RESUMO
[Figure: see text].
Assuntos
Aorta Torácica/transplante , Arteriosclerose/prevenção & controle , Monóxido de Carbono/farmacologia , Diferenciação Celular/efeitos dos fármacos , Músculo Liso Vascular/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Neointima , Células-Tronco/efeitos dos fármacos , Animais , Aorta Torácica/enzimologia , Aorta Torácica/patologia , Arteriosclerose/enzimologia , Arteriosclerose/genética , Arteriosclerose/patologia , Transplante de Medula Óssea , Células Cultivadas , Modelos Animais de Doenças , Heme Oxigenase-1/genética , Heme Oxigenase-1/metabolismo , Cinética , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Liso Vascular/enzimologia , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/enzimologia , Miócitos de Músculo Liso/patologia , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Células-Tronco/enzimologia , Células-Tronco/patologia , Quimeras de Transplante , Remodelação Vascular/efeitos dos fármacosRESUMO
Exposing donor mice to carbon monoxide (CO) protects transplanted islet allografts from immune rejection after transplantation (referred as the "donor" effect). In an attempt to understand the mechanisms of the donor effect of CO, we found that donor treatment with CO upregulates expression of peroxisome proliferator-activated receptor γ (PPARγ), a transcriptional regulator, in isolated islets. In this study, we evaluated whether PPARγ contributes to the survival and function of transplanted islets and whether PPARγ mediates the protective effect of CO in a major mismatch islet allogeneic transplantation model. BALB/c (H-2(d)) islets in which PPARγ activity was induced by its agonists, 15-deoxy-Δ(12-14)-prostaglandin J(2) (15d-PGJ(2)) or troglitazone were transplanted into C57BL/6 (H-2(b)) recipients that had been rendered diabetic by streptozotocin (STZ). Blood glucose levels of recipients were monitored to determine the function of transplanted islets. Our data indicated that PPARγ activation in islets led to a high percentage of BALB/c islets survived long-term in C57BL/6 recipients. Activation of PPARγ in the donor suppresses expressions of proinflammatory cytokines including tumor necrosis factor-α (TNF-α) and inducible nitric oxide synthase (iNOS) in transplanted islets. Blocking PPARγ activity by its antagonist, GW9662, abrogated the donor effect of CO in vivo and in vitro. Our data demonstrate that PPARγ plays a critical role in the survival and function of transplanted islets after transplantation in the recipient. The protective effects of CO are at least in part mediated by PPARγ.
Assuntos
Sobrevivência de Enxerto/fisiologia , Transplante das Ilhotas Pancreáticas/métodos , PPAR gama/metabolismo , Animais , Apoptose/imunologia , Apoptose/fisiologia , Monóxido de Carbono/farmacologia , Sobrevivência de Enxerto/efeitos dos fármacos , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Óxido Nítrico Sintase Tipo II/metabolismo , PPAR gama/agonistas , Análise de Sobrevida , Transplante Homólogo , Resultado do Tratamento , Fator de Necrose Tumoral alfa/biossíntese , Regulação para CimaRESUMO
We have previously demonstrated that bilirubin administration to the recipient induces tolerance towards islet cell transplants across a complete MHC mismatch in a mouse model. Here we assess the mechanisms of such protection. Bilirubin treatment of recipients improved function of islet allografts by suppressing expressions of proinflammatory and proapoptotic genes in those islets and by increasing Foxp3(+) T regulatory (Treg) cells at the site of transplanted islets at various days after transplantation. No prolongation of graft survival was observed in recipients treated with bilirubin when CD4(+)CD25(+) T cells were predepleted from those recipients, indicating that Treg cells are necessary for the protective effect of bilirubin. Adoptive transfer of Treg cells from tolerant mice into Rag1(-/-) recipients resulted in long-term acceptance of skin allografts in an alloantigen-specific manner, suggesting that Treg cells are sufficient to induce tolerance. In addition, bilirubin treatment promoted de novo generation of Treg cells in Rag1(-/-) recipients. Thus, bilirubin treatment to the recipients prolongs islet allograft survival via a Treg-dependent manner in which CD4(+)CD25(+) Treg cells are both necessary and sufficient for tolerance induction and graft acceptance. Bilirubin treatment promotes de novo generation of Treg cells that might account for the protective effects of bilirubin given to recipients.
