RESUMO
Hepcidin regulates iron homeostasis by controlling the level of ferroportin, the only membrane channel that facilitates export of iron from within cells. Binding of hepcidin to ferroportin induces the ubiquitination of ferroportin at multiple lysine residues and subsequently causes the internalization and degradation of the ligand-channel complex within lysosomes. The objective of this study was to identify components of the ubiquitin system that are involved in ferroportin degradation. A HepG2 cell line, which inducibly expresses ferroportingreen fluorescent protein (FPN-GFP), was established to test the ability of small interfering (siRNA) directed against components of the ubiquitin system to prevent BMP6- and exogenous hepcidin-induced ferroportin degradation. Of the 88 siRNA directed against components of the ubiquitin pathway that were tested, siRNA-mediated depletion of the alternative E1 enzyme UBA6 as well as the adaptor protein NDFIP1 prevented BMP6- and hepcidin-induced degradation of ferroportin in vitro. A third component of the ubiquitin pathway, ARIH1, indirectly inhibited ferroportin degradation by impairing BMP6-mediated induction of hepcidin. In mice, the AAV-mediated silencing of Ndfip1 in the murine liver increased the level of hepatic ferroportin and increased circulating iron. The results suggest that the E1 enzyme UBA6 and the adaptor protein NDFIP1 are involved in iron homeostasis by regulating the degradation of ferroportin. These specific components of the ubiquitin system may be promising targets for the treatment of iron-related diseases, including iron overload and anemia of inflammation.
Assuntos
Proteínas de Transporte de Cátions , Sobrecarga de Ferro , Proteínas de Membrana , Enzimas Ativadoras de Ubiquitina , Animais , Proteínas de Transporte/genética , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Hepcidinas/genética , Hepcidinas/metabolismo , Humanos , Ferro/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Proteólise , Enzimas Ativadoras de Ubiquitina/genética , Enzimas Ativadoras de Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , UbiquitinaçãoRESUMO
BACKGROUND AND OBJECTIVES: Carbon monoxide (CO) poisoning is responsible for nearly 50,000 emergency department visits and 1200 deaths per year. Compared to oxygen, CO has a 250-fold higher affinity for hemoglobin (Hb), resulting in the displacement of oxygen from Hb and impaired oxygen delivery to tissues. Optimal treatment of CO-poisoned patients involves the administration of hyperbaric 100% oxygen to remove CO from Hb and to restore oxygen delivery. However, hyperbaric chambers are not widely available and this treatment requires transporting a CO-poisoned patient to a specialized center, which can result in delayed treatment. Visible light is known to dissociate CO from carboxyhemoglobin (COHb). In a previous study, we showed that a system composed of six photo-extracorporeal membrane oxygenation (ECMO) devices efficiently removes CO from a large animal with CO poisoning. In this study, we tested the hypothesis that the application of hyperbaric oxygen to the photo-ECMO device would further increase the rate of CO elimination. STUDY DESIGN/MATERIAL AND METHODS: We developed a hyperbaric photo-ECMO device and assessed the ability of the device to remove CO from CO-poisoned human blood. We combined four devices into a "hyperbaric photo-ECMO system" and compared its ability to remove CO to our previously described photo-ECMO system, which was composed of six devices ventilated with normobaric oxygen. RESULTS: Under normobaric conditions, an increase in oxygen concentration from 21% to 100% significantly increased CO elimination from CO-poisoned blood after a single pass through the device. Increased oxygen pressure within the photo-ECMO device was associated with higher exiting blood PO2 levels and increased CO elimination. The system of four hyperbaric photo-ECMO devices removed CO from 1 L of CO-poisoned blood as quickly as the original, normobaric photo-ECMO system composed of six devices. CONCLUSION: This study demonstrates the feasibility and efficacy of using a hyperbaric photo-ECMO system to increase the rate of CO elimination from CO-poisoned blood. This technology could provide a simple portable emergency device and facilitate immediate treatment of CO-poisoned patients at or near the site of injury.
