RESUMO
AIMS/HYPOTHESIS: In type 1 diabetes, cardiovascular disease (CVD) and diabetic nephropathy progress in parallel, thereby potentiating the risk of premature death during their development. Since urinary liver-type fatty acid binding protein (L-FABP) predicts the progression of diabetic nephropathy, the aim of this study was to investigate whether urinary L-FABP also predicts cardiovascular outcomes and mortality. METHODS: We tested our hypothesis in a Finnish cohort of 2329 individuals with type 1 diabetes and a median follow-up of 14.1 years. The L-FABP to creatinine ratio was determined from baseline urine samples. The predictive value of urinary L-FABP was evaluated using Cox regression models, while its added predictive benefit for cardiovascular outcomes and mortality was evaluated using a panel of statistical indexes. RESULTS: Urinary L-FABP predicted incident stroke independently of traditional risk factors (HR 1.33 [95% CI 1.20, 1.49]) and after further adjustment for eGFR (HR 1.28 [95% CI 1.14, 1.44]) or AER (HR 1.24 [95% CI 1.06, 1.44]). In addition, it predicted mortality independently of traditional risk factors (HR 1.34 [95% CI 1.24, 1.45]), and after adjustment for eGFR (HR 1.29 [95% CI 1.18, 1.39]) or AER (HR 1.22 [95% CI 1.09, 1.36]). Urinary L-FABP was as good a predictor as eGFR or AER, and improved the AUC for both outcomes on top of traditional risk factors, with no reclassification benefit (integrated discrimination improvement/net reclassification improvement) for stroke or mortality when AER or eGFR were added to traditional risk factors. However, urinary L-FABP was not a predictor of other cardiovascular endpoints (coronary artery disease, peripheral vascular disease and overall CVD events) when adjusted for the AER. CONCLUSIONS/INTERPRETATION: Urinary L-FABP is an independent predictor of stroke and mortality in individuals with type 1 diabetes.
Assuntos
Diabetes Mellitus Tipo 1/metabolismo , Proteínas de Ligação a Ácido Graxo/metabolismo , Acidente Vascular Cerebral/metabolismo , Adulto , Idoso , Albuminúria/metabolismo , Biomarcadores/metabolismo , Creatinina/metabolismo , Nefropatias Diabéticas/metabolismo , Feminino , Taxa de Filtração Glomerular/fisiologia , Humanos , Masculino , Pessoa de Meia-Idade , Modelos de Riscos Proporcionais , Fatores de RiscoRESUMO
AIMS/HYPOTHESIS: The receptor for AGEs (RAGE) is linked to proinflammatory pathology in a range of tissues. The objective of this study was to assess the potential modulatory role of RAGE in diabetic retinopathy. METHODS: Diabetes was induced in wild-type (WT) and Rage (-/-) mice (also known as Ager (-/-) mice) using streptozotocin while non-diabetic control mice received saline. For all groups, blood glucose, HbA1c and retinal levels of methylglyoxal (MG) were evaluated up to 24 weeks post diabetes induction. After mice were killed, retinal glia and microglial activation, vasopermeability, leucostasis and degenerative microvasculature changes were determined. RESULTS: Retinal expression of RAGE in WT diabetic mice was increased after 12 weeks (p < 0.01) but not after 24 weeks. Rage (-/-) mice showed comparable diabetes but accumulated less MG and this corresponded to enhanced activity of the MG-detoxifying enzyme glyoxalase I in their retina when compared with WT mice. Diabetic Rage (-/-) mice showed significantly less vasopermeability, leucostasis and microglial activation (p < 0.05-0.001). Rage (-/-) mice were also protected against diabetes-related retinal acellular capillary formation (p < 0.001) but not against pericyte loss. CONCLUSIONS/INTERPRETATION: Rage (-/-) in diabetic mice is protective against many retinopathic lesions, especially those related to innate immune responses. Inhibition of RAGE could be a therapeutic option to prevent diabetic retinopathy.
