RESUMEN
Several inherited arrhythmias, including Brugada syndrome and arrhythmogenic cardiomyopathy, primarily affect the right ventricle and can lead to sudden cardiac death. Among many differences, right and left ventricular cardiomyocytes derive from distinct progenitors, prompting us to investigate how embryonic programming may contribute to chamber-specific conduction and arrhythmia susceptibility. Here, we show that developmental perturbation of Wnt signaling leads to chamber-specific transcriptional regulation of genes important in cardiac conduction that persists into adulthood. Transcriptional profiling of right versus left ventricles in mice deficient in Wnt transcriptional activity reveals global chamber differences, including genes regulating cardiac electrophysiology such as Gja1 and Scn5a. In addition, the transcriptional repressor Hey2, a gene associated with Brugada syndrome, is a direct target of Wnt signaling in the right ventricle only. These transcriptional changes lead to perturbed right ventricular cardiac conduction and cellular excitability. Ex vivo and in vivo stimulation of the right ventricle is sufficient to induce ventricular tachycardia in Wnt transcriptionally inactive hearts, while left ventricular stimulation has no effect. These data show that embryonic perturbation of Wnt signaling in cardiomyocytes leads to right ventricular arrhythmia susceptibility in the adult heart through chamber-specific regulation of genes regulating cellular electrophysiology.
Asunto(s)
Arritmias Cardíacas/etiología , Arritmias Cardíacas/metabolismo , Ventrículos Cardíacos/metabolismo , Ventrículos Cardíacos/fisiopatología , Proteínas Wnt/metabolismo , Vía de Señalización Wnt , Arritmias Cardíacas/diagnóstico , Arritmias Cardíacas/fisiopatología , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Biomarcadores , Biología Computacional/métodos , Simulación por Computador , Susceptibilidad a Enfermedades , Electrocardiografía , Elementos de Facilitación Genéticos , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Genotipo , Sistema de Conducción Cardíaco/fisiopatología , Humanos , Inmunohistoquímica , Mutación , Miocitos Cardíacos/metabolismo , Imagen Óptica , Fenotipo , Unión Proteica , Proteínas Represoras/metabolismo , Proteínas Wnt/genética , beta CateninaRESUMEN
A disintegrin and metalloproteinase domain 10 (Adam10), a member of the ADAM family of cell membrane-anchored proteins, has been linked to the regulation of the Notch, EGF, E-cadherin, and other signaling pathways. However, it is unclear what role Adam10 has in the kidney in vivo. In this study, we showed that Adam10 deficiency in ureteric bud (UB) derivatives leads to a decrease in urinary concentrating ability, polyuria, and hydronephrosis in mice. Furthermore, Adam10 deficiency led to a reduction in the percentage of aquaporin 2 (Aqp2)(+) principal cells (PCs) in the collecting ducts that was accompanied by a proportional increase in the percentage of intercalated cells (ICs). This increase was more prominent in type A ICs than in type B ICs. Foxi1, a transcription factor important for the differentiation of ICs, was upregulated in the Adam10 mutants. The observed reduction of Notch activity in Adam10 mutant collecting duct epithelium and the similar reduction of PC/IC ratios in the collecting ducts in mice deficient for mindbomb E3 ubiquitin protein ligase 1, a key regulator of the Notch and Wnt/receptor-like tyrosine kinase signaling pathways, suggest that Adam10 regulates cell fate determination through the activation of Notch signaling, probably through the regulation of Foxi1 expression. However, phenotypic differences between the Adam10 mutants, the Mib1 mutants, and the Foxi1 mutants suggest that the functions of Adam10 in determining the fate of collecting duct cells are more complex than those of a simple upstream factor in a linear pathway involving Notch and Foxi1.
Asunto(s)
Proteínas ADAM/metabolismo , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Riñón/metabolismo , Proteínas de la Membrana/metabolismo , Proteína ADAM10 , Animales , Apoptosis , Acuaporina 2/metabolismo , Cadherinas/metabolismo , Proliferación Celular , Células Epiteliales/citología , Factores de Transcripción Forkhead/metabolismo , Hidronefrosis/genética , Túbulos Renales/citología , Túbulos Renales Colectores/metabolismo , Ligandos , Ratones , Ratones Transgénicos , Mutación , Poliuria/genética , Receptores Notch/metabolismo , Transducción de Señal , Células Madre/citología , Regulación hacia Arriba , Vía de Señalización WntRESUMEN
PURPOSE: Congenital urinary tract obstruction is a leading cause of renal maldevelopment and pediatric kidney disease. Nonetheless, few groups have examined its molecular pathogenesis in humans. We evaluated the role of BMP-7, a protein required for renal injury repair and nephrogenesis, in disease progression in patients with obstructive uropathy. MATERIALS AND METHODS: Whole kidney and cell specific BMP-7 expression was examined in a murine model of unilateral ureteral obstruction and in patients with congenital ureteropelvic junction obstruction. Findings were correlated with molecular markers of renal injury and clinical parameters. RESULTS: Unilateral ureteral obstruction led to a dramatic decrease in BMP-7 expression in the proximal and distal tubules before the onset of significant loss of renal architecture and fibrosis, suggesting that this is a critical molecular event that drives early stage disease progression. Loss of BMP-7 expression then extended to the collecting ducts and glomeruli in end stage kidney disease. When translating these findings to patients with ureteropelvic junction obstruction, global loss of BMP-7 expression correlated with a decreased number of nephrons, loss of renal architecture, severe renal fibrosis and loss of kidney function. CONCLUSIONS: Given that BMP-7 has a critical role in renal injury repair and nephrogenesis, these findings show that cell specific changes in BMP-7 expression contribute to the onset of irreversible renal injury and impaired kidney development secondary to congenital urinary tract obstruction. Accordingly therapies that target these cell populations to restore BMP-7 activity may limit disease progression in patients with obstructive uropathy.
