transforming-growth-factor-beta and Liver-Diseases

Genetic alterations affecting transforming growth factor-β (TGF-β) signaling are exceptionally common in diseases and cancers of the gastrointestinal system. As a regulator of tissue renewal, TGF-β signaling and the downstream SMAD-dependent transcriptional events play complex roles in the transition from a noncancerous disease state to cancer in the gastrointestinal tract, liver, and pancreas. Furthermore, this pathway also regulates the stromal cells and the immune system, which may contribute to evasion of the tumors from immune-mediated elimination. Here, we review the involvement of the TGF-β pathway mediated by the transcriptional regulators SMADs in disease progression to cancer in the digestive system. The review integrates human genomic studies with animal models that provide clues toward understanding and managing the complexity of the pathway in disease and cancer.

Topics: Animals; Digestive System Neoplasms; Disease Progression; Gastrointestinal Diseases; Gene Expression Regulation, Neoplastic; Humans; Liver Diseases; Pancreatic Diseases; Receptors, Transforming Growth Factor beta; Signal Transduction; Smad Proteins; Transforming Growth Factor beta; Tumor Microenvironment

Almost all patients with chronic liver diseases (CLD) show altered bone metabolism. Depending on the etiology, this manifests in a severe osteoporosis in up to 75% of the affected patients. Due to high prevalence, the generic term hepatic osteodystrophy (HOD) evolved, describing altered bone metabolism, decreased bone mineral density, and deterioration of bone structure in patients with CLD. Once developed, HOD is difficult to treat and increases the risk of fragility fractures. Existing fractures affect the quality of life and, more importantly, long-term prognosis of these patients, which presents with increased mortality. Thus, special care is required to support the healing process. However, for early diagnosis (reduce fracture risk) and development of adequate treatment strategies (support healing of existing fractures), it is essential to understand the underlying mechanisms that link disturbed liver function with this bone phenotype. In the present review, we summarize proposed molecular mechanisms favoring the development of HOD and compromising the healing of associated fractures, including alterations in vitamin D metabolism and action, disbalances in transforming growth factor beta (TGF-β) and bone morphogenetic protein (BMP) signaling with histone deacetylases (HDACs) as secondary regulators, as well as alterations in the receptor activator of nuclear factor kappa B ligand (RANKL)-osteoprotegerin (OPG) system mediated by sclerostin. Based on these mechanisms, we give an overview on the limitations of early diagnosis of HOD with established serum markers.

Topics: Animals; Bone Diseases, Metabolic; Bone Morphogenetic Proteins; Calcium; Humans; Liver Diseases; Transforming Growth Factor beta; Vitamin D

Transforming growth factor β (TGF-β) belongs to a class of pleiotropic cytokines that are involved in the processes of embryonic development, wound healing, cell proliferation, and differentiation. Moreover, TGF-β is also regarded as a central regulator in the pathogenesis and development of various liver diseases because it contributes to almost all of the stages of disease progression. A range of liver cells are considered to secrete TGF-β ligands and express related receptors and, consequently, play a crucial role in the progression of liver disease via different signal pathways. In this manuscript, we review the role of the TGF-β signaling pathway in liver disease and the potential of targeting the TGF-β signaling in the pharmacological treatment of liver diseases.

Topics: Animals; Humans; Liver Diseases; Receptors, Transforming Growth Factor beta; Transforming Growth Factor beta

After hepatitis virus infection, plasma transforming growth factor (TGF)-β increases in either the acute or chronic inflammatory microenvironment. Although TGF-β is upregulated in patients with hepatocellular carcinoma, it is one of the most potent growth inhibitors for hepatocytes. This cytokine also upregulates extracellular matrix (ECM) production of hepatic stellate cells. Therefore, TGF-β is considered to be the major factor regulating liver carcinogenesis and accelerating liver fibrosis. Smad2 and Smad3 act as the intracellular mediators of TGF-β signal transduction pathway. We have generated numerous antibodies against individual phosphorylation sites in Smad2/3, and identified 3 types of phosphorylated forms (phospho-isoforms): COOH-terminally phosphorylated Smad2/3 (pSmad2C and pSmad3C), linker phosphorylated Smad2/3 (pSmad2L and pSmad3L) and dually phosphorylated Smad2/3 (pSmad2L/C and pSmad3L/C). These Smad phospho-isoforms are categorized into 3 groups: cytostatic pSmad3C signaling, mitogenic pSmad3L signaling and invasive/fibrogenic pSmad2L/C signaling. In this review, we describe differential regulation of TGF-β/Smad signaling after acute or chronic liver injuries. In addition, we consider how chronic inflammation associated with hepatitis virus infection promotes hepatic fibrosis and carcinogenesis (fibro-carcinogenesis), focusing on alteration of Smad phospho-isoform signaling. Finally, we show reversibility of Smad phospho-isoform signaling after therapy against hepatitis virus infection.

Topics: Animals; Gene Expression Regulation; Gene Expression Regulation, Neoplastic; Hepatitis, Viral, Human; Humans; Liver Diseases; Phosphorylation; Signal Transduction; Smad Proteins; Transforming Growth Factor beta

Curcumin is a principal polyphenolic curcuminoid extracted from turmeric rhizome, which has been used for treating inflammation of joints, ulcers, jaundice and other disorders in Asian traditional medicine. In recent years, many studies have indicated that curcumin plays important roles in treatment of liver diseases. Curcumin attenuates liver injury and non-alcoholic fatty liver disease by lowering the release of inflammation cytokines, minimizing oxidative stress, enhancing the sensitivity of insulin and altering lipid metabolism. Curcumin shows potent anti-fibrosis activity, contributing to inhibit the activation of hepatic stellate cells and reduce the deposition of extracellular matrix by its regulation of PPAR-γ, NF-ΚB and TGF-β signaling pathways. Moreover, curcumin exhibits anti-cancer effect by inducing G2/M phase cell cycle arrest and apoptosis in several hepatoma cell lines. However, poor water solubility and low bioavailability of curcumin limit its clinical applications. To overcome its limited systemic bioavailability, many new approaches have been explored to deliver curcumin effectively. This article focuses on advances in the effects of curcumin and its derivatives for treatment of liver injury, non-alcoholic fatty liver disease, liver fibrosis and hepatocarcinoma.

Topics: Apoptosis; Cell Cycle Checkpoints; Cell Line, Tumor; Curcumin; Hepatic Stellate Cells; Humans; Inflammation; Liver Diseases; NF-kappa B; Oxidative Stress; PPAR gamma; Signal Transduction; Transforming Growth Factor beta

A wide range of pathophysiological changes are involved in the progression of chronic liver disease (CLD). The multifunctional cytokine transforming growth factor β (TGFβ) can cross talk with other signaling pathways and mediate nearly every aspect of cell physiology from growth to differentiation and cell death. TGFβ can maintain immune response homeostasis and mediate wound-healing responses. Additionally, not only can TGFβ prevent the occurrence of hepatocarcinoma by regulating the proliferation of hepatic progenitor cells, TGFβ can also play important roles in blunting inflammation by interfering with tumor inhibition response generated by inflammatory mediators. The TGFβ/Smad signaling pathway is an important response that is mediated by TGFβ. This pathway also plays multiple functions in the progression of chronic liver disease. The pleiotropic effects of TGFβ and TGFβ/Smad signaling create challenges in the treatment of CLD under conditions that might interfere with the TGFβ signaling pathway. Detailed understanding of the pleiotropic roles of TGFβ signaling in CLD might assist in the selection of more accurate therapeutic approaches, and in the targeting of the right type of cell in the development of treatment strategies, and ultimately in achieving the desired therapeutic effects.

Topics: Animals; Cell Differentiation; Chronic Disease; Disease Progression; Endothelial Cells; Genetic Pleiotropy; Humans; Liver; Liver Diseases; Signal Transduction; Smad Proteins; Transforming Growth Factor beta

Transforming growth factor-β (TGF-β) is a central regulator in chronic liver disease contributing to all stages of disease progression from initial liver injury through inflammation and fibrosis to cirrhosis and hepatocellular carcinoma. Liver-damage-induced levels of active TGF-β enhance hepatocyte destruction and mediate hepatic stellate cell and fibroblast activation resulting in a wound-healing response, including myofibroblast generation and extracellular matrix deposition. Being recognised as a major profibrogenic cytokine, the targeting of the TGF-β signalling pathway has been explored with respect to the inhibition of liver disease progression. Whereas interference with TGF-β signalling in various short-term animal models has provided promising results, liver disease progression in humans is a process of decades with different phases in which TGF-β or its targeting might have both beneficial and adverse outcomes. Based on recent literature, we summarise the cell-type-directed double-edged role of TGF-β in various liver disease stages. We emphasise that, in order to achieve therapeutic effects, we need to target TGF-β signalling in the right cell type at the right time.

Topics: Animals; Chronic Disease; Disease Progression; Epithelial-Mesenchymal Transition; Humans; Liver Diseases; Liver Neoplasms; Signal Transduction; Transforming Growth Factor beta

Unbalanced Th1/Th2 T-cell responses in the liver are a characteristic of hepatic inflammation and subsequent liver fibrosis. The recently discovered Th17 cells, a subtype of CD4(+) T-helper cells mainly producing IL-17 and IL-22, have initially been linked to host defense against infections and to autoimmunity. Their preferred differentiation upon TGFβ and IL-6, two cytokines abundantly present in injured liver, makes a contribution of Th17 cells to hepatic inflammation very likely. Indeed, initial studies in humans revealed activated Th17 cells and Th17-related cytokines in various liver diseases. However, functional experiments in mouse models are not fully conclusive at present, and the pathogenic contribution of Th17 cells to liver inflammation might vary upon the disease etiology, for example, between infectious and autoimmune disorders. Understanding the chemokines and chemokine receptors promoting hepatic Th17 cell recruitment (possibly CCR6 or CCR4) might reveal new therapeutic targets interfering with Th17 migration or differentiation in liver disease.

Topics: Animals; Autoimmune Diseases; Autoimmunity; Cell Differentiation; Cell Movement; Chemical and Drug Induced Liver Injury; Concanavalin A; Disease Models, Animal; Humans; Infections; Inflammation; Interleukin-17; Interleukin-22; Interleukins; Liver; Liver Diseases; Mice; Receptors, Chemokine; T-Lymphocytes, Regulatory; Th17 Cells; Transforming Growth Factor beta

A large body of work has established roles for epithelial cells as important mediators of progressive fibrosis and carcinogenesis. Transforming growth factor-beta (TGF-beta) and pro-inflammatory cytokines are important inducers of fibro-carcinogenesis. TGF-beta signaling involves phosphorylation of Smad3 at middle linker and/or C-terminal regions. Reversible shifting of Smad3-dependent signaling between tumor-suppression and oncogenesis in hyperactive Ras-expressing epithelial cells indicates that Smad3 phosphorylated at the C-terminal region (pSmad3C) transmits a tumor-suppressive TGF-beta signal, while oncogenic activities such as cell proliferation and invasion are promoted by Smad3 phosphorylated at the linker region (pSmad3L). Notably, pSmad3L-mediated signaling promotes extracellular matrix deposition by activated mesenchymal cells. During progression of chronic liver diseases, hepatic epithelial hepatocytes undergo transition from the tumor-suppressive pSmad3C pathway to the fibrogenic/oncogenic pSmad3L pathway, accelerating liver fibrosis and increasing risk of hepatocellular carcinoma. c-Jun N-terminal kinase activated by pro-inflammatory cytokines is mediating this perturbed hepatocytic TGF-beta signaling. Thus, TGF-beta signaling of hepatocytes affected by chronic inflammation offers a general framework for understanding the molecular mechanisms of human fibro-carcinogenesis during progression of chronic liver diseases.

Topics: Chronic Disease; Disease Progression; Humans; Liver Diseases; Signal Transduction; Transforming Growth Factor beta

Hereditary hemorrhagic telangiectasia (HHT) is characterized by vessel alterations such as dilatation of postcapillary venules and arterio-venous communications, which account for the major clinical manifestations of the disease. Two types of HHT have been characterized HHT-1 and HHT-2, respectively, depending the former on endoglin mutations and the latter on activin receptor-like kinase 1 (ALK-1) mutations. Both endoglin and ALK-1 bind to the transforming growth factor (TGF) superfamily which, physiologically, regulates the activities of endothelial cells and also those related to the extracellular matrix. In this review, the salient features of TGF-beta will be outlined with special reference to its activity on the immune system and on tumorigenesis. Furthermore, the involvement of TGF-beta in the pathogenesis of some gastrointestinal diseases will be discussed and, in particular, in the course of liver disease, Helicobacter pylori infection and inflammatory bowel disease. In the light of these data and of animal model of HHT, the potential risk of developing other diseases in HHT patients will be discussed.

Topics: Gastrointestinal Diseases; Humans; Liver Diseases; Telangiectasia, Hereditary Hemorrhagic; Transforming Growth Factor beta

Transforming growth factor-beta1 (TGF-beta1) is released during the storage of blood components, particularly platelet concentrates, and transfusion recipients are exposed to high levels of TGF-beta1. Because TGF-beta1 is one of the most potent immunosuppressive cytokines known, understanding the immunobiologic functions of TGF-beta1 may be relevant for understanding the immunobiologic effects of transfusion. Our laboratory studies the biologic effects of TGF-beta1 in the immune system. Mice deficient in TGF-beta1 spontaneously develop autoimmunity, confirming the important role of this cytokinean an immune regulator. A few years ago, my laboratory made the observation that genetic background strongly affects the phenotype of TGF-beta1-/- mice. TGF-beta1-/- mice on the BALB/c background rapidly develop an aggressive T-cell-mediated hepatitis, whereas TGF-beta1-/- mice on the 129/CF-1 background do not. In this review, I summarize findings published or in press from our laboratory on disease pathogenesis in TGF-beta1-/- mice and then discuss some of the exciting (as-yet-unpublished) directions our laboratory is currently taking.

Topics: Animals; Autoimmune Diseases; Humans; Liver; Liver Diseases; Mice; Species Specificity; Th1 Cells; Transforming Growth Factor beta; Transforming Growth Factor beta1; Transfusion Reaction

Hepatic dendritic cells (DC) unquestionably play important roles in the induction and regulation of immune responses. Due to their paucity, functional characterisation of these important antigen presenting cells has been slow but use of DC growth factors (in particular GM-CSF and Flt3L) that markedly enhance their numbers has proved helpful in furnishing adequate study material. While there is growing evidence that DC function is affected in the pathogenesis of liver disease, most work to date has been performed on non-hepatic DC. Increasing knowledge of hepatic DC biology is likely to improve our understanding of disease pathogenesis and resistance to and therapy of liver disease.