Assuntos
Diabetes Mellitus Experimental/metabolismo , Retinopatia Diabética/metabolismo , Receptor para Produtos Finais de Glicação Avançada/metabolismo , Retina/metabolismo , Animais , Glicemia/metabolismo , Diabetes Mellitus Experimental/patologia , Retinopatia Diabética/patologia , Lactoilglutationa Liase/metabolismo , Leucostasia/metabolismo , Leucostasia/patologia , Masculino , Camundongos , Camundongos Knockout , Microvasos/metabolismo , Microvasos/patologia , Aldeído Pirúvico/metabolismo , Retina/patologiaRESUMO
AIMS/HYPOTHESIS: The aim of this study was to determine the protective effects of human insulin and its analogues, B28Asp human insulin (insulin aspart) and B29Lys(ε-tetradecanoyl),desB30 human insulin (insulin detemir), against glucose-induced lifespan reduction and neuronal damage in the model organism Caenorhabditis elegans and to elucidate the underlying mechanisms. METHODS: Nematodes were cultivated under high glucose (HG) conditions comparable with the situation in diabetic patients and treated with human insulin and its analogues. Lifespan was assessed and neuronal damage was evaluated with regard to structural and functional impairment. Additionally, the activity of glyoxalase-1 and superoxide dismutase (SOD) and the formation of reactive oxygen species (ROS) and AGEs were determined. RESULTS: Insulin and its analogues reversed the life-shortening effect of HG conditions and prevented the glucose-induced neuronal impairment. Insulin treatment under HG conditions was associated with reduced concentration of glucose, as well as a reduced formation of ROS and AGEs, and increased SOD activity. These effects were dependent on the Forkhead box O (FOXO) homologue abnormal dauer formation (DAF)-16. Furthermore, glyoxalase-1 activity, which was impaired under HG conditions, was restored by human insulin. This was essential for the insulin-induced lifespan extension under HG conditions, as no change in lifespan was observed following either suppression or overexpression of glyoxalase-1. CONCLUSIONS/INTERPRETATION: Human insulin and its analogues prevent the reduction in lifespan and neuronal damage caused by HG conditions. The effect of human insulin is mediated by a daf-2/insulin receptor and daf-16/FOXO-dependent pathway and is mediated by upregulation of detoxifying mechanisms.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Insulina Regular Humana/farmacologia , Lactoilglutationa Liase/metabolismo , Animais , Dano ao DNA , Reparo do DNA , Regulação da Expressão Gênica no Desenvolvimento , Longevidade , Transdução de SinaisRESUMO
OBJECTIVE: Dysregulation of inflammatory adipokines by the adipose tissue plays an important role in obesity-associated insulin resistance. Pathways leading to this dysregulation remain largely unknown. We hypothesized that the receptor for advanced glycation end products (RAGE) and the ligand N(ε)-(carboxymethyl)lysine (CML) are increased in adipose tissue and, moreover, that activation of the CML-RAGE axis plays an important role in obesity-associated inflammation and insulin resistance. APPROACH AND RESULTS: In this study, we observed a strong CML accumulation and increased expression of RAGE in adipose tissue in obesity. We confirmed in cultured human preadipocytes that adipogenesis is associated with increased levels of CML and RAGE. Moreover, CML induced a dysregulation of inflammatory adipokines in adipocytes via a RAGE-dependent pathway. To test the role of RAGE in obesity-associated inflammation further, we constructed an obese mouse model that is deficient for RAGE (ie, RAGE(-/-)/Leptr(Db-/-) mice). RAGE(-/-)/Leptr(Db-/-) mice displayed an improved inflammatory profile and glucose homeostasis when compared with RAGE(+/+)/Leptr(Db-/-) mice. In addition, CML was trapped in adipose tissue in RAGE(+/+)/Leptr(Db-/-) mice but not in RAGE(-/-)/Leptr(Db-/-). RAGE-mediated trapping in adipose tissue provides a mechanism underlying CML accumulation in adipose tissue and explaining decreased CML plasma levels in obese subjects. Decreased CML plasma levels in obese individuals were strongly associated with insulin resistance. CONCLUSIONS: RAGE-mediated CML accumulation in adipose tissue and the activation of the CML-RAGE axis are important mechanisms involved in the dysregulation of adipokines in obesity, thereby contributing to the development of obesity-associated insulin resistance.
Assuntos
Adipocinas/genética , Resistência à Insulina , Lisina/análogos & derivados , Obesidade/metabolismo , Receptores Imunológicos/fisiologia , Tecido Adiposo/metabolismo , Adulto , Animais , Células Cultivadas , Feminino , Humanos , Metabolismo dos Lipídeos , Lisina/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Receptor para Produtos Finais de Glicação AvançadaRESUMO
Promiscuity of pattern recognition receptors, such as receptor for advanced glycation end products (RAGE), allows for a complex regulatory network controlling inflammation. Scavenging of RAGE ligands by soluble RAGE treatment is effective in reducing delayed-type hypersensitivity (DTH), even in RAGE(-/-) mice by 50% (p < 0.001). This has led to the hypothesis that molecules scavenged by soluble RAGE bind to receptors other than RAGE. This study identifies CD166/ALCAM (ALCAM) as a close structural and functional homolog of RAGE, and it shows that binding of S100B to CD166/ALCAM induces dose- and time-dependent expression of members of the NF-κB family in wild type (WT) and RAGE(-/-) mouse endothelial cells. Blocking CD166/ALCAM expression using small interfering RNA completely inhibited S100B-induced NF-κB activation in RAGE(-/-), but not in WT cells. The in vivo significance of these observations was demonstrated by attenuation of DTH in WT and RAGE(-/-) animals pretreated with CD166/ALCAM small interfering RNA by 50% and 40%, respectively (p < 0.001). Experiments in ALCAM(-/-) animals displayed an only slight reduction of 16% in DTH, explained by compensatory reciprocal upregulation of RAGE in animals devoid of CD166/ALCAM, and vice versa. Consistently, ALCAM(-/-) mice, but not WT mice treated with RAGE small interfering RNA show a 35% reduction in DTH, and ALCAM(-/-) RAGE(-/-) double-knockout mice show a 27% reduction in DTH reaction. Thus, S100B is a proinflammatory cytokine bridging RAGE and CD166/ALCAM downstream effector mechanisms, both being compensatory upregulated after genetic deletion of its counterpart.