Assuntos
Bilirrubina/farmacologia , Transplante das Ilhotas Pancreáticas , Linfócitos T Reguladores/citologia , Animais , Antígenos CD4/metabolismo , Monóxido de Carbono/farmacologia , Caspase 12/genética , Caspase 12/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Sobrevivência de Enxerto , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Transplante de Pele , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Tolerância ao Transplante/imunologia , Transplante Homólogo , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Biliverdin reductase A (BVR) catalyzes the reduction of biliverdin (BV) to bilirubin (BR) in all cells. Others and we have shown that biliverdin is a potent anti-inflammatory molecule, however, the mechanism by which BV exerts its protective effects is unclear. We describe and elucidate a novel finding demonstrating that BVR is expressed on the external plasma membrane of macrophages (and other cells) where it quickly converts BV to BR. The enzymatic conversion of BV to BR on the surface by BVR initiates a signaling cascade through tyrosine phosphorylation of BVR on the cytoplasmic tail. Phosphorylated BVR in turn binds to the p85alpha subunit of phosphatidylinositol 3-kinase and activates downstream signaling to Akt. Using bacterial endotoxin (lipopolysaccharide) to initiate an inflammatory response in macrophages, we find a rapid increase in BVR surface expression. One of the mechanisms by which BV mediates its protective effects in response to lipopolysaccharide is through enhanced production of interleukin-10 (IL-10) the prototypical anti-inflammatory cytokine. IL-10 regulation is dependent in part on the activation of Akt. The effects of BV on IL-10 expression are lost with blockade of Akt. Inhibition of surface BVR with RNA interference attenuates BV-induced Akt signaling and IL-10 expression and in vivo negates the cytoprotective effects of BV in models of shock and acute hepatitis. Collectively, our findings elucidate a potentially important new molecular mechanism by which BV, through the enzymatic activity and phosphorylation of surface BVR (BVR)(surf) modulates the inflammatory response.
Assuntos
Membrana Celular/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/fisiologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Anti-Inflamatórios/farmacologia , Biliverdina/metabolismo , Humanos , Inflamação , Interleucina-10/biossíntese , Interleucina-10/metabolismo , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Modelos Biológicos , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/biossíntese , Frações Subcelulares/metabolismoRESUMO
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.
Assuntos
Doença , Heme Oxigenase-1/metabolismo , Homeostase , Animais , Bilirrubina/metabolismo , Biliverdina/metabolismo , Monóxido de Carbono/metabolismo , Indução Enzimática , Evolução Molecular , Heme/metabolismo , Heme Oxigenase-1/química , Humanos , Modelos Moleculares , Estrutura Terciária de ProteínaRESUMO
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.
Assuntos
Aorta/transplante , Imidazóis/uso terapêutico , Pirimidinas/uso terapêutico , Transplante Homólogo/patologia , Doenças Vasculares/prevenção & controle , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Animais , Antígenos CD4/análise , Antígenos CD8/análise , Sobrevivência de Enxerto , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Modelos Animais , Fosforilação , Ratos , Transplante Homólogo/imunologiaRESUMO
Bilirubin for decades was considered a potentially toxic waste product of heme degradation until the discovery that it is a potent antioxidant. Accumulating data from observations in humans and experimental studies indicate that the bile pigment may be protective against certain diseases. Based on our own observations that bilirubin induces cell cycle arrest in abnormally proliferating vascular smooth muscle cells and clinical observations describing a lesser incidence of cancer in healthy individuals with high normal or slightly elevated serum bilirubin levels, we hypothesized that bilirubin might suppress tumor cell proliferation in vitro and in vivo. As possible effectors we analyzed key proteins that are involved in cell cycle progression and apoptosis. In vivo, tumor growth was assessed in BALB/c nude mice bearing HRT-18 colon cancer xenografts that were treated with bilirubin. In vitro, we investigated the effect of bilirubin on various cell lines and the signaling pathways involved in bilirubin action on tumor cell proliferation in HRT-18 cells using western blots. Bilirubin potently inhibited tumor cell proliferation in vivo and acted cytostatic and pro-apoptotic in vitro. The signaling cascades responsible for this action involved induction of p53, p27, hypophosphorylation of the retinoblastoma tumor suppressor protein as well as caspase activation. These effects were dependent on ERK 1/2. Our study demonstrates that bilirubin may play a role in the defense against cancer by interfering with pro-cancerogenic signaling pathways.