Topics: Antigens, CD; Carcinoma, Hepatocellular; Cell Count; Chemotaxis; Dendritic Cells; Hepatitis, Viral, Human; Humans; Immune Tolerance; Immunity, Cellular; Leukocytes; Liver; Liver Diseases; Liver Neoplasms; Liver Transplantation; Lymph; Lymphoid Tissue; Phagocytosis; Phenotype; Stem Cells; T-Lymphocytes; Transforming Growth Factor beta

TGF-beta is a cytokine with varied properties and pleiotropic activity. It is released in an inactive form. To exhibit its biological activity, it requires binding to extracellular matrix proteins and, after that, proteolytic elimination of LAP (Latent Associated Protein) and LTBP (Latent TGF-beta Binding Protein). The process involves, among others, tissue transglutaminase, thrombin and plasmin. By stimulation of specific receptors, it influences transcription of some genes and translation of formed mRNA. Locally, it demonstrates proinflammatory properties whereas systemically, it has primarily a potent immunosuppressive effect. TGF-beta, by affecting proliferation, differentiation and migration of cells, as well as stimulation of extracellular matrix protein production, plays an important role in tissue regeneration and remodeling, but also in fibrosis. TGF-beta is also indispensable to maintain immune homeostasis of the organism. Reduced TGF-beta activity is considered to be responsible for development of autoimmune disorders in the course of several pathologic conditions. This cytokine plays an important role in the pathogenesis of chronic inflammatory processes taking place, among others, in inflammatory bowel diseases (IBD) and chronic hepatitis B and C. The paper presents a review of literature concerning diagnostic and prognostic value of TGF-beta level determinations in blood and tissue bioptates of patients with chronic non-specific enteritis and chronic hepatitis B and C.

Topics: Adult; Animals; Biomarkers; Child; Humans; Inflammation; Liver Diseases; Pediatrics; Prognosis; Protein Isoforms; Transforming Growth Factor beta

A variety of biological functions are regulated through extracellular signals. Amongst the best studied examples is growth control, which is achieved by the regulatory function of growth factors. In recent years it has become apparent that cell death (apoptosis) is controlled in a similar fashion.Apoptosis, firstly a morphologically defined process, is a highly controlled type of cell death that plays a critical role in embryonic development, deletion of autoreactive T-cells and adult tissue homoeostasis. There is increasing evidence that derangement of the apoptotic program is the underlying cause of a series of diseases including liver diseases. The deadly program can be initiated by ligand binding to membrane bound receptors such as CD95 (Fas), which is the most prominent cell death inducing member of the TNF receptor superfamily. The core of the subsequently activated intracellular machinery is formed by a set of proteases, namely caspases. Once activated, they orchestrate the complete destruction of the cellular skeleton leading to the typical apoptotic morphology. This review focuses on the underlying mechanism leading to derangement of the usually highly controlled apoptotic program in different liver diseases.

Topics: Animals; Antigens, CD; Apoptosis; Caspases; fas Receptor; Humans; Ligands; Liver Diseases; Receptors, Tumor Necrosis Factor; Receptors, Tumor Necrosis Factor, Type I; Transforming Growth Factor beta; Transforming Growth Factor beta1

Transforming growth factor (TGF-beta) is the protein playing a principal role in the intracellular signalling. The most important function is ability to stimulate synthesis of extracellular matrix proteins, what is responsible for wound formation and tissue reconstruction. The damage of hepatocytes is a signal for macrophages and platelets activation, resulting in release of TGF-beta and over-expression of genes responsible for morphologic and functional changes in Ito cells. They undergo transformation into myofibroblasts and become the source of extracellular matrix proteins, such as: collagens, fibronectin, laminin, entactin, tenascin, undulin. The consequence of their accumulation in the space of Disse and inside hepatic lobuli is fibrosis, which is the form of tissue healing in the place of necrosis. However continuous action of damaging agent leads to massive fibrosis and reconstruction of liver, what is clinically manifested as cirrhosis. The role of transforming growth factor beta in the pathogenesis of liver diseases and its possible use as an indicator of disease progression were discussed in this paper.

Topics: Disease Progression; Humans; Liver Diseases; Transforming Growth Factor beta

Cell death by apoptosis is thought to be involved in various pathophysiological situations involving the liver. Indeed, an understanding of apoptosis is becoming increasingly helpful for understanding disease and for patients' care. In this article, we review current scientific and clinical concepts of apoptosis, including death factors such as Fas ligand and tumor necrosis factor, apoptotic signal transduction mechanisms, and the role of intracellular proteinases called caspases. We also discuss apoptosis in the liver, as related to ischemia/reperfusion injury, cholestasis, and cancer, circumstances which physicians often face in the field of the liver surgery.

Topics: Apoptosis; Humans; Liver Diseases; Liver Neoplasms; Reperfusion Injury; Signal Transduction; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

TGF-beta 1 is a potent inhibitor of hepatocyte proliferation in vivo and in culture and an inducer of fibrogenesis. It is produced by non-parenchymal cells in normal, regenerating, neoplastic and pre-neoplastic liver. TGF-beta 2 and beta 3 are also found in liver non-parenchymal cells and the amounts of their mRNAs increase during liver regeneration. TGF-beta 2 has similar effects to TGF-beta 1. Membranes from normal adult rat liver bind TGF-beta 1 with kinetics consistent with the presence of a single high affinity binding site; membranes from livers that have been regenerating for 12-72 hours show high affinity binding sites not detected in livers of normal or sham-operated rats. Affinity labelling of membranes from normal and regenerating liver shows two receptor proteins with Mr 85,000 and 65,000. In contrast, a prominent band corresponding to a binding protein of Mr 280,000 is detected in membrane preparations of cultured liver epithelial cells. Although modulation of TGF-beta 1 receptors occurs during liver regeneration, it has not been possible to determine which receptor is responsible for the TGF-beta 1 effects in hepatocytes. Other studies have demonstrated a significant correlation between TGF-beta 1 mRNA expression and various indicators of fibrogenesis in patients with chronic liver disease. Thus in animals and humans TGF-beta 1 appears to play a major role in the pathogenesis of fibrosis in chronic liver disease.

Topics: Animals; Cell Division; Extracellular Matrix Proteins; Fibrosis; Gene Expression Regulation; Humans; Liver; Liver Diseases; Liver Neoplasms, Experimental; Liver Regeneration; Rats; Receptors, Cell Surface; Receptors, Transforming Growth Factor beta; RNA, Messenger; Transforming Growth Factor beta

Topics: Animals; Cytokines; Ephedrine; Hepatitis; Liver; Liver Diseases; Oxidative Stress; Rats; Rats, Sprague-Dawley; Transforming Growth Factor beta

The study aimed to investigate the potential attenuation effect of chitosan in liver ischemia/reperfusion injury (I/R), and its relevant protective mechanisms. Chitosan (200 mg/kg) has been administered orally for 30 days, later animals underwent liver 45 min ischemia and reperfusion for 60 min. Following treatment with chitosan, the levels of serum aminotransferases and lactate dehydrogenase were significantly reduced. Similarly, hepatic (GSH, SOD, CAT, GST and GPx) were enhanced, and the level of tissue malondialdehyde (MDA) was decreased. In addition, inflammatory cytokinesis (TNF-α and TGF-β) have recorded a significant decrease in their mRNA expression and protein levels using qPCR and ELISA respectively. Marked reduction of apoptosis has been indicated by the elevation in BCL2, and decreasing in BAX, Caspace-3 and Cytochrome-c expression levels, which furthermore confirmed by DNA fragmentation assay. The enhancement of the previous parameters resulted in a marked improvement in the liver architectures after chitosan administration. In conclusion, chitosan has proved its efficiency as an anti-inflammatory and antioxidant agent through its inhibitory effect of cytokines and reducing ROS respectively. In addition, chitosan could modulate the changes in histological structure and alleviate apoptosis induced by liver I/R, which recommend it as an efficient agent for protection against liver I/R injury.

Topics: Alanine Transaminase; Animals; Anti-Inflammatory Agents; Antioxidants; Apoptosis; Aspartate Aminotransferases; bcl-2-Associated X Protein; Caspase 3; Chitosan; Cytokines; Inflammation; Liver; Liver Diseases; Male; Malondialdehyde; Oxidative Stress; Protective Agents; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Wistar; Reactive Oxygen Species; Reperfusion Injury; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

We have evaluated the protective mechanism of tanshinone IIA in ischemia/reperfusion injury (IRI) induced by liver grafts, revealing novel supplementary immunotherapy for liver transplantation.. The tanshinone IIA preconditioning group (TP group) was pretreated with tanshinone IIA via intraperitoneal injection for 1 week before receiving orthotopic liver transplantation with hepatic arterial ischemia for 30min. The sham-operation group (SO group), control graft group (CG group) and IRI group were pretreated with an equivalent volume of normal saline. The IRI group and CG group received orthotopic liver transplantation with or without hepatic arterial ischemia. Rats were sacrificed at each time point, serum was collected for ELISA detection, and Kupffer cells (KCs) were isolated to extract total protein and RNA for western blotting and real-time PCR, respectively.. The levels of TNF-α and IL-4 in the TP group were significantly lower than those of in the IRI group; meanwhile the IL-10 and TGF-β levels were significantly higher than in the IRI group. The protein and mRNA expression levels of HMGB1 were significantly lower in TP group than in the IRI group at each time point. The TLR-4, Myd88, NLRP3 and p-NF-κb p65 expression levels in the TP groups were significantly lower than those in the IRI group, while the PTEN, PI3K and AKT phosphorylation levels in the TP groups were significantly higher than those in the IRI group.. Tanshinone IIA attenuates IRI caused by liver grafts via down-regulation of the HMGB1-TLR-4/NF-κb pathway in KCs and activation of PTEN/PI3K/AKT pathway, suggesting a potential role for prevention of liver cell IRI during liver transplantation.

Topics: Abietanes; Animals; HMGB1 Protein; Interleukin-10; Interleukin-4; Kupffer Cells; Liver; Liver Diseases; Liver Transplantation; Male; Myeloid Differentiation Factor 88; NF-kappa B; NLR Family, Pyrin Domain-Containing 3 Protein; Phosphatidylinositol 3-Kinases; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Signal Transduction; Toll-Like Receptor 4; Transcription Factor RelA; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Postnatal intake of an energy dense diet, the Western diet (WD), is a strong risk factor for liver fibrosis. Recently, adverse in utero conditions resulting in low birth weight (LBW) have also been associated with postnatal fibrosis development. We assessed the independent and possible synergistic effects of placental insufficiency-induced LBW and postnatal WD consumption on liver fibrosis in early adulthood, with a specific focus on changes in inflammation and apoptosis pathways in association with fibrogenesis. Male LBW (uterine artery ablation) and normal birth weight (NBW) guinea pig pups were fed either a control diet (CD) or WD from weaning to 150 days. Significant steatosis, mild lobular inflammation, apoptosis and mild stage 1 fibrosis (perisinusoidal or portal) were evident in WD-fed offspring (NBW/WD and LBW/WD). In LBW/CD versus NBW/CD offspring, increased transforming growth factor-beta 1 and matrix metallopeptidase mRNA and sma- and Mad-related protein 4 (SMAD4) were present in conjunction with minimal stage 1 portal fibrosis. Further, connective tissue growth factor mRNA was increased and miR-146a expression decreased in LBW offspring, irrespective of diet. Independent of birth weight, WD-fed offspring exhibited increased expression of fibrotic genes as well as elevated inflammatory and apoptotic markers. Moreover, the augmented expression of collagen, type III, alpha 1 and tumor necrosis factor-alpha was associated with increased recruitment of RNA polymerase II and enhanced histone acetylation (K9) to their respective promoters. These data support a role for both LBW and postnatal WD as factors contributing to hepatic fibrosis development in offspring through distinct pathways.

Topics: Animals; Apoptosis; Birth Weight; Collagen; Diet, High-Fat; Female; Fibrosis; Guinea Pigs; Histones; Liver; Liver Diseases; Male; Matrix Metalloproteinases; MicroRNAs; RNA Polymerase II; Smad Proteins; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Background and Objective. High-cholesterol diet (HCD) intends to increase the oxidative stress in liver tissues inducing hepatotoxicity. Rutin is a natural flavonoid (vitamin p) which is known to have antioxidative properties. The aim of the present study was to investigate the potential effects of Rutin on hypercholesterolemia-induced hepatotoxicity in rats. Materials and Methods. Male Wistar rats were divided into four groups: G-I control, G-II Rutin, G-III HCD, and G-IV Rutin + HCD. The liver functions and lipid profile were used to evaluate the HCD-induced hepatotoxicity. Quantitative real time-PCR was carried out to evaluate the expression levels of genes in TGF-β/Smad signaling pathway. Results. Rutin in combination with HCD showed a significant protective effect against hepatotoxicity. HCD caused significant increase in the mRNA expression of transforming growth factor beta (TGF-β), Mothers Against Decapentaplegic Homolog 2 (Smad-2), Mothers Against Decapentaplegic Homolog 4 (Smad-4), Bcl-2-binding component 3 (Bbc3), caspase-3, P53 and Interleukin-6 (IL-6) and decrease in the expression levels of Cyclin depended kinase inhibitor (P21) and Interleukin-3 (IL-3) in hepatic cells. Conclusion. TGF-β/Smad signaling pathway is involved in HCD-induced hepatotoxicity and Rutin inhibits the hepatotoxicity via suppressing this pathway. Therefore, Rutin might be considered as a protective agent for hepatotoxicity.

Topics: Alanine Transaminase; Animals; Aspartate Aminotransferases; Body Weight; Caspase 3; Cholesterol; Diet, High-Fat; Interleukins; Liver; Liver Diseases; Male; Organ Size; Rats, Wistar; Rutin; Smad Proteins; Transforming Growth Factor beta

Transforming growth factor β (TGF-β) is cytostatic towards damage-induced compensatory hepatocyte proliferation. This function is frequently lost during hepatocarcinogenesis, thereby switching the TGF-β role from tumour suppressor to tumour promoter. In the present study, we investigate Smad7 overexpression as a pathophysiological mechanism for cytostatic TGF-β inhibition in liver damage and hepatocellular carcinoma (HCC). Transgenic hepatocyte-specific Smad7 overexpression in damaged liver of fumarylacetoacetate hydrolase (FAH)-deficient mice increased compensatory proliferation of hepatocytes. Similarly, modulation of Smad7 expression changed the sensitivity of Huh7, FLC-4, HLE and HLF HCC cell lines for cytostatic TGF-β effects. In our cohort of 140 HCC patients, Smad7 transcripts were elevated in 41.4% of HCC samples as compared with adjacent tissue, with significant positive correlation to tumour size, whereas low Smad7 expression levels were significantly associated with worse clinical outcome. Univariate and multivariate analyses indicate Smad7 levels as an independent predictor for overall (P<0.001) and disease-free survival (P=0.0123). Delineating a mechanism for Smad7 transcriptional regulation in HCC, we identified cold-shock Y-box protein-1 (YB-1), a multifunctional transcription factor. YB-1 RNAi reduced TGF-β-induced and endogenous Smad7 expression in Huh7 and FLC-4 cells respectively. YB-1 and Smad7 mRNA expression levels correlated positively (P<0.0001). Furthermore, nuclear co-localization of Smad7 and YB-1 proteins was present in cancer cells of those patients. In summary, the present study provides a YB-1/Smad7-mediated mechanism that interferes with anti-proliferative/tumour-suppressive TGF-β actions in a subgroup of HCC cells that may facilitate aspects of tumour progression.

