RESUMEN
In clinical practice, extracorporeal circulation (ECC) is associated with coagulopathy and inflammation, eventually leading to organ injuries without preventive systemic pharmacological treatment. Relevant models are needed to reproduce the pathophysiology observed in humans and preclinical tests. Rodent models are less expensive than large models but require adaptations and validated comparisons to clinics. This study aimed to develop a rat ECC model and to establish its clinical relevance. One hour of veno-arterial ECC or a sham procedure were achieved on mechanically ventilated rats after cannulations with a mean arterial pressure objective > 60 mmHg. Five hours post-surgery, the rats' behavior, plasmatic/blood biomarkers, and hemodynamics were measured. Blood biomarkers and transcriptomic changes were compared in 41 patients undergoing on-pump cardiac surgery. Five hours post-ECC, the rats presented hypotension, hyperlactatemia, and behavioral alterations. The same patterns of marker measurements (Lactate dehydrogenase, Creatinine kinase, ASAT, ALAT, and Troponin T) were observed in both rats and human patients. Transcriptome analyses showed similarity in both humans and rats in the biological processes involved in the ECC response. This new ECC rat model seems to resemble both ECC clinical procedures and the associated pathophysiology, but with early organ injury corresponding to a severe phenotype. Although the mechanisms at stake in the post-ECC pathophysiology of rats or humans need to be described, this new rat model appears to be a relevant and costless preclinical model of human ECC.
Asunto(s)
Circulación Extracorporea , Insuficiencia Multiorgánica , Ratas , Humanos , Animales , Circulación Extracorporea/métodos , BiomarcadoresRESUMEN
Strong evidence suggests that endoplasmic reticulum stress plays a critical role in the pathogenesis of amyotrophic lateral sclerosis (ALS) through altered regulation of proteostasis. Robust preclinical findings demonstrated that guanabenz selectively inhibits endoplasmic reticulum stress-induced eIF2α-phosphatase, allowing misfolded protein clearance, reduces neuronal death and prolongs survival in in vitro and in vivo models. However, its safety and efficacy in patients with ALS are unknown. To address these issues, we conducted a multicentre, randomized, double-blind trial with a futility design. Patients with ALS who had displayed an onset of symptoms within the previous 18 months were randomly assigned in a 1:1:1:1 ratio to receive 64 mg, 32 mg or 16 mg of guanabenz or placebo daily for 6 months as an add-on therapy to riluzole. The purpose of the placebo group blinding was to determine safety but not efficacy. The primary outcome was the proportion of patients progressing to higher stages of disease within 6 months as measured using the ALS Milano-Torino staging system, compared with a historical cohort of 200 patients with ALS. The secondary outcomes were the rate of decline in the total revised ALS functional rating scale score, slow vital capacity change, time to death, tracheotomy or permanent ventilation and serum light neurofilament level at 6 months. The primary assessment of efficacy was performed using intention-to-treat analysis. The treatment arms using 64 mg and 32 mg guanabenz, both alone and combined, reached the primary hypothesis of non-futility, with the proportions of patients who progressed to higher stages of disease at 6 months being significantly lower than that expected under the hypothesis of non-futility and a significantly lower difference in the median rate of change in the total revised ALS functional rating scale score. This effect was driven by patients with bulbar onset, none of whom (0/18) progressed to a higher stage of disease at 6 months compared with those on 16 mg guanabenz (4/8; 50%), the historical cohort alone (21/49; 43%; P = 0.001) or plus placebo (25/60; 42%; P = 0.001). The proportion of patients who experienced at least one adverse event was higher in any guanabenz arm than in the placebo arm, with higher dosing arms having a significantly higher proportion of drug-related side effects and the 64 mg arm a significantly higher drop-out rate. The number of serious adverse events did not significantly differ between the guanabenz arms and the placebo. Our findings indicate that a larger trial with a molecule targeting the unfolded protein response pathway without the alpha-2 adrenergic related side-effect profile of guanabenz is warranted.
