RESUMEN
Fibrogenesis or scarring of the liver is a common consequence of all chronic liver diseases. Here we refine a quantitative trait locus that confers susceptibility to hepatic fibrosis by in silico mapping and show, using congenic mice and transgenesis with recombined artificial chromosomes, that the gene Hc (encoding complement factor C5) underlies this locus. Small molecule inhibitors of the C5a receptor had antifibrotic effects in vivo, and common haplotype-tagging polymorphisms of the human gene C5 were associated with advanced fibrosis in chronic hepatitis C virus infection. Thus, the mouse quantitative trait gene led to the identification of an unknown gene underlying human susceptibility to liver fibrosis, supporting the idea that C5 has a causal role in fibrogenesis across species.
Asunto(s)
Complemento C5/genética , Cirrosis Hepática/genética , Sitios de Carácter Cuantitativo , Animales , Mapeo Cromosómico , Complemento C5/metabolismo , Genotipo , Humanos , Ratones , Ratones Endogámicos , Polimorfismo GenéticoRESUMEN
UNLABELLED: In vivo knockdown of connective tissue growth factor (CTGF/CCN2) was recently shown to attenuate the formation of experimental liver fibrosis. The secreted, cysteine-rich growth factor is proposed to adversely modulate the binding of profibrogenic transforming growth factor beta (TGF-beta) and its natural antagonist bone morphogenetic protein (BMP) to their cognate receptors in several cellular systems, but the functionality of CTGF in modulation of the TGF-beta/BMP signaling pathways is still unknown. This study aims at characterizing a potentially differential modulating role of CTGF on TGF-beta- and BMP7-dependent transactivation of reporter gene [Ad-(CAGA)(12)-MLP-luc, Ad-hCTGF-luc, and Ad-(BRE)(2)-luc reporter gene] expression in rat hepatocytes. In this context, emphasis is also placed on the differential roles of Smad2 and Smad3 in the TGF-beta-dependent transactivation of the endogenous CTGF gene and the CTGF gene reporter, as investigated following adenoviral infection of wild-type and dominant negative Smad2/3 or treatment with the specific inhibitor of Smad3 or ALK5-specific (SB-431542) inhibitor. In this analysis, we found (1) a selective transcriptional activation of the CTGF promoter by Smad2 (but not Smad3); (2) the failure of BMP7 to inhibit the transcriptional activation of the Smad3-selective (CAGA)(12)-luc reporter by TGF-beta, as well as the failure of TGF-beta to inhibit the transcriptional activation of the Smad5-selective (BRE)(2)-luc reporter by BMP7; and (3) the sensitization of hepatocytes toward TGF-beta type I receptor (ALK5)/Smad2 and Smad3-mediated TGF-beta signaling by CTGF, whereas BMP type I receptor (ALK1)/Smad5-mediated BMP7 signaling is not modulated. CONCLUSION: CTGF acts as a Smad2-dependent sensitizer of TGF-beta actions that does not influence BMP7 signaling in hepatocytes.
Asunto(s)
Proteína Morfogenética Ósea 7/fisiología , Factor de Crecimiento del Tejido Conjuntivo/fisiología , Hepatocitos/fisiología , Proteína Smad2/fisiología , Factor de Crecimiento Transformador beta/metabolismo , Animales , Células Cultivadas , Masculino , Ratas , Ratas Sprague-Dawley , Transducción de Señal/fisiologíaRESUMEN
Latent transforming growth factor-beta binding proteins are a family of extracellular matrix proteins comprising four isoforms (LTBP-1, -2, -3, -4) with different structures, tissue expression patterns and affinity for TGF-beta. So far, respective knockout models have highlighted some essential functions for LTBP-2, LTBP-3 and LTBP-4, while the physiological significance of LTBP-1 is only superficially known. Here we report for the first time the generation and characterization of a mouse model lacking both the long and short LTBP-1 isoform. Surprisingly, respective mice are viable and fertile. However, detailed X-ray analysis of the skull revealed a modified facial profile. In addition, the gene disruption induces a reduced biological activity of TGF-beta that became evident in an experimental model of hepatic fibrogenesis in which the LTBP-1 knockout animals were less prone to hepatic fibrogenesis. Furthermore, comparative cDNA microarray gene expression profiling of cultured hepatic stellate cells confirmed that respective nulls were less receptive to cellular activation and transdifferentiation into myofibroblasts. Therefore, we conclude that LTBP-1 has essential functions in the control of TGF-beta activation.