Asunto(s)
Proteína Morfogenética Ósea 7/metabolismo , Enfermedades Renales/metabolismo , Túbulos Renales Distales/metabolismo , Túbulos Renales Proximales/metabolismo , Obstrucción Ureteral/metabolismo , Obstrucción Ureteral/patología , Animales , Antígenos CD/metabolismo , Antígenos de Diferenciación Mielomonocítica/metabolismo , Caspasa 3/metabolismo , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Humanos , Túbulos Renales Distales/citología , Túbulos Renales Proximales/citología , Ratones Endogámicos C57BLRESUMEN
PURPOSE: The calcineurin-NFAT signaling pathway regulates the transcription of genes important for development. It is impacted by various genetic and environmental factors. We investigated the potential role of NFAT induced transcriptional dysregulation in the pathogenesis of congenital abnormalities of the kidneys and urinary tract. MATERIALS AND METHODS: A murine model of conditional NFATc1 activation in the ureteric bud was generated and examined for histopathological changes. Metanephroi were also cultured in vitro to analyze branching morphogenesis in real time. RESULTS: NFATc1 activation led to defects resembling multicystic dysplastic kidney. These mutants showed severe disorganization of branching morphogenesis characterized by decreased ureteric bud branching and the disconnection of ureteric bud derivatives from the main collecting system. The orphan ureteric bud derivatives may have continued to induce nephrogenesis and likely contributed to the subsequent formation of blunt ended filtration units and cysts. The ureter also showed irregularities consistent with impaired epithelial-mesenchymal interaction. CONCLUSIONS: This study reveals the profound effects of NFAT signaling dysregulation on the ureteric bud and provides insight into the pathogenesis of multicystic dysplastic kidney. Our results suggest that the obstruction hypothesis and the bud theory may not be mutually exclusive to explain the pathogenesis of multicystic dysplastic kidney. Ureteric bud dysfunction such as that induced by NFAT activation can disrupt ureteric bud-metanephric mesenchyma interaction, causing primary defects in branching morphogenesis, subsequent dysplasia and cyst formation. Obstruction of the main collecting system can further enhance these defects, producing the pathological changes associated with multicystic dysplastic kidney.
Asunto(s)
Riñón Displástico Multiquístico/embriología , Factores de Transcripción NFATC/fisiología , Organogénesis/fisiología , Uréter/embriología , Animales , Calcineurina/fisiología , Ratones , Transducción de Señal/fisiología , Transcripción GenéticaRESUMEN
BACKGROUND: Mutations in the gene MTARC1 (mitochondrial amidoxime-reducing component 1) protect carriers from metabolic dysfunction-associated steatohepatitis (MASH) and cirrhosis. MTARC1 encodes the mARC1 enzyme, which is localized to the mitochondria and has no known MASH-relevant molecular function. Our studies aimed to expand on the published human genetic mARC1 data and to observe the molecular effects of mARC1 modulation in preclinical MASH models. METHODS AND RESULTS: We identified a novel human structural variant deletion in MTARC1, which is associated with various biomarkers of liver health, including alanine aminotransferase levels. Phenome-wide Mendelian Randomization analyses additionally identified novel putatively causal associations between MTARC1 expression, and esophageal varices and cardiorespiratory traits. We observed that protective MTARC1 variants decreased protein accumulation in in vitro overexpression systems and used genetic tools to study mARC1 depletion in relevant human and mouse systems. Hepatocyte mARC1 knockdown in murine MASH models reduced body weight, liver steatosis, oxidative stress, cell death, and fibrogenesis markers. mARC1 siRNA treatment and overexpression modulated lipid accumulation and cell death consistently in primary human hepatocytes, hepatocyte cell lines, and primary human adipocytes. mARC1 depletion affected the accumulation of distinct lipid species and the expression of inflammatory and mitochondrial pathway genes/proteins in both in vitro and in vivo models. CONCLUSIONS: Depleting hepatocyte mARC1 improved metabolic dysfunction-associated steatotic liver disease-related outcomes. Given the functional role of mARC1 in human adipocyte lipid accumulation, systemic targeting of mARC1 should be considered when designing mARC1 therapies. Our data point to plasma lipid biomarkers predictive of mARC1 abundance, such as Ceramide 22:1. We propose future areas of study to describe the precise molecular function of mARC1, including lipid trafficking and subcellular location within or around the mitochondria and endoplasmic reticulum.