Assuntos
Intoxicação por Monóxido de Carbono , Monóxido de Carbono , Animais , Intoxicação por Monóxido de Carbono/complicações , Intoxicação por Monóxido de Carbono/terapia , Carboxihemoglobina , Hemoglobinas , Humanos , Oxigênio , Fototerapia/métodosRESUMO
BACKGROUND AND OBJECTIVES: Carbon monoxide (CO) inhalation is the leading cause of poison-related deaths in the United States. CO binds to hemoglobin (Hb), displaces oxygen, and reduces oxygen delivery to tissues. The optimal treatment for CO poisoning in patients with normal lung function is the administration of hyperbaric oxygen (HBO). However, hyperbaric chambers are only available in medical centers with specialized equipment, resulting in delayed therapy. Visible light dissociates CO from Hb with minimal effect on oxygen binding. In a previous study, we combined a membrane oxygenator with phototherapy at 623 nm to produce a "mini" photo-ECMO (extracorporeal membrane oxygenation) device, which improved CO elimination and survival in CO-poisoned rats. The objective of this study was to develop a larger photo-ECMO device ("maxi" photo-ECMO) and to test its ability to remove CO from a porcine model of CO poisoning. STUDY DESIGN/MATERIALS AND METHODS: The "maxi" photo-ECMO device and the photo-ECMO system (six maxi photo-ECMO devices assembled in parallel), were tested in an in vitro circuit of CO poisoning. To assess the ability of the photo-ECMO device and the photo-ECMO system to remove CO from CO-poisoned blood in vitro, the half-life of COHb (COHb-t1/2 ), as well as the percent COHb reduction in a single blood pass through the device, were assessed. In the in vivo studies, we assessed the COHb-t1/2 in a CO-poisoned pig under three conditions: (1) While the pig breathed 100% oxygen through the endotracheal tube; (2) while the pig was connected to the photo-ECMO system with no light exposure; and (3) while the pig was connected to the photo-ECMO system, which was exposed to red light. RESULTS: The photo-ECMO device was able to fully oxygenate the blood after a single pass through the device. Compared to ventilation with 100% oxygen alone, illumination with red light together with 100% oxygen was twice as efficient in removing CO from blood. Changes in gas flow rates did not alter CO elimination in one pass through the device. Increases in irradiance up to 214 mW/cm2 were associated with an increased rate of CO elimination. The photo-ECMO device was effective over a range of blood flow rates and with higher blood flow rates, more CO was eliminated. A photo-ECMO system composed of six photo-ECMO devices removed CO faster from CO-poisoned blood than a single photo-ECMO device. In a CO-poisoned pig, the photo-ECMO system increased the rate of CO elimination without significantly increasing the animal's body temperature or causing hemodynamic instability. CONCLUSION: In this study, we developed a photo-ECMO system and demonstrated its ability to remove CO from CO-poisoned 45-kg pigs. Technical modifications of the photo-ECMO system, including the development of a compact, portable device, will permit treatment of patients with CO poisoning at the scene of their poisoning, during transit to a local emergency room, and in hospitals that lack HBO facilities.
Assuntos
Intoxicação por Monóxido de Carbono , Venenos , Animais , Monóxido de Carbono , Intoxicação por Monóxido de Carbono/terapia , Carboxihemoglobina/metabolismo , Humanos , Fototerapia/métodos , Ratos , SuínosRESUMO
Atherosclerotic cardiovascular disease is a major burden on global health and a leading cause of death worldwide. The pathophysiology of this chronic disease is complex, involving inflammation, lipoprotein oxidation and accumulation, plaque formation, and calcification. In 1981, Dr. Jerome Sullivan formulated the 'Iron Hypothesis', suggesting that higher levels of stored iron promote cardiovascular diseases, whereas iron deficiency may have an atheroprotective effect. This hypothesis has stimulated research focused on clarifying the role of iron in the development of atherosclerosis. However, preclinical and clinical studies have produced contradictory results and the observation that patients with hemochromatosis do not appear to have an increased risk of atherosclerosis seemed incongruous with Sullivan's initial hypothesis. The 'paradox' of systemic iron overload not being accompanied by an increased risk for atherosclerosis led to a refinement of the iron hypothesis focusing on intracellular macrophage iron. More recent in vitro and animal studies have elucidated the complex signaling pathways regulating iron, with a particular focus on hepcidin, the master regulator of body iron homeostasis. Bone morphogenetic protein (BMP) signaling is the major pathway that is required for induction of hepcidin expression in response to increasing levels of iron. Strong links between iron homeostasis, BMP signaling, inflammation and atherosclerosis have been established in both mechanistic and human studies. This review summarizes the current understanding of the role of iron homeostasis and hepcidin in the development of atherosclerosis and discusses the BMP-hepcidin-ferroportin axis as a novel therapeutic target for the treatment of cardiovascular disease.