Asunto(s)
Cara/anatomía & histología , Proteínas de Unión a TGF-beta Latente/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Animales , Células Cultivadas , Regulación hacia Abajo , Proteínas de Unión a TGF-beta Latente/deficiencia , Proteínas de Unión a TGF-beta Latente/genética , Cirrosis Hepática/genética , Cirrosis Hepática/metabolismo , Cirrosis Hepática/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Análisis de Secuencia por Matrices de Oligonucleótidos , Fenotipo , ARN Mensajero/genética , Regulación hacia Arriba/genéticaRESUMEN
Cysteine-rich FGF receptor (CFR) was originally identified as a FGF2 receptor and found to be identical to Golgi complex-localized glycoprotein-1 (GLG1), also known as MG-160, and to a murine E-selectin ligand-1 (ESL-1). Although CFR is a 150-kDa integral membrane glycoprotein that is primarily located in the cis-medial Golgi complex, a substantial proportion of CFR is secreted but the underlying mechanism is unknown. CFR contains several possible furin-like proprotein convertase (PC) and matrix metalloproteinase cleavage sites. Cells expressing CFR were treated with the furin protease inhibitor decanoyl-Arg-Val-Lys-Arg-chloromethylketone (decCMK) or the MMP-inhibitor GM6001. In the presence of furin-like PC inhibitor, secretion of CFR was almost completely inhibited. Secretion was not affected by the GM6001 inhibitor. The secreted forms were further characterized by creating different mutant CFR proteins with N-terminal and C-terminal tags. Immunoblot analysis and immunofluorescence indicated, that successive endoproteolytical processing of CFR which takes place in the Golgi complex is essential for secretion. Secreted CFR bound to heparan sulphate proteoglycan (HSPG) could trap FGFs and thereby directly competing with tyrosine kinase receptors for FGF binding.
Asunto(s)
Proproteína Convertasas/metabolismo , Procesamiento Proteico-Postraduccional , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Sialoglicoproteínas/metabolismo , Clorometilcetonas de Aminoácidos/farmacología , Animales , Células COS , Pollos , Chlorocebus aethiops , Dipéptidos/farmacología , Factores de Crecimiento de Fibroblastos/metabolismo , Furina/antagonistas & inhibidores , Furina/metabolismo , Humanos , Metaloendopeptidasas/antagonistas & inhibidores , Mutación , Proproteína Convertasas/antagonistas & inhibidores , Receptores de Factores de Crecimiento de Fibroblastos/genética , Sialoglicoproteínas/genéticaRESUMEN
BACKGROUND: Recently, we identified hepatocytes as the major cellular source of profibrogenic connective tissue growth factor (CTGF/CCN2) in the liver. Based on reports of a hepatoprotective effect of coffee consumption, we were the first to provide evidence that caffeine suppresses transforming growth factor (TGF)-beta dependent and -independent CTGF expression in hepatocytes in vitro and in vivo, thus suggesting this xanthine-alkaloid as a potential therapeutic agent. AIM: This study aims at comparing the inhibitory capacities of caffeine and its three demethylated derivates paraxanthine, theophylline and theobromine on CTGF expression in hepatocytes and hepatic stellate cells (HSC). RESULTS: Our data suggest paraxanthine as the most important pharmacological repressor of hepatocellular CTGF expression among the caffeine-derived metabolic methylxanthines with an inhibitory dosage (ID)50 of 1.15 mM, i.e. 3.84-fold lower than what is observed for caffeine. In addition, paraxanthine displayed the least cell toxicity as proven by the water-soluble tetrazolium-1 cell vitality assay. However, caffeine or any of the metabolites did not inhibit CTGF expression in HSC. At the toxicological threshold concentration of 1 mM for paraxanthine, we observed an inhibition of hepatocellular CTGF synthesis by 44%, which was strongly reverted in the presence of the specific competitive cyclic adenosine monophosphate inhibitor Rp-adenosine 3',5-cyclic monophosphorothioate triethylammonium salt. Furthermore, CTGF protein expression induced by various concentrations of TGF-beta (0.13-1 ng/ml) is still reduced by, on average, 27%/45% in the presence of paraxanthine (1.25 mM/2.5 mM). CONCLUSION: Our data provide an evidence-based suggestion of the caffeine-derived primary metabolite paraxanthine as a potentially powerful antifibrotic drug by its inhibitory effect on (hepatocellular) CTGF synthesis.
Asunto(s)
Cafeína/química , Factor de Crecimiento del Tejido Conjuntivo/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Hepatocitos/metabolismo , Teofilina/farmacología , Animales , Western Blotting , Células Cultivadas , Cartilla de ADN/genética , Electroforesis en Gel de Poliacrilamida , Concentración 50 Inhibidora , Masculino , Estructura Molecular , Ratas , Ratas Sprague-Dawley , Análisis de Regresión , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Teobromina/farmacología , Teofilina/análisisRESUMEN
BACKGROUND: Platelet-derived growth factor-BB (PDGF-BB) is a profibrotic factor in liver fibrosis through its ability to stimulate hepatic stellate cells (HSC). The liver-derived serine protease factor VII activating protease (FSAP) regulates the activities of PDGF-BB in a cell-specific manner. AIMS: Our aim was to determine the influence of FSAP on the activation of HSC and to analyse the regulation of FSAP in hepatic fibrogenesis. METHODS: The effect of FSAP on PDGF-stimulated p42/p44 mitogen-activated protein kinase (MAPK) activation in primary rat HSC was determined by Western blotting. Migration and proliferation of HSC was evaluated in Boyden chamber experiments and (3)H-thymidine incorporation assays respectively. Expression of FSAP was analysed in a CCl(4) mouse model of liver fibrosis by Western blot, quantitative real-time polymerase chain reaction and immunohistochemistry. RESULTS: FSAP inhibited PDGF-BB-stimulated p42/p44 MAPK phosphorylation, proliferation and migration of HSC. FSAP mRNA expression level was increased 3 h after CCl(4) application and decreased after 18 h and, in established fibrosis, after chronic CCl(4) administration. In parallel, there was a decrease in the circulating FSAP protein in chronic fibrosis. Concurrently, the homogenous hepatic expression pattern of FSAP was disturbed. Immunohistochemistry revealed a decrease of FSAP in hepatocytes in inflammatory and fibrotic lesions. CONCLUSIONS: Our results demonstrate an inhibitory effect of FSAP on PDGF-mediated activation of HSC. In addition, FSAP expression is transiently increased in acute-phase reaction but decreased during chronic fibrogenesis, which in turn may influence PDGF-BB availability and myofibroblast activity.
