RESUMEN
Acetaminophen (APAP)-induced liver injury is one of the most prevalent causes of acute liver failure (ALF). We assessed the role of the bone morphogenetic protein (BMP) type I receptors ALK2 and ALK3 in APAP-induced hepatotoxicity. The molecular mechanisms that regulate the balance between cell death and survival and the response to oxidative stress induced by APAP was assessed in cultured human hepatocyte-derived (Huh7) cells treated with pharmacological inhibitors of ALK receptors and with modulated expression of ALK2 or ALK3 by lentiviral infection, and in a mouse model of APAP-induced hepatotoxicity. Inhibition of ALK3 signalling with the pharmacological inhibitor DMH2, or by silencing of ALK3, showed a decreased cell death both by necrosis and apoptosis after APAP treatment. Also, upon APAP challenge, ROS generation was ameliorated and, thus, ROS-mediated JNK and P38 MAPK phosphorylation was reduced in ALK3-inhibited cells compared to control cells. These results were also observed in an experimental model of APAP-induced ALF in which post-treatment with DMH2 after APAP administration significantly reduced liver tissue damage, apoptosis and oxidative stress. This study shows the protective effect of ALK3 receptor inhibition against APAP-induced hepatotoxicity. Furthermore, findings obtained from the animal model suggest that BMP signalling might be a new pharmacological target for the treatment of ALF.
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Enfermedad Hepática Crónica Inducida por Sustancias y Drogas , Enfermedad Hepática Inducida por Sustancias y Drogas , Morfolinas , Ratones , Animales , Humanos , Acetaminofén/efectos adversos , Especies Reactivas de Oxígeno/metabolismo , Enfermedad Hepática Crónica Inducida por Sustancias y Drogas/metabolismo , Hígado/metabolismo , Hepatocitos/metabolismo , Estrés Oxidativo , Enfermedad Hepática Inducida por Sustancias y Drogas/genética , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Ratones Endogámicos C57BLRESUMEN
BACKGROUND AND AIMS: The molecular mechanisms driving non-alcoholic fatty liver disease (NAFLD) are poorly understood; however, microRNAs might play a key role in these processes. We hypothesize that let-7d-5p could contribute to the pathophysiology of NAFLD and serve as a potential diagnostic biomarker. METHODS: We evaluated let-7d-5p levels and its targets in liver biopsies from a cross-sectional study including patients with NAFLD and healthy donors, and from a mouse model of NAFLD. Moreover, the induction of let-7d-5p expression by fatty acids was evaluated in vitro. Further, we overexpressed let-7d-5p in vitro to corroborate the results observed in vivo. Circulating let-7d-5p and its potential as a NAFLD biomarker was determined in isolated extracellular vesicles from human plasma by RT-qPCR. RESULTS: Our results demonstrate that hepatic let-7d-5p was significantly up-regulated in patients with steatosis, and this increase correlated with obesity and a decreased expression of AKT serine/threonine kinase (AKT), insulin-like growth factor 1 (IGF1), IGF-I receptor (IGF1R) and insulin receptor (INSR). These alterations were corroborated in a NAFLD mouse model. In vitro, fatty acids increased let-7d-5p expression, and its overexpression decreased AKT, IGF-IR and IR protein expression. Furthermore, let-7d-5p hindered AKT phosphorylation in vitro after insulin stimulation. Finally, circulating let-7d-5p significantly decreased in steatosis patients and receiver operating characteristic (ROC) analyses confirmed its utility as a diagnostic biomarker. CONCLUSIONS: Our results highlight the emerging role of let-7d-5p as a potential therapeutic target for NAFLD since its overexpression impairs hepatic insulin signalling, and also, as a novel non-invasive biomarker for NAFLD diagnosis.