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Hígado Graso , Hepatocitos , Animales , Humanos , Ratones , Adipocitos , Biomarcadores , Ceramidas , Análisis de la Aleatorización MendelianaRESUMEN
Megsin is a serine protease inhibitor (Serpin) that has known expression in kidney mesangial cells. Here, we report the generation and characterization of a bacterial artificial chromosome (BAC) transgene expressing Cre under the control of Megsin regulatory elements. When crossed to the ROSA26R-lacZ reporter mice, the Megsin-Cre transgene mediates loxP recombination primarily in the skin, forestomach, and esophagus, but surprisingly not in the mesangial cells. Within the skin, cells in all epidermal layers and the hair follicle cells expressed Cre. This transgene also has uniform expression in the epithelium of the forestomach and esophagus. Conditional deletion of Adam10, a gene known to have important functions in skin development, by using this Megsin-Cre transgene led to severe skin defects. In addition, these mutants appear to have reduced folds and surface area in the forestomach. These results show that the Megsin-Cre transgene can mediate loxP-recombination in all epidermal layers of the skin, the hair follicle cells, as well as in the epithelium of the forestomach and esophagus, all of which have known expression of various keratins. This Megsin-Cre transgene can serve as a new tool for conditional genetic manipulation to study development and diseases in the skin and the upper digestive tract.
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Epitelio/metabolismo , Queratinas/genética , Serpinas/genética , Transgenes , Animales , Cromosomas Artificiales Bacterianos/genética , Marcación de Gen/métodos , Genes Reporteros/genética , Ingeniería Genética/métodos , Integrasas/genética , Queratinas/metabolismo , Ratones , Ratones Transgénicos , Especificidad de Órganos , Regiones Promotoras Genéticas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Transcripción GenéticaRESUMEN
Transgenic mice overexpressing SUR1 and gain of function Kir6.2[∆N30, K185Q] K(ATP) channel subunits, under cardiac α-myosin heavy chain (αMHC) promoter control, demonstrate arrhythmia susceptibility and premature death. Pregnant mice, crossed to carry double transgenic progeny, which harbor high levels of both overexpressed subunits, exhibit the most extreme phenotype and do not deliver any double transgenic pups. To explore the fetal lethality and embryonic phenotype that result from K(ATP) overexpression, wild type (WT) and K(ATP) overexpressing embryonic cardiomyocytes were isolated, cultured and voltage-clamped using whole cell and excised patch clamp techniques. Whole mount embryonic imaging, Hematoxylin and Eosin (H&E) and α smooth muscle actin (αSMA) immunostaining were used to assess anatomy, histology and cardiac development in K(ATP) overexpressing and WT embryos. Double transgenic embryos developed in utero heart failure and 100% embryonic lethality by 11.5 days post conception (dpc). K(ATP) currents were detectable in both WT and K(ATP)-overexpressing embryonic cardiomyocytes, starting at early stages of cardiac development (9.5 dpc). In contrast to adult cardiomyocytes, WT and K(ATP)-overexpressing embryonic cardiomyocytes exhibit basal and spontaneous K(ATP) current, implying that these channels may be open and active under physiological conditions. At 9.5 dpc, live double transgenic embryos demonstrated normal looping pattern, although all cardiac structures were collapsed, probably representing failed, non-contractile chambers. In conclusion, K(ATP) channels are present and active in embryonic myocytes, and overexpression causes in utero heart failure and results in embryonic lethality. These results suggest that the K(ATP) channel may have an important physiological role during early cardiac development.