Topics: Animals; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Cells, Cultured; Female; Gene Expression Regulation, Neoplastic; Hep G2 Cells; Hepatocytes; Humans; Liver Diseases; Liver Neoplasms; Male; Mice, 129 Strain; Mice, Knockout; Mice, Transgenic; Microscopy, Confocal; Middle Aged; Multivariate Analysis; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; Smad7 Protein; Survival Analysis; Transforming Growth Factor beta; Y-Box-Binding Protein 1

Collagen triple helix repeat containing-1 (Cthrc1) is a documented specific inhibitor of TGF-β signaling. Based on this observation, we developed the hypothesis that knocking in/knocking out the Cthrc1 gene in murine models of cholestasis would alter the natural history of cholestatic fibrosis. To study this thesis, we studied two murine models of fibrosis, first, common bile duct ligation (CBDL) and second, feeding of 3, 5-diethoxy-carbonyl-1, 4-dihydrocollidine (DDC). In both models, we administered well-defined adenoviral vectors that expressed either Cthrc1 or, alternatively, a short hairpin RNA (shRNA)-targeting Cthrc1 either before or after establishment of fibrosis. Importantly, when Cthrc1 gene expression was enhanced, we noted a significant improvement of hepatic fibrosis, both microscopically and by analysis of fibrotic gene expression. In contrast, when Cthrc1 gene expression was deleted, there was a significant exacerbation of fibrosis. To identify the mechanism of action of these significant effects produced by knocking in/knocking out Cthrc gene expression, we thence studied the interaction of Cthrc1 gene expression using hepatic stellate cells (HSCs) and human LX-2 cells. Importantly, we demonstrate that Cthrc1 is induced by TGF-β1 via phospho-Smad3 binding to the promoter with subsequent transcription activation. In addition, we demonstrate that Cthrc1 inhibits TGF-β signaling by accelerating degradation of phospho-Smad3 through a proteosomal pathway. Importantly, the anti-fibrotic effects can be recapitulated with a truncated fragment of Cthrc1. In conclusion, our findings uncover a critical negative feedback regulatory loop in which TGF-β1 induces Cthrc1, which can attenuate fibrosis by accelerating degradation of phospho-Smad3.

Topics: Animals; Autoimmune Diseases; Cell Line; Cholestasis, Intrahepatic; Extracellular Matrix Proteins; Gene Expression Regulation; Genetic Therapy; Hepatic Stellate Cells; Humans; Liver Cirrhosis; Liver Diseases; Mice; Mice, Knockout; Pyridines; Smad3 Protein; Transforming Growth Factor beta

This study was designed to investigate the effect of recombinant sTGFβ1RII and sIL13Rα2 receptor proteins on schistosomiasis japonica, hepatic fibrosis and the expression of SMAD3 and STAT6. The proteins sTGFβ1RII and sIL13Rα2 were expressed in Escherichiacoli, purified using affinity chromatography and characterized by Western blotting. Female BALB/C mice (48) were randomly divided into eight groups and infected with Schistosoma japonicum. Five weeks after infection, test groups were injected with the recombinant proteins at different doses. Eight weeks after infection, lung and hepatic tissue samples were obtained and stained with hematoxylin and eosin (HE) and Masson's trichrome. Immunohistochemical staining was used to detect the expression of SMAD3 and STAT6. The recombinant proteins sTGFβ1RII and sIL13Rα2 were successfully expressed, purified, and characterized. The granuloma area, hepatic hydroxyproline (HYP) level and hepatic fibrosis of the protein therapeutic groups were significantly smaller than those of the positive control group (P<0.01). Treatment with sTGFβ1RII was more effective when the protein was administered for 4weeks rather than 2 (P<0.01). Hepatic fibrosis in the groups using a low dose of protein sTGFβ1 was lower that of the combination group (P<0.05). The expression level of STAT6 was significantly lower in groups treated with sIL13Rα2 than in groups not treated with the protein (P<0.01). The recombinant proteins TGFβ1RII and sIL13Rα2 were able to decrease granuloma area and hepatic fibrosis in schistosomiasis japonica, and also reduced the expression of the signal transduction proteins SMAD3 and STAT6. The proteins were more effective when used in combination than when applied singly.

Topics: Animals; Disease Models, Animal; Eukaryotic Initiation Factors; Extracellular Matrix Proteins; Female; Granuloma; Hydroxyproline; Interleukin-13; Interleukin-13 Receptor alpha2 Subunit; Intracellular Signaling Peptides and Proteins; Liver; Liver Cirrhosis; Liver Diseases; Lung; Mice; Mice, Inbred BALB C; Pulmonary Fibrosis; Random Allocation; Real-Time Polymerase Chain Reaction; Recombinant Proteins; Schistosomiasis japonica; Signal Transduction; Smad Proteins; STAT6 Transcription Factor; Transforming Growth Factor beta

The regenerative response to drug- and toxin-induced liver injury induces changes to the hepatic stroma, including the extracellular matrix. Although the extracellular matrix is known to undergo changes during the injury response, its impact on maintaining hepatocyte function and viability in this process remains largely unknown. We demonstrate that recovery from toxin-mediated injury is impaired in mice deficient in a key liver extracellular matrix molecule, type XVIII collagen, and results in rapid death. The type-XVIII-collagen-dependent response to liver injury is mediated by survival signals induced by α1β1 integrin, integrin linked kinase and the Akt pathway, and mice deficient in either α1β1 integrin or hepatocyte integrin linked kinase also succumb to toxic liver injury. These findings demonstrate that type XVIII collagen is an important functional component of the liver matrix microenvironment and is crucial for hepatocyte survival during injury and stress.

Topics: Animals; Carbon Tetrachloride; Cell Death; Collagen Type XVIII; Extracellular Matrix; Gene Expression Regulation; Hepatocytes; Integrin alpha1beta1; Liver; Liver Diseases; Mice; Protein Binding; Signal Transduction; Survival Analysis; Transforming Growth Factor beta

The aim of the present study was to establish a progressive steatohepatitis mouse model because few reported animal models of non-alcoholic steatohepatitis (NASH) show the progression from fatty liver to steatohepatitis. C57BL/6N mice were fed a high-fat diet (HFD) to develop obesity and were either administered carbon tetrachloride (CCl4 ) eight times (0.05 mL/kg, s.c., once, followed by 0.1 mL/kg, s.c., seven times) or not. Serum parameters and hepatic histopathology were examined. In a separate experiment, CCl4 was administered subcutaneously from 0 to eight times to HFD-fed obese mice to investigate progressive changes. Markers of oxidative stress, inflammation and apoptosis, as well as histopathological changes in the liver, were analysed. The HFD-fed obese mice showed fatty liver but not steatohepatitis. In contrast, HFD-fed mice administered CCl4 eight times showed histopathological features of steatohepatitis (fatty liver, inflammation, hepatocellular ballooning and fibrosis) and increased serum alanine aminotransferase levels. However, the multiple administration of CCl4 to obese mice reduced the ratio of reduced glutathione to oxidized glutathione, superoxide dismutase activity and mitochondrial DNA copy number, leading to the development of chronic oxidative stress, increased numbers of apoptotic cells and increased levels of both tumour necrosis factor-α and transforming growth factor-β mRNA. The resulting inflammation led to increased hydroxyproline content in the liver and fibrosis. The present study demonstrates that multiple administration of CCl4 to HFD-fed obese mice induces chronic oxidative stress that triggers inflammation and apoptosis and leads to the development of fibrosis in the liver, resulting in progression from fatty liver to steatohepatitis. This murine model will be useful in the research of hepatic disorders.

Topics: Alanine Transaminase; Animals; Apoptosis; Carbon Tetrachloride; Diet, High-Fat; Disease Models, Animal; DNA Copy Number Variations; DNA, Mitochondrial; Fatty Liver; Fibrosis; Glutathione; Glutathione Disulfide; Inflammation; Liver; Liver Diseases; Male; Mice; Mice, Inbred C57BL; Non-alcoholic Fatty Liver Disease; Obesity; Oxidative Stress; RNA, Messenger; Superoxide Dismutase; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Human autosomal recessive polycystic kidney disease (ARPKD) produces kidneys which are massively enlarged due to multiple cysts, hypertension, and congenital hepatic fibrosis characterized by dilated bile ducts and portal hypertension. The PCK rat is an orthologous model of human ARPKD with numerous fluid-filled cysts caused by stimulated cellular proliferation in the renal tubules and hepatic bile duct epithelia, with interstitial fibrosis developed in the liver. We previously reported that a peroxisome proliferator activated receptor (PPAR)-γ full agonist ameliorated kidney and liver disease in PCK rats. Telmisartan is an angiotensin receptor blocker (ARB) used widely as an antihypertensive drug and shows partial PPAR-γ agonist activity. It also has nephroprotective activity in diabetes and renal injury and prevents the effects of drug-induced hepatotoxicity and hepatic fibrosis. In the present study, we determined whether telmisartan ameliorates progression of polycystic kidney and fibrocystic liver disease in PCK rats. Five male and 5 female PCK and normal control (+/+) rats were orally administered 3 mg/kg telmisartan or vehicle every day from 4 to 20 weeks of age. Treatment with telmisartan decreased blood pressure in both PCK and +/+ rats. Blood levels of aspartate amino transferase, alanine amino transferase and urea nitrogen were unaffected by telmisartan treatment. There was no effect on kidney disease progression, but liver weight relative to body weight, liver cystic area, hepatic fibrosis index, expression levels of Ki67 and TGF-β, and the number of Ki67- and TGF-β-positive interstitial cells in the liver were significantly decreased in telmisartan-treated PCK rats. Therefore, telmisartan ameliorates congenital hepatic fibrosis in ARPKD, possibly through the inhibition of signaling cascades responsible for cellular proliferation and interstitial fibrosis in PCK rats. The present results support the potential therapeutic use of ARBs for the treatment of fibrocystic liver disease in ARPKD patients.

Topics: Angiotensin II; Animals; Benzimidazoles; Benzoates; Blood Pressure; Cell Proliferation; Cysts; Disease Models, Animal; Extracellular Signal-Regulated MAP Kinases; Female; Hepatocytes; Humans; Kidney Function Tests; Liver; Liver Diseases; Liver Function Tests; Male; Organ Size; Polycystic Kidney, Autosomal Recessive; Rats; Rats, Sprague-Dawley; Receptor, Angiotensin, Type 1; Signal Transduction; Telmisartan; Transforming Growth Factor beta

Sulforaphane (SFN) is a dietary isothiocyanate that exerts chemopreventive effects via NF-E2-related factor 2 (Nrf2)-mediated induction of antioxidant/phase II enzymes, such as heme oxygenase-1 (HO-1) and NAD(P)H quinone oxidoreductase 1 (NQO1). This work was undertaken to evaluate the effects of SFN on hepatic fibrosis and profibrotic transforming growth factor (TGF)-β/Smad signaling, which are closely associated with oxidative stress. SFN suppressed TGF-β-enhanced expression of α-smooth muscle actin (α-SMA), a marker of hepatic stellate cell (HSC) activation, and profibrogenic genes such as type I collagen, fibronectin, tissue inhibitor of matrix metalloproteinase (TIMP)-1, and plasminogen activator inhibitor (PAI)-1 in hTERT, an immortalized human HSC line. SFN inhibited TGF-β-stimulated activity of a PAI-1 promoter construct and (CAGA)(9) MLP-Luc, an artificial Smad3/4-specific reporter, in addition to reducing phosphorylation and nuclear translocation of Smad3. Nrf2 overexpression was sufficient to inhibit the TGF-β/Smad signaling and PAI-1 expression. Conversely, knockdown of Nrf2, but not inhibition of HO-1 or NQO1 activity, significantly abolished the inhibitory effect of SFN on (CAGA)(9) MLP-Luc activity. However, inhibition of NQO1 activity reversed repression of TGF-β-stimulated expression of type I collagen by SFN, suggesting the involvement of antioxidant activity of SFN in the suppression of Smad-independent fibrogenic gene expression. Finally, SFN treatment attenuated the development and progression of early stage hepatic fibrosis induced by bile duct ligation in mice, accompanied by reduced expression of type I collagen and α-SMA. Collectively, these results show that SFN elicits an antifibrotic effect on hepatic fibrosis through Nrf2-mediated inhibition of the TGF-β/Smad signaling and subsequent suppression of HSC activation and fibrogenic gene expression.

Topics: Active Transport, Cell Nucleus; Animals; Cell Line; Enzyme Induction; Extracellular Matrix Proteins; Fibrosis; Gene Expression; Genes, Reporter; Humans; Isothiocyanates; Liver Diseases; Luciferases; Male; Mice; Mice, Inbred C57BL; NF-E2-Related Factor 2; Phosphorylation; Promoter Regions, Genetic; Protein Binding; Serpin E2; Signal Transduction; Smad Proteins; Sulfoxides; Thiocyanates; Transforming Growth Factor beta

In autosomal recessive polycystic kidney disease (ARPKD), progressive enlargement of fluid-filled cysts is due to aberrant proliferation of tubule epithelial cells and transepithelial fluid secretion leading to extensive nephron loss and interstitial fibrosis. Congenital hepatic fibrosis associated with biliary cysts/dilatations is the most common extrarenal manifestation in ARPKD and can lead to massive liver enlargement. Peroxisome proliferator-activated receptor γ (PPAR-γ), a member of the ligand-dependent nuclear receptor superfamily, is expressed in a variety of tissues, including the kidneys and liver, and plays important roles in cell proliferation, fibrosis, and inflammation. In the current study, we determined that pioglitazone (PIO), a PPAR-γ agonist, decreases polycystic kidney and liver disease progression in the polycystic kidney rat, an orthologous model of human ARPKD. Daily treatment with 10 mg/kg PIO for 16 wk decreased kidney weight (% of body weight), renal cystic area, serum urea nitrogen, and the number of Ki67-, pERK1/2-, and pS6-positive cells in the kidney. There was also a decrease in liver weight (% of body weight), liver cystic area, fibrotic index, and the number of Ki67-, pERK1/2-, pERK5-, and TGF-β-positive cells in the liver. Taken together, these data suggest that PIO inhibits the progression of polycystic kidney and liver disease in a model of human ARPKD by inhibiting cell proliferation and fibrosis. These findings suggest that PPAR-γ agonists may have therapeutic value in the treatment of the renal and hepatic manifestations of ARPKD.