Assuntos
Aterosclerose/metabolismo , Doenças Cardiovasculares/metabolismo , Hepcidinas/metabolismo , Homeostase , Ferro/metabolismo , Macrófagos/metabolismo , Animais , Aterosclerose/sangue , Doenças Cardiovasculares/sangue , Células Espumosas/metabolismo , Hepcidinas/genética , Humanos , Ferro/sangueRESUMO
BACKGROUND: Cancer is a life-threatening disease that causes every fourth death. It is often hard to determine the time point of progression. Therefore, biomarkers for cancer entities that indicate disease progression or aggressiveness and thereby guide therapeutic decisions are required. Unfortunately, reliable biomarkers are rare. In this study, the potential of serum hepcidin and serum GDF-15 as biomarkers that correlate with patient's survival in the two entities upper urinary tract urothelial carcinomas (UUTUC) and renal cell carcinoma (RCC) were analyzed. METHODS: In this retrospective study n = 38 patients suffering from UUTUC, n = 94 patients suffering from RCC and n = 21 patients without infections or cancer, all hospitalized at the University Hospital Muenster, were included. Serum samples of patients were retrospectively analyzed. Serum hepcidin and GDF-15 levels were measured and correlated to aggressiveness and progression of the disease as well as patient's outcome. RESULTS: For both entities, UUTUC and RCC, serum hepcidin levels as well as serum GDF-15 levels were increased compared to sera of controls. High serum hepcidin and GDF-15 levels were associated with metastases and cancer relapse. Also, in both entities, the overall survival was decreased in patients with increased serum hepcidin and GDF-15 levels. Hence, high serum hepcidin and GDF-15 levels correlated with patient's outcome. CONCLUSION: To conclude, the data of this study show a correlation of high serum hepcidin and GDF-15 levels with aggressiveness and progression of the disease and demonstrate potential prognostic properties of serum hepcidin and GDF-15 levels. The data support the further assessment of serum hepcidin and GDF-15 as prognostic markers in RCC and UUTUC.
Assuntos
Biomarcadores Tumorais/sangue , Carcinoma de Células Renais/sangue , Carcinoma de Células de Transição/sangue , Fator 15 de Diferenciação de Crescimento/sangue , Hepcidinas/sangue , Neoplasias Renais/sangue , Recidiva Local de Neoplasia/diagnóstico , Neoplasias Ureterais/sangue , Idoso , Carcinoma de Células Renais/mortalidade , Carcinoma de Células Renais/patologia , Carcinoma de Células de Transição/mortalidade , Carcinoma de Células de Transição/patologia , Progressão da Doença , Feminino , Humanos , Neoplasias Renais/mortalidade , Neoplasias Renais/patologia , Masculino , Pessoa de Meia-Idade , Prognóstico , Estudos Retrospectivos , Neoplasias Ureterais/mortalidade , Neoplasias Ureterais/patologiaRESUMO
Although the relationship between hereditary hemochromatosis and mutations in the HFE gene was discovered more than 20 years ago, information on the in vivo regulation of HFE protein expression is still limited. The purpose of the study was to determine the response of liver HFE protein content to iron deficiency in mice and rats by immunoblotting. Attempts to visualize the HFE protein in whole liver homogenates were unsuccessful; however, HFE could be detected in liver microsomes or in plasma membrane-enriched fractions. Five-week-old male C57BL/6 mice fed an iron-deficient diet for 4 wk presented with a significant decrease in liver iron content and liver Hamp expression, as well as with a significant decrease in liver HFE protein content. Rats fed an iron-deficient diet for 4 wk also displayed significant decrease in liver Hamp expression and liver HFE protein content. These results suggest that the downregulation of HFE-dependent signaling may contribute to decreased Hamp gene expression in states of prolonged iron deficiency. It has recently been proposed that HFE protein could be a potential target of matriptase-2, a hepatocyte protease mutated in iron-refractory iron deficiency anemia. However, immunoblot analysis of HFE protein in the livers from Tmprss6-mutated mask mice did not show evidence of matriptase-2-dependent HFE protein cleavage. In addition, no indication of HFE protein cleavage was seen in iron-deficient rats, whereas the full-length matriptase-2 protein content in the same animals was significantly increased. These results suggest that HFE is probably not a major physiological target of matriptase-2. NEW & NOTEWORTHY Feeding of iron-deficient diet for 4 wk decreased liver HFE protein content in both mice and rats, suggesting that decreased HFE-dependent signaling may contribute to hepcidin downregulation in iron deficiency. There was no difference in HFE protein band appearance between matriptase-2-mutated mask mice and wild-type mice, indicating that HFE is probably not a major physiological substrate of matriptase-2-mediated protease activity in vivo.