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Resistencia a la Insulina , MicroARNs , Enfermedad del Hígado Graso no Alcohólico , Animales , Humanos , Ratones , Biomarcadores , Estudios Transversales , Ácidos Grasos , Insulina , MicroARNs/genética , MicroARNs/metabolismo , Enfermedad del Hígado Graso no Alcohólico/patología , Proteínas Proto-Oncogénicas c-aktRESUMEN
BACKGROUND & AIMS: Advanced hepatic fibrosis is the main risk factor of liver-related morbidity and mortality in patients with chronic liver disease. In this study, we assessed the potential role of bone morphogenetic protein 8A (BMP8A) as a novel target involved in liver fibrosis progression. METHODS: Histological assessment and BMP8A expression were determined in different murine models of hepatic fibrosis. Furthermore, serum BMP8A was measured in mice with bile duct ligation (BDL), in 36 subjects with histologically normal liver (NL) and in 85 patients with biopsy-proven non-alcoholic steatohepatitis (NASH): 52 with non- or mild fibrosis (F0-F2) and 33 with advanced fibrosis (F3-F4). BMP8A expression and secretion was also determined in cultured human hepatocyte-derived (Huh7) and human hepatic stellate (LX2) cells stimulated with transforming growth factor êµ (TGFêµ). RESULTS: Bmp8a mRNA levels were significantly upregulated in livers from fibrotic mice compared to control animals. Notably, serum BMP8A levels were also elevated in BDL mice. In addition, in vitro experiments showed increased expression and secretion to the culture supernatant of BMP8A in both Huh7 and LX2 cells treated with TGFêµ. Noteworthy, we found that serum BMP8A levels were significantly higher in NASH patients with advanced fibrosis than in those with non- or mild fibrosis. In fact, the AUROC of circulating BMP8A concentrations to identify patients with advanced fibrosis (F3-F4) was 0.74 (pË0.0001). Moreover, we developed an algorithm based on serum BMP8A levels that showed an AUROC of 0.818 (pË0.0001) to predict advanced fibrosis in NASH patients. CONCLUSION: This study provides experimental and clinical evidence indicating that BMP8A is a novel molecular target linked to liver fibrosis and introduces an efficient algorithm based on serum BMP8A levels to screen patients at risk for advanced hepatic fibrosis.
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La infección crónica por el virus de la hepatitis C (VHC) está asociada con la resistencia a la insulina y la diabetes tipo 2. El objetivo general de este estudio fue evaluar los efectos del sofosbuvir sobre la resistencia a la insulina inducida por el VHC. Para ello, se realizó un estudio clínico que incluyó 42 pacientes con VHC resistentes a la insulina, que fueron tratados con diferentes regímenes antivirales basados en sofosbuvir. Asimismo, se utilizó una línea de hepatocitos humanos que expresan un replicón del VHC de manera estable para determinar los mecanismos moleculares implicados en la acción de la insulina regulada por sofosbuvir. Todos los pacientes alcanzaron una respuesta virológica sostenida después del tratamiento con sofosbuvir y se observó una reducción significativa en los marcadores de daño hepático, así como en el estadio de fibrosis. El índice de resistencia a la insulina (HOMA) mejoró significativamente a lo largo del estudio. A nivel molecular, el tratamiento con sofosbuvir mejoró la activación de la cascada de señalización de la insulina tras la estimulación con dicha hormona en los hepatocitos con VHC, y, en consecuencia, revirtió la expresión elevada de genes gluconeogénicos, el aumento de la producción de glucosa y la deficiencia de la síntesis de glucógeno en estas células. En conclusión, estos resultados sugieren que el sofosbuvir mejora la respuesta deficiente a la insulina originada por la infección del VHC, proporcionando novedosas evidencias en cuanto a los mecanismos moleculares implicados en la sensibilización a la insulina inducida por este tratamiento. (AU)