Assuntos
Molécula de Adesão de Leucócito Ativado/fisiologia , Antígenos CD/fisiologia , Glicoproteínas/fisiologia , Hipersensibilidade Tardia/imunologia , Mediadores da Inflamação/antagonistas & inibidores , Mediadores da Inflamação/fisiologia , Fatores de Crescimento Neural/fisiologia , Peptídeos/fisiologia , Proteínas S100/fisiologia , Antígeno AC133 , Molécula de Adesão de Leucócito Ativado/química , Animais , Antígenos CD/química , Células Cultivadas , Relação Dose-Resposta Imunológica , Endotélio Vascular/imunologia , Endotélio Vascular/metabolismo , Endotélio Vascular/patologia , Glicoproteínas/antagonistas & inibidores , Glicoproteínas/química , Humanos , Hipersensibilidade Tardia/metabolismo , Hipersensibilidade Tardia/prevenção & controle , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fatores de Crescimento Neural/biossíntese , Fatores de Crescimento Neural/química , Peptídeos/antagonistas & inibidores , Peptídeos/química , Subunidade beta da Proteína Ligante de Cálcio S100 , Proteínas S100/biossíntese , Proteínas S100/química , Relação Estrutura-Atividade , Regulação para Cima/imunologiaRESUMO
Absence of the leucine zipper transcription factor p45NF-E2 results in thrombocytopenia, impaired placental vascularization and intrauterine growth restriction (IUGR) in mice. The mechanism underlying the p45NF-E2-dependent placental defect and IUGR remains unknown. Here, we show that the placental defect and IUGR of p45NF-E2 (Nfe2) null mouse embryos is unrelated to thrombocytopenia, establishing that embryonic platelets and platelet-released mediators are dispensable for placentation. Rather, p45NF-E2, which was hitherto thought to be specific to hematopoietic cells, is expressed in trophoblast cells, where it is required for normal syncytiotrophoblast formation, placental vascularization and embryonic growth. Expression of p45NF-E2 in labyrinthine trophoblast cells colocalizes with that of Gcm1, a transcription factor crucial for syncytiotrophoblast formation. In the absence of p45NF-E2, the width of syncytiotrophoblast layer 2 and the expression of Gcm1 and Gcm1-dependent genes (Synb and Cebpa) are increased. In vitro, p45NF-E2 deficiency results in spontaneous syncytiotrophoblast formation, which can be reversed by Gcm1 knockdown. Increased Gcm1 expression in the absence of p45NF-E2 is dependent on enhanced protein acetylation, including post-translational modification of Gcm1. Increasing and inhibiting acetylation in the placenta of wild-type control embryos phenocopies and corrects, respectively, the changes observed in p45NF-E2-deficient embryos. These studies identify a novel function of p45NF-E2 during placental development: in trophoblast cells, p45NF-E2 represses Gcm1 and syncytiotrophoblast formation via acetylation.
Assuntos
Desenvolvimento Embrionário , Subunidade p45 do Fator de Transcrição NF-E2/metabolismo , Neovascularização Fisiológica , Neuropeptídeos/metabolismo , Placenta/irrigação sanguínea , Trofoblastos/metabolismo , Acetilação , Animais , Células Cultivadas , Imunoprecipitação da Cromatina , Proteínas de Ligação a DNA , Embrião de Mamíferos/citologia , Embrião de Mamíferos/embriologia , Embrião de Mamíferos/metabolismo , Feminino , Retardo do Crescimento Fetal , Técnicas de Introdução de Genes , Células Gigantes/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Subunidade p45 do Fator de Transcrição NF-E2/genética , Neuropeptídeos/genética , Placenta/metabolismo , Placentação , Reação em Cadeia da Polimerase , Gravidez , Processamento de Proteína Pós-Traducional , Trombocitopenia , Fatores de TranscriçãoRESUMO
UNLABELLED: The receptor for advanced glycation endproducts (RAGE) is a multiligand receptor and member of the immunoglobulin superfamily. RAGE is mainly involved in tissue damage and chronic inflammatory disorders, sustaining the inflammatory response upon engagement with damage-associated molecular pattern molecules (DAMPs) such as S100 proteins and high-mobility group box 1 (HMGB1). Enhanced expression of RAGE and its ligands has been demonstrated in distinct tumors and several studies support its crucial role in tumor progression and metastasis by still unknown mechanisms. Here we show that RAGE supports hepatocellular carcinoma (HCC) formation in the Mdr2(-/-) mouse model, a prototype model of inflammation-driven HCC formation, which mimics the human pathology. Mdr2(-/-) Rage(-/-) (dKO) mice developed smaller and fewer HCCs than Mdr2(-/-) mice. Interestingly, although in preneoplastic Mdr2(-/-) livers RAGE ablation did not affect the onset of inflammation, premalignant dKO livers showed reduced liver damage and fibrosis, in association with decreased oval cell activation. Oval cells expressed high RAGE levels and displayed reduced proliferation upon RAGE silencing. Moreover, stimulation of oval cells with HMGB1 promoted an ERK1/2-Cyclin D1-dependent oval cell proliferation in vitro. Finally, genetic and pharmacologic blockade of RAGE signaling impaired oval cell activation in an independent mouse model of oval cell activation, the choline deficient ethionine-supplemented dietary regime. CONCLUSION: Our data identified a novel function of RAGE in regulating oval cell activation and tumor development in inflammation-associated liver carcinogenesis.