Assuntos
Anemia Ferropriva/metabolismo , Proteína da Hemocromatose/metabolismo , Deficiências de Ferro , Fígado/metabolismo , Anemia Ferropriva/genética , Animais , Feminino , Proteína da Hemocromatose/genética , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteólise , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismoRESUMO
BACKGROUND: Inflammatory stimuli induce the hepatic iron regulatory hormone hepcidin, which contributes to anaemia of inflammation (AI). Hepcidin expression is regulated by the bone morphogenetic protein (BMP) and the interleukin-6 (IL-6) signalling pathways. Prior results indicate that the BMP type I receptor ALK3 is mainly involved in the acute inflammatory hepcidin induction four and 72 h after IL-6 administration. In this study, the role of ALK3 in a chronic model of inflammation was investigated. The intact, heat-killed bacterium Brucella abortus (BA) was used to analyse its effect on the development of inflammation and hypoferremia in mice with hepatocyte-specific Alk3-deficiency (Alk3fl/fl; Alb-Cre) compared to control (Alk3fl/fl) mice. RESULTS: An iron restricted diet prevented development of the iron overload phenotype in mice with hepatocyte-specific Alk3 deficiency. Regular diet leads to iron overload and increased haemoglobin levels in these mice, which protects from the development of AI per se. Fourteen days after BA injection Alk3fl/fl; Alb-Cre mice presented milder anaemia (Hb 16.7 g/dl to 11.6 g/dl) compared to Alk3fl/fl control mice (Hb 14.9 g/dl to 8.6 g/dl). BA injection led to an intact inflammatory response in all groups of mice. In Alk3fl/fl; Alb-Cre mice, SMAD1/5/8 phosphorylation was reduced after BA as well as after infection with Staphylococcus aureus. The reduction of the SMAD1/5/8 signalling pathway due to hepatocyte-specific Alk3 deficiency partly suppressed the induction of STAT3 signalling. CONCLUSION: The results reveal in vivo, that 1) hepatocyte-specific Alk3 deficiency partly protects from AI, 2) the development of hypoferremia is partly dependent on ALK3, and 3) the ALK3/BMP/hepcidin axis may serve as a possible therapeutic target to attenuate AI.