Chronic hepatitis C virus (HCV) infection is associated with insulin resistance and type 2 diabetes. The overall aim of this study was to evaluate the effects of sofosbuvir (SOF) on HCV-induced insulin resistance. Clinical parameters were recorded and insulin resistance index (HOMA) calculated from 42 insulin-resistant HCV-patients who underwent SOF-based regimens, at baseline, at the end of treatment (EoT), and at one year after the EoT. Likewise, Huh7 cells expressing full-length HCV replicons were used to elucidate the molecular mechanisms involved in insulin action regulated by SOF. All patients reached a sustained virological response after SOF treatment and, as expected, a significant reduction in liver damage markers and fibrosis stage was observed at the EoT that remained one year later. HOMA significantly improved throughout the study time period. Besides, an increase of total cholesterol, low-density lipoprotein cholesterol and apolipoprotein B levels were maintained over time after the EoT. At the molecular level, SOF treatment improved the activation of the insulin signalling cascade after stimulation with the hormone in HCV-hepatocytes and, accordingly, reversed the elevated expression of gluconeogenic genes, the increased glucose production and the impairment of glycogen synthesis induced by HCV. Furthermore, SOF challenge induced an increase of insulin receptor substrate 1 (IRS1) content parallel to a reduction in its serine phosphorylation in HCV-hepatocytes. These results provide novel evidence about the molecular mechanisms involved in the hepatic insulin sensitization induced by SOF treatment involving the recovery of IRS1 protein levels as a hallmark of SOF effects. (AU)
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Humanos , Hepacivirus , Antivirales , Sofosbuvir , Resistencia a la Insulina , HepatocitosRESUMEN
BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) is the commonest cause of chronic liver disease worldwide, being non-alcoholic steatohepatitis (NASH) its most clinically relevant form. Given the risks associated with taking a liver biopsy, the design of accurate non-invasive methods to identify NASH patients is of upmost importance. BMP2 plays a key role in metabolic homeostasis; however, little is known about its involvement in NAFLD onset and progression. This study aimed to elucidate the impact of BMP2 in NAFLD pathophysiology. METHODS: Hepatic and circulating levels of BMP2 were quantified in serum and liver specimens from 115 biopsy-proven NAFLD patients and 75 subjects with histologically normal liver (NL). In addition, BMP2 content and release was determined in cultured human hepatocytes upon palmitic acid (PA) overload. RESULTS: We found that BMP2 expression was abnormally increased in livers from NAFLD patients than in subjects with NL and this was reflected in higher serum BMP2 levels. Notably, we observed that PA upregulated BMP2 expression and secretion by human hepatocytes. An algorithm based on serum BMP2 levels and clinically relevant variables to NAFLD showed an AUROC of 0.886 (95%CI, 0.83-0.94) to discriminate NASH. We used this algorithm to develop SAN (Screening Algorithm for NASH): a SAN < 0.2 implied a low risk and a SAN ≥ 0.6 indicated high risk of NASH diagnosis. CONCLUSION: This proof-of-concept study shows BMP2 as a new molecular target linked to NAFLD and introduces SAN as a simple and efficient algorithm to screen individuals at risk for NASH.
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La infección crónica por el virus de la hepatitis C (VHC) está asociada con la resistencia a la insulina y la diabetes tipo 2. El objetivo general de este estudio fue evaluar los efectos del sofosbuvir sobre la resistencia a la insulina inducida por el VHC. Para ello, se realizó un estudio clínico que incluyó 42 pacientes con VHC resistentes a la insulina, que fueron tratados con diferentes regímenes antivirales basados en sofosbuvir. Asimismo, se utilizó una línea de hepatocitos humanos que expresan un replicón del VHC de manera estable para determinar los mecanismos moleculares implicados en la acción de la insulina regulada por sofosbuvir. Todos los pacientes alcanzaron una respuesta virológica sostenida después del tratamiento con sofosbuvir y se observó una reducción significativa en los marcadores de daño hepático, así como en el estadio de fibrosis. El índice de resistencia a la insulina (HOMA) mejoró significativamente a lo largo del estudio. A nivel molecular, el tratamiento con sofosbuvir mejoró la activación de la cascada de señalización de la insulina tras la estimulación con dicha hormona en los hepatocitos con VHC, y, en consecuencia, revirtió la expresión elevada de genes gluconeogénicos, el aumento de la producción de glucosa y la deficiencia de la síntesis de glucógeno en estas células. En conclusión, estos resultados sugieren que el sofosbuvir mejora la respuesta deficiente a la insulina originada por la infección del VHC, proporcionando novedosas evidencias en cuanto a los mecanismos moleculares implicados en la sensibilización a la insulina inducida por este tratamiento