Topics: Animals; Blood Urea Nitrogen; Cell Proliferation; Disease Models, Animal; Disease Progression; Female; Ki-67 Antigen; Liver Cirrhosis; Liver Diseases; Male; Mitogen-Activated Protein Kinase Kinases; Pioglitazone; Polycystic Kidney, Autosomal Recessive; PPAR gamma; Rats; Rats, Sprague-Dawley; Thiazolidinediones; Transforming Growth Factor beta

Earlier studies conducted by our laboratory have shown that suppression of transforming growth factor-beta (TGFbeta)-mediated upregulation of connective tissue growth factor (CTGF) by iloprost resulted in a greatly diminished oval cell response to 2-acetylaminofluorene/partial hepatectomy (2AAF/PH) in rats. We hypothesized that this effect is due to decreased activation of hepatic stellate cells. To test this hypothesis, we maintained rats on a diet supplemented with 2% L-cysteine as a means of inhibiting stellate cell activation during the oval cell response to 2AAF/PH. In vitro experiments show that L-cysteine did, indeed, prevent the activation of stellate cells while exerting no direct effect on oval cells. Desmin immunostaining of liver sections from 2AAF/PH animals indicated that maintenance on the L-cysteine diet resulted in an 11.1-fold decrease in the number of activated stellate cells within the periportal zones. The total number of cells proliferating in the periportal zones of livers from animals treated with L-cysteine was drastically reduced. Further analyses showed a greater than fourfold decrease in the magnitude of the oval cell response in animals maintained on the L-cysteine diet as determined by immunostaining for both OV6 and alpha-fetoprotein (AFP). Global liver expression of AFP as measured by real-time PCR was shown to be decreased 4.7-fold in the L-cysteine-treated animals. These data indicate that the activation of hepatic stellate cells is required for an appropriate oval cell response to 2AAF/PH.

Topics: 2-Acetylaminofluorene; alpha-Fetoproteins; Animals; Connective Tissue Growth Factor; Cysteine; Hepatectomy; Hepatic Stellate Cells; Liver; Liver Diseases; Liver Regeneration; Male; Rats; Rats, Inbred F344; Stem Cells; Transforming Growth Factor beta

Translational studies have explored the therapeutic effects of stem cells, raising hopes for the treatment of numerous diseases. Here, we evaluated the therapeutic effect of chorionic plate-derived mesenchymal stem cells (CP-MSCs) isolated from human placenta and transplanted into rats with carbon tetrachloride (CCl(4))-injured livers. CP-MSCs were analyzed for hepatocyte-specific gene expression, indocyanine green (ICG) uptake, glycogen storage, and urea production following hepatogenic differentiation. PKH26-labeled CP-MSCs were directly transplanted into the livers of rats that had been exposed to CCl(4) (1.6 g/kg, twice per week for 9 weeks). Blood and liver tissue were analyzed at 1, 2, and 3 weeks post-transplantation. The expression of type I collagen (Col I) and matrix metalloproteinases (MMPs) was analyzed in rat T-HSC/Cl-6 hepatic stellate cells co-cultured with CP-MSCs following exposure to TGF-β. The expression levels of α-smooth muscle actin (α-SMA) and Col I were lower in transplanted (TP) rats than in non-transplanted (Non-TP) animals (P < 0.05), whereas the expression levels of albumin and MMP-9 were increased. TP rats exhibited significantly higher uptake/excretion of ICG than non-TP rats (P < 0.005). In addition, collagen synthesis in T-HSC/Cl-6 cells exposed to TGF-β was decreased by co-culture with CP-MSCs, which triggered the activation of MMP-2 and MMP-9. These results contribute to our understanding of the potential pathophysiological roles of CP-MSCs, including anti-fibrotic effects in liver disease, and provide a foundation for the development of new cell therapy-based strategies for the treatment of difficult-to-treat liver diseases.

Topics: Animals; Blotting, Western; Carbon Tetrachloride; Cell Differentiation; Cell Line; Cells, Cultured; Chemical and Drug Induced Liver Injury; Enzyme-Linked Immunosorbent Assay; Female; Flow Cytometry; Humans; Liver Diseases; Male; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Placenta; Pregnancy; Rats; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction; Transforming Growth Factor beta

In liver wound healing, transforming growth factor-beta (TGF-beta) plays a critical role in stellate cell activation as well as signaling cascades in the fibrogenic response to injury. We postulate that the TGF-beta-dependent downstream signaling pathway may vary according to the mechanism of stellate cell activation; this study was undertaken to ascertain whether the downstream signaling pathways mediated by TGF-beta vary in different liver injury models. We measured Smad3 and MAP kinase activation after isolating stellate cells from rat livers injured by either bile duct ligation (BDL) or repeated carbon tetrachloride (CCl(4)) administration. Phospho-Smad3 was dramatically up-regulated in stellate cells after CCl(4) injury, but not after BDL-induced injury. TGF-beta signaling in stellate cells activated after BDL was mediated prominently through ERK activation, whereas activation induced by CCl(4) injury or culture led to a cross-signaling mechanism involving both Smad3 and p38. The divergent Smad signaling pathways observed appeared to be attributable to the differential regulation of the early growth response gene-1 (Egr-1), an apparent negative transcriptional factor for Smad3 in our system. In addition, inhibition of ERK activation in stellate cells from BDL-injured liver led to a decrease in expression of endothelin-converting enzyme-1, a critical regulator of endothelin-1. We speculate that TGF-beta signaling proceeds through differential signaling pathways depending on the mechanism of liver injury that leads to stellate cell activation.

Topics: Animals; Aspartic Acid Endopeptidases; Bile Ducts; Carbon Tetrachloride; Cells, Cultured; Electrophoretic Mobility Shift Assay; Endothelin-Converting Enzymes; Enzyme Activation; Extracellular Signal-Regulated MAP Kinases; Hepatocytes; Humans; Immunoblotting; Immunohistochemistry; Immunoprecipitation; Ligation; Liver; Liver Diseases; Metalloendopeptidases; Rats; Signal Transduction; Smad3 Protein; Transforming Growth Factor beta

The cytokines IL-10 and TGF-beta regulate immunity and inflammation. IL-10 is known to suppress the extent of hepatic damage caused by parasite ova during natural infection with Schistosoma mansoni, but the role of TGF-beta is less clear. Cytokine blockade studies in mice revealed that anti-IL-10R mAb treatment during acute infection modestly increased cytokine production and liver damage, whereas selective anti-TGF-beta mAb treatment had marginal effects. In contrast, mice administered both mAbs developed severe hepatic inflammation, with enlarged, necrotic liver granulomas, cachexia, and >80% mortality by 8 wk postinfection, despite increased numbers of CD4(+)CD25(+)Foxp3(+) T regulatory cells. Blocking both IL-10 and TGF-beta at the onset of egg production also significantly increased IL-4, IL-6, TNF, IFN-gamma, and IL-17 production and markedly increased hepatic, peritoneal, and splenic neutrophilia. In contrast, coadministration of anti-IL-10R and TGF-beta mAbs had little effect upon parasite ova-induced intestinal pathology or development of alternatively activated macrophages, which are required to suppress intestinal pathology. This suggests that inflammation is controlled during acute S. mansoni infection by two distinct, organ-specific mechanisms: TGF-beta and IL-10 redundantly suppress hepatic inflammation while intestinal inflammation is regulated by alternatively activated macrophages.

Topics: Animals; Enzyme-Linked Immunosorbent Assay; Flow Cytometry; Interleukin-10; Liver Diseases; Macrophage Activation; Macrophages; Male; Mice; Mice, Inbred BALB C; Reverse Transcriptase Polymerase Chain Reaction; Schistosomiasis; Transforming Growth Factor beta

Phyllanthus rheedii Wight. (Euphorbiaceae) has been used by Muthuvan tribes of Kerala for curing liver diseases. The present study was conducted to assess the hepatoprotective activity of this plant. The ethanol extract of Phyllanthus rheedii was pharmacologically analysed for its preventive effect in d-galactosamine (d-GalN) induced liver damage in rats.. The levels of hepatotoxicity in various groups were quantified by different parameters of liver damage viz. serum levels of ALT, AST, LDH, GGT, ALP and total bilirubin. The effect of extract on the expression levels of pro-inflammatory cytokines like TNF-alpha, and TGF-beta were analysed by RT-PCR. Histological changes in the liver were evaluated by hematoxylin and eosin staining of paraffin processed liver sections. The antioxidant and choleretic activity of the extract were also analysed.. Comparison of serum values of control and extract treated groups have revealed that the d-GalN induced alterations in the serum and liver markers were normalized in extract treatment groups showing hepatoprotective activity of the extract. The extract also prevented the toxin induced histological changes in liver. The mRNA levels of TGF-beta and TNF-alpha in the liver were up regulated by the hepatotoxin. The extract treatment has normalized this change, giving light to the probable mechanism of action of the extract. It has showed marked antioxidant and choleretic activity. Preliminary phytochemical screening has revealed the presence of tannins, flavanoids and phenolics as major components.. This study concluded the ethanol extract of P. rheedii could be a promissory candidate for drug development and validated the tribal claim.

Topics: Animals; Chemical and Drug Induced Liver Injury; Ethanol; Female; Galactosamine; Gene Expression Regulation; Liver; Liver Diseases; Male; Phyllanthus; Phytotherapy; Plant Extracts; Rats; Rats, Wistar; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Transforming growth factor-beta (TGF-beta) signalling is induced in liver as a consequence of damage and contributes to wound healing with transient activation, whereas it mediates fibrogenesis with long-term up-regulation in chronic disease. Smad-dependent TGF-beta effects are blunted by antagonistic Smad7, which is transcriptionally activated as an immediate early response upon initiation of TGF-beta signalling in most cell types, thereby providing negative feedback regulation. Smad7 can be induced by other cytokines, e.g. IFN-gamma, leading to a crosstalk of these signalling pathways. Here we report on a novel mouse strain, denoted S7DeltaE1, with a deletion of exon I from the endogenous smad7 gene. The mice were viable and exhibited normal adult liver architecture. To obtain insight into Smad7-depend-ent protective effects, chronic liver damage was induced in mice by carbon tetrachloride (CCI4) administration. Subsequent treatment, elevated serum liver enzymes indicated enhanced liver damage in mice lacking functional Smad7. CCI4-dependent Smad2 phosphorylation was pronounced in S7DeltaE1 mice and accompanied by increased numbers of alpha-smooth muscle actin positive 'activated' HSCs. There was evidence for matrix accumulation, with elevated collagen deposition as assessed morphometrically in Sirius red stained tissue and confirmed with higher levels of hydroxyproline in S7DeltaE1 mice. In addition, the number of CD43 positive infiltrating lymphocytes as well as of apoptotic hepatocytes was increased. Studies with primary hepatocytes from S7DeltaE1 and wild-type mice indicate that in the absence of functional Smad7 protein, hepatocytes are more sensitive for TGF-beta effects resulting in enhanced cell death. Furthermore, S7DeltaE1 hepatocytes display increased oxidative stress and cell damage in response to CCI4, as measured by reactive oxygen species production, glutathione depletion, lactate dehydrogenase release and lipid peroxidation. Using an ALK-5 inhibitor all investigated CCI4 effects on hepatocytes were blunted, confirming participation of TGF-beta signalling. We conclude that Smad7 mediates a protective effect from adverse TGF-beta signalling in damaged liver, re-iterating its negative regulatory loop on signalling.

Topics: Actins; Animals; Apoptosis; Carbon Tetrachloride; Collagen; Disease Models, Animal; Fibrosis; Genes, Reporter; Immunohistochemistry; Inflammation; Liver Cirrhosis; Liver Diseases; Luciferases; Mice; Mice, Mutant Strains; Phosphorylation; Recombinant Proteins; Signal Transduction; Smad2 Protein; Smad7 Protein; Transforming Growth Factor beta; Up-Regulation

Recently, synthesis and secretion of connective tissue growth factor (CTGF)/CYR61/CTGF/NOV-family member 2 (CCN2) in cultures of hepatocytes were shown, which are sensitively up-regulated by exogenous TGF-beta. In this study TGF-beta-dependent CTGF/CCN2 expression in hepatocytes cultured under completely TGF-beta-free conditions was analysed by Western-blots, metabolic labelling, and CTGF-reporter gene assays. In alkaline phosphatase monoclonal anti-alkaline phosphatase complex (APAAP)-staining of cultured hepatocytes it was demonstrated that latent TGF-beta within the hepatocytes becomes rapidly detectable during culture indicating an intracellular demasking of the mature TGF-beta antigen. Subsequent signaling to theCTGF/CCN2 promoter occurs via p-Smad2, whereas p-Smad3 does not seem to be involved. Cycloheximide did not abolish the rapid immunocytochemical appearance of mature TGF-beta, but calpain inhibitors partially suppressed intracellular TGF-beta activation and subsequently CTGF up-regulation. Calpain treatment had the reverse effect. None of the inhibitors of extracellular TGF-beta signalling was effective in the reduction of spontaneous CTGF synthesis, but intracellularly acting Alk 4-/Alk 5-specific inhibitor SB-431542 was able to diminish CTGF expression. The assumption that latent intracellular TGF-beta is activated by calpains during culture-induced stress or injurious conditions in the liver in vivo was further validated by a direct effect of calpains on the activation of recombinant latent TGF-beta. In conclusion, these data are the first to suggest the possibility of intracrine TGF-beta signalling due to calpain-dependent intracellular proteolytic activation leading to transcriptional activation of CTGF/CCN2 as a TGF-beta-sensitive reporter gene. This mechanism might be deleterious for keeping long-term hepatocyte cultures due to TGF-beta-induced apoptosis and, further, might be of relevance for induction of apoptosis or epithelial-mesenchymal transition of hepatocytes in injured liver.