Assuntos
Carcinoma Hepatocelular/etiologia , Neoplasias Hepáticas/etiologia , Receptores Imunológicos/fisiologia , Células-Tronco/fisiologia , Subfamília B de Transportador de Cassetes de Ligação de ATP/deficiência , Animais , Carcinoma Hepatocelular/patologia , Transformação Celular Neoplásica , Proteína HMGB1/metabolismo , Inflamação/metabolismo , Cirrose Hepática/patologia , Neoplasias Hepáticas/patologia , Masculino , Camundongos , Camundongos Knockout , Receptor para Produtos Finais de Glicação Avançada , Receptores Imunológicos/biossíntese , Células-Tronco/patologia , Membro 4 da Subfamília B de Transportadores de Cassetes de Ligação de ATPRESUMO
Data providing direct evidence for a causative link between endothelial dysfunction, microvascular disease and diabetic end-organ damage are scarce. Here we show that activated protein C (APC) formation, which is regulated by endothelial thrombomodulin, is reduced in diabetic mice and causally linked to nephropathy. Thrombomodulin-dependent APC formation mediates cytoprotection in diabetic nephropathy by inhibiting glomerular apoptosis. APC prevents glucose-induced apoptosis in endothelial cells and podocytes, the cellular components of the glomerular filtration barrier. APC modulates the mitochondrial apoptosis pathway via the protease-activated receptor PAR-1 and the endothelial protein C receptor EPCR in glucose-stressed cells. These experiments establish a new pathway, in which hyperglycemia impairs endothelial thrombomodulin-dependent APC formation. Loss of thrombomodulin-dependent APC formation interrupts cross-talk between the vascular compartment and podocytes, causing glomerular apoptosis and diabetic nephropathy. Conversely, maintaining high APC levels during long-term diabetes protects against diabetic nephropathy.
Assuntos
Apoptose , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Experimental/prevenção & controle , Nefropatias Diabéticas/patologia , Nefropatias Diabéticas/prevenção & controle , Endotélio Vascular/patologia , Podócitos/patologia , Proteína C/fisiologia , Substituição de Aminoácidos/genética , Animais , Apoptose/genética , Linhagem Celular Transformada , Células Cultivadas , Citoproteção/genética , Diabetes Mellitus Experimental/enzimologia , Diabetes Mellitus Experimental/genética , Nefropatias Diabéticas/enzimologia , Nefropatias Diabéticas/genética , Endotélio Vascular/enzimologia , Ativação Enzimática/genética , Humanos , Glomérulos Renais/irrigação sanguínea , Glomérulos Renais/enzimologia , Glomérulos Renais/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Camundongos Transgênicos , Microcirculação/enzimologia , Microcirculação/patologia , Podócitos/enzimologia , Proteína C/biossíntese , Proteína C/genética , Transdução de Sinais/genética , Trombomodulina/fisiologiaRESUMO
Neutrophilic inflammation, which often persists over days despite appropriate antibiotic therapy, contributes substantially to brain damage in bacterial meningitis. We hypothesized that persistent inflammation is the consequence of a vicious cycle in which inflammation-induced cell injury leads to the release of endogenous danger molecules (e.g. high mobility group box 1) that drive the inflammatory response, causing further damage. The present study aimed to assess the mechanisms of high mobility group box 1 protein release and its functional relevance for the development and progression of pneumococcal meningitis. High mobility group box 1 was found in large quantities in cerebrospinal fluid samples of patients and mice with pneumococcal meningitis (predominantly in advanced stages of the disease). By using macrophages, we demonstrated that the release of high mobility group box 1 from macrophages following pneumococcal challenge is passive in nature and probably not connected with inflammasome- and oxidative stress-dependent inflammatory cell death forms. In a mouse meningitis model, treatment with the high mobility group box 1 antagonists ethyl pyruvate or Box A protein had no effect on the development of meningitis, but led to better resolution of inflammation during antibiotic therapy, which was accompanied by reduced brain pathology and better disease outcome. Additional experiments using gene-deficient mice and murine neutrophils provided evidence that high mobility group box 1 acts as a chemoattractant for neutrophils in a receptor for advanced glycosylation end products-dependent fashion. In conclusion, the present study implicated high mobility group box 1, likely released from dying cells, as a central propagator of inflammation in pneumococcal meningitis. Because persistent inflammation contributes to meningitis-associated brain damage, high mobility group box 1 may represent a promising target for adjunctive therapy of this disease.
Assuntos
Progressão da Doença , Proteína HMGB1/fisiologia , Mediadores da Inflamação/fisiologia , Meningite Pneumocócica/metabolismo , Meningite Pneumocócica/patologia , Animais , Linhagem Celular , Inflamação/etiologia , Inflamação/metabolismo , Inflamação/patologia , Masculino , Meningite Pneumocócica/etiologia , Camundongos , Camundongos Endogâmicos C57BLRESUMO
INTRODUCTION: The role of reactive carbonyl species, such as methylglyoxal (MG), has been overlooked within the context of the sepsis syndrome. The aims of this study were to assess the impact of MG formation in different inflammatory settings and to evaluate its use for early diagnosis as well as prognosis of the sepsis syndrome. METHODS: In total, 120 patients in three groups were enrolled in this observational clinical pilot study. The three groups included patients with septic shock (n = 60), postoperative controls (n = 30), and healthy volunteers (n = 30). Plasma samples from patients with septic shock were collected at sepsis onset and after 24 hours and 4, 7, 14, and 28 days. Plasma samples from postoperative controls were collected prior to surgery, immediately following the end of the surgical procedure as well as 24 hours later and from healthy volunteers once. Plasma levels of MG were determined by high-performance liquid chromatography. Additionally, plasma levels of procalcitonin, C-reactive protein, soluble CD14 subtype, and interleukin-6 were determined. RESULTS: Patients with septic shock showed significantly higher plasma levels of MG at all measured times, compared with postoperative controls. MG was found to identify patients with septic shock more effectively-area under the curve (AUC): 0.993-than procalcitonin (AUC: 0.844), C-reactive protein (AUC: 0.791), soluble CD14 subtype (AUC: 0.832), and interleukin-6 (AUC: 0.898) as assessed by receiver operating characteristic (ROC) analysis. Moreover, plasma levels of MG in non-survivors were significantly higher than in survivors (sepsis onset: *P = 0.018 for 90-day survival; **P = 0.008 for 28-day survival). Plasma levels of MG proved to be an early predictor for survival in patients with septic shock (sepsis onset: ROC-AUC 0.710 for 28-day survival; ROC-AUC 0.686 for 90-day survival). CONCLUSIONS: MG was identified as a marker for monitoring the onset, development, and remission of sepsis and was found to be more useful than routine diagnostic markers. Further studies are required to determine the extent of MG modification in sepsis and whether targeting this pathway could be therapeutically beneficial to the patient. TRIAL REGISTRATION: German Clinical Trials Register DRKS00000505. Registered 8 November 2010.