Asunto(s)
Agonistas de Receptores Adrenérgicos alfa 2/uso terapéutico , Esclerosis Amiotrófica Lateral/tratamiento farmacológico , Esclerosis Amiotrófica Lateral/metabolismo , Guanabenzo/uso terapéutico , Respuesta de Proteína Desplegada/fisiología , Agonistas de Receptores Adrenérgicos alfa 2/farmacología , Anciano , Esclerosis Amiotrófica Lateral/diagnóstico , Método Doble Ciego , Femenino , Guanabenzo/farmacología , Humanos , Masculino , Persona de Mediana Edad , Respuesta de Proteína Desplegada/efectos de los fármacosRESUMEN
Oculopharyngeal muscular dystrophy (OPMD) is a rare late onset genetic disease leading to ptosis, dysphagia and proximal limb muscles at later stages. A short abnormal (GCN) triplet expansion in the polyA-binding protein nuclear 1 (PABPN1) gene leads to PABPN1-containing aggregates in the muscles of OPMD patients. Here we demonstrate that treating mice with guanabenz acetate (GA), an FDA-approved antihypertensive drug, reduces the size and number of nuclear aggregates, improves muscle force, protects myofibers from the pathology-derived turnover and decreases fibrosis. GA targets various cell processes, including the unfolded protein response (UPR), which acts to attenuate endoplasmic reticulum (ER) stress. We demonstrate that GA increases both the phosphorylation of the eukaryotic translation initiation factor 2α subunit and the splicing of Xbp1, key components of the UPR. Altogether these data show that modulation of protein folding regulation is beneficial for OPMD and promote the further development of GA or its derivatives for treatment of OPMD in humans. Furthermore, they support the recent evidences that treating ER stress could be therapeutically relevant in other more common proteinopathies.
Asunto(s)
Guanabenzo/farmacología , Distrofia Muscular Oculofaríngea/tratamiento farmacológico , Proteína I de Unión a Poli(A)/genética , Proteína 1 de Unión a la X-Box/genética , Empalme Alternativo/efectos de los fármacos , Empalme Alternativo/genética , Animales , Modelos Animales de Enfermedad , Estrés del Retículo Endoplásmico/efectos de los fármacos , Fibrosis/tratamiento farmacológico , Fibrosis/genética , Fibrosis/patología , Humanos , Ratones , Distrofia Muscular Oculofaríngea/genética , Distrofia Muscular Oculofaríngea/patología , Fosforilación/efectos de los fármacos , Agregado de Proteínas/efectos de los fármacos , Agregado de Proteínas/genética , Pliegue de Proteína , Respuesta de Proteína Desplegada/efectos de los fármacosRESUMEN
Oculopharyngeal muscular dystrophy (OPMD) is an autosomal dominant disease characterized by the progressive degeneration of specific muscles. OPMD is due to a mutation in the gene encoding poly(A) binding protein nuclear 1 (PABPN1) leading to a stretch of 11 to 18 alanines at N-terminus of the protein, instead of 10 alanines in the normal protein. This alanine tract extension induces the misfolding and aggregation of PABPN1 in muscle nuclei. Here, using Drosophila OPMD models, we show that the unfolded protein response (UPR) is activated in OPMD upon endoplasmic reticulum stress. Mutations in components of the PERK branch of the UPR reduce muscle degeneration and PABPN1 aggregation characteristic of the disease. We show that oral treatment of OPMD flies with Icerguastat (previously IFB-088), a Guanabenz acetate derivative that shows lower side effects, also decreases muscle degeneration and PABPN1 aggregation. Furthermore, the positive effect of Icerguastat depends on GADD34, a key component of the phosphatase complex in the PERK branch of the UPR. This study reveals a major contribution of the ER stress in OPMD pathogenesis and provides a proof-of-concept for Icerguastat interest in future pharmacological treatments of OPMD.