Assuntos
Bilirrubina/fisiologia , Proliferação de Células/efeitos dos fármacos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Animais , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Bilirrubina/farmacologia , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/fisiologia , Linhagem Celular , Linhagem Celular Tumoral , Citostáticos/farmacologia , Ativação Enzimática , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Transplante de Neoplasias , Fosforilação , Transdução de Sinais , Transplante Heterólogo , Proteínas Supressoras de Tumor/fisiologiaRESUMO
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.
Assuntos
Bilirrubina/uso terapêutico , Biliverdina/uso terapêutico , Transplante de Órgãos/fisiologia , Animais , Bilirrubina/metabolismo , Biliverdina/metabolismo , Citoproteção , Rejeição de Enxerto/enzimologia , Rejeição de Enxerto/genética , Heme/química , Heme/metabolismo , Heme Oxigenase-1/metabolismo , Heme Oxigenase-1/fisiologia , Heme Oxigenase-1/uso terapêutico , Humanos , Transplante das Ilhotas Pancreáticas , Modelos Biológicos , Estrutura Molecular , Traumatismo por Reperfusão/tratamento farmacológico , Traumatismo por Reperfusão/prevenção & controle , Transplante HomólogoRESUMO
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.
Assuntos
Bilirrubina/farmacologia , Monóxido de Carbono/farmacologia , Heme Oxigenase (Desciclizante)/metabolismo , Tolerância Imunológica , Ilhotas Pancreáticas/imunologia , Linfócitos T Reguladores/imunologia , Animais , Fatores de Transcrição Forkhead/metabolismo , Ilhotas Pancreáticas/metabolismo , Transplante das Ilhotas Pancreáticas , Teste de Cultura Mista de Linfócitos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Crescimento Transformador beta/metabolismo , Transplante HomólogoRESUMO
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.
Assuntos
Rejeição de Enxerto/enzimologia , Heme Oxigenase-1/fisiologia , Transplante de Órgãos , Animais , Rejeição de Enxerto/prevenção & controle , Heme Oxigenase-1/metabolismo , Camundongos , Traumatismo por Reperfusão/prevenção & controleRESUMO
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.
Assuntos
Monóxido de Carbono/farmacologia , Diabetes Mellitus Experimental/terapia , Sobrevivência de Enxerto/fisiologia , Transplante das Ilhotas Pancreáticas , Receptor 4 Toll-Like/antagonistas & inibidores , Adenoviridae/genética , Animais , Western Blotting , Rejeição de Enxerto/imunologia , Rejeição de Enxerto/prevenção & controle , Tolerância Imunológica/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Knockout , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Linfócitos T/citologia , Receptor 4 Toll-Like/metabolismo , Transplante HomólogoRESUMO
Adenosine and heme oxygenase-1 (HO-1) exert a wide range of anti-inflammatory and immunomodulatory actions, making them crucial regulatory molecules. Despite the diversity in their modes of action, the similarity of biological effects of adenosine and HO-1 led us to hypothesize a possible interrelationship between them. We assessed a potential role for HO-1 in the ability of adenosine or 5'-N-ethylcarboxamidoadenosine (NECA), a stable adenosine analog, to modify the response of LPS-stimulated macrophages. Adenosine and NECA markedly induced HO-1 and blocked LPS-induced TNF-alpha production via adenosine A2aR-mediated signaling; blocking of HO-1 by RNA interference abrogated the effects of adenosine and NECA on TNF-alpha. HO-1 overexpression or exposure to carbon monoxide (CO), a product of HO-1 enzymatic activity, resulted in augmented A2aR mRNA and protein levels in RAW264.7 cells and primary macrophages. The induction of A2aR expression by HO-1 or CO resulted in an increase in the sensitivity to the anti-inflammatory effects of adenosine and NECA, which was lost in macrophages isolated from A2aR-deficient mice. Moreover, a decrease in cAMP levels upon NECA stimulation of naive macrophages was counterbalanced by CO exposure to up-regulate A2aR levels. This implies adenosine receptor isoform switch as a selective modification in macrophage phenotype. Taken together, these data suggest the existence of a positive feedback loop among adenosine, HO-1, CO, and the A2aR in the chronological resolution of the inflammatory response.