Assuntos
Aldeído Pirúvico/sangue , Choque Séptico/sangue , Choque Séptico/diagnóstico , Idoso , Biomarcadores/sangue , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Projetos PilotoRESUMO
Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are common disorders in patients requiring critical care. The clinical management of these disorders is difficult and unrewarding, and thus they are among the most common causes of death in intensive care units. The activation and damage of pulmonary endothelium comprise the hallmark of ALI/ARDS. Therefore, the recruitment of circulating endothelial progenitor cells (EPCs) to these lesions may exert a beneficial effect on the clinical course of ALI/ARDS. Consequently, cell-based therapies using stem cells to regenerate lung tissue have emerged as potential novel treatment strategies. Although initial studies suggested implantations of exogenously administered bone marrow-derived progenitor cells into damaged vessel walls, recent evidence indicates that this is rather a rare occurrence with uncertain physiologic significance. In the past few years, different populations of progenitor cells were identified, with different functional capacities. This review (1) highlights the different populations of EPCs identified or administered in different models of ALI/ARDS, (2) reports on whether beneficial effects of EPCs could be demonstrated, and (3) puts the conflicting results of different studies into perspective.
Assuntos
Lesão Pulmonar Aguda , Terapia Baseada em Transplante de Células e Tecidos , Células Endoteliais , Síndrome do Desconforto Respiratório , Transplante de Células-Tronco , Células-Tronco , Lesão Pulmonar Aguda/metabolismo , Lesão Pulmonar Aguda/patologia , Lesão Pulmonar Aguda/terapia , Animais , Células da Medula Óssea , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Células Endoteliais/transplante , Humanos , Regeneração , Síndrome do Desconforto Respiratório/metabolismo , Síndrome do Desconforto Respiratório/patologia , Síndrome do Desconforto Respiratório/terapia , Células-Tronco/metabolismo , Células-Tronco/patologiaRESUMO
Neuropathic pain can develop as an agonizing sequela of diabetes mellitus and chronic uremia. A chemical link between both conditions of altered metabolism is the highly reactive compound methylglyoxal (MG), which accumulates in all cells, in particular neurons, and leaks into plasma as an index of the severity of the disorder. The electrophilic structure of this cytotoxic ketoaldehyde suggests TRPA1, a receptor channel deeply involved in inflammatory and neuropathic pain, as a molecular target. We demonstrate that extracellularly applied MG accesses specific intracellular binding sites of TRPA1, activating inward currents and calcium influx in transfected cells and sensory neurons, slowing conduction velocity in unmyelinated peripheral nerve fibers, and stimulating release of proinflammatory neuropeptides from and action potential firing in cutaneous nociceptors. Using a model peptide of the N terminus of human TRPA1, we demonstrate the formation of disulfide bonds based on MG-induced modification of cysteines as a novel mechanism. In conclusion, MG is proposed to be a candidate metabolite that causes neuropathic pain in metabolic disorders and thus is a promising target for medicinal chemistry.
Assuntos
Canais de Cálcio/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neuralgia/metabolismo , Nociceptores/metabolismo , Aldeído Pirúvico/metabolismo , Canais de Cátion TRPC/metabolismo , Canais de Potencial de Receptor Transitório/metabolismo , Potenciais de Ação , Animais , Sítios de Ligação , Canais de Cálcio/genética , Células HEK293 , Humanos , Mediadores da Inflamação/metabolismo , Camundongos , Camundongos Mutantes , Proteínas do Tecido Nervoso/genética , Neuralgia/dietoterapia , Neuralgia/genética , Neuralgia/patologia , Neurônios/metabolismo , Neurônios/patologia , Neuropeptídeos/metabolismo , Nociceptores/patologia , Ratos , Canal de Cátion TRPA1 , Canais de Cátion TRPC/genética , Canais de Potencial de Receptor Transitório/genéticaRESUMO
We recently demonstrated that the bZip transcription factor nuclear factor erythroid-derived 2 (Nfe2) represses protein acetylation and expression of the transcription factor glial cell missing 1 (Gcm1) in trophoblast cells, preventing excess syncytiotrophoblast formation and permitting normal placental vascularization and embryonic growth. However, the Gcm1 promoter lacks a Nfe2-binding site and hence the mechanisms linking Nfe2 and Gcm1 expression remained unknown. Here we show that Nfe2 represses JunD DNA-binding activity to the Gcm1 promoter during syncytiotrophoblast differentiation. Interventional studies using knockdown and knockin approaches show that enhanced JunD DNA-binding activity is required for increased expression of Gcm1 and syncytiotrophoblast formation as well as impaired placental vascularization and reduced growth of Nfe2(-/-) embryos. Induction of Gcm1 expression requires binding of JunD to the -1441 site within the Gcm1 promoter, which is distinct from the -1314 site previously shown to induce Gcm1 expression by other bZip transcription factors. Nfe2 modulates JunD binding to the Gcm1 promoter via acetylation, as reducing JunD acetylation using the histone acetyltransferase inhibitor curcumin reverses the increased JunD DNA-binding activity observed in the absence of Nfe2. This identifies a novel mechanism through which bZip transcription factors interact. Within the placenta this interaction regulates Gcm1 expression, syncytiotrophoblast formation, placental vascularization, and embryonic growth.