Asunto(s)
Distrofia Muscular Oculofaríngea , Animales , Distrofia Muscular Oculofaríngea/genética , Distrofia Muscular Oculofaríngea/metabolismo , Distrofia Muscular Oculofaríngea/patología , Músculo Esquelético/metabolismo , Respuesta de Proteína Desplegada , Núcleo Celular/metabolismo , Estrés del Retículo Endoplásmico , DrosophilaRESUMEN
Venesection has been proposed as a treatment for hepatic iron overload in a number of chronic liver disorders that are not primarily linked to mutations in iron metabolism genes. Our aim was to analyse the impact of venesection on iron mobilisation in a mouse model of secondary iron overload. C57Bl/6 mice were given oral iron supplementation with or without phlebotomy between day 0 (D0) and D22, and the results were compared to controls without iron overload. We studied serum and tissue iron parameters, mRNA levels of hepcidin1, ferroportin, and transferrin receptor 1, and protein levels of ferroportin in the liver and spleen. On D0, animals with iron overload displayed elevations in iron parameters and hepatic hepcidin1 mRNA. By D22, in the absence of phlebotomies, splenic iron had increased, but transferrin saturation had decreased. This was associated with high hepatic hepcidin1 mRNA, suggesting that iron bioavailability decreased due to splenic iron sequestration through ferroportin protein downregulation. After 22days with phlebotomy treatments, control mice displayed splenic iron mobilisation that compensated for the iron lost due to phlebotomy. In contrast, phlebotomy treatments in mice with iron overload caused anaemia due to inadequate iron mobilisation. In conclusion, our model of secondary iron overload led to decreased plasma iron associated with an increase in hepcidin expression and subsequent restriction of iron export from the spleen. Our data support the importance of managing hepcidin levels before starting venesection therapy in patients with secondary iron overload that are eligible for phlebotomy.
Asunto(s)
Antibacterianos/farmacología , Péptidos Catiónicos Antimicrobianos/farmacología , Sobrecarga de Hierro/patología , Sobrecarga de Hierro/terapia , Hierro/farmacocinética , Bazo/metabolismo , Animales , Western Blotting , Proteínas de Transporte de Catión/genética , Proteínas de Transporte de Catión/metabolismo , Modelos Animales de Enfermedad , Hepcidinas , Hierro/sangre , Sobrecarga de Hierro/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Flebotomía , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Bazo/patología , Distribución TisularRESUMEN
PURPOSE: To quantify hepatic and splenic iron load, which is a critical issue for iron overload disease diagnosis. MRI is useful to noninvasively determine liver iron concentration, but not proven to be adequate for robust evaluation of splenic iron load. We evaluated the usefulness of MRI-derived parameters to determine splenic iron concentration in mice. MATERIALS AND METHODS: A mouse model of experimental iron load was used. Multi-echo spin-echo images of liver and spleen were acquired at 4.7 Tesla. The parameters were tested at all echoes with and without an external reference. Splenic and hepatic iron concentrations were determined using biochemical assay as the gold standard. RESULTS: Our results show that (i) use of an internal or external reference is essential; (ii) optimal echo times were TE = 19.5 ms and TE = 32.5 ms for the liver and spleen, respectively; (iii) in the liver, the relationship between biochemical and MRI iron concentration determinations is logarithmic; (iv) in the spleen, the best relationship is an inverse function. CONCLUSION: A single spin-echo sequence allows robust estimation of hepatic and splenic iron content. Parameters classically used for hepatic iron concentration cannot be applied to splenic iron determination, which requires both the specific sequence and the adapted fitting function.
Asunto(s)
Sobrecarga de Hierro/diagnóstico , Hierro/metabolismo , Bazo/metabolismo , Animales , Modelos Animales de Enfermedad , Hierro/análisis , Hígado/química , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Músculo Esquelético/química , Músculo Esquelético/metabolismo , Distribución Aleatoria , Sensibilidad y Especificidad , Bazo/química , Estadísticas no ParamétricasRESUMEN
Thalassemia associates anemia and iron overload, two opposite stimuli regulating hepcidin gene expression. We characterized hepatic hepcidin expression in 10 thalassemia major and 13 thalassemia intermedia patients. Hepcidin mRNA levels were decreased in the thalassemia intermedia group which presented both lower hemoglobin and higher plasma soluble transferrin receptor levels. There was no relationship between hepcidin mRNA levels and those of genes controlling iron metabolism, including HFE, hemojuvelin, transferrin receptor-2 and ferroportin. These results underline the role of erythropoietic activity on hepcidin decrease in thalassemic patients and suggest that mRNA modulations of other studied genes do not have a significant impact.