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Pérdida del Embrión/genética , Genes Letales , Canales KATP/genética , Miocardio/metabolismo , Animales , Femenino , Expresión Génica , Corazón/fisiopatología , Atrios Cardíacos/enzimología , Atrios Cardíacos/metabolismo , Atrios Cardíacos/fisiopatología , Canales KATP/metabolismo , Ratones , Ratones Transgénicos , EmbarazoRESUMEN
The role of axial structures, especially the notochord, in metanephric kidney development has not been directly examined. Here, we showed that disruption of the notochord and floor plate by diphtheria toxin (DTA)-mediated cell ablation did not disrupt nephrogenesis, but resulted in kidney fusions, resembling horseshoe kidneys in humans. Axial disruptions led to more medially positioned metanephric mesenchyme (MM) in midgestation. However, neither axial disruption nor the ensuing positional shift of the MM affected the formation of nephrons and other structures within the kidney. Response to Shh signaling was greatly reduced in midline cell populations in the mutants. To further ascertain the molecular mechanism underlying these abnormalities, we specifically inactivated Shh in the notochord and floor plate. We found that depleting the axial source of Shh was sufficient to cause kidney fusion, even in the presence of the notochord. These results suggested that the notochord is dispensable for nephrogenesis but required for the correct positioning of the metanephric kidney. Axial Shh signal appears to be critical in conferring the effects of axial structures on kidney positioning along the mediolateral axis. These studies also provide insights into the pathogenesis of horseshoe kidneys and how congenital kidney defects can be caused by signals outside the renal primordia.
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Regulación del Desarrollo de la Expresión Génica , Proteínas Hedgehog/metabolismo , Riñón/embriología , Transducción de Señal , Animales , Animales Recién Nacidos , Tipificación del Cuerpo , Toxina Diftérica/metabolismo , Toxina Diftérica/farmacología , Embrión de Mamíferos/embriología , Embrión de Mamíferos/metabolismo , Desarrollo Embrionario , Proteínas Hedgehog/genética , Inmunohistoquímica , Hibridación in Situ , Mesodermo/metabolismo , Ratones , Ratones Transgénicos , Modelos Biológicos , Mutación , Notocorda/citología , Notocorda/fisiología , TransgenesRESUMEN
Tetraspanins have gained increased attention due to their functional versatility. But the universal cellular mechanism that governs such versatility remains unknown. Herein we present the evidence that tetraspanins CD81 and CD82 regulate the formation and/or development of cell membrane protrusions. We analyzed the ultrastructure of the cells in which a tetraspanin is either overexpressed or ablated using transmission electron microscopy. The numbers of microvilli on the cell surface were counted, and the radii of microvillar tips and the lengths of microvilli were measured. We found that tetraspanin CD81 promotes the microvillus formation and/or extension while tetraspanin CD82 inhibits these events. In addition, CD81 enhances the outward bending of the plasma membrane while CD82 inhibits it. We also found that CD81 and CD82 proteins are localized at microvilli using immunofluorescence. CD82 regulates microvillus morphogenesis likely by altering the plasma membrane curvature and/or the cortical actin cytoskeletal organization. We predict that membrane protrusions embody a common morphological phenotype and cellular mechanism for, at least some if not all, tetraspanins. The differential effects of tetraspanins on microvilli likely lead to the functional diversification of tetraspanins and appear to correlate with their functional propensity.
Asunto(s)
Membrana Celular/fisiología , Proteína Kangai-1/fisiología , Tetraspanina 28/fisiología , Animales , Línea Celular , Membrana Celular/metabolismo , Membrana Celular/ultraestructura , Proteína Kangai-1/genética , Proteína Kangai-1/metabolismo , Ratones , Ratones Mutantes , Microvellosidades/fisiología , Microvellosidades/ultraestructura , Tetraspanina 28/genética , Tetraspanina 28/metabolismoRESUMEN
PURPOSE: We genetically disrupted the wolffian duct in mice to study the affected organogenesis processes and to test the hypothesis that cell loss can be the developmental basis for a wide spectrum of congenital anomalies in the kidney and urinary tract. MATERIALS AND METHODS: We used Hoxb7-Cre transgenic lines (HC1 and HC2) to induce diphtheria toxin production from a ROSA(DTA) allele, disrupting the wolffian duct and derivatives. RESULTS: The first set of mutants (HC1;ROSA(DTA/+)) exhibited agenesis of the kidney, ureter and reproductive tracts. The second set of mutants (HC2;ROSA(DTA/+)) exhibited diverse defects, including renal agenesis/hypoplasia, hydronephrosis, hydroureter, ureter-vas deferens fistulas in males and ureter-oviduct/uterus fistulas in females. The phenotypic differences correspond to the degree of apoptosis induced caudal truncation of the wolffian duct, which is less severe and more variable in HC2;ROSA(DTA/+) mice. Whenever the wolffian duct failed to reach the urogenital sinus, the ureter failed to separate from the wolffian duct, suggesting that ureteral migration along the common nephric duct to the cloaca and the subsequent common nephric duct degeneration constitute the only pathway for separating the ureter and wolffian duct derivatives. CONCLUSIONS: The diverse and severe defects observed emphasize the central role of the wolffian duct in providing progenitors and signals for urogenital development. These results also indicate that the quantitative difference in cell death induced caudal truncation of the wolffian duct can lead to a wide range of qualitatively distinct defects, and that cell death can serve as a single etiological cause of a wide spectrum of congenital kidney and urinary tract defects.