Assuntos
Adenosina/metabolismo , Monóxido de Carbono/metabolismo , Heme Oxigenase-1/fisiologia , Macrófagos/imunologia , Receptor A2A de Adenosina/metabolismo , Adenosina/farmacologia , Adenosina-5'-(N-etilcarboxamida)/farmacologia , Animais , Células Cultivadas , Regulação para Baixo , Heme Oxigenase-1/antagonistas & inibidores , Heme Oxigenase-1/genética , Interleucina-10/metabolismo , Interleucina-12/metabolismo , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Camundongos , Interferência de RNA , RNA Mensageiro/metabolismo , Receptor A2A de Adenosina/genética , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
The most salient feature of carbon monoxide (CO)-mediated cytoprotection is the suppression of inflammation and cell death. One of the important cellular targets of CO is the macrophage (mphi). Many studies have shown that exposure of mphi to CO results in the generation of an antiinflammatory phenotype; however, these reports have ignored the effect of CO alone on the cell before stimulation. Most investigations have focused on the actions of CO in modulating the response to noxious stimuli. We demonstrate here that exposure of mphi to CO results in a significant and transient burst of reactive oxygen species (ROS) arising from the mitochondria (mitochondria-deficient mphi do not respond to CO to produce ROS). The ROS promote rapid activation and stabilization of the transcription factor hypoxia-inducible factor 1alpha (HIF-1alpha), which regulates expression of genes involved in inflammation, metabolism, and cell survival. The increase in HIF-1alpha expression induced by CO results in regulated expression of TGF-beta, a potent antiinflammatory cytokine. CO-induced HIF-1alpha and TGF-beta expression are necessary to prevent anoxia/reoxygenation-induced apoptosis in mphi. Furthermore, blockade of HIF-1alpha using RNA interference and HIF-1alpha-cre-lox mphi resulted in a loss of TGF-beta expression and CO-induced protection. A similar mechanism of CO-induced protection was operational in vivo to protect against lung ischemia-reperfusion injury. Taken together, we conclude that CO conditions the mphi via a HIF-1alpha and TGF-beta-dependent mechanism and we elucidate the earliest events in mphi signaling that lead to and preserve cellular homeostasis at the site of injury.
Assuntos
Monóxido de Carbono/farmacologia , Citoproteção/efeitos dos fármacos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Precondicionamento Isquêmico , Animais , Apoptose/efeitos dos fármacos , Hipóxia Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Interleucina-10/metabolismo , Pulmão/efeitos dos fármacos , Pulmão/patologia , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Camundongos , Mitocôndrias/efeitos dos fármacos , Oxigênio/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Traumatismo por Reperfusão/induzido quimicamente , Termodinâmica , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismoRESUMO
BACKGROUND: Arteriovenous grafts often fail due to stenosis caused by venous anastomotic intimal hyperplasia (IH) and vascular smooth muscle cell (VSMC) proliferation. We examined the effects of inhaled carbon monoxide (CO), a product of heme-oxygenase-1 degradation of heme, on IH in a porcine arteriovenous graft model. MATERIALS AND METHODS: Eighteen Yorkshire pigs were divided into three groups (N = 6/group): (1) CO 100 ppm preoperatively for 1 h; (2) CO 250 ppm preoperatively for 1 h and intraoperatively; and (3) air-treated controls. Animals underwent end-to-side placement of polytetrafluoroethylene grafts connecting the common femoral artery and vein in both groins. Intimal thickness of the venous anastomosis at 30 days was measured blinded. The effect of CO on pig VSMC proliferation was studied in cell culture using [(3)H]thymidine incorporation. RESULTS: Pigs in the group receiving CO 250 ppm showed significantly less IH compared to animals in the group receiving 100 ppm and the air-treated group (267.5 +/- 21.4, 824 +/- 145.8, and 914.8 +/- 133.7 pixels, respectively, P < 0.0001). This effect was not observed when comparing the 100 ppm group to the air-treated group. COHb levels were significantly elevated in the 100 ppm and 250 ppm compared to air-treated pigs (5.8 +/- 0.47, 13.2 +/- 1.0 versus 2.3 +/- 0.11%, respectively, P < 0.001). Oxygen saturation, respiratory rate, and hemodynamics were not significantly different between the groups. CO induced VSMC growth arrest compared to air in vitro (11.9 +/- 4 versus 20.3 +/- 5 10(3) counts/min/well, P < 0.01). CONCLUSION: A single exposure to a low concentration of inhaled CO (250 ppm) confers protection against intimal proliferation of VSMCs when given perioperatively in a clinically relevant model of arteriovenous grafts. These data are the first to suggest, in a clinically relevant model, the potential role for CO in clinical applications.