Asunto(s)
Anemia/complicaciones , Péptidos Catiónicos Antimicrobianos/biosíntesis , Regulación de la Expresión Génica , Hierro/metabolismo , Hígado/metabolismo , Talasemia beta/complicaciones , Adulto , Anciano , Proteínas de Transporte de Catión/biosíntesis , Femenino , Proteínas Ligadas a GPI , Proteína de la Hemocromatosis , Hepcidinas , Antígenos de Histocompatibilidad Clase I/biosíntesis , Humanos , Masculino , Proteínas de la Membrana/biosíntesis , Persona de Mediana Edad , Receptores de Transferrina/biosíntesisRESUMEN
Disruption of the insulin-PI3K-Akt signalling pathway in kidney podocytes causes endoplasmic reticulum (ER) stress, leading to podocyte apoptosis and proteinuria in diabetic nephropathy. We hypothesised that by improving insulin sensitivity we could protect podocytes from ER stress. Here we use established activating transcription factor 6 (ATF6)- and ER stress element (ERSE)-luciferase assays alongside a novel high throughput imaging-based C/EBP homologous protein (CHOP) assay to examine three models of improved insulin sensitivity. We find that by improving insulin sensitivity at the level of the insulin receptor (IR), either by IR over-expression or by knocking down the negative regulator of IR activity, protein tyrosine-phosphatase 1B (PTP1B), podocytes are protected from ER stress caused by fatty acids or diabetic media containing high glucose, high insulin and inflammatory cytokines TNFα and IL-6. However, contrary to this, knockdown of the negative regulator of PI3K-Akt signalling, phosphatase and tensin homolog deleted from chromosome 10 (PTEN), sensitizes podocytes to ER stress and apoptosis, despite increasing Akt phosphorylation. This indicates that protection from ER stress is conferred through not just the PI3K-Akt pathway, and indeed we find that inhibiting the MEK/ERK signalling pathway rescues PTEN knockdown podocytes from ER stress.
Asunto(s)
Estrés del Retículo Endoplásmico , Fosfatidilinositol 3-Quinasas/metabolismo , Podocitos/fisiología , Receptor de Insulina/metabolismo , Transducción de Señal , Animales , Apoptosis , Células Cultivadas , Insulina/metabolismo , Ratones , Fosfohidrolasa PTEN/metabolismo , Fosforilación , Podocitos/citología , Proteína Tirosina Fosfatasa no Receptora Tipo 1/metabolismoRESUMEN
Normal iron metabolism is highly regulated and takes a crucial role in the maintenance of cell functions. The plasmatic iron bioavailability control is a key step of this metabolism which involves numerous proteins implicated at various levels, including the digestive iron absorption by enterocytes, and iron release from macrophages. These two phenomenons are modulated in a coordonated fashion by the plasmatic level of hepcidin, a peptide mainly synthetized by the liver, secreted in plasma and modulating the expression of ferroportin, the cellular exporter of iron, and thus the iron egress. Numerous factors are able to modulate the hepcidin expression, including iron status, erythropoietic activity, inflammation and hepatic status which are already identified. Abnormalities occurring in the regulation of hepcidin expression may favour the development of iron metabolism disturbance, including systemic iron overload or relative iron deficiency. The use of hepcidin for diagnostic purpose or as a therapeutic target remains to be determined.