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Sistema Urinario/anomalías , Animales , Apoptosis/genética , Muerte Celular/genética , Femenino , Riñón/anomalías , Masculino , Ratones , Conductos Mesonéfricos/anomalíasRESUMEN
Atrial fibrillation (AF) is the most common cardiac arrhythmia, yet the molecular signature of the vulnerable atrial substrate is not well understood. Here, we delineated a distinct transcriptional signature in right versus left atrial cardiomyocytes (CMs) at baseline and identified chamber-specific gene expression changes in patients with a history of AF in the setting of end-stage heart failure (AF+HF) that are not present in heart failure alone (HF). We observed that human left atrial (LA) CMs exhibited Notch pathway activation and increased ploidy in AF+HF but not in HF alone. Transient activation of Notch signaling within adult CMs in a murine genetic model is sufficient to increase ploidy in both atrial chambers. Notch activation within LA CMs generated a transcriptomic fingerprint resembling AF, with dysregulation of transcription factor and ion channel genes, including Pitx2, Tbx5, Kcnh2, Kcnq1, and Kcnip2. Notch activation also produced distinct cellular electrophysiologic responses in LA versus right atrial CMs, prolonging the action potential duration (APD) without altering the upstroke velocity in the left atrium and reducing the maximal upstroke velocity without altering the APD in the right atrium. Our results support a shared human/murine model of increased Notch pathway activity predisposing to AF.
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Potenciales de Acción , Fibrilación Atrial/patología , Biomarcadores/metabolismo , Regulación de la Expresión Génica , Atrios Cardíacos/patología , Insuficiencia Cardíaca/patología , Miocitos Cardíacos/patología , Animales , Fibrilación Atrial/genética , Atrios Cardíacos/metabolismo , Insuficiencia Cardíaca/genética , Humanos , Ratones , Miocitos Cardíacos/metabolismo , Receptores Notch/genética , Receptores Notch/metabolismo , TranscriptomaRESUMEN
Tbx18 is a T-Box transcription factor that has specific expression and indispensible function in the lower urinary tract. Here, we report the generation and characterization of a bacterial artificial chromosome (BAC) transgene expressing Cre under the control of Tbx18 regulatory elements. When crossed to the ROSA26R-lacZ reporter mice, the Tbx18-Cre transgene mediates loxP recombination in the mesenchymal derivatives in the lower urinary tract, especially in the smooth muscle cells (SMCs) and the stromal cells. There is no expression of this transgene in the urothelium or in the kidney. This Tbx18-Cre transgene recapitulates the endogenous Tbx18 expression in the urinary system and can be used for the study of the development, physiology, and diseases in the urinary tract. Its additional expression in the epicardium, limb, vibrissae, and other structures would be useful for studies in the relevant fields.
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Embrión de Mamíferos/metabolismo , Integrasas/genética , Recombinación Genética , Sistema Urinario/metabolismo , Animales , Sitios de Unión , Embrión de Mamíferos/embriología , Femenino , Regulación del Desarrollo de la Expresión Génica , Técnicas Genéticas , Integrasas/metabolismo , Riñón/embriología , Riñón/metabolismo , Operón Lac/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Ratones Transgénicos , Reacción en Cadena de la Polimerasa , Proteínas de Dominio T Box/genética , Factores de Tiempo , Transgenes/genética , Sistema Urinario/embriología , Urotelio/embriología , Urotelio/metabolismoRESUMEN
Although epithelial morphogenesis is tightly controlled by intrinsic genetic programs, the microenvironment in which epithelial cells proliferate and differentiate also contributes to the morphogenetic process. The roles of the physical microenvironment in epithelial morphogenesis, however, have not been well dissected. In this study, we assessed the impact of the microenvironment on epithelial cyst formation, which often marks the beginning or end step of morphogenesis of epithelial tissues and the pathological characteristic of some diseases. Previous studies have demonstrated that Madin-Darby canine kidney (MDCK) epithelial cells form cysts when grown in a three-dimensional (3D) extracellullar matrix (ECM) environment. We have now further demonstrated that the presence of ECM in the 3D scaffold is required for the formation of properly polarized cysts. Also, we have found that the full interface of epithelial cells with the ECM environment (in-3D) is not essential for cyst formation, since partial contact (on-3D) is sufficient to induce cystogenesis. In addition, we have defined the minimal ECM environment or the physical threshold for cystogenesis under the on-3D condition. Only above the threshold can the morphological cues from the ECM environment induce cyst formation. Moreover, cyst formation under the on-3D condition described in this study defines a novel and more feasible model to analyze in vitro morphogenesis. Finally, we have found that, during cystogenesis, MDCK cells generate basal microprotrusions and produce vesicle-like structures to the basal extracellular space, which are specific to and correlated with cyst formation. For the first time, we have systematically and quantitatively elucidated the microenvironmental determinants for epithelial cystogenesis.