Chronic hepatitis C virus (HCV) infection is associated with insulin resistance and type 2 diabetes. The overall aim of this study was to evaluate the effects of sofosbuvir (SOF) on HCV-induced insulin resistance. Clinical parameters were recorded and insulin resistance index (HOMA) calculated from 42 insulin-resistant HCV-patients who underwent SOF-based regimens, at baseline, at the end of treatment (EoT), and at one year after the EoT. Likewise, Huh7 cells expressing full-length HCV replicons were used to elucidate the molecular mechanisms involved in insulin action regulated by SOF. All patients reached a sustained virological response after SOF treatment and, as expected, a significant reduction in liver damage markers and fibrosis stage was observed at the EoT that remained one year later. HOMA significantly improved throughout the study time period. Besides, an increase of total cholesterol, low-density lipoprotein cholesterol and apolipoprotein B levels were maintained over time after the EoT. At the molecular level, SOF treatment improved the activation of the insulin signalling cascade after stimulation with the hormone in HCV-hepatocytes and, accordingly, reversed the elevated expression of gluconeogenic genes, the increased glucose production and the impairment of glycogen synthesis induced by HCV. Furthermore, SOF challenge induced an increase of insulin receptor substrate 1 (IRS1) content parallel to a reduction in its serine phosphorylation in HCV-hepatocytes. These results provide novel evidence about the molecular mechanisms involved in the hepatic insulin sensitization induced by SOF treatment involving the recovery of IRS1 protein levels as a hallmark of SOF effects
Asunto(s)
Humanos , Masculino , Femenino , Adulto , Persona de Mediana Edad , Sofosbuvir/farmacología , Antivirales/farmacología , Resistencia a la Insulina , Hepacivirus/efectos de los fármacos , Hepatitis B Crónica/tratamiento farmacológico , Estudios Retrospectivos , Muestreo Aleatorio Simple , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/virología , Hepatitis B Crónica/metabolismo , Técnica del Anticuerpo Fluorescente , Inmunoprecipitación , Western Blotting , Reacción en Cadena en Tiempo Real de la Polimerasa , Células Cultivadas , Carga Viral , Glucosa/metabolismo , Proteínas Sustrato del Receptor de Insulina/análisis , Proteínas Sustrato del Receptor de Insulina/efectos de los fármacos , Resultado del Tratamiento , Respuesta Virológica SostenidaRESUMEN
Nocturnal intermittent hypoxia (IH) featuring obstructive sleep apnea (OSA) dysregulates hepatic lipid metabolism and might contribute to the development of non-alcoholic fatty liver disease (NAFLD) observed in OSA patients. However, further research is required to better understanding the molecular mechanisms underlying IH-induced hepatic lipid accumulation. Therefore, the aim of the present study was to determine the effects of OSA on hepatic CD36 expression and the impact of IH by using a mouse model of OSA. Histological analysis, lipid content and CD36 expression were assessed in livers from subjects who underwent liver biopsy and polygraphic study during sleep, and in livers from mice submitted to chronic IH mimicking OSA. Among those who presented OSA features, NAFLD were significantly more frequent than in control subjects with normal respiratory function (77.8 vs. 36.4%, respectively), and showed more severe liver disease. Interestingly, CD36 expression was significantly overexpressed within the liver of OSA patients with respect to controls, and a significant positive correlation was observed between hepatic levels of CD36 and the values of two well-known respiratory parameters that characterized OSA: apnea/hypopnea index (AHI) and oxygen desaturation index (ODI). Moreover, hepatic lipid accumulation as well as induction of hepatic lipogenic genes, and CD36 mRNA and protein expression were significantly higher in livers from mice exposed to IH conditions for 8 weeks than in their corresponding littermates. This study provides novel evidence that IH featuring OSA could contribute to NAFLD setup partly by upregulating hepatic CD36 expression.