Asunto(s)
Células Estrelladas Hepáticas/metabolismo , Cirrosis Hepática/metabolismo , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Serina Endopeptidasas/metabolismo , Animales , Becaplermina , Movimiento Celular/fisiología , Proliferación Celular/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Células Estrelladas Hepáticas/efectos de los fármacos , Humanos , Inmunohistoquímica , Cirrosis Hepática/patología , Ratones , Ratones Endogámicos BALB C , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-sis , Ratas , Ratas Sprague-Dawley , Serina Endopeptidasas/farmacología , Estadísticas no ParamétricasRESUMEN
BACKGROUND/AIMS: Current knowledge confers a crucial role to connective tissue growth factor (CTGF/CCN2) in hepatic fibrogenesis. Hepatocytes are likely to be the major cellular source of CTGF in the liver in which CTGF is sensitively upregulated by TGF-beta. Recently, we demonstrated that the methylxanthine derivate caffeine leads to an upregulation of peroxisome proliferator activated receptor gamma (PPARgamma) expression in hepatocytes, thus sensitizing these cells to the well-known inhibitory effect of 15-deoxy-Delta(12,14)-prostaglandin J(2) (15-d-PGJ(2)) on CTGF expression. However, upregulation of the receptor alone is not sufficient per se; its physiological ligand 15-d-PGJ(2) is required to exert an inhibitory effect on transforming growth factor-beta (TGF-beta) target genes such as CTGF. METHODS: This study compared serum concentrations of 15-d-PGJ(2) in Caucasian patients with fibrotic liver diseases (n=289), Caucasian controls (n=136) and Caucasian non-liver disease (NLD) sick (n=307), as well as of Chinese patients with hepatocellular carcinoma (HCC) (n=43) and Chinese healthy controls (n=63) in order to characterize their suitability for therapeutic approaches with PPARgamma-inducing (i.e. CTGF inhibitory) drugs such as caffeine. RESULTS: The presented data showed that Caucasian patients with ongoing hepatic fibrogenesis (mean 6.2+/-5.9 microg/L) displayed strikingly higher serum concentrations of 15-d-PGJ(2) than healthy probands (mean 2.3+/-1.0) and Caucasian patients with NLD (mean 2.7+/-1.4 microg/L). Similar results were found in Chinese patients with fully developed HCC (mean 1.3+/-0.7 microg/L) compared with Chinese healthy controls (mean 0.4+/-0.2 microg/L). CONCLUSIONS: In conclusion, our data thus proposed an increased suitability of these patient groups for therapeutic approaches with drugs inducing PPARgamma expression, such as methylxanthine derivates.
Asunto(s)
Factor de Crecimiento del Tejido Conjuntivo/antagonistas & inhibidores , Cirrosis Hepática/sangre , Cirrosis Hepática/metabolismo , PPAR gamma/metabolismo , Prostaglandina D2/análogos & derivados , Adulto , Anciano , Pueblo Asiatico , Cafeína/farmacología , Femenino , Hepatocitos/metabolismo , Humanos , Cirrosis Hepática/patología , Masculino , Persona de Mediana Edad , Prostaglandina D2/sangre , Prostaglandina D2/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Población BlancaRESUMEN
Leukocytes and tumor cells use E-selectin binding ligands to attach to activated endothelial cells expressing E-selectin during inflammation or metastasis. The cysteine-rich fibroblast growth factor receptor (CFR) represents the main E-selectin ligand (ESL-1) on granulocytes and its expression is exclusively modified by alpha(1,3)-fucosyltransferases IV or VII (FucT4 and FucT7). Hepatic stellate cells (HSC) are pericytes of liver sinusoidal endothelial cells. The activation of HSC and transdifferentiation into a myofibroblastic phenotype is involved in the repair of liver tissue injury, liver regeneration and angiogenesis of liver metastases. In the present study, we demonstrated that HSC expressed CFR together with FucT7 and exhibited a functional E-selectin binding activity on their cell surface. Since HSC appear to be oxygen-sensing cells, the expression of E-selectin binding activity was analyzed in HSC under a hypoxic atmosphere. While the expression of the glycoprotein CFR was unaffected by hypoxia, the cell-associated E-selectin binding activity decreased. However, under the same conditions, mRNA expression of the modifying enzyme FucT7 increased. The loss of E-selectin binding activity, therefore, appears to be neither the result of a reduced expression of the modifying transferase nor the expression of the backbone glycoprotein. After the transient transfection of HSC with CFR cDNA, the E-selectin binding activity (ESL-1) was efficiently released into the supernatant. Therefore, we hypothesize that under hypoxia, ESL-1 is shed from activated HSC. Our findings provide a novel perspective on the function of HSC in liver metastasis and inflammatory liver diseases.