Topics: Alkaline Phosphatase; Animals; Calpain; Cells, Cultured; Connective Tissue Growth Factor; Enzyme Inhibitors; Hepatocytes; Humans; Intracellular Space; Liver Diseases; Male; Models, Biological; Phosphorylation; Rats; Rats, Sprague-Dawley; Signal Transduction; Smad Proteins; Time Factors; Transcriptional Activation; Transforming Growth Factor beta

Acute liver injury induced by administration of carbon tetrachloride (CCl4) was shown to be a model of wound-repair in rat liver. Albumin gene expression was significantly reduced at 24 h post injection with CCl4, but recovered at 48 h. We also observed significant and transient expression of bone morphogenic protein-2 (BMP-2) at 6-24 h post treatment. This expression was also shown with depletion of Kupffer cell by GdCl3, and immunostaining with anti-BMP-2 antibody showed BMP-2-producing cells interspersed in intralobular spaces of injured liver. These observations suggest that BMP-2 secreted from oval-like cells plays important roles in the wound healing response of injured liver.

Topics: Animals; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Carbon Tetrachloride Poisoning; Chemical and Drug Induced Liver Injury; Guanidine; Hepatocytes; Kupffer Cells; Lipopolysaccharide Receptors; Liver Diseases; Liver Regeneration; Male; Rats; Rats, Wistar; Transforming Growth Factor beta

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) accumulates and remains stable in the fatty tissues and liver of rodents for a long time. Considering the pronounced difference between species, long-term, low dose hepatic effects of TCDD were investigated after subcutaneous administration of TCDD into rhesus monkeys during pregnancy. Macroscopic and histopathological examination of the liver carried out 4 y after TCDD administration demonstrated intrahepatic focal fatty changes, infarction, hemorrhage, microthrombi-formation, sinusoidal ectasia, small hepatocyte hyperplasia, and increased number of alpha-smooth muscle actin (alpha-SMA)-positive cells. An electron microscopic study disclosed sinusoidal endothelial cell degeneration and injury in the liver of TCDD-treated monkeys. Western blot analysis showed downregulation of aryl hydrocarbon receptor (AhR) protein expression and decreased level of vascular endothelial (VE) cadherin but increased expression levels of CYP1A1 and transforming growth factor beta (TGF-beta) protein in the liver tissues. These changes observed in TCDD-exposed monkeys indicated sinusoidal endothelial cell injury and impairment in intrasinusoidal microcirculation. Infarction, focal fatty change, and microthrombi-formation are considered to be closely associated with intrahepatic circulatory impairment. Increased number of alpha-SMA-positive cells and decreased level of VE cadherin expression in the liver tissues might also be associated with sinusoidal endothelial cell injury. In addition, downregulation of AhR expression and increased CYP1A1 protein levels in the liver were consistent with persistent effects of TCDD. Although it has been reported that TCDD induced endothelial cell injury, this is the first report to describe vascular disorders and protein expression in the liver after injection with TCDD in a primate model.

Topics: Animals; Antigens, CD; Blotting, Northern; Cadherins; Chemical and Drug Induced Liver Injury; Cytochrome P-450 CYP1A1; Endothelial Cells; Fatty Liver; Female; Hemorrhage; Infarction; Injections, Subcutaneous; Liver Diseases; Macaca mulatta; Microscopy, Electron; Muscle, Smooth; Polychlorinated Dibenzodioxins; Pregnancy; Receptors, Aryl Hydrocarbon; Thrombosis; Time Factors; Transforming Growth Factor beta

Regulatory T-cells (Treg) are natural suppressors of autoimmunity. Previous studies indicate that immunosuppressive drugs, especially calcineurin-inhibitors, may interfere with Treg homeostasis. Inflammatory bowel disease (IBD) can relapse or develop de novo after liver transplantation. IBD is associated with a relative deficiency of Treg. The aim of this study was to determine the effect of long-term immunosuppression on the presence of Treg in the noninflamed colonic mucosa of liver transplant recipients.. Colonic biopsies of normal mucosa of 36 liver transplant recipients on different types of immunosuppression and 11 controls were studied. Treg marker Foxp3 and Treg products transforming growth factor-beta (TGF-beta) and interleukin-10 (IL-10) were studied by quantitative polymerase chain reaction (Q-PCR) and immunohistochemistry. TGF-beta-induced Smad-protein 3 and 7 were studied by Q-PCR.. No significant differences between controls and patients were observed in IL-10, TGF-beta, and Smad expression. Mucosal Foxp3 mRNA levels and Foxp3+CD3+ cells were significantly reduced in transplant recipients using prednisone/azathioprine/tacrolimus compared with controls but no direct relationship between Foxp3 expression and 1 specific drug was detected.. These results challenge the hypothesis that calcineurin-induced reduction of Treg or TGF-beta expression predisposes nontransplanted tissue to inflammation, but indicate that combined immunosuppression hampers Treg development in the intestine.

Topics: Adult; Aged; Autoimmunity; Biopsy; CD3 Complex; Colon; Disease Progression; Forkhead Transcription Factors; Gene Expression; Graft Rejection; Humans; Immunohistochemistry; Immunosuppression Therapy; Immunosuppressive Agents; Interleukin-10; Intestinal Mucosa; Liver Diseases; Liver Transplantation; Male; Middle Aged; Reverse Transcriptase Polymerase Chain Reaction; RNA; Smad3 Protein; Smad7 Protein; T-Lymphocytes, Regulatory; Transforming Growth Factor beta

The purpose of the present paper was to assess expression of proliferation, fibrosis and apoptosis markers in different phases of chronic liver diseases.. Sixty-six adults with chronic liver diseases (chronic hepatitis C, n = 48; chronic hepatitis B, n = 10; alcohol chronic liver disease, n = 8) treated at the Department of Infectious Diseases and Hepatology from 1999 to 2001, composed the study group. Liver biopsy specimens were used for immunohistochemical assessment of expression of Ki-67, transforming growth factor beta1 (TGF-beta1) and B-cell lymphoma-leukemia-2 (Bcl-2). Grade of liver inflammation and stage of fibrosis were evaluated according to the Scheuer scale.. Expression of Ki-67 in hepatocytes was most intensive in patients with grade 2 and 3 inflammation. The expression in patients with grade 4 inflammation was low. The expression of Ki-67 in lymphocytes was most intensive in patients with grade 2 inflammation. Expression of TGF-beta1 in hepatocytes reached a maximum in patients with grade 2 or 3 inflammation and dropped in patients with grade 4 inflammation. There was a statistically significant correlation between stage of fibrosis and expression of TGF-beta1 in liver stromal cells. A very strong correlation was found between the expression of Bcl-2 in bile ductules epithelium and the grade of inflammation (P = 0.006). The expression of Bcl-2 in hepatocytes was observed only in patients with very intense liver inflammation (grade 3) and in patients with stage 3 or 4 fibrosis.. Processes of proliferation, fibrosis and apoptosis are not directly correlated to progression of liver disease. Expression of studied markers can be used for analysis of dynamics of these processes.

Topics: Adult; Apoptosis; Cell Proliferation; Chronic Disease; Gene Expression; Hepatocytes; Humans; Ki-67 Antigen; Liver; Liver Cirrhosis; Liver Diseases; Proto-Oncogene Proteins c-bcl-2; Transforming Growth Factor beta; Transforming Growth Factor beta1

To transfer human HGF gene into the liver of rats by direct electroporation as a means to prevent radiation-induced liver damage.. Rat whole liver irradiation model was accomplished by intra-operative approach. HGF plasmid was injected into liver and transferred by electroporation using a pulse generator. Control rats (n = 8) received electrogene therapy (EGT) vehicle plasmid and another 8 rats received HGF-EGT 100 mug 48 h before WLIR. Expression of HGF in liver was examined by RT-PCR and ELISA methods. Apoptosis was determined by TUNEL assay. Histopathology was evaluated 10 wk after whole liver irradiation.. Marked decrease of apoptotic cells and down-regulation of transforming growth factor-beta 1 (TGF-beta1) mRNA were observed in the HGF-EGT group 2 d after liver irradiation compared to control animals. Less evidence of radiation-induced liver damage was observed morphologically in liver specimen 10 wk after liver irradiation and longer median survival time was observed from HGF-EGT group (14 wk) compared to control rats (5 wk). (P = 0.031).. For the first time it has been demonstrated that HGF-EGT would prevent liver from radiation-induced liver damage by preventing apoptosis and down-regulation of TGF-beta1.

Topics: Animals; Apoptosis; Down-Regulation; Electroporation; Female; Genetic Therapy; Hepatocyte Growth Factor; Humans; Liver Diseases; Radiation Injuries; Rats; Rats, Sprague-Dawley; Transforming Growth Factor beta; Transforming Growth Factor beta1

Schistosomiasis mansoni disseminated worm eggs in mice and humans induce granulomatous inflammations and cumulative fibrosis causing morbidity and possibly mortality. In this study, intrahepatic and I.V. injections of a double-stranded oligodeoxynucleotide decoy containing the TGF-beta regulatory element found in the distal promoter of the COL1A1 gene into worm-infected mice suppressed TGF-beta1, COL1A1, tissue inhibitor of metalloproteinase-1, and decreased COL3A1 mRNAs to a lesser extent. Sequence comparisons within the mouse genome found homologous sequences within the COL3A1, TGF-beta1, and TIMP-1 5' flanking regions. Cold competition gel mobility shift assays using these homologous sequences with 5' and 3' flanking regions found in the natural COL1A1 gene showed competition. Competitive gel mobility assays in a separate experiment showed no competition using a 5-base mutated or scrambled sequence. Explanted liver granulomas from saline-injected mice incorporated 10.45 +/- 1.7% (3)H-proline into newly synthesized collagen, whereas decoy-treated mice showed no collagen synthesis. Compared with the saline control schistosomiasis mice phosphorothioate double-stranded oligodeoxynucleotide treatment decreased total liver collagen content (i.e. hydroxy-4-proline) by 34%. This novel molecular approach has the potential to be employed as a novel antifibrotic treatment modality.

Topics: Animals; Collagen; Collagen Type I; Collagen Type I, alpha 1 Chain; Consensus Sequence; DNA; Female; Fibroblasts; Granuloma; Hydroxyproline; Liver; Liver Cirrhosis; Liver Diseases; Mice; Mice, Inbred C57BL; Mutation; Myocytes, Smooth Muscle; Oligodeoxyribonucleotides; Oligonucleotides; Schistosomiasis mansoni; Sequence Homology, Nucleic Acid; Signal Transduction; Tissue Inhibitor of Metalloproteinase-1; Transfection; Transforming Growth Factor beta

To study the effect of IL-10 on the expression of growth factors--transforming growth factor-beta1 (TGF-beta1), epidermal growth factor (EGF), hepatocyte growth factor (HGF) and platelet-derived growth factor (PDGF) of hepatic stellate cells (HSCs) of hepatic fibrosis rat and the anti-fibrogenic role of exogenous IL-10.. Hepatic fibrosis was induced by CCl(4) administration intra-peritoneally. Sixty clean male Sprague-Dawley (SD) rats were randomly divided into three groups: normal control group (GN, 8 rats), hepatic fibrosis model group (GC, 28 rats) and IL-10 treated group (GI, 24 rats). At the beginning of the 7th and 11th wk, rats in each group were routinely perfused with pronase E and type IV collagenase through a portal vein catheter and the suspension obtained from the liver was spun by centrifugation with 11% Nycodenz density gradient to isolate HSCs. Histological examination was used to determine the degree of hepatic fibrosis. RT-PCR was employed to analyze mRNA expression from freshly isolated cells. Immunocytochemistry was performed to detect protein expression in primary cultured HSCs.. Rat hepatic fibrosis was developed with the increase of injection frequency of CCl(4), and HSCs were successfully isolated. At the 7th and 11th wk, TGF-beta1, EGF, and HGF mRNA in GC increased obviously compared with GN (P = 0.001/0.042, 0.001/0.001, 0.001/0.001) and GI (P = 0.001/0.007, 0.002/0.001, 0.001/0.001). For TGF-beta1, no difference was observed between GI and GN. For EGF, mRNA level in GI increased compared with GN during the 7th wk (P = 0.005) and 11th wk (P = 0.049). For HGF, mRNA level in GI decreased compared with GN at the 7th wk (P = 0.001) and 11th wk (P = 0.021). Between these two time points, TGF-beta1 expression at the 7th wk was higher than that of the 11th wk (P = 0.049), but for EGF, the former was lower than the latter (P = 0.022). As for PDGF mRNA, there was no significant difference between these groups, but difference seemed to exist in protein levels. Results by immunocytochemistry of TGF-beta1 and EGF were paralleled with the above findings.. The expression of TGF-beta1, EGF and HGF increased in HSC of hepatic fibrosis rat and decreased after treatment with IL-10. IL-10 plays an anti-fibrogenic role by suppressing growth factors expression.

Topics: Animals; Base Sequence; Carbon Tetrachloride Poisoning; DNA Primers; Epidermal Growth Factor; Gene Expression Regulation; Hepatocyte Growth Factor; Interleukin-10; Liver Diseases; Male; Rats; Rats, Sprague-Dawley; RNA, Messenger; Transforming Growth Factor beta; Transforming Growth Factor beta1

Hemorrhage initiates an inflammatory response that induces the systemic release of cytokines and sequestration of polymorphonuclear neutrophils. Sequestered polymorphonuclear neutrophils release proteases, including matrix metalloproteinases (MMPs) that degrade elements of the extracellular matrix, contributing to the morbidity and mortality seen from hemorrhage. Activation of MMPs may be associated with changes in transforming growth factor beta1 (TGF-beta1) and caspase-3 signaling pathways. In this study, the authors examined hemorrhage-induced changes in the expression of rat hepatic MMP-9, tissue inhibitor of metalloproteinase-1 (TIMP-l), TGF-beta1, and caspase-3 activities in the presence and absence of the MMP inhibitor hydroxamate.. Hemorrhagic shock was induced in fasted, anesthetized, and cannulated rats by rapid phlebotomy to a mean arterial pressure level of 40 mm Hg, maintained for 90 minutes by withdrawal and infusion of blood, followed by a resuscitation period of lactated Ringer's infusion. Rats received either hydroxamate (25 mg/kg) or vehicle by gavage before hemorrhage. Twenty-four hours after resuscitation, plasma and liver samples were collected. Liver MMP-9, TGF-beta1, and caspase-3 levels were quantified by Western immunoblotting. Plasma glutamic oxaloacetic transaminase (GOT) and plasma glutamic pyruvic transaminase (GPT) were determined enzymatically.. Plasma GOT, plasma GPT, and liver MMP-9, TGF-beta1, and caspase-3 levels were all significantly elevated at 24 hours postresuscitation when compared with the control values. Hepatic TIMP-1, an in vivo inhibitor of MMP-9, was unaltered at 24 hours. Hydroxamate treatment reduced GOT, GPT, MMP-9, TGF-beta1, and caspase-3 levels at 24 hours. The mortality of hemorrhaged untreated rats was 29% after 24 hours, and pretreatment with hydroxamate reduced mortality to 0%.. These results indicate the beneficial effects of MMP inhibitor in preventing an increase in GOT, GPT, MMP-9, TGF-beta1, and caspase-3 activity with the potential for improvement of hepatic injury due to hemorrhage.