RESUMEN
BACKGROUND & AIMS: Molecular mechanisms by which hypoxia might contribute to hepatosteatosis, the earliest stage in non-alcoholic fatty liver disease (NAFLD) pathogenesis, remain still to be elucidated. We aimed to assess the impact of hypoxia-inducible factor 2α (HIF2α) on the fatty acid translocase CD36 expression and function in vivo and in vitro. METHODS: CD36 expression and intracellular lipid content were determined in hypoxic hepatocytes, and in hypoxic CD36- or HIF2α -silenced human liver cells. Histological analysis, and HIF2α and CD36 expression were evaluated in livers from animals in which von Hippel-Lindau (Vhl) gene is inactivated (Vhlf/f -deficient mice), or both Vhl and Hif2a are simultaneously inactivated (Vhlf/f Hif2α/f -deficient mice), and from 33 biopsy-proven NAFLD patients and 18 subjects with histologically normal liver. RESULTS: In hypoxic hepatocytes, CD36 expression and intracellular lipid content were augmented. Noteworthy, CD36 knockdown significantly reduced lipid accumulation, and HIF2A gene silencing markedly reverted both hypoxia-induced events in hypoxic liver cells. Moreover livers from Vhlf/f -deficient mice showed histologic characteristics of non-alcoholic steatohepatitis (NASH) and increased CD36 mRNA and protein amounts, whereas both significantly decreased and NASH features markedly ameliorated in Vhlf/f Hif2αf/f -deficient mice. In addition, both HIF2α and CD36 were significantly overexpressed within the liver of NAFLD patients and, interestingly, a significant positive correlation between hepatic transcript levels of CD36 and erythropoietin (EPO), a HIF2α -dependent gene target, was observed in NAFLD patients. CONCLUSIONS: This study provides evidence that HIF2α drives lipid accumulation in human hepatocytes by upregulating CD36 expression and function, and could contribute to hepatosteatosis setup.
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Enfermedad del Hígado Graso no Alcohólico , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Antígenos CD36/genética , Ácidos Grasos , Humanos , Hipoxia , Hígado , RatonesRESUMEN
Insulin receptor substrate 2 (IRS2) is a key downstream mediator of insulin and insulin-like growth factor 1 (IGF1) signalling pathways and plays a major role in liver metabolism. The aim of this study was to investigate whether IRS2 had an impact on the hepatic fibrotic process associated with cholestatic injury. Bile duct ligation (BDL) was performed in wild-type (WT) and Irs2-deficient (IRS2KO) female mice. Histological and biochemical analyses, together with fibrogenic and inflammatory responses were evaluated in livers from mice at 3, 7 and 28â days following BDL. We also explored whether activation of human hepatic stellate cells (HSCs) induced by IGF1 was modulated by IRS2. IRS2KO mice displayed reduced disruption of liver histology, such hepatocyte damage and excess deposition of extracellular matrix components, compared with WT mice at 3 and 7â days post-BDL. However, no histological differences between genotypes were found at 28â days post-BDL. The less pro-inflammatory profile of bile acids accumulated in the gallbladder of IRS2KO mice after BDL corresponded with the reduced expression of pro-inflammatory markers in these mice. Stable silencing of IRS2 or inhibition of ERK1/2 reduced the activation of human LX2 cells and also reduced induction of MMP9 upon IGF1 stimulation. Furthermore, hepatic MMP9 expression was strongly induced after BDL in WT mice, but only a slight increase was found in mice lacking IRS2. Our results have unravelled the signalling pathway mediated by IGF1R-IRS2-ERK1/2-MMP9 as a key axis in regulating HSC activation, which might be therapeutically relevant for targeting liver fibrosis.
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Colestasis/complicaciones , Proteínas Sustrato del Receptor de Insulina/genética , Cirrosis Hepática/genética , Animales , Conductos Biliares/patología , Línea Celular , Colestasis/patología , Femenino , Células Estrelladas Hepáticas/patología , Humanos , Factor I del Crecimiento Similar a la Insulina/metabolismo , Cirrosis Hepática/etiología , Cirrosis Hepática/patología , Ratones , Ratones Noqueados , Transducción de SeñalRESUMEN
Hepatic vitamin D receptor (VDR) expression is increased in patients with nonalcoholic fatty liver (NAFL) and is required for liver steatosis in an NAFL mouse model. However, how hepatocyte VDR is involved in setting up steatosis remains unclear. The authors transduced human hepatocyte-derived cells with an adenoviral vector encoding human VDR and found that angiopoietin-like protein 8 (ANGPTL8) expression was increased upon VDR activation by vitamin D or lithocholic acid. The mRNA levels of hepatic VDR- and vitamin D-related genes [cytochrome P450 (CYP) 2R1, CYP27A1, and CYP3A4] were higher in NAFL patients compared with normal liver subjects. Noteworthy, hepatic ANGPTL8 mRNA and protein levels were elevated in NAFL patients, and its mRNA correlated with VDR mRNA and with the steatosis grade. Moreover, increases in serum conjugated bile acids, including the VDR agonist glycine-lithocholic acid, were observed in NAFL patients. Additionally, free fatty acids and insulin were able to up-regulate both VDR and ANGPTL8 mRNA in human hepatocytes, whereas ANGPTL8 gene knockdown attenuated free fatty acids-induced triglyceride accumulation in these cells. In conclusion, activated VDR up-regulates ANGPTL8 expression, contributing to triglyceride accumulation in human hepatocytes. Moreover, hepatic ANGPTL8 mRNA positively correlates with VDR mRNA content and the grade of steatosis in NAFL patients, suggesting that this novel pathway may play a key role in the pathogenesis of hepatosteatosis.