Asunto(s)
Quimiotaxis de Leucocito/fisiología , Células Estrelladas Hepáticas/metabolismo , Neoplasias Hepáticas/metabolismo , Receptores de Factores de Crecimiento de Fibroblastos/biosíntesis , Sialoglicoproteínas/biosíntesis , Animales , Northern Blotting , Western Blotting , Hipoxia de la Célula , Selectina E/genética , Selectina E/metabolismo , Técnica del Anticuerpo Fluorescente , Fucosiltransferasas/genética , Fucosiltransferasas/metabolismo , Expresión Génica , Humanos , Neoplasias Hepáticas/secundario , Masculino , Metástasis de la Neoplasia/patología , ARN Mensajero/análisis , Ratas , Ratas Sprague-Dawley , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
In differentiated smooth muscle cells (SMC) the regulation of SMC marker genes (e.g. alpha-smooth muscle actin) is mainly conducted by the serum response factor (SRF) and accessory co-factors like myocardin. A number of SMC markers are also expressed in activated hepatic stellate cells which are the main cellular effectors in liver fibrogenesis. In the present study we found that during cellular activation and transdifferentiation the SRF transcription factor is up-regulated by transforming growth factor-beta, accumulated in the nucleus, and exhibited increased DNA-binding activity. These observations were accompanied by a forced expression of the SRF co-activator myocardin. Specific targeting of SRF by small interference RNA resulted in diminished contents of alpha-smooth muscle actin. Therefore, we conclude that hepatic stellate cells retain differentiation capacity to evolve characteristics that are typical for cells of the cardiac and smooth muscle lineages.
Asunto(s)
Hepatocitos/citología , Hepatocitos/metabolismo , Factor de Respuesta Sérica/metabolismo , Factor de Crecimiento Transformador beta/farmacología , Regulación hacia Arriba/efectos de los fármacos , Animales , Biomarcadores/metabolismo , Diferenciación Celular/efectos de los fármacos , ADN/metabolismo , Hepatocitos/efectos de los fármacos , Masculino , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/metabolismo , Proteínas Nucleares/metabolismo , Unión Proteica/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Transactivadores/metabolismoRESUMEN
Platelet-derived growth factor (PDGF) has an essential role in liver fibrogenesis, as PDGF-B and -D both act as potent mitogens on culture-activated hepatic stellate cells (HSCs). Induction of PDGF receptor type-beta (PDGFR beta) in HSC is well documented in single-dose carbon tetrachloride (CCl(4))-induced acute liver injury. Of the newly discovered isoforms PDGF-C and -D, only PDGF-D shows significant upregulation in bile duct ligation (BDL) models. We have now investigated the expression of PDGF isoforms and receptors in chronic liver injury in vivo after long-term CCl(4) treatment and demonstrated that isolated hepatocytes have the requisite PDGF signaling pathways, both in the naive state and when isolated from CCl(4)-treated rats. In vivo, PDGF gene expression showed upregulation of all PDGF isoforms and receptors, with values peaking at 4 weeks and decreasing to near basal levels by 8 and 12 weeks. Interestingly, PDGF-C increased significantly when compared to BDL-models. PDGF-A, PDGF-C and PDGF receptor type-alpha (PDGFR alpha) correlated closely with inflammation and steatosis. Immunohistochemistry revealed expression of PDGF-B, -C and -D in areas corresponding to centrilobular necrosis, inflammation and fibrosis, whereas PDGF-A localized in regenerative hepatocytes. PDGFR beta was identified along the fibrotic septa, whereas PDGFR alpha showed positive staining in fibrotic septa and regenerative hepatocytes. Despite a significant decline of PDGF isoforms, hepatocyte regeneration peaked at 8 weeks. A marked difference in the degree of fibrosis was observed amongst the individual animals. In summary, PDGF expression in liver damage primarily parallels mesenchymal cell proliferation and extracellular matrix production, rather than hepatocyte regeneration. We conclude that PDGF levels in chronic liver injury peak at 4 weeks after onset of injury, and that the outcome of chronic toxic liver injury strongly depends on the individual capacity for tissue regeneration in the weeks following the peak of PDGF expression.