Assuntos
Antimetabólitos/farmacologia , Prótese Vascular , Monóxido de Carbono/farmacologia , Oclusão de Enxerto Vascular/tratamento farmacológico , Oclusão de Enxerto Vascular/prevenção & controle , Administração por Inalação , Animais , Implante de Prótese Vascular , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Artéria Femoral , Veia Femoral , Masculino , Músculo Liso Vascular/citologia , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/patologia , Politetrafluoretileno , Sus scrofa , Túnica Íntima/efeitos dos fármacos , Túnica Íntima/patologiaRESUMO
We have recently shown that the natural bile pigment bilirubin has antiproliferative effects on vascular smooth muscle cells (VSMCs). Bilirubin is the end product of heme catabolism mediated by heme oxygenases and has for decades been considered a toxic waste product of our bodies. However, 14 separate studies and a meta-analysis have documented an inverse correlation between atherosclerosis and the levels of bilirubin in normal individuals. Having high normal or supranormal levels of bilirubin is associated with less atherosclerotic-type disease as compared with that in individuals with low normal levels of bilirubin. This combined with experimental data showing anti-atherosclerotic properties of the enzyme heme oxygenase-1 encouraged us to hypothesize that bilirubin and its precursor biliverdin, would act to ameliorate components of atherosclerosis, in a manner similar to what has been shown with HO-1. Both did so in an animal model of restenosis in which vascular smooth muscle cell proliferation leads to intimal proliferation and causes narrowing of the vessels. We also analyzed the antiproliferative effects of the bile pigments in an in vitro system where bilirubin/biliverdin caused p53 dependent cell cycle arrest by hypophosphorylation of the retinoblastoma tumor suppressor protein in growth factor stimulated VSMCs.
Assuntos
Aterosclerose/tratamento farmacológico , Bilirrubina/uso terapêutico , Biliverdina/uso terapêutico , Animais , Aterosclerose/patologia , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Meios de Cultura Livres de Soro/farmacologia , Masculino , Camundongos , Ratos , Ratos Endogâmicos LewRESUMO
Inflammation and immunoregulatory cytokines play a central role in alcohol-induced liver damage. We previously reported that acute alcohol treatment augments IL-10 and inhibits TNF-alpha production in monocytes. Heme oxygenase-1 (HO-1), a stress-inducible protein, also regulates IL-10 and TNF-alpha production. Here, we report that augmentation of LPS-induced IL-10 production by alcohol was prevented by inhibition of HO-1 activity. Acute ethanol increased LPS-induced enzyme activity and RNA levels of HO-1, and DNA binding of AP-1, a transcription factor essential in HO-1 regulation. LPS-induced phospho-p38 MAPK levels were augmented by ethanol treatment and the p38 inhibitor, SB203580, prevented both the ethanol-induced increase in IL-10 production and the inhibitory effect of ethanol on TNF-alpha production. Ethanol-induced down-regulation of TNF-alpha production was abrogated by inhibition of HO-1. We found that LPS-induced activation of NF-kappaB, a regulator of TNF-alpha, was inhibited by both ethanol treatment and HO-1 activation, but the ethanol-induced inhibition of NF-kappaB was HO-1 independent. In LPS-challenged mice in vivo, both acute alcohol administration and HO-1 activation augmented IL-10 and inhibited TNF-alpha serum levels. These results show that 1) acute alcohol augments HO-1 activation in monocytes, 2) HO-1 activation plays a role in alcohol-induced augmentation of IL-10 production likely via increased p38 MAPK activation, and 3) HO-1 activation is involved in attenuation of TNF-alpha production by alcohol independent of inhibition of NF-kappaB activation by alcohol. Thus, HO-1 activation is a key mediator of the anti-inflammatory effects of acute alcohol on monocytes.