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Proteínas Similares a la Angiopoyetina/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Hepatocitos/patología , Enfermedad del Hígado Graso no Alcohólico/patología , Hormonas Peptídicas/metabolismo , Receptores de Calcitriol/metabolismo , Adulto , Proteína 8 Similar a la Angiopoyetina , Proteínas Similares a la Angiopoyetina/genética , Estudios de Casos y Controles , Células Cultivadas , Ácidos Grasos no Esterificados/farmacología , Femenino , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Humanos , Insulina/farmacología , Masculino , Persona de Mediana Edad , Enfermedad del Hígado Graso no Alcohólico/tratamiento farmacológico , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Hormonas Peptídicas/genética , Receptores de Calcitriol/genética , Triglicéridos/metabolismoRESUMEN
OBJECTIVES: Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease in Western countries. Protein tyrosine phosphatase 1B (PTP1B), a negative modulator of insulin and cytokine signaling, is a therapeutic target for type 2 diabetes and obesity. We investigated the impact of PTP1B deficiency during NAFLD, particularly in non-alcoholic steatohepatitis (NASH). METHODS: NASH features were evaluated in livers from wild-type (PTP1BWT) and PTP1B-deficient (PTP1BKO) mice fed methionine/choline-deficient diet (MCD) for 8 weeks. A recovery model was established by replacing MCD to chow diet (CHD) for 2-7 days. Non-parenchymal liver cells (NPCs) were analyzed by flow cytometry. Oval cells markers were measured in human and mouse livers with NASH, and in oval cells from PTP1BWT and PTP1BKO mice. RESULTS: PTP1BWT mice fed MCD for 8 weeks exhibited NASH, NPCs infiltration, and elevated Fgf21, Il6 and Il1b mRNAs. These parameters decreased after switching to CHD. PTP1B deficiency accelerated MCD-induced NASH. Conversely, after switching to CHD, PTP1BKO mice rapidly reverted NASH compared to PTP1BWT mice in parallel to the normalization of serum triglycerides (TG) levels. Among NPCs, a drop in cytotoxic natural killer T (NKT) subpopulation was detected in PTP1BKO livers during recovery, and in these conditions M2 macrophage markers were up-regulated. Oval cells markers (EpCAM and cytokeratin 19) significantly increased during NASH only in PTP1B-deficient livers. HGF-mediated signaling and proliferative capacity were enhanced in PTP1BKO oval cells. In NASH patients, oval cells markers were also elevated. CONCLUSIONS: PTP1B elicits a dual role in NASH progression and reversion. Additionally, our results support a new role for PTP1B in oval cell proliferation during NAFLD.