Assuntos
Diferenciação Celular , DNA/metabolismo , Subunidade p45 do Fator de Transcrição NF-E2/metabolismo , Proteínas Proto-Oncogênicas c-jun/metabolismo , Trofoblastos/citologia , Trofoblastos/metabolismo , Acetilação , Animais , Proteínas de Ligação a DNA , Feminino , Células HEK293 , Humanos , Camundongos , Neovascularização Fisiológica , Neuropeptídeos/genética , Placenta/citologia , Placenta/embriologia , Placenta/metabolismo , Gravidez , Regiões Promotoras Genéticas/genética , Ligação Proteica , Fator de Transcrição AP-1/metabolismo , Fatores de TranscriçãoRESUMO
Urinary tract obstruction during nephron development causes tubular apoptosis, tubular atrophy, and interstitial fibrosis. Leukocyte recruitment is critical in the development of obstructive nephropathy leading to interstitial inflammation and renal fibrosis. RAGE, the receptor of advanced glycation end products, is implicated in chronic inflammation and has been recently identified as a novel receptor for the ß2-integrin Mac-1, cooperating with ICAM-1 and thereby directly mediating leukocyte recruitment in vivo. Here, we studied the role of RAGE and ICAM-1 in a model of unilateral ureteral obstruction in neonatal mice. Interestingly, the number of infiltrating leukocytes was independent of RAGE and ICAM-1 in the ureteral obstructed neonatal kidney. By contrast, galectin-3, a marker for profibrogenic M2 macrophages, was strongly reduced in ureteral obstructed RAGE and RAGE-Icam1 knockout mice. Snail expression and loss of E-cadherin but not NF-κB activation were attenuated in both knockout models. Epithelial cell cycle arrest at G2/M, which mediates kidney fibrosis, and transforming growth factor-ß expression were reduced in ureteral obstructed RAGE knockout mice. Thus, RAGE and ICAM-1 promote renal fibrosis in the developing kidney upon ureteral obstruction. Combined RAGE- and ICAM-1-blocking strategies may prove beneficial in neonatal obstructive nephropathy.
Assuntos
Células Epiteliais/metabolismo , Nefropatias/etiologia , Rim/metabolismo , NF-kappa B/metabolismo , Receptores Imunológicos/metabolismo , Obstrução Ureteral/complicações , Animais , Animais Recém-Nascidos , Apoptose , Caderinas/metabolismo , Proliferação de Células , Quimiotaxia de Leucócito , Modelos Animais de Doenças , Células Epiteliais/patologia , Transição Epitelial-Mesenquimal , Fibrose , Pontos de Checagem da Fase G2 do Ciclo Celular , Galectina 3/metabolismo , Genótipo , Molécula 1 de Adesão Intercelular/genética , Molécula 1 de Adesão Intercelular/metabolismo , Rim/crescimento & desenvolvimento , Rim/patologia , Nefropatias/metabolismo , Nefropatias/patologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , Receptor para Produtos Finais de Glicação Avançada , Receptores Imunológicos/deficiência , Receptores Imunológicos/genética , Transdução de Sinais , Fatores de Transcrição da Família Snail , Fatores de Tempo , Fatores de Transcrição/metabolismo , Obstrução Ureteral/metabolismo , Obstrução Ureteral/patologiaRESUMO
Whereas it is generally perceived to be harmful, enhanced coagulation activation can also convey salutary effects. The high prevalence of the prothrombotic factor V Leiden (FVL) mutation in whites has been attributed to a positive selection pressure (eg, resulting from reduced blood loss or improved survival in sepsis). The consequences of enhanced coagulation activation, as observed in FVL carriers, on microvascular diabetic complications remain unknown. We therefore investigated the role of FVL in diabetic nephropathy. In heterozygous or homozygous diabetic FVL mice, albuminuria and indices of diabetic nephropathy were reduced compared with diabetic wild-type mice. This was associated with reduced glomerular apoptosis and preservation of podocytes in diabetic FVL-positive mice. In vitro, low-dose thrombin (50pM) prevented, whereas high-dose thrombin (20nM) aggravated, glucose-induced apoptosis in podocytes. In diabetic patients, the FVL mutation, but not the plasminogen activator inhibitor-1 4G/5G polymorphism, is associated with reduced albuminuria, which is consistent with a nephroprotective role of low but sustained thrombin generation. Consistently, anticoagulation of diabetic FVL-positive mice with hirudin abolished the nephroprotective effect. These results identify a nephroprotective function of low but sustained thrombin levels in FVL carriers, supporting a dual, context-dependent function of thrombin in chronic diseases.