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Células Epiteliales/citología , Riñón/citología , Riñón/crecimiento & desarrollo , Morfogénesis , Animales , Agregación Celular , Comunicación Celular , Técnicas de Cultivo de Célula , Diferenciación Celular , Línea Celular , Polaridad Celular , Medios de Cultivo , Perros , Células Epiteliales/ultraestructura , Epitelio/crecimiento & desarrollo , Epitelio/ultraestructura , Matriz Extracelular , Concentración de Iones de Hidrógeno , Imagenología Tridimensional , Riñón/ultraestructura , Microscopía Confocal , Microscopía ElectrónicaRESUMEN
BACKGROUND: Although methylation-mediated inactivation of expression of RASSF1A, a candidate tumor suppressor gene, has been observed in several human cancers, the data concerning alteration of RASSF1A expression and methylation in Chinese primary gastric cancer are scarce. Moreover, direct evidence showing the association between protein expression of RASSF1A and primary human cancers is lacking. The aim of this study was to investigate RASSF1A expression in tissue of primary gastric cancer (GC) at mRNA and protein levels, and to establish the possible relationship between DNA methylation status and protein expression of RASSF1A in Chinese. METHODS: Fifty-four patients with primary gastric cancers were included in the study of RASSF1A mRNA expression and methylation status between the cancer tissue and the corresponding adjacent normal tissue. 20 out of 54 patients were included for study of RASSF1A protein expression. The expression of RASSF1A at mRNA and protein levels was determined by RT-PCR and Western-blotting, respectively. The RASSF1A promoter methylation was detected by methylation-specific PCR. RESULTS: RASSF1A mRNA and protein expressions in GC were reduced significantly with comparison to the corresponding normal tissues (OD value: 0.2589 +/- 0.2407 vs 0.5448 +/- 0.2971, P < 0.0001; 0.1874 +/- 0.0737 vs 0.6654 +/- 0.2201, P < 0.0001, respectively). Methylation frequency of RASSF1A in primary GC is higher than that in the corresponding normal tissues (66.7% vs. 14.8%, P < 0.0001). Furthermore, RASSF1A mRNA expression in methylation group of GC was further reduced when compared to the unmethylation group of GC (0.1384 +/- 0.1142 vs. 0.5018 +/- 0.2463, P < 0.0001). CONCLUSION: Expression of RASSF1A was reduced in tissue of GC at mRNA and protein levels. Diminished expression of RASSF1A was associated with the promoter methylation.
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Pueblo Asiatico/genética , Biomarcadores de Tumor/análisis , Metilación de ADN , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor/genética , Biopsia con Aguja , Estudios de Casos y Controles , China/epidemiología , ADN de Neoplasias/análisis , Femenino , Mucosa Gástrica/metabolismo , Mucosa Gástrica/patología , Regulación Neoplásica de la Expresión Génica , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Probabilidad , Pronóstico , Regiones Promotoras Genéticas , Valores de Referencia , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Medición de Riesgo , Sensibilidad y Especificidad , Neoplasias Gástricas/etnología , Análisis de Supervivencia , Proteínas Supresoras de Tumor/genéticaRESUMEN
Sinomenine is a pure alkaloid extracted from the Chinese medical plant Sinomenium acutum. It was demonstrated that sinomenine had anti-inflammatory and immunosuppressive effects in the previous studies. The aim of the present study was to evaluate therapeutic effects of sinomenine on 2, 4, 6-trinitrobenzene sulfonic acid (TNBS) induced colitis in mice. Two hours following colonic instillation of TNBS, sinomenine with several doses (30, 100, 200 mg/kg) was given by gastric gavage once daily for 7 days. Comparing with the saline-treated mice with TNBS-induced colitis, sinomenine (100 mg/kg and 200 mg/kg)-treated mice with TNBS-induced colitis were shown improvements of weight loss, macroscopic score, histological score, and myeloperoxidase activity. Moreover, treatments with sinomenine (100 mg/kg and 200 mg/kg) decreased the up-regulated mRNA and protein levels of tumour necrosis factor-alpha(TNF-alpha) and interferon-gamma (IFN-gamma) caused by TNBS. Our findings suggest that sinomenine attenuates TNBS-induced colitis in mice and the therapeutic mechanism might be related to the reduction of up-regulated colonic TNF-alpha and IFN-gamma production caused by TNBS.