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Enfermedad del Hígado Graso no Alcohólico/metabolismo , Proteína Tirosina Fosfatasa no Receptora Tipo 1/metabolismo , Animales , Células Cultivadas , Colina/administración & dosificación , Dieta/efectos adversos , Molécula de Adhesión Celular Epitelial/sangre , Factores de Crecimiento de Fibroblastos/sangre , Humanos , Interleucina-1beta/sangre , Interleucina-6/sangre , Queratina-19/sangre , Hígado/metabolismo , Hígado/patología , Macrófagos/metabolismo , Masculino , Metionina/administración & dosificación , Metionina/deficiencia , Ratones , Ratones Endogámicos C57BL , Enfermedad del Hígado Graso no Alcohólico/etiología , Proteína Tirosina Fosfatasa no Receptora Tipo 1/genéticaRESUMEN
Limbal stem cells are responsible for the continuous renewal of the corneal epithelium. The destruction or dysfunction of these stem cells or their niche induces limbal stem cell deficiency (LSCD) leading to visual loss, chronic pain, and inflammation of the ocular surface. To restore the ocular surface in cases of bilateral LSCD, an extraocular source of stem cells is needed to avoid dependence on allogeneic limbal stem cells that are difficult to obtain, isolate, and culture. The aim of this work was to test the tolerance and the efficacy of human adipose tissue-derived mesenchymal stem cells (hAT-MSCs) to regenerate the ocular surface in two experimental models of LSCD that closely resemble different severity grades of the human pathology. hAT-MSCs transplanted to the ocular surface of the partial and total LSCD models developed in rabbits were well tolerated, migrated to inflamed tissues, reduced inflammation, and restrained the evolution of corneal neovascularization and corneal opacity. The expression profile of the corneal epithelial cell markers CK3 and E-cadherin, and the limbal epithelial cell markers CK15 and p63 was lost in the LSCD models, but was partially recovered after hAT-MSC transplantation. For the first time, we demonstrated that hAT-MSCs improve corneal and limbal epithelial phenotypes in animal LSCD models. These results support the potential use of hAT-MSCs as a novel treatment of ocular surface failure due to LSCD. hAT-MSCs represent an available, non-immunogenic source of stem cells that may provide therapeutic benefits in addition to reduce health care expenses. Stem Cells 2017;35:2160-2174.
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Córnea/fisiopatología , Células Madre Mesenquimatosas/metabolismo , Nicho de Células Madre/fisiología , Animales , Células Cultivadas , Humanos , ConejosRESUMEN
The transplantation of limbal epithelial stem cells (LESCs) cultured in vitro is a great advance in the treatment of patients suffering from LESC deficiency. However, the optimal technique for LESC isolation from a healthy limbal niche has not yet been established. Our aim was to determine which isolation method renders the highest recovery of functional LESCs from the human limbus. To achieve this purpose, we compared limbal primary cultures (LPCs) obtained from explants and cell suspensions on plastic culture plates. Cell morphology was observed by phase contrast and transmission electron microscopy. LESC, corneal epithelial cell, fibroblast, endothelial cell, melanocyte, and dendritic cell markers were analyzed by real time by reverse transcription polymerase chain reaction and/or immunofluorescence. In addition, colony forming efficiency (CFE) and the presence of holoclones, meroclones, and paraclones were studied. We observed that LPC cells obtained from both methods had cuboidal morphology, desmosomes, and prominent intermediate filaments. The expression of LESC markers (K14, K15, ABCG2, p63α) was similar or higher in LPCs established through cell suspensions, except the expression of p63α mRNA, and there were no significant differences in the expression of corneal epithelial markers (K3, K12). Endothelial cell (PECAM), melanocyte (MART-1), and dendritic cell (CD11c) proteins were not detected, while fibroblast-protein (S100A4) was detected in all LPCs. The CFE was significantly higher in LPCs from cell suspensions. Cells from confluent LPCs produced by explants generated only paraclones (100%), while the percentage of paraclones from LPCs established through cell suspensions was 90% and the remaining 10% were meroclones. In conclusion, LPCs established from cell suspensions have a cell population richer in functional LESCs than LPCs obtained from explants. These results suggest that in a clinical situation in which it is possible to choose between either of the isolation techniques from the donor limbal tissue, then the cell suspension is probably the best option as long as the cells are expanded following our culture conditions.