Topics: Alanine Transaminase; Animals; Aspartate Aminotransferases; Cardiopulmonary Resuscitation; Caspase 3; Caspases; Disease Models, Animal; Hemorrhage; Hydroxamic Acids; Liver Diseases; Male; Matrix Metalloproteinase 9; Matrix Metalloproteinase Inhibitors; Rats; Rats, Sprague-Dawley; Reference Values; Transforming Growth Factor beta; Transforming Growth Factor beta1

To determine the effects of dominant-negative TGF-beta receptor expression during liver regeneration in rats with dimethylnitrosamine (DMN)-induced liver injury.. Rats were first treated with DMN for 3 weeks, and then intravenously injected once with AdTbeta-TR, AdLacZ, or saline. Serial changes in hepatocyte proliferation and apoptosis were evaluated by immunohistochemistry using anti-Ki67 antibody, and TUNEL staining, respectively. The mRNA expression of regeneration factors (HGF, TGF-alpha, EGF, and IGF-I) and IL-6 were evaluated by real-time PCR and northern blotting.. Anti-TGF-beta molecular intervention up-regulated hepatocyte proliferation and inhibited apoptosis. In the AdTbeta-TR-treated rats, EGF and IGF-I mRNA expression levels were significantly increased at day 1 and remained high for 3 days after gene transfer; TGF-alpha mRNA expression levels were significantly increased at 2 to 5 days after gene transfer; HGF mRNA expression levels were significantly up-regulated at day 2 only after gene transfer; while IL-6 mRNA expression level tended to increase at day 1, but decreased thereafter.. In rats with DMN-induced liver injury, anti-TGF-beta molecular intervention therapy stimulates proliferation and reduces apoptosis of hepatocytes, and also up-regulates the transcription of various growth factors.

Topics: Animals; Apoptosis; Blotting, Northern; Cell Division; Chemical and Drug Induced Liver Injury; Chronic Disease; Dimethylnitrosamine; Gene Transfer Techniques; Growth Substances; Immunohistochemistry; In Situ Nick-End Labeling; Interleukin-6; Liver Diseases; Liver Regeneration; Male; Rats; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Transcription, Genetic; Transforming Growth Factor beta; Up-Regulation

Transforming growth factor beta 1 (TGFbeta1) is a major fibrogenic cytokine, the expression of which is increased in the livers of children with cystic fibrosis liver disease (CFLD) and in the bronchoalveolar lavage fluid of patients with cystic fibrosis pulmonary disease (CFPD). The purpose of our study was to investigate the usefulness of plasma TGFbeta1 as a noninvasive marker of CFLD and CFPD, or both and to investigate the contribution of platelet-derived TGFbeta1 to plasma TGFbeta1 by correlating the latter with platelet factor 4 (PF4).. Three groups of patients with cystic fibrosis were studied: 1) those with CFLD, 2) those with CF and no liver disease (CFNLD), and 3) those with CFPD. Controls were healthy adolescents and adults. Plasma TGFbeta1 was assayed using the R and D Quantikine quantitative sandwich enzyme immunoassay technique and PF4 (American Bioproducts ELISA kit).. Plasma TGFbeta1 was markedly increased in CFPD (15 +/- 3 ng/mL) versus healthy adults (1 +/- 0 ng/mL; P < 0.004. The PF4 values followed a similar pattern: 105 +/- 8 ng/mL in CFPD versus 12 +/- 4 ng/mL (P < 0.0001). Plasma TGFbeta1 values in CFLD did not differ from CFNLD patients of comparable age nor from values for healthy adolescents. Plasma TGFbeta1 values were strongly correlated with values for PF4: r = 0.543, P < 0.0001.. Plasma TGFbeta1 is not a useful marker of CFLD. The increased plasma TGFbeta1 and PF4 in CFPD patients are of interest both as possible noninvasive markers of pulmonary fibrosis and because the increased values suggest that platelet activation may play a pathogenetic role in CFPD.

Topics: Adult; Biomarkers; Blood Platelets; Bronchoalveolar Lavage Fluid; Case-Control Studies; Cystic Fibrosis; Female; Humans; Liver Diseases; Lung Diseases; Male; Platelet Activation; Platelet Factor 4; Transforming Growth Factor beta; Transforming Growth Factor beta1

Genomic ablation of hepatocyte-specific fibroblast growth factor receptor (FGFR)4 in mice revealed a role of FGF signaling in cholesterol and bile acid metabolism and hepatolobular restoration in response to injury without effect on liver development or hepatocyte proliferation. Although the potential role of all 23 FGF polypeptides in the liver is still unclear, the most widely studied prototypes, FGF1 and FGF2, are present and have been implicated in liver cell growth and function in vitro. To determine whether FGF1 and FGF2 play a role in response to injury and fibrosis, we examined the impact of both acute and chronic exposure to carbon tetrachloride (CCl(4)) in the livers of FGF1- and FGF2-deficient mice. After acute CCl(4) exposure, FGF1(-/-)FGF2(-/-) mice exhibited an accelerated release of serum alanine aminotransferase similar to FGFR4 deficiency, but no effect on overall hepatolobular restoration or bile acid metabolism. FGF1(-/-)FGF2(-/-) mice exhibited a normal increase in alpha-smooth muscle actin and desmin associated with activation and migration of hepatic stellate cells to damage, but a reduced level of hepatic stellate cell-derived matrix collagen alpha1(I) synthesis. Liver fibrosis resulting from chronic CCl(4) exposure was markedly decreased in the livers of FGF1/FGF2-deficient mice. These results suggest an agonist role for FGF1 and FGF2 in specifically insult-induced liver matrix deposition and hepatic fibrogenesis and a potential target for the prevention of hepatic fibrosis.

Topics: Actins; Animals; Bile Acids and Salts; Carbon Tetrachloride; Chemical and Drug Induced Liver Injury; Collagen Type I; Drug Administration Schedule; Extracellular Matrix; Fibroblast Growth Factor 1; Fibroblast Growth Factor 2; Hepatectomy; Liver; Liver Cirrhosis; Liver Diseases; Liver Regeneration; Mice; Mice, Knockout; Muscle, Smooth; Time Factors; Transforming Growth Factor beta; Transforming Growth Factor beta1

Hepatocellular carcinoma is associated with liver fibrosis. Murine schistosomiasis infection offers a model to study hepatic fibrogenesis. Single-stranded phosphorothiate oligodeoxynucleotides containing the TGF-beta regulatory element have been shown to regulate the transcription of this gene and effectively inhibit collagen synthesis in primary fibroblasts isolated from schistosomiasis-induced hepatic granulomas. While the single-stranded oligos did not decrease collagen and non-collagen protein synthesis below control levels, their double-stranded modified and unmodified counterparts did. Competitive cold oligodeoxynucleotide gel mobility shift analysis using control fibroblast nuclear extract demonstrated that the single-stranded oligos diminished binding of the TGF-beta activator protein to the TGF-beta regulatory element while the double-stranded oligos totally inhibited this binding. TGF-beta element containing single-stranded phosphorothioate oligodeoxynucleotides and their double-stranded counterparts may be successful therapeutic agents to inhibit hepatic fibrogenesis and associated hepatocellular carcinoma.

Topics: Animals; Collagen; Female; Fibroblasts; Granuloma; Liver Cirrhosis; Liver Diseases; Liver Neoplasms; Mice; Mice, Inbred CBA; Oligodeoxyribonucleotides; Rats; Schistosomiasis mansoni; Transforming Growth Factor beta; Wound Healing

The response to the liver damage caused by portacaval shunt (PCS) is characterized by low-grade hyperplasia and atrophy. To clarify mechanisms of this dissociation, we correlated the expression of 'hepatotrophic factors' and the antihepatotrophic and proapoptotic peptide, transforming growth factor (TGF)-beta, with the pathologic changes caused by PCS in rats.. PCS was created by side-to-side anastomosis between the portal vein and inferior vena cava, with ligation of the hilar portal vein. Hepatic growth mediators were measured to 2 months.. The decrease in the liver/body weight ratio during the first 7 days which stabilized by day 15, corresponded to parenchymal cell apoptosis and increases in hepatic TGF-beta concentration that peaked at 1.4 x baseline at 15 days before returning to control levels by day 30. Variable increases in the concentrations of growth promoters (hepatocyte growth factor, TGF-alpha and augmenter of liver regeneration) also occurred during the period of hepatocellular apoptosis.. The development of hepatic atrophy was associated with changes in TGF-beta concentration, and occurred despite increased expression of multiple putative growth promoters. The findings suggest that apoptosis set in motion by TGF-beta constrains the amount of hepatocyte proliferation independently from control of liver volume.

Topics: Animals; Apoptosis; Atrophy; Cell Division; Gene Expression; Growth Substances; Hepatic Artery; Liver; Liver Diseases; Male; Organ Size; Portacaval Shunt, Surgical; Rats; Rats, Inbred Lew; RNA, Messenger; Transforming Growth Factor alpha; Transforming Growth Factor beta

During chronic liver injury, transforming growth factor beta (TGF-beta) plays a prominent role in stimulating liver fibrogenesis by myofibroblast-like cells derived from hepatic stellate cells (HSCs). On the other hand, Smad 7 was recently shown to antagonize the TGF-beta-induced activation of signal-transducing Smads (2 and 3). In this study, we investigated the regulatory mechanisms of the TGF-beta signals in rat HSCs during acute liver injury and myofibroblasts (MFBs) during chronic liver injury, focusing on the roles of Smad 2 and antagonistic Smad 7. In acute liver injury, HSC-derived TGF-beta increased plasminogen activator inhibitor type 1 (PAI-1) and alpha2(I) procollagen (COL1A2) transcripts. Smad 2 in HSCs during liver injury and primary cultured HSCs were activated by an autocrine mechanism, because high levels of Smad 2 phosphorylation and induction of PAI-1 transcript by TGF-beta were observed in HSCs. Thereafter, Smad 7 induced by TGF-beta negatively regulated the Smad 2 action. These results indicated that endogenous TGFbeta-mediated Smad 7 in HSCs terminated the fibrotic signals mediated by signal-transducing Smads, and might be involved in the transient response to autocrine TGF-beta signal after acute liver injury. By contrast, Smad 7 was not induced by the autocrine TGF-beta signal, and constitutive Smad 2 activation was observed in MFBs throughout chronic liver injury, although Smad 7 could inhibit the TGF-beta signal requiring Smad 2 phosphorylation by activated TGF-beta receptor in cultured MFBs. This constitutive phosphorylation of Smad 2 by endogenous TGF-beta under a low level of Smad 7 could be involved in the progression of liver fibrosis.

Topics: Acute Disease; Animals; Carbon Tetrachloride; Cell Division; Cell Line; Cells, Cultured; Chemical and Drug Induced Liver Injury; Chronic Disease; Collagen; Collagen Type I; DNA-Binding Proteins; Fibroblasts; Gene Expression; Hepatocytes; Liver; Liver Diseases; Male; Phosphorylation; Plasminogen Activator Inhibitor 1; Promoter Regions, Genetic; Rats; Rats, Wistar; RNA, Messenger; Signal Transduction; Smad2 Protein; Smad7 Protein; Trans-Activators; Transfection; Transforming Growth Factor beta

Liver disease causes significant morbidity and mortality from multilobular cirrhosis in patients with cystic fibrosis. Abnormal bile transport and biliary fibrosis implicate abnormal biliary physiology in the pathogenesis of cystic fibrosis-associated liver disease (CFLD), yet the mediators linking biliary events to fibrosis remain unknown. Activated hepatic stellate cells (HSCs) are the pre-eminent mediators of fibrosis in a range of hepatic disorders. The dominant stimulus for matrix production by HSCs is the cytokine transforming growth factor (TGF)-beta(1). In CFLD, the role of HSCs and the source of TGF-beta(1) have not been evaluated. Liver biopsy tissue obtained from 38 children with CFLD was analyzed. Activated HSCs, identified by co-localization of procollagen alpha(1)(I) mRNA and alpha-smooth muscle actin, were demonstrated as the cellular source of excess collagen production in the fibrosis surrounding the bile ducts and the advancing edge of scar tissue. TGF-beta protein and TGF-beta(1) mRNA expression were shown to be predominantly expressed by bile duct epithelial cells. TGF-beta(1) expression was significantly correlated with both hepatic fibrosis and the percentage of portal tracts showing histological abnormalities associated with CFLD. This study demonstrates a definitive role for HSCs in fibrogenesis associated with CFLD and establishes a potential mechanism for the induction of HSC collagen gene expression through the production of TGF-beta(1) by bile duct epithelial cells.

Topics: Actins; Adolescent; Child; Child, Preschool; Collagen; Cystic Fibrosis; Female; Gene Expression; Humans; Immunohistochemistry; In Situ Hybridization; Infant; Keratins; Liver; Liver Diseases; Male; Muscle, Smooth; Procollagen; RNA, Messenger; Transforming Growth Factor beta; Transforming Growth Factor beta1

The acute-phase response (APR) represents a systemic reaction of the organism to multiple nonspecific inflammatory stimuli. In general, it is protective for the host, and hepatocytes are the main cells responding with alterations in the expression of a set of liver-specific proteins named the acute-phase proteins. We have previously shown that although a turpentine-induced APR is not fibrogenic per se, it enhances collagen deposition in rats treated with CCl(4) and up-regulates expression of hepatic alpha1(I) collagen and tissue inhibitor of metalloproteinases 1 (TIMP-1) messenger RNAs (mRNAs). In this report we extended our studies and showed that turpentine induced, in a time-dependent manner, expression of alpha1(I) and alpha1(IV) collagens, TIMP-1, and matrix-metalloproteinase 2 (MMP-2) mRNAs. We further showed that expression of these mRNAs occurs in hepatic stellate cells, but not in hepatocytes obtained 6 hours after the induction of an APR episode. These changes were accompanied by increased blood levels of tumor necrosis factor alpha (TNF-alpha) and interleukin 6 (IL-6) without noticeable immediate changes in the expression of their respective mRNAs in the liver. In contrast to CCl(4)-induced liver damage, turpentine alone, whether administered as a single dose or as a weekly dose for 3 weeks did not up-regulate expression of transforming growth factor beta1 (TGF-beta1) mRNA and did not result in excess collagen deposition. Overall, these findings suggest that collagen deposition in the livers of rats with repeated APR episodes may be enhanced only when given together with a fibrogenic stimulus that activates hepatic stellate cells (HSCs) and/or up-regulates TGF-beta1 mRNA expression.