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Antiinflamatorios , Colitis/tratamiento farmacológico , Morfinanos/uso terapéutico , Ácido Trinitrobencenosulfónico , Animales , Peso Corporal/efectos de los fármacos , Colitis/inducido químicamente , Colitis/patología , Colon/enzimología , Colon/patología , Ensayo de Inmunoadsorción Enzimática , Femenino , Interferón gamma/biosíntesis , Ratones , Ratones Endogámicos BALB C , Morfinanos/farmacología , Peroxidasa/metabolismo , ARN Mensajero/biosíntesis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
AIM: N-acetyltransferase 2 (NAT2) is an important enzyme catalyzing N-acetylation of sulfasalazine (SASP). The aim of the present study was to investigate associations of the genotypes of NAT2 with inflammatory bowel disease (IBD), and with adverse effects of SASP, which is used as the first-line treatment of IBD. PATIENTS AND METHODS: The wildtype allele (NAT2*4) and three variant alleles (NAT2*5B, NAT2*6A and NAT*7B) of the NAT2 gene were determined in 101 patients with IBD (84 patients with ulcerative colitis and 17 patients with Crohn's disease) and 109 healthy controls by the polymerase chain reaction-restriction fragment length polymorphism method. Sixty-eight patients with IBD treated with SASP were followed, and their adverse reactions were recorded. RESULTS: Eleven patients (16%) experienced adverse effects from SASP, including nine cases of sulfapyridine (SP) dose-related adverse effects and two cases of hypersensitivity (skin rash). Patients with the slow acetylator genotypes without the NAT2*4 allele experienced adverse effects more frequently (36%) than those with the fast acetylator genotypes with at least one NAT2*4 allele (11%), but the results were not significantly different (OR of 0.26, 95% CI 0.065 to 1.004; P=0.051). However, those with the slow acetylator genotypes experienced more SP dose-related adverse effects than those with the fast acetylator genotypes (36% versus 8%, OR of 0.17, 95% CI 0.039 to 0.749; P=0.019). CONCLUSIONS: The NAT2 gene polymorphism was not associated with susceptibility to IBD in Chinese populations, but the NAT2 slow acetylator genotypes were significantly associated with SP dose-related adverse effects of SASP in the treatment of IBD.
Asunto(s)
Antiinflamatorios no Esteroideos/efectos adversos , Arilamina N-Acetiltransferasa/genética , Enfermedades Inflamatorias del Intestino/tratamiento farmacológico , Enfermedades Inflamatorias del Intestino/genética , Sulfasalazina/efectos adversos , Acetilación , Acetiltransferasas/metabolismo , Adolescente , Adulto , Anciano , Alelos , Antiinflamatorios no Esteroideos/uso terapéutico , Arilamina N-Acetiltransferasa/metabolismo , Femenino , Predisposición Genética a la Enfermedad , Genotipo , Humanos , Enfermedades Inflamatorias del Intestino/metabolismo , Masculino , Persona de Mediana Edad , Polimorfismo Genético , Sulfasalazina/uso terapéuticoRESUMEN
Ulcerative colitis (UC) is characterized by chronic intestinal inflammation as a result of an exaggerated T cell response. Cytotoxic T lymphocyte associated antigen-4 (CTLA-4), expressed mainly in activated T cells, inhibits T cell activation and proliferation by combining B7 through competing CD28 and maintains immune homeostasis. Polymorphisms of the CTLA-4 gene are known to be associated with several autoimmune diseases. The aim of this study was to investigate the association between the CTLA-4 gene microsatellite polymorphism and UC in Chinese patients. Unrelated 100 Chinese patients with UC and 140 healthy controls were studied. The (AT) repeats in the 3' untranslated region of exon 4 of the CTLA-4 gene were amplified by allele-specific polymerase chain reaction (PCR). The amplified products were electrophoresed on a 12% polyacrylamide gel, followed by silver staining. Twenty alleles were found in Chinese patients and healthy controls. The 122-bp allele was increased in UC compared with healthy controls (9.5% vs 0.7%, P = 0.0001/Pc = 0.002, OR = 14.591, 95%CI 3.357-63.420). The frequency of the longer alleles (>or=118 bp) of UC was higher than that in healthy controls (26% vs 4%, P = 0.0001/Pc = 0.0002, OR = 7.644, 95%CI 3.950-14.792), but was not associated with location and severity of the disease. Furthermore, the longer alleles were not associated with haplotypes of C-318T/A+49G of the CTLA-4 gene in Chinese patients with UC. The longer alleles of the CTLA-4 gene microsatellite polymorphism were strongly associated with UC in Chinese patients.