Asunto(s)
Intoxicación por Tetracloruro de Carbono/metabolismo , Hepatocitos/metabolismo , Cirrosis Hepática/metabolismo , Regeneración Hepática/fisiología , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Animales , Células Cultivadas , Cirrosis Hepática/inducido químicamente , Masculino , Isoformas de Proteínas/metabolismo , Ratas , Ratas Sprague-Dawley , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/metabolismoRESUMEN
BACKGROUND AND AIMS: Transforming growth factor beta1 (TGFbeta1) acts as an important profibrogenic cytokine in liver fibrogenesis. The aim of this study was to explore the association between TGFbeta1 gene polymorphisms and liver cirrhosis. METHODS: Totally 118 Chinese suffering from liver cirrhosis induced by HBV infection and 104 healthy controls were recruited. The polymorphisms at positions -988, -800, -509 and codon10, codon25, codon263 of the TGFbeta1 gene were genotyped by ARMS-PCR or LightCycler. Enzyme immunoassay was used for TGFbeta1 measurement. The promoter activities and DNA-binding capacities containing -509C>T were analyzed by reporter gene and EMSA. RESULTS: The allele frequencies of CAT -509 and of T at codon10 were elevated in patients at severe Child-Pugh grade. Elevated concentrations of TGFbeta1 were observed in patients, especially in those with -509CC/CT and codon10 TT/TC. Flanking sequences containing -509C showed higher promoter activities than -509T. EMSA showed one nucleotide change at -509C>T influenced nuclear protein binding to the putative binding site. CONCLUSIONS: The C allele at -509 and the T allele at codon10 could play important roles in progression of liver cirrhosis. The C allele at -509 mediates higher transcriptional activity than the T allele providing a potential explanation for the clinical findings.
Asunto(s)
Variación Genética , Cirrosis Hepática/genética , Transcripción Genética , Factor de Crecimiento Transformador beta1/genética , Adulto , Anciano , Alelos , Sustitución de Aminoácidos/genética , Animales , Línea Celular , Femenino , Frecuencia de los Genes , Hepatitis B Crónica/genética , Hepatitis B Crónica/patología , Humanos , Cirrosis Hepática/patología , Masculino , Ratones , Persona de Mediana Edad , Células 3T3 NIH , Mutación PuntualRESUMEN
Aging is associated with low-grade inflammation on the one hand and mild zinc deficiency on the other. These conditions contribute to decreased immune functions, resulting in increased incidences of infections and autoimmune diseases. The aim of this study was to give more insight into the question, to what extent is low-grade inflammation caused by zinc deficient status. Here we report the effect of improved intracellular zinc status on low-grade inflammatory activity in 19 healthy elderly subjects. Our experiments show that adjustment of labile zinc by moderate zinc supplementation reduces spontaneous cytokine release and defects in termination of inflammatory activity. This results in reduced amounts of unspecific preactivated T cells and leads to improved T cell response upon mitogenic stimulation. Therefore, in contrast to other anti-inflammatory drugs, zinc does not suppress, but improves immune reaction upon pathogen invasion. These results suggest that mildly zinc-deficient, healthy elderly subjects might benefit from moderate zinc supplementation due to a more balanced immune response with reduced incidences of infections and autoimmune diseases.
Asunto(s)
Anciano , Suplementos Dietéticos , Mediadores de Inflamación/metabolismo , Linfocitos T/efectos de los fármacos , Zinc/farmacología , Adulto , Anciano de 80 o más Años , Proteínas de Transporte de Catión/genética , Células Cultivadas , Citocinas/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Absorción Intestinal/genética , Leucocitos/efectos de los fármacos , Leucocitos/metabolismo , Lipopolisacáridos/farmacología , Masculino , Linfocitos T/fisiología , Zinc/metabolismoRESUMEN
Connective tissue growth factor (CTGF=CCN2), one of six members of cysteine-rich, secreted, heparin-binding proteins with a modular structure, is recognized as an important player in fibrogenic pathways as deduced from findings in non-hepatic tissues and emerging results from liver fibrosis. Collectively, the data show strongly increased expression in fibrosing tissues and transforming growth factor (TGF-beta)-stimulated expression in hepatocytes, biliary epithelial cells and stellate cells. Functional activity as a mediator of fibre-fibre, fibre-matrix and matrix-matrix interactions, as an enhancer of profibrogenic TGF-beta and several secondary effects owing to TGF-beta enhancement, and as a down-modulator of the bioactivity of bone morphogenetic protein-7 has been proposed. By changing the activity ratio of TGF-beta to its antagonist bone-morphogenetic protein-7, CTGF is proposed as a fibrogenic master switch for epithelial-mesenchymal transition. Consequently, knockdown of CTGF considerably attenuates experimental liver fibrosis. The spill-over of CTGF from the liver into the blood stream proposes this protein as a non-invasive reporter of TGF-beta bioactivity in this organ. Indeed, CTGF-levels in sera correlate significantly with fibrogenic activity. The data suggest CTGF as a multifaceted regulatory protein in fibrosis, which offers important translational aspects for diagnosis and follow-up of hepatic fibrogenesis and as a target for therapeutic interventions. In addition, CTGF-promoter polymorphism might be of importance as a prognostic genetic marker to predict the progression of fibrosis.