Assuntos
Apoptose/genética , Coagulação Sanguínea/fisiologia , Nefropatias Diabéticas/genética , Fator V/genética , Podócitos/patologia , Animais , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Nefropatias Diabéticas/metabolismo , Nefropatias Diabéticas/patologia , Fator V/metabolismo , Genótipo , Glucose/efeitos adversos , Humanos , Hiperglicemia/complicações , Immunoblotting , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Camundongos , Camundongos Endogâmicos C57BL , Mutação de Sentido IncorretoRESUMO
Clearance of apoptotic cells by macrophages and other phagocytic cells, called efferocytosis, is a central process in the resolution of inflammation. Although the receptor for advanced glycation end products (RAGE) has been shown to participate in a variety of acute and chronic inflammatory processes in the lungs and other organs, a role for RAGE in efferocytosis has not been reported. In the present studies, we examined the potential involvement of RAGE in efferocytosis. Macrophages from transgenic RAGE(-/-) mice showed a decreased ability to engulf apoptotic neutrophils and thymocytes. Pretreatment of RAGE(+/+) macrophages with advanced glycation end products, which competitively bind to RAGE, or Abs against RAGE diminished phagocytosis of apoptotic cells. Overexpression of RAGE in human embryonic kidney 293 cells resulted in an increased ability to engulf apoptotic cells. Furthermore, we found that incubation with soluble RAGE enhances phagocytosis of apoptotic cells by both RAGE(+/+) and RAGE(-/-) macrophages. Direct binding of RAGE to phosphatidylserine (PS), an "eat me" signal highly expressed on apoptotic cells, was shown by using solid-phase ELISA. The ability of RAGE to bind to PS on apoptotic cells was confirmed in an adhesion assay. Decreased uptake of apoptotic neutrophils by macrophages was found under in vivo conditions in the lungs and peritoneal cavity of RAGE(-/-) mice. These results demonstrate a novel role for RAGE in which it is able to enhance efferocytosis through binding to PS on apoptotic cells.
Assuntos
Apoptose/imunologia , Macrófagos/imunologia , Fagócitos/imunologia , Receptores Imunológicos/imunologia , Animais , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Produtos Finais de Glicação Avançada/metabolismo , Produtos Finais de Glicação Avançada/farmacologia , Células HEK293 , Humanos , Interleucina-10/imunologia , Interleucina-10/metabolismo , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neutrófilos/imunologia , Neutrófilos/metabolismo , Fagócitos/metabolismo , Fagocitose/efeitos dos fármacos , Fagocitose/imunologia , Fosfatidilserinas/imunologia , Fosfatidilserinas/metabolismo , Receptor para Produtos Finais de Glicação Avançada , Receptores Imunológicos/genética , Receptores Imunológicos/metabolismo , Timo/citologia , Timo/imunologia , Timo/metabolismoRESUMO
Methylglyoxal (MG), the major dicarbonyl substrate of the enzyme glyoxalase 1 (GLO1), is a reactive metabolite formed via glycolytic flux. Decreased GLO1 activity in situ has been shown to result in an accumulation of MG and increased formation of advanced glycation endproducts, both of which can accumulate during physiological aging and at an accelerated rate in diabetes and other chronic degenerative diseases. To determine the physiological consequences which result from elevated MG levels and the role of MG and GLO1 in aging, wound healing in young (≤12 weeks) and old (≥52 weeks) wild-type mice was studied. Old mice were found to have a significantly slower rate of wound healing compared to young mice (74.9 ± 2.2 vs. 55.4 ± 1.5% wound closure at day 6; 26% decrease; p < 0.0001). This was associated with decreases in GLO1 transcription, expression and activity. The importance of GLO1 was confirmed in mice by inhibition of GLO1. Direct application of MG to the wounds of young mice, decreased wound healing by 24% compared to untreated mice, whereas application of BSA modified minimally by MG had no effect. Treatment of either young or old mice with aminoguanidine, a scavenger of free MG, significantly increased wound closure by 16% (66.8 ± 1.6 vs. 77.2 ± 3.1%; p < 0.05) and 64% (40.4 ± 7.9 vs. 66.4 ± 5.2%; p < 0.05), respectively, by day 6. As a result of the aminoguanidine treatment, the overall rate of wound healing in the old mice was restored to the level observed in the young mice. These findings were confirmed in vitro, as MG reduced migration and proliferation of fibroblasts derived from young and old, wild-type mice. The data demonstrate that the balance between MG and age-dependent GLO1 downregulation contributes to delayed wound healing in old mice.