Assuntos
Anti-Inflamatórios não Esteroides/administração & dosagem , Etanol/administração & dosagem , Heme Oxigenase-1/fisiologia , Interleucina-10/biossíntese , Monócitos/enzimologia , Monócitos/imunologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Células Cultivadas , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/imunologia , Feminino , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Monócitos/efeitos dos fármacosRESUMO
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.
Assuntos
Monóxido de Carbono/metabolismo , Monóxido de Carbono/uso terapêutico , Animais , Anti-Inflamatórios/uso terapêutico , Biliverdina/metabolismo , Colite/tratamento farmacológico , Ferritinas/metabolismo , Heme/metabolismo , Heme Oxigenase-1/metabolismo , Humanos , Modelos BiológicosRESUMO
Carbon monoxide (CO) suppresses proinflammatory responses in macrophages reacting to LPS. We hypothesize that CO acts by inducing a molecule(s) that suppresses the inflammatory response to subsequent stress. Exposure of macrophages to CO alone in vitro produced a brief burst of mitochondrial-derived ROS, which led to expression of PPARgamma. PPARgamma expression proved essential for mediating the anti-inflammatory effects of CO. Blocking the CO-mediated increase in ROS generation prevented PPARgamma induction, and blocking PPARgamma prevented CO's anti-inflammatory effects. In a model of acute lung injury in mice, CO blocked expression of Egr-1, a central mediator of inflammation, and decreased tissue damage; inhibition of PPARgamma abrogated both effects. These data identify the mitochondrial oxidases as an (perhaps the) initial cellular target of CO and demonstrate that CO upregulates expression of PPARgamma via the mitochondria, which assures that a subsequent stress stimulus will lead to a cytoprotective as opposed to a proinflammatory phenotype.
Assuntos
Monóxido de Carbono/farmacologia , Inflamação/prevenção & controle , Estresse Oxidativo/efeitos dos fármacos , PPAR alfa/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Animais , Western Blotting , Monóxido de Carbono/imunologia , Modelos Animais de Doenças , Proteína 1 de Resposta de Crescimento Precoce/efeitos dos fármacos , Proteína 1 de Resposta de Crescimento Precoce/imunologia , Eletroforese em Gel de Poliacrilamida , Ensaio de Desvio de Mobilidade Eletroforética , Expressão Gênica/efeitos dos fármacos , Inflamação/imunologia , Pulmão/efeitos dos fármacos , Pulmão/patologia , Pneumopatias/imunologia , Pneumopatias/metabolismo , Lesão Pulmonar , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Camundongos , Mitocôndrias/efeitos dos fármacos , Oxigenases de Função Mista/efeitos dos fármacos , Estresse Oxidativo/imunologia , PPAR alfa/imunologia , PPAR alfa/metabolismo , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , TransfecçãoRESUMO
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.
Assuntos
Heme Oxigenase-1/imunologia , Tolerância ao Transplante , Animais , Biliverdina/metabolismo , Biliverdina/farmacologia , Monóxido de Carbono/metabolismo , Monóxido de Carbono/farmacologia , Rejeição de Enxerto , Transplante de Coração/imunologia , Heme/metabolismo , CamundongosRESUMO
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.