Asunto(s)
Factor de Crecimiento del Tejido Conjuntivo/metabolismo , Cirrosis Hepática/metabolismo , Animales , Biomarcadores/metabolismo , Factor de Crecimiento del Tejido Conjuntivo/sangre , Regulación de la Expresión Génica , Humanos , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
BACKGROUND/AIMS: Haemoglobin-depleted erythrocyte ghosts have been recommended as vesicle carriers of drugs with hepatotropic properties. However, the influence of liver injury on ghost elimination and targeting has not been reported so far. METHODS: Human and rat ghosts were prepared and loaded with model substances, and the basic parameters were characterized. Ghosts were injected intravenously into rats with acute, subacute and chronic liver injuries. Elimination from circulation, organ distribution and cellular targeting was measured. The uptake of ghosts by liver macrophages/Kupffer cells was determined in cell culture. RESULTS: Ghosts are strong hepatotropic carriers with a recovery of 90% in normal liver. Kupffer cells are the almost exclusive target cell type. Hepatotropic properties remain in rats with chronic liver diseases, but are reduced by 60-70% in acute liver damage as a result of decline of phagocytosis of macrophages/Kupffer cells. Although the uptake of ghosts per gram liver tissue in chronic liver injury was also reduced by about 40%, the increase of liver mass and of macrophages/Kupffer cells compensated for the reduced phagocytotic activity. In subacute injury, the uptake per gram liver tissue was only moderately reduced. CONCLUSION: Drug targeting with ghosts might be feasible in chronic and subacute liver injuries, e.g. fibrogenesis and tumours, because the content of ingested ghosts is released by Kupffer cells into the micro-environment, providing the uptake by and pharmacological effects on adjacent cells.
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Sistemas de Liberación de Medicamentos/métodos , Membrana Eritrocítica/metabolismo , Membrana Eritrocítica/trasplante , Hígado/lesiones , Alanina Transaminasa/metabolismo , Fosfatasa Alcalina/metabolismo , Animales , Aspartato Aminotransferasas/metabolismo , Células Cultivadas , Glutamato Deshidrogenasa/metabolismo , Humanos , Inyecciones Intravenosas , Macrófagos del Hígado/metabolismo , Hígado/metabolismo , Macrófagos/metabolismo , Masculino , Neuraminidasa , Ratas , Ratas Sprague-Dawley , TecnecioRESUMEN
BACKGROUND/AIMS: Up to now, the effect of activin A on the expression of the important transforming growth factor (TGF)-beta downstream modulator connective tissue growth factor (CTGF) is not known, but might be of relevance for the functional effects of this cytokine on several liver cell types. METHODS: In this study, activin A-dependent CTGF expression in hepatocytes (PC) primed by exogenous activin A and in PC maintained under complete activin-free culture conditions was analysed by Western blots, metabolic labelling, gene silencing, reverse transcriptase-polymerase chain reaction (RT-PCR) and CTGF reporter gene assays. This study was supplemented by immunocytochemical staining of activin A and CTGF in PC of injured liver. RESULTS: Using alkaline phosphatase alpha-alkaline phosphatase staining, it is demonstrated that activin A becomes increasingly detectable during the course of CCl(4)-liver damage. Addition of activin A to cultured PC induced CTGF protein expression via phosphorylation of Smad2 and Smad3. This induction can be inhibited by the antagonist follistatin and alpha-activin A antibody respectively. When PC were cultured under serum(i.e. activin A)-free culture conditions, a time-dependent increase of activin expression during the course of the culture was proven by RT-PCR. Silencing of inhibin beta(A) gene expression under serum-free conditions by small interfering RNAs greatly suppressed CTGF synthesis and the phosphorylations of Smad2 and Smad3. However, both the extracellularly acting follistatin and the alpha-activin A antibody could not inhibit spontaneous CTGF expression, which, however, was achieved by the cell-permeable TGF-beta Alk4/Alk5 receptor-kinase-inhibitor SB431542. CONCLUSIONS: In conclusion, the results point to activin A as an inducer of CTGF synthesis in PC. Intracellular activin A contributes to spontaneous CTGF expression in PC independent of exogenous activin A, which is proposed to occur via Alk4/Alk5-receptors. The findings might be important for many actions of activin A on the liver.