Topics: Acute-Phase Reaction; Animals; Carbon Tetrachloride; Chemical and Drug Induced Liver Injury; Collagen; Interleukin-6; Irritants; Liver; Liver Diseases; Male; Matrix Metalloproteinase 2; Rats; Rats, Inbred F344; Rats, Sprague-Dawley; RNA, Messenger; Time Factors; Tissue Inhibitor of Metalloproteinase-1; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Necrosis Factor-alpha; Turpentine

Transforming growth factor beta (TGF-beta) is the most potent profibrogenic mediator in liver fibrosis. Although Smad proteins have been identified as intracellular mediators in the TGF-beta signaling pathway, the function of individual Smad proteins remains poorly understood. The aim of this study was to explore the contribution of Smad3 in mediating TGF-beta responses in a model of acute liver injury in vivo and in culture-activated hepatic stellate cells (HSCs). Wild-type, Smad3 heterozygous or Smad3 homozygous knockout mice were treated with a single intragastric administration of CCl(4). After 72 hours, the induction of hepatic collagen alpha1(I) and alpha2(I) messenger RNA (mRNA) levels in Smad3 knockout mice was only 42% and 64%, respectively, of the levels induced in wild-type mice. However, smooth muscle alpha-actin (alpha-SMA) was expressed at a slightly higher level in livers from knockout mice compared with wild-type mice. In culture-activated HSCs from Smad3 knockout mice, collagen alpha1(I) mRNA was 73% of wild-type HSCs, but alpha-SMA expression was the same. HSCs from knockout mice showed a higher proliferation rate than wild-type HSCs. Smad3-deficient HSCs did not form TGF-beta1-induced Smad-containing DNA-binding complexes. In conclusion, (1) maximal expression of collagen type I in activated HSCs requires Smad3 in vivo and in culture; (2) Smad3 is not necessary for HSC activation as assessed by alpha-SMA expression; (3) Smad3 is necessary for inhibition of proliferation of HSCs, which might be TGF-beta-dependent; and (4) Smad3 is required for TGF-beta1-mediated Smad-containing DNA-binding complex formation in cultured HSCs.

Topics: Actins; Animals; Carbon Tetrachloride; Cattle; Cell Division; Cells, Cultured; Chemical and Drug Induced Liver Injury; Collagen; DNA; DNA-Binding Proteins; Enzyme Activation; Fetal Blood; Hepatocytes; In Situ Hybridization; Liver Diseases; Mice; Mice, Knockout; Mitogen-Activated Protein Kinases; Muscle, Smooth; Platelet-Derived Growth Factor; RNA, Messenger; Smad3 Protein; Trans-Activators; Transforming Growth Factor beta

The aim of the study was to evaluate the possible association between plasma concentrations of transforming growth factor-beta1 (TGF-beta1) and the degree of hepatic dysfunction in patients with chronic liver diseases. TGF-beta1 was measured with an enzyme immunoassay in plasma from 21 patients with chronic active hepatitis and 40 patients with liver cirrhosis. Normal values were obtained from a group of 13 healthy volunteers. Results were analysed with respect to aetiology and the degree of liver insufficiency as evaluated by the Child-Pugh classification. The mean plasma concentration of TGF-beta1 in patients (36.9+/-2.8 ng/ml) was twice that found in normal volunteers (18.3+/-1.6 ng/ml). The highest values were observed in patients with alcoholic liver cirrhosis (44.4+/-4.7 ng/ml). Plasma TGF-beta1 showed a statistically significant positive correlation with the degree of liver insufficiency. These results indicate the possible use of plasma TGF-beta1 measurement as a good marker of liver function impairment. Further observation of patients involved in this study may help to evaluate its possible prognostic value in chronic liver diseases.

Topics: Chronic Disease; Female; Humans; Liver; Liver Diseases; Male; Middle Aged; Transforming Growth Factor beta

Intraperitoneal injection of streptococcal cell walls (SCW) into Lewis rats results in dissemination of SCW to the liver, spleen, bone marrow, and peripheral joints. The uptake of SCW by Kupffer cells in the liver initiates a chain of events largely mediated by T lymphocytes and macrophages. Local synthesis and secretion of cytokines and growth factors in response to the persistent SCW lead to the evolution and maintenance of a chronic T cell-dependent granulomatous response and result in granuloma formation and irreversible hepatic fibrosis. In an attempt to impede the development of the chronic granulomatous lesions in the liver, we injected a plasmid DNA encoding TGF-beta 1 i.m. to the SCW animals to determine the effect of TGF-beta 1 gene transfer on the course of liver inflammation and fibrosis. A single injection of plasmid DNA encoding TGF-beta 1 resulted in virtual abolition of the development of the SCW-induced hepatic granuloma formation and matrix expansion. TGF-beta 1 DNA not only reduced key proinflammatory cytokines including TNF-alpha, IL-1 beta, IFN-gamma, and IL-18, but also inhibited both CXC and CC chemokine production, thereby blocking inflammatory cell recruitment and accumulation in the liver. Moreover, TGF-beta 1 gene delivery inhibited its own expression in the liver tissue, which is otherwise up-regulated in SCW-injected animals. Our study suggests that TGF-beta 1 gene transfer suppresses hepatic granuloma formation by blocking the recruitment of inflammatory cells to the liver, and thus may provide a new approach to the control of hepatic granulomatous and fibrotic diseases.

Topics: Animals; Cell Movement; Cell Wall; Cytokines; DNA; Extracellular Matrix Proteins; Female; Gene Transfer Techniques; Granuloma; Immunosuppressive Agents; Injections, Intramuscular; Injections, Intraperitoneal; Leukocytes; Liver Diseases; Rats; Rats, Inbred Lew; RNA, Messenger; Streptococcus pyogenes; Transforming Growth Factor beta

Latent transforming growth factor beta binding protein (LTBP), a component of the extracellular matrix (ECM) of various tissues, is important for the secretion of TGF-beta and, furthermore, for the storage of TGF-beta in ECM. The proteolytic cleavage of LTBP is assumed to be the prerequisite for the activation of TGF-beta. We investigated the mRNA expression pattern of the three LTBP isoforms (LTBP-1, -2, -3) and the protein distribution of the components of the large latent TGF-beta complex, namely LTBP-1 and -2, latency-associated protein (LAP), and TGF-beta, in human liver using reverse-transcription polymerase chain reaction (RT-PCR) and immunohistochemical alkaline phosphatase anti-alkaline phosphatase (APAAP) staining. Parts of explanted livers diagnosed as hepatitis B, hepatitis C, primary biliary cirrhosis (PBC), and primary sclerosing cholangitis (PSC) and normal liver tissue were examined. LTBP transcripts were detected in the same manner in all liver specimens. Interestingly, we found a new splice variant of LTBP-1 (LTBP-1D), in which the sequence coding for the proteinase-sensitive hinge region is deleted. The corresponding parts of the human LTBP-2 and LTBP-3 cDNA coding for the hinge region were sequenced and show neither similar proteinase cleavage sites nor deleted cDNA sequences. The proposed proteinase cleavage site of mouse LTBP-3 seems not to be conserved in the human LTBP-3 gene. By immunohistochemistry, LTBP-1, -2, and LAP were detectable in normal and diseased livers and showed a different staining pattern for both LTBP isoforms. By contrast, TGF-beta showed a spotted staining pattern in diseased livers only, predominantly in the area of parenchymal cells that are close to fibrotic tissue. This strongly suggests the release of active TGF-beta from preexisting latent complexes. The LTBP-1D splice variant, which is probably less sensitive against proteolytic degradation and therefore may protect TGF-beta from activation, may have importance for modulating the biological activity of TGF-beta in normal and diseased liver.

Topics: Amino Acid Sequence; Animals; Base Sequence; Carrier Proteins; Endopeptidases; Gene Expression Regulation; Humans; Intracellular Signaling Peptides and Proteins; Latent TGF-beta Binding Proteins; Liver; Liver Diseases; Mice; Molecular Sequence Data; RNA Splicing; RNA, Messenger; Sequence Alignment; Transforming Growth Factor beta

The increased deposition of extracellular matrix proteins in the liver is a key factor in the morbidity and mortality of alcoholic liver disease (ALD). This increased fibrosis may be due to a superabundance of profibrogenic factors such as transforming growth factor-beta (TGF-beta). The original peptide is now called TGF-beta 1, and two other isoforms have been recognized in humans (TGF-beta 2 and TGF-beta 3). It was the aim of the present study to determine the expression of the TGF-beta isoforms in different stages of ALD. Thirty patients with ALD had percutaneous liver biopsies performed for diagnostic purposes. They were grouped by clinical findings and by liver histology into four groups: I, steatosis; II, fibrosis; III, hepatitis; and IV, cirrhosis. An unused portion of each biopsy sample was used to evaluate the gene expression of TGF-beta 1, TGF-beta 2, and TGF-beta 3 by reverse transcription polymerase chain reaction (RT-PCR). The expression of all isoforms from patients was significantly greater than their expression in controls. No significant correlation was determined between TGF-beta isoform expression and liver function test results. When the different isoforms were grouped by histology, increased expression with more severe disease was found; however, differences existed among the isoforms. In ALD, all TGF-beta isoforms were increased and their expression was significantly greater in patients with more active and advanced disease. RT-PCR is an effective method for evaluating gene expression in clinical samples which often provide a limited amount of tissue.

Topics: Adult; Aged; DNA Primers; Female; Gene Expression Regulation, Neoplastic; Humans; Liver Cirrhosis, Alcoholic; Liver Diseases; Male; Middle Aged; Polymerase Chain Reaction; Transcription, Genetic; Transforming Growth Factor beta

Although several liver diseases of childhood, particularly biliary atresia (BA) and cystic fibrosis (CF) liver disease (CFLD) are characterized by hepatic fibrosis, the pathogenesis of this process is incompletely understood. The cytokine transforming growth factor-beta1 (TGF-beta1) has been implicated in hepatic fibrosis in experimental animals, in which both the hepatic expression and plasma concentration of this cytokine are increased. The objective of our study was to determine whether there are similar alterations of TGF-beta1 in patients with hepatic fibrosis secondary to either BA and/or CFLD. The study design was as follows. In study 1, plasma TGF-beta1 was assessed by ELISA in 9 children with BA undergoing liver transplantation, 11 patients with CFLD, and appropriate control subjects. In study 2, hepatic expression of TGF-beta1 protein (assessed immunohistochemically) and hepatic fibrosis were scored semiquantitatively, on a 1-3 scale, by blinded investigators, in archival liver biopsy specimens from 10 children with BA, 10 with CFLD, and from 10 older children with normal hepatic histology, as well as in 4 patients with liver diseases of various etiologies. Simultaneous plasma and liver TGF-beta1 studies were performed in 8 patients with liver disease. Results were as follows. Plasma TGF-beta1 values were inversely correlated with age in healthy subjects (r=-0.54, p < 0.0001). The plasma TGF-beta1 protein of children with BA was decreased (13+/-2 ng/mL) compared with values for healthy children (42+/-6 ng/mL, n=10, p < 0.005). Similarly, the plasma TGF-beta1 concentration in patients with CFLD was also decreased compared with values for children with CF and normal serum liver profiles (n=14) (2+/-1 ng/mL versus 12+/-1, p < 0.05). However, the plasma TGF-beta1 concentration was increased in two patients with other types of liver disease. The hepatic expression of TGF-beta1 was increased in the presence of hepatic fibrosis in all types of liver diseases studied. Forty-six percent of patients had both marked hepatic fibrosis and marked TGF-beta1 labeling; 86% of samples without fibrosis showed no TGF-beta1 labeling, p=0.007. In conclusion, these studies have established the association of hepatic TGF-beta1 protein and hepatic fibrosis in several common liver diseases of childhood. Our data also suggest that, in children, plasma TGF-beta1 does not appear to be a useful marker of hepatic expression of this cytokine.

Topics: Adult; Biomarkers; Child; Child, Preschool; Humans; Infant; Liver; Liver Diseases; Liver Transplantation; Transforming Growth Factor beta

Our aim was to assess the relationship between transforming growth factor beta 1 (TGF-beta 1) and alpha-fetoprotein (AFP) levels in hepatocellular carcinoma (HCC).. Urinary TGF-beta 1 and serum AFP were determined in 123 HCC patients, 50 patients with chronic liver disease (CLD), and 50 healthy controls.. Both TGF-beta 1 and AFP levels were higher in HCC patients than in CLD patients or controls (each, P = 0.0001). There was a negative correlation between TGF-beta 1 and logAFP (r = -0.196, P = 0.029). Multivariate analysis indicated that TGF-beta 1 and AFP were associated with an increased risk of HCC development. By receiver-operating characteristic curve analysis, determination of AFP and TGF-beta 1 in parallel significantly increased the sensitivity and diagnostic accuracy in detecting HCC.. Increased urinary TGF-beta 1 level can be used as a complementary marker to AFP for detection of HCC with low AFP production.

Topics: Adult; alpha-Fetoproteins; Biomarkers, Tumor; Carcinoma, Hepatocellular; Case-Control Studies; Female; Humans; Liver Diseases; Liver Neoplasms; Male; Middle Aged; Multivariate Analysis; Predictive Value of Tests; ROC Curve; Sensitivity and Specificity; Transforming Growth Factor beta

To test the hypothesis that inflammation in hepatitis C follows mechanisms common to immune-activated pathways, the distributions of T and B cells, adhesion molecules and transforming growth factor-beta (TGF-beta) were assessed in liver biopsies with chronic inflammation due to hepatitis C (HCV, n = 8) and other causes (non-HCV, n = 10). Frozen sections were immunostained using primary antibodies to CD2, CD20, CD4, CD8, intercellular adhesion molecule (ICAM-1), vascular cell adhesion molecule (VCAM)-1, HLA-DR, lymphocyte function-associated antigen (LFA)-1, and TGF-beta. Inflammatory cells positive for each immunophenotypic marker were counted, and positive staining for adhesion molecules, HLA-DR and TGF beta was graded in triads and lobules and compared in HCV and non-HCV biopsies. In all biopsies, T cells were more frequent than B cells, both in triads and lobules. CD20+, CD4+, CD8+ and LFA-1+ cells were increased in HCV compared to non-HCV biopsies. Portal lymphoid aggregates were present in 6 of 8 HCV biopsies and 3 of 10 non-HCV biopsies. Aggregates consisted of CD20+, CD4+, CD8+ and LFA-1+ cells, and ICAM-1 and VCAM-1 were increased. Sinusoidal lining cells in HCV biopsies and non-HCV biopsies with inflammation expressed HLA-DR, ICAM-1, and CD4. TGF-beta was increased in foci of necrosis. Inflammation in chronic HCV involves common immune-mediated cellular effector pathways and the inflammation in the portal triads represents aggregation of both T and B cells, mediated in part by upregulation of adhesion molecules on portal stromal cells; this is possibly in response to antigens draining from necroinflammatory foci in the lobules. TGF-beta is increased in active necroinflammatory foci, but not in portal lymphoid aggregates.