Assuntos
Envelhecimento/fisiologia , Lactoilglutationa Liase/fisiologia , Cicatrização/fisiologia , Envelhecimento/genética , Envelhecimento/metabolismo , Animais , Células Cultivadas , Regulação para Baixo , Fibroblastos/efeitos dos fármacos , Fibroblastos/fisiologia , Guanidinas/farmacologia , Lactoilglutationa Liase/antagonistas & inibidores , Lactoilglutationa Liase/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Aldeído Pirúvico/metabolismo , Aldeído Pirúvico/farmacologia , Cicatrização/efeitos dos fármacos , Cicatrização/genéticaRESUMO
During infection, vertebrates develop "sickness syndrome," characterized by fever, anorexia, behavioral withdrawal, acute-phase protein responses, and inflammation. These pathophysiological responses are mediated by cytokines, including TNF and IL-1, released during the innate immune response to invasion. Even in the absence of infection, qualitatively similar physiological syndromes occur following sterile injury, ischemia reperfusion, crush injury, and autoimmune-mediated tissue damage. Recent advances implicate high-mobility group box 1 (HMGB1), a nuclear protein with inflammatory cytokine activities, in stimulating cytokine release. HMGB1 is passively released during cell injury and necrosis, or actively secreted during immune cell activation, positioning it at the intersection of sterile and infection-associated inflammation. To date, eight candidate receptors have been implicated in mediating the biological responses to HMGB1, but the mechanism of HMGB1-dependent cytokine release is unknown. Here we show that Toll-like receptor 4 (TLR4), a pivotal receptor for activation of innate immunity and cytokine release, is required for HMGB1-dependent activation of macrophage TNF release. Surface plasmon resonance studies indicate that HMGB1 binds specifically to TLR4, and that this binding requires a cysteine in position 106. A wholly synthetic 20-mer peptide containing cysteine 106 from within the cytokine-stimulating B box mediates TLR4-dependent activation of macrophage TNF release. Inhibition of TLR4 binding with neutralizing anti-HMGB1 mAb or by mutating cysteine 106 prevents HMGB1 activation of cytokine release. These results have implications for rationale, design, and development of experimental therapeutics for use in sterile and infectious inflammation.
Assuntos
Citocinas/biossíntese , Proteína HMGB1/química , Proteína HMGB1/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Receptor 4 Toll-Like/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Células CHO , Cricetinae , Cricetulus , Cisteína/química , Primers do DNA/genética , Proteína HMGB1/genética , Proteína HMGB1/imunologia , Humanos , Imunidade Inata , Técnicas In Vitro , Antígeno 96 de Linfócito/metabolismo , Ativação de Macrófagos , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Ligação Proteica , Ratos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Receptor 4 Toll-Like/deficiência , Receptor 4 Toll-Like/genéticaRESUMO
The receptor for advanced glycation end products (RAGE) is a member of the immunoglobulin superfamily that has multiple ligands and is implicated in the pathogenesis of various diseases, including diabetic complications, neurodegenerative disorders, and inflammatory responses. However, the role of RAGE in normal physiology is largely undefined. Here, we present evidence for a role of RAGE in osteoclast maturation and function, which has consequences for bone remodeling. Mice lacking RAGE had increased bone mass and bone mineral density and decreased bone resorptive activity in vivo. In vitro-differentiated RAGE-deficient osteoclasts exhibited disrupted actin ring and sealing zone structures, impaired maturation, and reduced bone resorptive activity. Impaired signaling downstream of alphavbeta3 integrin was observed in RAGE(-/-) bone marrow macrophages and precursors of OCs. These results demonstrate a role for RAGE in osteoclast actin cytoskeletal reorganization, adhesion, and function, and suggest that the osteosclerotic-like phenotype observed in RAGE knockout mice is due to a defect in osteoclast function.
Assuntos
Osso e Ossos/fisiologia , Osteoclastos/fisiologia , Receptores Imunológicos/fisiologia , Actinas/fisiologia , Animais , Osso e Ossos/citologia , Osso e Ossos/metabolismo , Osso e Ossos/patologia , Adesão Celular/fisiologia , Diferenciação Celular/fisiologia , Células Cultivadas , Citoesqueleto/fisiologia , Regulação para Baixo/fisiologia , Produtos Finais de Glicação Avançada , Integrina alfaVbeta3/antagonistas & inibidores , Integrina alfaVbeta3/biossíntese , Integrina alfaVbeta3/fisiologia , Fator Estimulador de Colônias de Macrófagos/antagonistas & inibidores , Fator Estimulador de Colônias de Macrófagos/fisiologia , Macrófagos/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Osteoclastos/citologia , Osteoclastos/metabolismo , Osteoclastos/patologia , Receptor para Produtos Finais de Glicação Avançada , Receptores Imunológicos/deficiência , Receptores Imunológicos/genéticaRESUMO
Psychosocial stress has been shown to be a contributing factor in the development of atherosclerosis. Although the underlying mechanisms have not been elucidated entirely, it has been shown previously that the transcription factor nuclear factor-κB (NF-κB) is an important component of stress-activated signaling pathway. In this study, we aimed to decipher the mechanisms of stress-induced NF-κB-mediated gene expression, using an in vitro and in vivo model of psychosocial stress. Induction of stress led to NF-κB-dependent expression of proinflammatory (tissue factor, intracellular adhesive molecule 1 [ICAM-1]) and protective genes (manganese superoxide dismutase [MnSOD]) via p50, p65 or cRel. Selective inhibition of the different subunits and the respective kinases showed that inhibition of cRel leads to the reduction of atherosclerotic lesions in apolipoprotein(-/-) (ApoE(-/-)) mice via suppression of proinflammatory gene expression. This observation may therefore provide a possible explanation for ineffectiveness of antioxidant therapies and suggests that selective targeting of cRel activation may provide a novel approach for the treatment of stress-related inflammatory vascular disease.