Assuntos
Anemia/metabolismo , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/deficiência , Inflamação/metabolismo , Anemia/complicações , Animais , Brucella abortus , Brucelose/complicações , Eritropoese , Feminino , Hepatócitos/metabolismo , Inflamação/etiologia , Mediadores da Inflamação/metabolismo , Camundongos Endogâmicos C57BL , Fosforilação , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Proteína Smad1/metabolismo , Proteína Smad5/metabolismo , Proteína Smad8/metabolismoAssuntos
Proteína Morfogenética Óssea 2/metabolismo , Proteína Morfogenética Óssea 6/metabolismo , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/metabolismo , Citocinas/metabolismo , Hepcidinas/metabolismo , Proteínas Musculares/metabolismo , Animais , Células Cultivadas , Células HEK293 , Hepatócitos/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Multimerização ProteicaRESUMO
Nitric Oxide (NO) is administered as gas for inhalation to induce selective pulmonary vasodilation. It is a safe therapy, with few potential risks even if administered at high concentration. Inhaled NO gas is routinely used to increase systemic oxygenation in different disease conditions. The administration of high concentrations of NO also exerts a virucidal effect in vitro. Owing to its favorable pharmacodynamic and safety profiles, the familiarity in its use by critical care providers, and the potential for a direct virucidal effect, NO is clinically used in patients with coronavirus disease-2019 (COVID-19). Nevertheless, no device is currently available to easily administer inhaled NO at concentrations higher than 80 parts per million (ppm) at various inspired oxygen fractions, without the need for dedicated, heavy, and costly equipment. The development of a reliable, safe, inexpensive, lightweight, and ventilator-free solution is crucial, particularly for the early treatment of non-intubated patients outside of the intensive care unit (ICU) and in a limited-resource scenario. To overcome such a barrier, a simple system for the non-invasive NO gas administration up to 250 ppm was developed using standard consumables and a scavenging chamber. The method has been proven safe and reliable in delivering a specified NO concentration while limiting nitrogen dioxide levels. This paper aims to provide clinicians and researchers with the necessary information on how to assemble or adapt such a system for research purposes or clinical use in COVID-19 or other diseases in which NO administration might be beneficial.
Assuntos
Tratamento Farmacológico da COVID-19 , Óxido Nítrico/uso terapêutico , Ventiladores Mecânicos , Administração por Inalação , Cuidados Críticos , Humanos , Unidades de Terapia Intensiva , Óxido Nítrico/administração & dosagem , Dispositivos de Proteção Respiratória , SARS-CoV-2RESUMO
RATIONALE: Inhalation of nitric oxide (NO) exerts selective pulmonary vasodilation. Nitric oxide also has an antimicrobial effect on a broad spectrum of pathogenic viruses, bacteria and fungi. OBJECTIVES: The aim of this study was to investigate the effect of inhaled NO on bacterial burden and disease outcome in a murine model of Klebsiella pneumonia. METHODS: Mice were infected with Klebsiella pneumoniae and inhaled either air alone, air mixed with constant levels of NO (at 80, 160, or 200 parts per million (ppm)) or air intermittently mixed with high dose NO (300 ppm). Forty-eight hours after airway inoculation, the number of viable bacteria in lung, spleen and blood was determined. The extent of infiltration of the lungs by inflammatory cells and the level of myeloperoxidase activity in the lungs were measured. Atomic force microscopy was used to investigate a possible mechanism by which nitric oxide exerts a bactericidal effect. MEASUREMENTS AND MAIN RESULTS: Compared to control animals infected with K. pneumoniae and breathed air alone, intermittent breathing of NO (300 ppm) reduced viable bacterial counts in lung and spleen tissue. Inhaled NO reduced infection-induced lung inflammation and improved overall survival of mice. NO destroyed the cell wall of K. pneumoniae and killed multiple-drug resistant K. pneumoniae in-vitro. CONCLUSIONS: Intermittent administration of high dose NO may be an effective approach to the treatment of pneumonia caused by K. pneumoniae.
Assuntos
Klebsiella pneumoniae , Pneumonia , Animais , Antibacterianos , Modelos Animais de Doenças , Pulmão , Camundongos , Óxido NítricoRESUMO
The mammalian brain is highly vulnerable to oxygen deprivation, yet the mechanism underlying the brain's sensitivity to hypoxia is incompletely understood. Hypoxia induces accumulation of hydrogen sulfide, a gas that inhibits mitochondrial respiration. Here, we show that, in mice, rats, and naturally hypoxia-tolerant ground squirrels, the sensitivity of the brain to hypoxia is inversely related to the levels of sulfide:quinone oxidoreductase (SQOR) and the capacity to catabolize sulfide. Silencing SQOR increased the sensitivity of the brain to hypoxia, whereas neuron-specific SQOR expression prevented hypoxia-induced sulfide accumulation, bioenergetic failure, and ischemic brain injury. Excluding SQOR from mitochondria increased sensitivity to hypoxia not only in the brain but also in heart and liver. Pharmacological scavenging of sulfide maintained mitochondrial respiration in hypoxic neurons and made mice resistant to hypoxia. These results illuminate the critical role of sulfide catabolism in energy homeostasis during hypoxia and identify a therapeutic target for ischemic brain injury.