Asunto(s)
Hierro/metabolismo , Péptidos Catiónicos Antimicrobianos/genética , Eritrocitos/metabolismo , Hepcidinas , Humanos , Absorción Intestinal , Hierro/sangre , Modelos Biológicos , Valores de ReferenciaRESUMEN
During the inflammatory process, hepcidin overexpression favours the development of anaemia of chronic diseases which represents the second most common form of anaemia worldwide. The identification of therapeutic agents decreasing hepcidin expression is therefore an important goal. The aim of this study was to target the STAT3 signalling involved in the development of increased hepcidin expression related to chronic inflammation. In a co-culture model associating mouse hepatocytes and rat liver epithelial cells, the mRNA levels of hepcidin1, albumin, aldolase B, Cyp3a4, Stat3, Smad4 and iron regulatory genes were measured by real-time PCR. STAT3 and phosphorylated SMAD1/5/8 proteins were analysed by Western blot. At variance of hepatocyte pure culture, co-culture provided high levels of hepcidin1 mRNA, reaching 400% of the freshly isolated hepatocyte values after 6 days of culture. Hepcidin expression was associated with the maintenance of hepatocyte phenotype, STAT3 phosphorylation and functional BMP/SMAD pathway. Stat3 siRNAs inhibited the hepcidin1 mRNA expression. STAT3 inhibitors, including curcumin, AG490 and a peptide (PpYLKTK), reduced hepcidin1 mRNA expression even when cells were additionally exposed to IL-6. Hepcidin1 mRNA was expressed at high levels by hepatocytes in the co-culture model, and STAT3 pathway activation was controlled through STAT3 inhibitors. Such inhibitors could be useful to prevent anaemia related to hepcidin overexpression during chronic inflammation.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/metabolismo , Regulación de la Expresión Génica , Hepatocitos/metabolismo , Factor de Transcripción STAT3/antagonistas & inhibidores , Factor de Transcripción STAT3/metabolismo , Animales , Péptidos Catiónicos Antimicrobianos/genética , Células Cultivadas , Técnicas de Cocultivo , Regulación de la Expresión Génica/efectos de los fármacos , Hepatocitos/citología , Hepcidinas , Masculino , Ratones , Ratones Endogámicos C57BL , Fosforilación , Ratas , Factor de Transcripción STAT3/genéticaRESUMEN
Iron absorption, distribution, use, and storage are thought to be tightly regulated since altered iron stores may lead to cellular damage and disease. HFE, the hereditary hemochromatosis gene product, is expressed in the crypts of the duodenum, but the molecular mechanism by which it contributes to the inhibition of iron absorption is still unknown. In this study we aimed to identify transcriptional profiles in the duodenal epithelium of Hfe(-/-) mice. We used dedicated microarrays to compare gene expression among the duodenum of Hfe(-/-) mice, induced iron overload mice, and control mice. We found 151 differentially expressed genes and unknown sequences between Hfe(-/-) mice and normal littermates. Gene profiling revealed a gene subset more specific for Hfe inactivation. The functional annotation of upregulated genes highlighted that mucus production and cell maintenance may account for the influence of Hfe on epithelium integrity and luminal iron uptake.
Asunto(s)
Duodeno/metabolismo , Hemocromatosis/genética , Antígenos de Histocompatibilidad Clase I/genética , Mucosa Intestinal/metabolismo , Hierro/metabolismo , Proteínas de la Membrana/genética , Animales , Perfilación de la Expresión Génica , Proteína de la Hemocromatosis , Hierro/sangre , Sobrecarga de Hierro/metabolismo , Hígado/metabolismo , Ratones , Ratones Noqueados , Análisis de Secuencia por Matrices de OligonucleótidosRESUMEN
Hereditary hemochromatosis (HH), a common autosomal recessive disorder due to a mutation in HFE, which encodes an atypical MHC class I glycoprotein, is characterized by excessive absorption of dietary iron. Little is known however of the apparently complex pathophysiology of HFE involvement in the process of iron influx. Here, in order to tackle the issue in vivo, we decided to target HFE expression exclusively to the relevant tissue, intestinal epithelium. This was achieved by putting HFE under transcriptional control of the rat fatty acid binding protein (Fabpi) promoter. Quite unexpectedly, Fabpi-HFE mice had significantly elevated serum transferrin saturation levels in comparison to those of normal littermates. By a careful, layer by layer analysis of transgene expression along the crypt-villus axis, we were able to affirm that the ectopic expression of transgenic HFE in the differentiated villi enterocytes was responsible for ferric hyperabsorption, a phenomenon exacerbated in the absence of endogenous HFE expression, which we assessed by crossing the transgene onto an HFE(-/-) (knockout) background. This forced dichotomy between the absence of HFE in the crypt and expression in the villi provides experimental support that HFE functions as a "gatekeeper," regulating the cross-talk between the crypt and villi enterocytes and thereby modulating the avidity of mature enterocytes for dietary iron.