Topics: Adult; Aged; Antigens, CD; Biomarkers; Biopsy; Female; Hepatitis C, Chronic; Humans; Immunohistochemistry; Intercellular Adhesion Molecule-1; Liver Diseases; Lymphocyte Function-Associated Antigen-1; Male; Middle Aged; Transforming Growth Factor beta; Vascular Cell Adhesion Molecule-1

Hepatic silicosis, cirrhosis, liver cell adenoma, and carcinomas developed in nude mice (NCr-Nu) given quartz by the subcutaneous and intraperitoneal routes. Syrian golden hamsters (15:16 EHS:cr) given quartz by both routes developed extensive fibrosis and cirrhosis and had higher morbidity and mortality rates after 3 months. Crystalline silica (quartz) induces fibrosis, adenomas, and carcinomas in the lungs of Fisher 344 rats, but certain strains of mice and hamsters are resistant to quartz-induced pulmonary carcinogenesis. Pulmonary fibrosis, however, is minimal in mice and absent in hamsters who received quartz intratracheally. To determine whether species differences are due to organ-specific rather than species-specific factors, susceptibility of the liver to quartz toxicity was investigated in nude mice and hamsters. The present study shows that the differential manifestations of quartz toxicity by these rodent species are dependent on factors that are organ-specific rather than host-specific. At 3 months, hepatocytes in mice were immunostained with intracellular transforming growth factor (TGF) beta 1 (LC 1-30) but not with TGF-beta 1 latency-associated peptide (LAP) protein (266-278); at 12 months, hepatocytes were immunostained with TGF-beta 1 LAP (266-278) but not with TGF-beta 1 (LC1-30). The hepatocytes of hamsters at 3 months showed immunoreactivities to TGF-beta 1 LAP (266-278) and TGF-beta 1 (LC1-30); immunostaining to TGF-beta 1 (LC1-30) was detected in nonparenchymal cells. Extracellular TGF-beta 1 (CC1-30) was detected in the silicotic granulomas and fibrous tissue in livers of both species. Quartz-induced liver carcinoma did not express TGF-beta 1 LAP (266-278) and LC (1-30) proteins, but these were detected in the cells of the adenoma in the same liver. Control animals showed no hepatic lesions nor immunoreactivity to TGF-beta 1. The spatial and temporal patterns of expression of TGF-beta 1, TGF-beta 2, TGF-beta receptor type II messenger RNAs (mRNAs), and TGF-beta 1 proteins in the different hepatic lesions suggests that TGF-beta isoforms may play a role in the pathogenesis of quartz-induced fibrosis, cirrhosis, liver cell adenoma, and carcinoma.

Topics: Adenoma, Liver Cell; Animals; Blotting, Northern; Chemical and Drug Induced Liver Injury; Cricetinae; Female; Immunohistochemistry; Liver; Liver Cirrhosis, Experimental; Liver Diseases; Liver Neoplasms, Experimental; Male; Mesocricetus; Mice; Mice, Nude; Microscopy, Electron; Microscopy, Immunoelectron; Quartz; Receptors, Transforming Growth Factor beta; RNA, Messenger; Silicosis; Transforming Growth Factor beta

Ito cells (lipocytes, stellate cells) are regarded as the principle matrix-producing cell of the liver and have been shown recently to express glial fibrillary acidic protein, an intermediate filament typically found in glia cells of the nervous system. The present study examines 1) whether Ito cells of rat liver express central nervous system typical adhesion molecules, namely, neural cell adhesion molecule (N-CAM), in a cell-type-specific manner and 2) whether N-CAM expression is affected by activation of Ito cells in vitro and during rat liver injury in vivo. As assessed by reverse transcriptase polymerase chain reaction, Northern blotting, Western blotting, and immunocytochemistry of freshly isolated and cultivated hepatic cells, N-CAM expression was restricted to Ito cells and was absent in hepatocytes, Kupffer cells, and sinusoidal endothelial cells. Ito cells expressed predominantly N-CAM-coding transcripts of 6.1 and 4.8 kb in size and 140-kd isoforms of the N-CAM protein, which was localized on the cell surface membrane of Ito cells. In parallel to glial fibrillary acidic protein down-regulation and smooth muscle alpha-actin up-regulation, N-CAM expression was increased during in vitro transformation of Ito cells from resting to activated (myofibroblast-like) cells and by the fibrogenic mediator transforming growth factor-beta 1. By immunohistochemistry, N-CAM was detected in normal rat liver in the portal field as densely packed material and in a spot as well as fiber-like pattern probably representing nerve structures. However, after liver injury, N-CAM expression became detectable in mesenchymal cells within and around the necrotic area and within fibrotic septae. In serially cut tissue sections, N-CAM-positive cells were predominantly co-distributed with smooth muscle alpha-actin-positive cells rather than glial fibrillary acidic protein-positive cells, especially in fibrotic livers. The experimental results illustrate that N-CAM positivity in the liver cannot be solely ascribed to nerve endings as, among the different types of resident liver cells, Ito cells specifically express N-CAM in vitro and presumably in vivo. In addition to its role as potential cell-type-specific marker protein for activated Ito cells, the induction of N-CAM expression might illustrate a mechanism by which mesenchymal cell proliferation might be inhibited when tissue repair is concluded.

Topics: Actins; Animals; Base Sequence; Biomarkers; Cell Adhesion Molecules, Neuronal; Cells, Cultured; DNA Primers; Gene Expression Regulation; Glial Fibrillary Acidic Protein; Liver; Liver Diseases; Molecular Sequence Data; Polymerase Chain Reaction; Rats; Rats, Wistar; RNA-Directed DNA Polymerase; Transforming Growth Factor beta; Up-Regulation

Growth factors have been implicated in the pathogenesis of liver fibrosis, a major determinant of the clinical course of chronic liver disease. The aim of this study was to study the relationship of growth factor expression to inflammation and fibrosis in a variety of human liver diseases.. We studied by in situ hybridization the expression of transforming growth factor (TGF) beta 1, platelet-derived growth factor (PDGF) A and PDGF-B, and procollagen type I (pro-I) messenger RNAs (mRNAs) in liver diseases of various etiologies.. Pro-I mRNA was expressed by mesenchymal cells at sites of inflammation and scarring, where TGF-beta 1 immunoreactivity was often found, and by perisinusoidal cells. TGF-beta 1 and PDGF-A mRNAs were expressed mainly by mononuclear cells and proliferating ductular cells. TGF-beta 1 mRNA was also expressed by perisinusoidal cells. PDGF-A gene expression was more common than that of PDGF-B. Pro-I and TGF-beta 1 expression correlated with both ductular proliferation and tissue inflammation, whereas PDGF-A and PDGF-B only correlated with ductular proliferation.. Our data suggest that TGF-beta 1 and PDGF are involved in human liver inflammation and fibrosis. The expression of growth factor mRNAs in proliferating ductular cells may indicate a role for these cells in liver fibrogenesis and may help explain the pathophysiology of conditions such as biliary atresia progressing to fibrosis despite the absence of marked inflammation.

Topics: Gene Expression; Growth Substances; Humans; Immunohistochemistry; In Situ Hybridization; Liver; Liver Diseases; Platelet-Derived Growth Factor; Procollagen; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-sis; Reference Values; RNA, Messenger; Transforming Growth Factor beta

To investigate the effects of transforming growth factor beta 1 (TGF-beta 1) on regeneration and induction of apoptosis of liver cell and bile duct in various liver diseases.. Formalin fixed paraffin wax sections of 18 liver tissue samples were obtained by needle biopsy, surgery, or necropsy; these included six liver cirrhosis, three obstructive jaundice; five fulminant hepatitis, one subacute hepatitis, and three normal liver. Expression of TGF-beta 1, apoptosis related Le(y) antigen, Fas antigen, a receptor for tumour necrosis factor, and biotin nick end labelling with terminal deoxynucleotidyl transferase mediated dUTP (TUNEL) for locating DNA fragmentation, was investigated histochemically.. TGF-beta 1 was expressed in areas of atypical bile duct proliferation, where bile duct continuously proliferated from liver cells. In occlusive jaundice and fulminant hepatitis, TUNEL was positive in nuclei and cytoplasm of metaplastic cells which formed incomplete bile ducts, and these cells appeared to extend from TGF-beta 1 expressing liver cells. Fas antigen was found only on the cell membrane of proliferated bile duct in fulminant hepatitis, which differed from TGF-beta 1 and TUNEL positive areas. Le(y) antigen was expressed in liver cell and bile duct at the areas with atypical bile duct proliferation, but its coexpression with TUNEL was rare.. TGF-beta 1 plays a role in the arrest of liver cell regeneration and atypical bile duct proliferation, and in areas of rapidly progressing atypical bile duct proliferation, such as in fulminant hepatitis or bile retention. Apoptosis appears to be induced by TGF-beta 1. This phenomenon may account for the inadequate hepatic regeneration that occurs with liver disease.

Topics: Apoptosis; Cholestasis; Hepatitis; Humans; Immunoenzyme Techniques; Liver Cirrhosis; Liver Diseases; Liver Regeneration; Transforming Growth Factor beta

Intraperitoneal injection of Group A streptococcal cell wall (SCW) fragments into female Lewis rats results in the induction of an acute hepatic inflammation that progresses to granulomatous lesions. Kupffer cells have been shown to rapidly clear circulating SCW which triggers production of TGF-beta. In this study, we examined Kupffer cells for the expression of TGF-beta receptors to determine if these cells might be modulated in an autocrine/paracrine fashion by TGF-beta during SCW-hepatic inflammation. By receptor crosslinking and subsequent SDS-PAGE analysis we demonstrate that Kupffer cells express Type I TGF-beta receptors, but not Types II and III. Scatchard analysis indicated a receptor density of approximately 1100 receptors per cell. Functionally, TGF-beta was found to be chemotactic for Kupffer cells in vitro and this chemotactic response was higher in cells isolated from rats 1-21 days post SCW-injection. Although TGF-beta 1 mRNA is constitutively expressed by Kupffer cells, in vitro stimulation of the cultures with purified TGF-beta augments the expression of TGF-beta 1 mRNA and protein synthesis suggesting autocrine/paracrine regulation. These results indicate that TGF beta secreted by Kupffer cells during SCW-induced hepatic inflammation may amplify its own expression and regulate Kupffer cell functions relevant to the formation of granulomatous lesions within the liver.

Topics: Animals; Cell Wall; Cells, Cultured; Chemotaxis; DNA Probes; Granuloma; Kupffer Cells; Liver Diseases; Male; Rats; Rats, Inbred Lew; Receptors, Cell Surface; Receptors, Transforming Growth Factor beta; RNA, Messenger; Streptococcal Infections; Streptococcus pyogenes; Transforming Growth Factor beta

We recently demonstrated that transforming growth factor-beta 1 stimulates collagen synthesis in hepatic cells in vitro, and that the synthesis of this cytokine is markedly increased in two rodent models of hepatic fibrosis. In the present study, we investigated the association of transforming growth factor-beta 1 (TFG-beta 1) gene expression in human liver disease. Sixteen patients with active liver disease had percutaneous liver biopsies performed for diagnostic purposes. Total RNA was extracted from an unused portion of each biopsy and then subjected to hybridization analysis with the following human cDNA clones: albumin, pro alpha 1 (I) collagen, and TGF-beta 1. Surgical liver biopsy specimens from two patients without hepatic disease were used as controls. When compared to controls, the patients with active liver disease had a 19% decrease in albumin, a 97% increase in type I collagen, and a 120% increase in transforming growth factor-beta 1 mRNA levels. Moreover, steady-state levels of TGF-beta 1 and procollagen mRNAs were significantly correlated. Nuclear run-on assays showed that livers from two patients with fibrosis had TGF-beta 1 transcription rates that were more than 2-fold higher than rates in control livers. These findings indicate that transforming growth factor-beta 1 gene expression is significantly enhanced in man during active liver disease.

Topics: Actins; Adult; Biopsy; Blotting, Northern; Cell Nucleus; Female; Gene Expression; Humans; Liver; Liver Diseases; Male; Middle Aged; Procollagen; RNA; RNA, Messenger; Serum Albumin; Transcription, Genetic; Transforming Growth Factor beta

Hepatic granulomas are induced by intraperitoneal injection of streptococcal cell walls (SCW) into Lewis rats. Kupffer cells rapidly clear SCW from the blood, and the authors examined Kupffer cells further for a role in SCW-hepatic inflammation. Isolated Kupffer cells cultured with SCW secreted high levels of tumor necrosis factor alpha (TNF alpha), interleukin-1 (IL-1), transforming growth factor beta (TGF beta), and prostaglandin E2 (PGE2). SCW transiently induced increased steady-state levels of IL-1 beta and TNF alpha mRNA; in contrast, constitutive expression of TGF beta 1 mRNA in Kupffer cells was not affected by SCW. Low concentrations of SCW induced the accumulation of intracellular IL-1 and TGF beta bioactivity, with intracellular IL-1 bioactivity remaining high through at least 72 hours of culture. Kupffer cells isolated 1, 7, and 21 days after SCW injection did not express IL-1 beta or TNF alpha mRNA greater than control levels and exhibited marked hyporesponsiveness to secondary in vitro stimulation with SCW or LPS. SCW transiently induces Kupffer cells to secrete a variety of soluble mediators that contribute to hepatic inflammation by inducing leukocyte recruitment and activation and fibroproliferation. The transient nature of the Kupffer cell response and the hyporesponsiveness to secondary stimulation may be a mechanism by which the hepatic inflammation is negatively regulated.

Topics: Animals; Cell Wall; Cells, Cultured; Cytokines; Dinoprostone; Granuloma; Inflammation; Kupffer Cells; Liver Diseases; Rats; Rats, Inbred Lew; RNA, Messenger; Streptococcus; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Topics: Humans; Liver; Liver Cirrhosis; Liver Diseases; Procollagen; RNA, Messenger; Transforming Growth Factor beta