Asunto(s)
Activinas/metabolismo , Factor de Crecimiento del Tejido Conjuntivo/metabolismo , Hepatocitos/metabolismo , Comunicación Paracrina , Animales , Comunicación Autocrina , Tetracloruro de Carbono/toxicidad , Células Cultivadas , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Inmunohistoquímica , Masculino , Ratas , Ratas Sprague-Dawley , Proteína Smad2/metabolismo , Proteína smad3/metabolismo , Regulación hacia ArribaRESUMEN
BACKGROUND: Gc-globulin or vitamin D binding protein is a highly expressed, multifunctional and polymorphic serum protein, which also serves as the major transporter for vitamin D metabolites in the circulation. The present study was performed to analyze the interaction between gc-globulin of hepatocytes and hepatic stellate cells, the most important fat-/retinol-storing cell type in the liver, which spontaneously transdifferentiates to myofibroblasts in culture. METHODS: Hepatic stellate cells and hepatocytes were isolated by the pronase/collagenase reperfusion method, hepatocytes by collagenase reperfusion of the organ. Gc-globulin expression was monitored by immunocytochemistry, immunoblotting, RT-PCR, metabolic labelling with [(35)S]-methionine, and its intracellular binding to alpha-smooth-muscle actin was investigated by co-immunoprecipitation. Cytoskeletal stainings of gc-globulin and alpha-smooth-muscle actin in hepatic stellate cells and the identification of the receptors megalin/gp330, HCAM/CD44, cubilin and annexin A2 were performed with confocal immunocytochemistry, immunoblotting and/or FACS-analysis. RESULTS: Hepatocytes synthesize and secrete gc-globulin as shown by RT-PCR and [(35)S]-methionine labelling, which could be suppressed by cycloheximide. Also, a strong signal for gc-globulin was detected in the immunoblot of native hepatic stellate cell lysates. However, no mRNA for gc-globulin was found in this cell type, which suggests no active synthesis by hepatic stellate cells. Hepatic stellate cells were tested positively for the presence of known gc-globulin interacting receptors megalin/gp330, HCAM/CD44, cubilin and annexin A2. Inhibition of the megalin/gp330 receptor by a competitive, neutralizing antibody resulted in decreased intracellular availability of gc-globulin in hepatic stellate cells. The latter effect was enhanced by additional incubation of hepatic stellate cells with EDTA for complexing Ca(2+), suggesting a Ca(2+)-dependent internalization of gc-globulin into hepatic stellate cells via the megalin/gp300 receptor. This was supported by confocal microscopy which showed a co-localization of gc-globulin with the multifunctional megalin/gp330 receptor on this cell type. Inside hepatic stellate cells, a linkage between gc-globulin and alpha-smooth muscle actin filaments of hepatic stellate cells was detected by immunocytochemistry. Intracellular binding of gc-globulin to alpha-smooth-muscle actin filaments was confirmed by co-immunoprecipitation. CONCLUSION: We give evidence to the expression of the megalin/gp330 receptor on hepatic stellate cells and that this receptor is involved in the Ca(2+)-dependent internalization of gc-globulin into hepatic stellate cells, a protein synthesized and secreted into the extracellular space and circulation by hepatocytes. Inside hepatic stellate cells, it co-localizes with and binds to alpha-smooth muscle actin filaments. Under consideration of the available literature, these findings propose a participation of gc-globulin in hepatic vitamin D metabolism as well as in hepatic stellate cell stability and apoptosis as important mechanisms of liver regeneration.
Asunto(s)
Calcio/metabolismo , Hepatocitos/metabolismo , Proteína 2 Relacionada con Receptor de Lipoproteína de Baja Densidad/metabolismo , Proteína de Unión a Vitamina D/biosíntesis , Animales , Secuencia de Bases , Células Cultivadas , Cartilla de ADN , Inmunoprecipitación , Masculino , Microscopía Fluorescente , Unión Proteica , Ratas , Ratas Sprague-Dawley , Proteína de Unión a Vitamina D/metabolismoRESUMEN
BACKGROUND: Adult-type hypolactasia is a genetically determined inability to digest lactose after weaning. Two single-nucleotide polymorphisms (C-13910T, G-22018A) located upstream of the lactase gene (LCT) within the gene MCM6 are associated with the lactase persistence/non-persistence trait in patients of European descent. Therefore, the genotyping of these SNPs has been established as a diagnostic tool for adult-type hypolactasia. We have recently shown that several novel allelic variants located in close proximity to the C-13910T SNP interfere with the diagnostic accuracy of real-time PCR-based genotyping methods. METHODS: We describe here the validation of a comprehensive reverse-hybridization teststrip-based assay for the detection of common and novel LCT SNPs (C-13907G, C-13910T, T-13913C, G-13914A, T-13915G, and G-22018A). This assay is based on multiplex DNA amplification and ready-to-use membrane teststrips containing variant-specific oligonucleotide probes immobilized as an array of parallel lines. RESULTS: We evaluated the novel reverse-hybridization StripAssay on 125 DNA samples in comparison to LightCycler analysis and sequencing. The outcome of StripAssay genotyping was found to be completely concordant with that obtained by sequencing. CONCLUSIONS: The StripAssay represents an accurate and robust screening tool to identify multiple LCT/MCM6 variants in a rapid manner. It overcomes diagnostic pitfalls that were reported and allows the simultaneous genotyping of closely spaced LCT variant sites in a single-step diagnostic approach.