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Técnicas de Cultivo de Célula/métodos , Epitelio Corneal/ultraestructura , Limbo de la Córnea/ultraestructura , Células Madre/ultraestructura , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores/metabolismo , Separación Celular , Células Cultivadas , Epitelio Corneal/metabolismo , Femenino , Humanos , Limbo de la Córnea/metabolismo , Masculino , Microscopía Electrónica de Transmisión , Persona de Mediana Edad , Células Madre/metabolismo , Donantes de TejidosRESUMEN
OBJECTIVE: To evaluate knowledge of living wills and attitudes to these documents in the populations of two basic health areas in Toledo (Spain). METHOD: We performed a descriptive, cross-sectional study in primary care centers. Participants consisted of the populations of two basic health areas selected by systematic stratified sampling. Inclusion criteria comprised age between 18 and 80 years, and absence of mental or terminal illnesses. The participants were contacted by telephone before a personal interview was carried out. Data on demographic variables and knowledge of living wills and attitudes to these documents were recorded. RESULTS: A total of 395 participants were interviewed (58% women) with a mean age of 46.22 years (SD: 17.13). The vast majority (88.6%) were unaware of living wills, with no significant differences in sex or religious beliefs. Most (67.8%) would sign a living will in the case of terminal illness whereas 56.3% would sign at the present moment, with no significant differences in sex or religious beliefs. More than half of the men (57.5%) would agree to modify the living will if requested by relatives versus 42.6% of the women. More than three-quarters (76.2%) believed that information on living wills was insufficient. One-third (34.5%) would allow a relative's organs to be donated even without the relative leaving express wishes, while 49.2% would donate their own organs. CONCLUSION: There was a huge lack of awareness on living wills in our population, which nevertheless proved to be highly receptive to, and in agreement with, the implications of these documents after receiving information on the topic.
Asunto(s)
Conocimientos, Actitudes y Práctica en Salud , Voluntad en Vida , Estudios Transversales , Femenino , Humanos , Masculino , Persona de Mediana EdadRESUMEN
Objetivo. Conocer el grado de conocimientos y actitudes de la población acerca del documento de voluntades anticipadas (DVA) en 2 zonas básicas de salud de Toledo. Método. Estudio descriptivo transversal realizado en atención primaria. Los participantes fueron la población de 2 zonas básicas de salud, seleccionadas mediante muestreo sistemático estratificado. Los criterios de inclusión fueron: edad entre 18 y 80 años, no tener enfermedad mental ni terminal. Se realizó entrevista personal, previa cita telefónica, recogiéndose variables sociodemográficas y datos acerca de conocimientos y actitudes ante el DVA. Resultados. Se entrevistó a 395 personas, siendo el 58% mujeres y la edad media de 46,22 años (desviación estándar = 17,13). El 88,8% no conocía el DVA, sin diferencias significativas respecto al sexo ni a creencias religiosas. Un 67,8% firmaría el DVA en caso de enfermedad terminal, mientras que el 56,3% lo firmaría en el momento actual, sin diferencias por sexo ni por creencias en ambos casos. Accedería a modificarlo a petición de sus familiares el 57,5% de los varones frente al 42,6% de las mujeres. El 76,2% consideró que no hay información suficiente acerca del DVA. Un 34,5% accedería a donar los órganos de un familiar que no hubiera dispuesto nada acerca del tema, frente al 49,2% que sí donaría sus propios órganos. Conclusiones. Hay un gran desconocimiento acerca de este tema en la población del presente estudio, que sinembargo se muestra muy receptiva y conforme con lo que implica el DVA tras recibir información
Objective. To evaluate knowledge of living wills and attitudes to these documents in the populations of two basic health areas in Toledo (Spain). Method. We performed a descriptive, cross-sectional study in primary care centers. Participants consisted of the populations of two basic health areas selected by systematic stratified sampling. Inclusion criteria comprised age between 18 and 80 years, and absence of mental or terminal illnesses. The participants were contacted by telephone before a personal interview was carried out. Data on demographic variables and knowledge of living wills and attitudes to these documents were recorded. Results. A total of 395 participants were interviewed (58% women) with a mean age of 46.22 years (SD: 17.13). The vast majority (88.6%) were unaware of living wills, with no significant differences in sex or religious beliefs. Most (67.8%) would sign a living will in the case of terminal illness whereas 56.3% would sign at the present moment, with no significant differences in sex or religious beliefs. More than half of the men (57.5%) would agree to modify the living will if requested by relatives versus 42.6% of the women. More than three-quarters (76.2%) believed that information on living wills was insufficient. One-third (34.5%) would allow a relatives organs to be donated even without the relative leaving express wishes, while 49.2% would donate their own organs. Conclusion. There was a huge lack of awareness on living wills in our population, which nevertheless proved to behighly receptive to, and in agreement with, the implications of these documents after receiving information on the topic