirubin to only the donor frequently led to long-term survival of DBA/2 islets in B6AF1 recipients and significantly prolonged graft survival of BALB/c islets in C57BL/6 recipients. Donor treatment with bilirubin up-regulated mRNA expression of protective genes such as HO-1 and bcl-2 and suppressed proinflammatory and proapoptotic genes including monocyte chemoattractant protein-1 and caspase-3 and -8 in the islet grafts before transplantation. Furthermore, treatment of only the donor suppressed the expression of proinflammatory cytokines including TNF-alpha, inducible nitric oxide synthase, monocyte chemoattractant protein-1, and other proapoptotic and proinflammatory genes normally seen in the islets after transplantation. Donor treatment also reduced the number of macrophages that infiltrated the islet grafts in the recipients. Preincubation of betaTC3 cells with bilirubin also protected the cells from lipid peroxidation. Our data suggests that the potent antioxidant and antiinflammatory actions of bilirubin may contribute to islet survival.

Donor treatment with carbon monoxide can yield islet allograft survival and tolerance.

Treatment of animals or certain cells with carbon monoxide (CO), a product of heme degradation by heme oxygenase-1 (HO-1), has potent anti-inflammatory and antiapoptotic effects that contribute to the survival of transplanted organs. We report here that inducing HO-1 in, or administering CO to, only the donor can be used in a therapeutic manner to sustain the survival of transplanted allogeneic islets. Similar treatments of only the islets or only the recipient are also salutary. Administering CO only to the donor frequently leads to long-term survival of those islets in untreated allogeneic recipients, which are then antigen-specifically tolerant. Several proinflammatory and proapoptotic genes that are strongly induced in islets after transplantation in the untreated situation were significantly suppressed after administering CO to the donor without further treatment. These included tumor necrosis factor-alpha, inducible nitric oxide synthase, monocyte chemoattractant protein-1, granzyme B, and Fas/Fas ligand, all of which contribute to the pathogenesis of the rejection of transplanted islets. This correlated with a lesser infiltration of recipient macrophages into the transplanted islets. Our present findings show that induction of HO-1 in, or administration of CO to, only the donor, islets, or the recipient or combinations of such treatments improve allogeneic islet survival.

Heme oxygenase-1 and transplantation tolerance.

Tolerance is the ultimate goal of transplantation: the lack of any need for immunosuppression with long-term survival of the transplanted tissue and a full immunological response to all antigens other than those on the transplant. In honor of my dear friend and colleague, Prof. Dr. Jon van Rood, I summarize here our work in this area making use of heme oxygenase-1 (HO-1) and the products that degradation of heme by HO-1 generates.

Heme oxygenase-1 modulates the allo-immune response by promoting activation-induced cell death of T cells.

Heme oxygenase-1 (HO-1), which degrades heme into three products (carbon monoxide, free iron, and biliverdin), plays a protective role in many models of disease via its anti-inflammatory, anti-apoptotic, and anti-proliferative actions. Overexpression of HO-1 has been shown to suppress immune responses and prolong the survival of allografts; however, the underlying mechanism is not clear. We demonstrate two "new" properties of HO-1 that mediate activation induced cell death (AICD) of allo-antigen-responsive murine CD4+ T cells, resulting in immunomodulation. First, it functions in vivo and in vitro to "boost" the proliferative response of CD4+ T cells to allo-antigens in the early phase of allo-antigen-driven immune responses. This "boosting" effect is accompanied with a significant increase of activation markers and IL-2 production. Second, it exerts a pro-apoptotic effect in those activated T cells after the initial burst of proliferation. We further show that the AICD effect is mediated through the Fas/CD95-FasL signal transduction pathway. Correlating with the above-mentioned findings is the observed prolongation of mouse heart graft survival when HO-1 is expressed in vivo in both donor and recipient. In conclusion, induction of HO-1 expression accelerates clonal deletion of peripheral alloreactive CD4+ T cells by promoting AICD, which is presumably a key mechanism for its immunomodulatory effects such as in prolonging the survival of transplanted organs.

Carbon monoxide protects pancreatic beta-cells from apoptosis and improves islet function/survival after transplantation.

Pancreatic islets transplanted to treat autoimmune type 1 diabetes often fail to function (primary nonfunction), likely because of islet beta-cell apoptosis. We show that carbon monoxide (CO), a product of heme oxygenase activity, protects beta-cells from apoptosis. Protection is mediated through guanylate cyclase activation, generation of cyclic GMP (cGMP), and activation of cGMP-dependent protein kinases. This antiapoptotic effect is still observed when beta-cells are exposed to CO for 1 h before the apoptotic stimulus. In a similar manner, mouse islets exposed to CO for just 2 h function significantly better after transplantation than islets not exposed to CO. These findings suggest a potential therapeutic application for CO in improving islet function/survival after transplantation in humans.