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Lactasa/genética , Intolerancia a la Lactosa/diagnóstico , Hibridación de Ácido Nucleico/métodos , Alelos , Austria/epidemiología , Frecuencia de los Genes , Genotipo , Humanos , Intolerancia a la Lactosa/epidemiología , Intolerancia a la Lactosa/genética , Polimorfismo de Nucleótido SimpleRESUMEN
The pathophysiology of liver injury has attracted the interest of experimentalists and clinicians over many centuries. With the discovery of liver-specific pericytes - formerly called fat-storing cells, Ito-cells, lipocytes, and currently designated as hepatic stellate cells (HSC) - the insight into the cellular and molecular pathobiology of liver fibrosis has evolved and the pivotal role of HSC as a precursor cell-type for extracellular matrix-producing myofibroblasts has been established. Although activation and transdifferentiation of HSC to myofibroblasts is still regarded as the pathogenetic key mechanism of fibrogenesis, recent studies point to a prominent heterogeneity of the origin of myofibroblasts. Currently, the generation of matrix-synthesizing fibroblasts by epithelial-mesenchymal transition, by influx of bone marrow-derived fibrocytes into damaged liver tissue, and by differentiation of circulating monocytes to fibroblasts after homing in the injured liver are discussed as important complementary mechanisms to enlarge the pool of (myo-)fibroblasts in the fibrosing liver. Among the molecular mediators, transforming growth factor-beta (TGF-beta) plays a central role, which is controlled by the bone-morphogenetic protein (BMP)-7, an important antagonist of TGF-beta action. The newly discovered pathways supplement the linear concept of HSC activation to myofibroblasts, point to fibrosis as a systemic response involving extrahepatic organs and reactions, add further evidence to a more or less uniform concept of organ fibrosis in general (e.g. liver, lung, kidney), and offer innovative approaches for the development of non-invasive biomarkers and antifibrotic trials.
Asunto(s)
Cirrosis Hepática/patología , Hígado/patología , Células de la Médula Ósea/patología , Movimiento Celular , Transdiferenciación Celular , Células Epiteliales/patología , Proteínas de la Matriz Extracelular/metabolismo , Fibroblastos/patología , Historia del Siglo XIX , Historia del Siglo XX , Historia Antigua , Humanos , Hígado/efectos de los fármacos , Hígado/metabolismo , Cirrosis Hepática/tratamiento farmacológico , Cirrosis Hepática/historia , Cirrosis Hepática/metabolismo , Monocitos/patología , Pericitos/patología , Transducción de SeñalRESUMEN
OBJECTIVE: There is growing evidence that hypertensive pregnancy complications and other adverse pregnancy outcomes are associated with the presence of inherited or acquired thrombophilias. As hemolysis, elevated liver enzymes, low platelets (HELLP) syndrome is one of the most severe forms of pre-eclampsia we aimed to assess the prevalence of the factor V Leiden, the prothrombin 20210G >A mutation and the methylenetetrahydrofolate reductase (MTHFR) 677C >T polymorphism in women with HELLP syndrome and in their fetuses from the same index pregnancy. DESIGN: The study was performed retrospectively in a case-control design. SAMPLE: Seventy-one mother-child pairs with HELLP syndrome and 79 control mother-child pairs with uncomplicated pregnancies were included in the study. METHODS: Genotyping of the three thrombophilic mutations was performed using the LightCycler technology. The chi-squared test was used for statistical analysis. Main outcome measures were maternal and fetal genotypes and their correlation with clinical parameters. RESULTS: Maternal heterozygosity for factor V Leiden was significantly more prevalent in the HELLP group than in controls (OR 4.45, 95% CI 1.31-15.31). No significant association was observed for maternal prothrombin mutation or MTHFR polymorphism (p=0.894, p=0.189, respectively). The fetal genotype was not associated with HELLP syndrome for any of the three mutations investigated. Analysis of gene-gene interactions and genotype-phenotype correlation with respect to clinical parameters and perinatal outcome revealed no further differences. CONCLUSIONS: Our study confirms that women heterozygous for factor V Leiden have an increased risk of developing HELLP syndrome, while the most frequent mutations of the prothrombin and MTHFR gene do not play a major role in the pathogenesis of HELLP syndrome.
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Factor V/genética , Síndrome HELLP/genética , Resistencia a la Proteína C Activada/genética , Adulto , Estudios de Casos y Controles , Femenino , Genotipo , Humanos , Embarazo , Estudios Retrospectivos , Población BlancaRESUMEN
BACKGROUND: In addition to the activation of hepatic stellate cells TGF-beta govern apoptosis and growth control of hepatocytes in liver injury. In non-parenchymal cells, TGF-beta induces plasminogen activator inhibitor 1 (PAI-1) and connective tissue growth factor (CTGF) expression, which are involved in extra cellular matrix formation. Both genes were also regulated by glucocorticoids, which in certain cases showed antagonistic effects to the TGF-beta-Smad 3 pathway. The purpose of our work was to investigate the influence of TGF-beta and dexamethasone on PAI-1 and CTGF expression and secretion in primary hepatocytes. RESULTS: By examining PAI-1 and CTGF mRNA and protein expression in cell lysates and cell-conditioned media under the influence of TGF-beta and dexamethasone, we analysed signalling pathways controlling their expression. TGF-beta and dexamethasone significantly co-induce PAI-1 and CTGF protein expression. On the other hand, we showed that TGF-beta diminished a glucocorticoid receptor dependent luciferase reporter signal in Hep-G2. Inhibition of Erk downstream activation decreased TGF-beta induced CTGF and PAI-1 expression to a basal level. PAI-1 was directly secreted by hepatocytes, whereas secretion of CTGF was retarded. CONCLUSION: The data provide evidence that beside the TGF-beta-Smad 3 pathway CTGF and PAI-1 expression is additionally dependent on Erk activity in hepatocytes giving new insights into regulation of the profibrogenic proteins.