Assuntos
Lesões Encefálicas/metabolismo , Encéfalo/metabolismo , Sulfeto de Hidrogênio/metabolismo , Quinona Redutases/metabolismo , Animais , Encéfalo/patologia , Lesões Encefálicas/genética , Células Cultivadas , Feminino , Hipóxia , Masculino , Potencial da Membrana Mitocondrial , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Knockout , Mitocôndrias/metabolismo , NAD/metabolismo , Quinona Redutases/genética , Interferência de RNA , Ratos Sprague-DawleyRESUMO
Non-alcoholic fatty liver disease (NAFLD) affects over 30% of adults in the United States. Bone morphogenetic protein (BMP) signaling is known to contribute to hepatic fibrosis, but the role of BMP signaling in the development of NAFLD is unclear. In this study, treatment with either of two BMP inhibitors reduced hepatic triglyceride content in diabetic (db/db) mice. BMP inhibitor-induced decrease in hepatic triglyceride levels was associated with decreased mRNA encoding Dgat2, an enzyme integral to triglyceride synthesis. Treatment of hepatoma cells with BMP2 induced DGAT2 expression and activity via intracellular SMAD signaling. In humans we identified a rare missense single nucleotide polymorphism in the BMP type 1 receptor ALK6 (rs34970181;R371Q) associated with a 2.1-fold increase in the prevalence of NAFLD. In vitro analyses revealed R371Q:ALK6 is a previously unknown constitutively active receptor. These data show that BMP signaling is an important determinant of NAFLD in a murine model and is associated with NAFLD in humans.
Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Hepatopatia Gordurosa não Alcoólica/metabolismo , Hepatopatia Gordurosa não Alcoólica/patologia , Transdução de Sinais , Animais , Biomarcadores/sangue , Linhagem Celular Tumoral , Diacilglicerol O-Aciltransferase/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Metabolismo dos Lipídeos/efeitos dos fármacos , Camundongos , Hepatopatia Gordurosa não Alcoólica/sangue , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Pirazóis/farmacologia , Pirazóis/uso terapêutico , Pirimidinas/farmacologia , Pirimidinas/uso terapêutico , Transdução de Sinais/efeitos dos fármacos , Proteínas Smad/metabolismoRESUMO
Hereditary hemochromatosis is the most common autosomal recessive genetic disorder of the iron metabolism. Iron accumulation in various organs, especially in liver and pancreas leads to diseases and may cause organ failure. In this study, methods for elemental bioimaging by means of quantitative micro X-ray fluorescence analysis (µXRF) and laser ablation-inductively coupled plasma-triple quadrupole mass spectrometry (LA-ICP-TQMS) were developed and applied to investigate the pathophysiological development of iron accumulation in murine tissue based on animals with an iron-overload phenotype caused by a hepatocyte-specific genetic mutation. The use of an external calibration with matrix-matched gelatin standards enables the quantification of iron by means of µXRF without the typically used fundamental parameters method or Monte Carlo simulation, which becomes more imprecise when analyzing thin tissue sections. A fast, non-destructive screening of the iron concentration and distribution with a spatial resolution of 25 µm in liver samples of iron-overload mice was developed. For improved limits of detection and higher spatial resolution down to 4 µm, LA-ICP-TQMS was used with oxygen as reaction gas. By monitoring the mass shift of 56Fe to 56Fe16O, a limit of detection of 0.5 µg/g was obtained. With this method, liver and pancreas samples of iron-overload mice as well as control mice were successfully analyzed. The high spatial resolution enabled the analysis of the iron distribution in different liver lobules. Compared to the established Prussian blue staining, both developed methods proved to be superior due to the possibility of direct iron quantification in the tissues.