Asunto(s)
Antígenos de Diferenciación/genética , Colitis Ulcerosa/genética , Predisposición Genética a la Enfermedad , Repeticiones de Microsatélite/genética , Adolescente , Adulto , Anciano , Antígenos CD , Pueblo Asiatico , Antígeno CTLA-4 , China/epidemiología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Polimorfismo GenéticoRESUMEN
OBJECTIVE: To investigate RASSF1A expression in tissue of primary gastric cancer (GC) at mRNA and protein levels and analyze the relationship between DNA methylation status and RASSF1A expression in GC. METHODS: RASSF1A mRNA expression was determined with RT-PCR in 54 specimens of cancer tissue and adjacent normal tissue. RASSF1A promoter methylation was detected with methylation-specific PCR. The expression of RASSF1A protein level was determined with Western blot in tissue of GC in 20 patients. RESULTS: RASSF1A mRNA and protein expressions in GC were reduced significantly than those in corresponding normal tissue (A value: 0.2589 +/- 0.2407 vs 0.5448 +/- 0.2971, P < 0.0001; 0.1874 +/- 0.0737 vs 0.6654 +/- 0.2201, P < 0.0001, respectively). Methylation frequency of RASSF1A in primary GC was higher than that in corresponding normal tissue (66.7% vs 14.8%, P < 0.0001). Furthermore, RASSF1A mRNA expression in methylation group of GC was much reduced than that in unmethylation group of GC (0.1384 +/- 0.1142 vs 0.5018 +/- 0.2463, P < 0.0001). CONCLUSION: Expression of RASSF1A is reduced in the tissue of GC at mRNA and protein levels. Low expression of RASSF1A is associated with hypermethylation of the promoter of RASSF1A gene.
Asunto(s)
Metilación de ADN , Neoplasias Gástricas/metabolismo , Proteínas Supresoras de Tumor/biosíntesis , Adulto , Anciano , Anciano de 80 o más Años , Islas de CpG , Humanos , Persona de Mediana Edad , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Neoplasias Gástricas/patología , Proteínas Supresoras de Tumor/genéticaRESUMEN
OBJECTIVE: Inflammatory bowel disease (IBD) is characterized by the T-cell excessive activation of intestinal mucosa. Cytotoxic T-lymphocyte associated antigen 4 (CTLA-4) is a negative regulator of T-lymphocyte activation. The aim of the present study is to investigate the association between CTLA-4 promoter -1722 (T/C), -1661 (A/G) polymorphisms and ulcerative colitis (UC) in Han Chinese, in Hubei province of central China. METHODS: Eighty-seven patients with UC and 116 healthy controls were genotyped for CTLA-4 promoter -1722 and -1661 polymorphisms with a method of polymerase chain reaction based restriction fragment length polymorphism. RESULTS: The frequency of "A/G + G/G" genotype at the -1661 site was statistically higher in UC patients than in healthy controls (34.5% vs 15.5%, P = 0.002, OR = 2.865, 95% CI = 1.467-5.596). The frequency of the G allele at the -1661 site was also significantly higher in UC patients than in the controls (19.0% vs 8.2%, P = 0.002, OR = 2.624, 95% CI = 1.435-4.796). However, the distribution of the genotypes at -1722 site was not significantly different between the UC patients and the controls. CONCLUSION: The G allele of CTLA-4 promoter -1661 polymorphism showed a highly significant association with UC in Han Chinese of the central China.
Asunto(s)
Antígenos CD/genética , Antígenos de Diferenciación/genética , Colitis Ulcerosa/genética , Polimorfismo Genético , Linfocitos T Citotóxicos/inmunología , Adulto , Alelos , Antígeno CTLA-4 , Estudios de Casos y Controles , Colitis Ulcerosa/inmunología , Femenino , Frecuencia de los Genes , Genotipo , Humanos , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de Restricción , Regiones Promotoras GenéticasRESUMEN
Emerging evidence has highlighted the pivotal role of microvasculature injury in the development and progression of renal fibrosis. Angiopoietin-1 (Ang-1) is a secreted vascular growth factor that binds to the endothelial-specific Tie2 receptor. Ang-1/Tie2 signaling is critical for regulating blood vessel development and modulating vascular response after injury, but is dispensable in mature, quiescent vessels. Although dysregulation of vascular endothelial growth factor (VEGF) signaling has been well studied in renal pathologies, much less is known about the role of the Ang-1/Tie2 pathway in renal interstitial fibrosis. Previous studies have shown contradicting effects of overexpressing Ang-1 systemically on renal tubulointerstitial fibrosis when different engineered forms of Ang-1 are used. Here, we investigated the impact of site-directed expression of native Ang-1 on the renal fibrogenic process and peritubular capillary network by exploiting a conditional transgenic mouse system [Pax8-rtTA/(TetO)7 Ang-1] that allows increased tubular Ang-1 production in adult mice. Using a murine unilateral ureteral obstruction (UUO) fibrosis model, we demonstrate that targeted Ang-1 overexpression attenuates myofibroblast activation and interstitial collagen I accumulation, inhibits the upregulation of transforming growth factor ß1 and subsequent phosphorylation of Smad 2/3, dampens renal inflammation, and stimulates the growth of peritubular capillaries in the obstructed kidney. Our results suggest that Ang-1 is a potential therapeutic agent for targeting microvasculature injury in renal fibrosis without compromising the physiologically normal vasculature in humans.