Heme oxygenase-1 genotype and restenosis after balloon angioplasty: a novel vascular protective factor.

OBJECTIVES: We investigated the association of the heme oxygenase-1 (HO-1) promoter genotype with the inflammatory response and restenosis after balloon angioplasty. BACKGROUND: Heme oxygenase-1, which is induced by balloon angioplasty, can inhibit neointima formation and vascular remodeling. A dinucleotide repeat in the HO-1 gene promoter shows a length polymorphism that modulates HO-1 gene transcription. Short (<25 guanosine thymidine [GT]) repeats are associated with a 10-fold greater up-regulation of HO-1 than are longer repeats. METHODS: We studied 381 consecutive patients who underwent femoropopliteal balloon angioplasty (n = 210) and comparison groups with femoropopliteal stenting (n = 68) and lower limb angiography (n = 103). C-reactive protein (CRP) was measured at baseline, 24, and 48 h. We evaluated patency at six months by duplex sonography and assessed the association of the length of GT repeats in the HO-1 gene promoter with postintervention CRP and restenosis. RESULTS: Restenosis within six months was found in 74 patients (35%) after balloon angioplasty and in 21 patients (31%) after stenting. After balloon angioplasty, carriers of the short length (<25 GT) dinucleotide repeats had a lower postintervention CRP at 24 h (p = 0.009) and 48 h (p < 0.001) and a reduced risk for restenosis (adjusted relative risk 0.43, 95% confidence interval: 0.24 to 0.71, p < 0.001) compared with patients with longer alleles. After stenting or angiography, we found no association between the HO-1 genotype with CRP or restenosis. CONCLUSIONS: The HO-1 promoter genotype that controls the degree of HO-1 up-regulation in response to stress stimuli is associated with the postintervention inflammatory response and the restenosis risk after balloon angioplasty.

Heme oxygenase-1-generated biliverdin ameliorates experimental murine colitis.

BACKGROUND: Heme oxygenase-1 (HO-1) seems to have an important protective role in acute and chronic inflammation. The products of heme catalysis, biliverdin/bilirubin, carbon monoxide (CO), and iron (that induces apoferritin) mediate the beneficial effects of HO-1. Blockade of HO-1 activity results in exacerbation of experimental colitis. We tested whether HO-1 has protective effects in the development of colitis and determined that specific enzymatic products of HO-1 are responsible for these effects. METHODS: Colitis was induced by oral administration of dextran sodium sulfate (5%) to C57BL/6 mice for 7 days. HO-1 was up-regulated by cobalt-protoporphyrin (5 mg/kg, intraperitoneally). Biliverdin, exogenous CO, or the iron chelator desferrioxamine was administered to other groups. RESULTS: Cobalt-protoporphyrin treatment resulted in significant up-regulation of HO-1 protein in mucosal and submucosal cells. Induction of HO-1 was associated with significantly less loss of body weight in mice with induced colitis (-12% versus -22% in the control animals, P < 0.001). Development of diarrhea and gastrointestinal hemorrhage was substantially delayed in animals in which HO-1 was induced, and mucosal injury was significantly attenuated. Administration of CO or desferrioxamine alone had no significant effects, whereas enhanced protection with lesser evidence of bowel inflammation was observed with systemic biliverdin administration (50 micromol/kg, 3 times per day, intraperitoneally). CONCLUSIONS: We conclude that heightened HO-1 expression or administration of biliverdin ameliorates dextran sodium sulfate-induced experimental colitis. Novel therapeutic strategies based on HO-1 and/or biliverdin administration may have use in inflammatory bowel disease.

Biliverdin, a natural product of heme catabolism, induces tolerance to cardiac allografts.

Biliverdin, a product of heme oxygenase-1 (HO-1) enzymatic action, is converted into bilirubin, which has been considered a waste product in the past. We now show that administration of biliverdin has a salutary effect in organ transplantation. A brief course of treatment with biliverdin leads to long-term survival of H-2 incompatible heart allografts. Furthermore, those recipients harboring long-surviving (>100 days) allografts were tolerant to donor antigens indicated by the acceptance of second donor strain hearts but not third-party grafts. Treatment with biliverdin decreased intragraft leukocyte infiltration and inhibited T cell proliferation. Likely related to tolerance induction, biliverdin interferes with T cell signaling by inhibiting activation of nuclear factor of activated T cells (NFAT) and nuclear factor kappaB (NF-kappaB), two transcription factors involved in interleukin-2 (IL-2) transcription and T cell proliferation, as well as suppressing Th1 interferon-gamma (IFN-gamma) production in vitro. These findings support the potential use of biliverdin, a natural product, in transplantation and other T cell mediated immune disorders.