Assuntos
Modelos Animais de Doenças , Fluorescência , Hemocromatose/diagnóstico por imagem , Ferro/análise , Animais , Hemocromatose/metabolismo , Ferro/metabolismo , Sobrecarga de Ferro/metabolismo , Terapia a Laser , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Raios XRESUMO
Mutations in HFE, the most common cause of hereditary hemochromatosis, lead to iron overload. The iron overload is characterized by increased iron uptake due to lower levels of the hepatic, iron regulatory hormone hepcidin. HFE was cloned 21 years ago, but the signaling pathway is still unknown. Because bone morphogenetic protein (BMP) signaling is impaired in patients with hereditary hemochromatosis, and the interaction of HFE and the BMP type I receptor ALK3 was suggested in vitro, in vivo experiments were performed. In vivo, hepatocyte-specific Alk3-deficient and control mice were injected with either AAV2/8-Hfe-Flag or PBS. HFE overexpression in control mice results in increased hepatic hepcidin levels, p-Smad1/5 levels, and iron deficiency anemia, whereas overexpression of HFE in hepatocyte-specific Alk3-deficient mice results in no change in hepcidin, p-Smad1/5 levels, or blood parameters. These results indicate that HFE signals predominantly via ALK3 to induce hepcidin in vivo.
RESUMO
Preoperative anemia occurs in about one third of patients who undergo elective surgery and is associated with an impaired outcome. Therefore, screening of preoperative anemia was established in the context of a multidisciplinary Patient Blood Management (PBM) program at the University Hospital of Muenster, Germany. Anemic patients without contraindications were treated with intravenous (IV) iron (ferric carboxymaltose) to increase their hemoglobin (Hgb) levels and hence to treat anemia prior to surgery. Interestingly, we detected a large variability in the response of Hgb levels after IV iron administration. Systemic iron homeostasis is mainly regulated by the hepatic hormone hepcidin, which regulates the cell surface expression of the sole known iron exporter ferroportin. The objective of this retrospective pilot study was to analyze the potential of hepcidin to predict the response of anemic patients to preoperative IV iron treatment measured as increase in Hgb. Serum samples of non-anemic (n = 48), untreated anemic (n = 64) and anemic patients treated with IV iron (n = 79), in total 191 patients, were collected between October 2014 until June 2016. Serum hepcidin levels were determined and data were analyzed retrospectively. The analysis revealed at first a correlation between serum hepcidin levels and the parameters of the iron status. Second, patients treated with IV iron showed a noticeably higher increase in their delta Hgb level between PBM consultation and surgery (0.45g/dl [0.05, 1.05] compared to patients without IV iron (0.1g/dl [-0.48, 0.73], *p = 0.03). Patients were then grouped into 'non-responders', defined as delta Hgb <0.6g/dl and 'responders', with delta Hgb ≥0.6g/dl between the day of IV iron treatment and the day of surgery. Within normal ranges and clinically unapparent, a statistically noticeable difference between responders and non-responders was found for CRP and leukocytes. Serum hepcidin levels were higher in the group of non-responders (10.6ng/ml [3.93, 34.77]) compared to responders (2.1ng/ml [0.25, 7.97], *p = 0.04). To conclude, the data of this retrospective pilot study indicate that hepcidin might be a promising biomarker to predict a patient`s responsiveness to IV iron in preoperative anemia treatment. Prospective studies have to investigate serum hepcidin levels as a biomarker to guide physician`s decision on IV iron substitution.
Assuntos
Anemia/terapia , Hepcidinas/metabolismo , Ferro/metabolismo , Administração Intravenosa , Adulto , Idoso , Idoso de 80 Anos ou mais , Anemia/sangue , Anemia Ferropriva/sangue , Anemia Ferropriva/terapia , Biomarcadores Farmacológicos/sangue , Feminino , Alemanha , Hemoglobinas/metabolismo , Hepcidinas/análise , Hepcidinas/sangue , Humanos , Ferro/administração & dosagem , Ferro/sangue , Masculino , Pessoa de Meia-Idade , Projetos Piloto , Cuidados Pré-Operatórios/métodos , Estudos Prospectivos , Estudos RetrospectivosRESUMO
The bone morphogenetic protein (BMP) type I receptors ALK2 and ALK3 are essential for expression of hepcidin, a key iron regulatory hormone. In mice, hepatocyte-specific Alk2 deficiency leads to moderate iron overload with periportal liver iron accumulation, while hepatocyte-specific Alk3 deficiency leads to severe iron overload with centrilobular liver iron accumulation and a more marked reduction of basal hepcidin levels. The objective of this study was to investigate whether the two receptors have additive roles in hepcidin regulation. Iron overload in mice with hepatocyte-specific Alk2 and Alk3 (Alk2/3) deficiency was characterized and compared to hepatocyte-specific Alk3 deficient mice. Co-immunoprecipitation studies were performed to detect the formation of ALK2 and ALK3 homodimer and heterodimer complexes in vitro in the presence and absence of ligands. The iron overload phenotype of hepatocyte-specific Alk2/3-deficient mice was more severe than that of hepatocyte-specific Alk3-deficient mice. In vitro co-immunoprecipitation studies in Huh7 cells showed that ALK3 can homodimerize in absence of BMP2 or BMP6. In contrast, ALK2 did not homodimerize in either the presence or absence of BMP ligands. However, ALK2 did form heterodimers with ALK3 in the presence of BMP2 or BMP6. ALK3-ALK3 and ALK2-ALK3 receptor complexes induced hepcidin expression in Huh7 cells. Our data indicate that: (I) ALK2 and ALK3 have additive functions in vivo, as Alk2/3 deficiency leads to a greater degree of iron overload than Alk3 deficiency; (II) ALK3, but not ALK2, undergoes ligand-independent homodimerization; (III) the formation of ALK2-ALK3 heterodimers is ligand-dependent and (IV) both receptor complexes functionally induce hepcidin expression in vitro.
Assuntos
Receptores de Ativinas Tipo I/genética , Proteína Morfogenética Óssea 2/genética , Proteína Morfogenética Óssea 6/genética , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/genética , Hepcidinas/genética , Sobrecarga de Ferro/genética , Ferro/metabolismo , Receptores de Ativinas Tipo I/deficiência , Animais , Proteína Morfogenética Óssea 2/metabolismo , Proteína Morfogenética Óssea 6/metabolismo , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/deficiência , Linhagem Celular Tumoral , Feminino , Regulação da Expressão Gênica , Hepatócitos/metabolismo , Hepatócitos/patologia , Hepcidinas/metabolismo , Humanos , Sobrecarga de Ferro/metabolismo , Sobrecarga de Ferro/patologia , Fígado/metabolismo , Fígado/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , Ligação Proteica , Multimerização Proteica , Índice de Gravidade de Doença , Transdução de SinaisRESUMO
Intravenous iron supplementation is an effective therapy in iron deficiency anemia (IDA), but controversial in anemia of inflammation (AI). Unbound iron can be used by bacteria and viruses for their replication and enhance the inflammatory response. Nowadays available high molecular weight iron complexes for intravenous iron substitution, such as ferric carboxymaltose, might be useful in AI, as these pharmaceuticals deliver low doses of free iron over a prolonged period of time. We tested the effects of intravenous iron carboxymaltose in murine AI: Wild-type mice were exposed to the heat-killed Brucella abortus (BA) model and treated with or without high molecular weight intravenous iron. 4h after BA injection followed by 2h after intravenous iron treatment, inflammatory cytokines were upregulated by BA, but not enhanced by iron treatment. In long term experiments, mice were fed a regular or an iron deficient diet and then treated with intravenous iron or saline 14 days after BA injection. Iron treatment in mice with BA-induced AI was effective 24h after iron administration. In contrast, mice with IDA (on iron deficiency diet) prior to BA-IA required 7d to recover from AI. In these experiments, inflammatory markers were not further induced in iron-treated compared to vehicle-treated BA-injected mice. These results demonstrate that intravenous iron supplementation effectively treated the murine BA-induced AI without further enhancement of the inflammatory response. Studies in humans have to reveal treatment options for AI in patients.