Jozsef Dudas

Changes of gene expression of iron regulatory proteins during turpentine oil-induced acute-phase response in the rat

In the present study, turpentine oil was injected in the hind limb muscle of the rat to stimulate an acute-phase response (APR). The changes in the gene expression of cytokines and proteins known to be involved in the iron regulatory pathway were then studied in the liver and in extra-hepatic tissue. In addition to the strong upregulation of interleukin-6 (IL-6) and IL-1β observed in the inflamed muscle, an upregulation of the genes for IL1-β and tumor necrosis factor-α, but not IL-6, were detectable in the liver. Hepatic Hepc gene expression increased to a maximum at 6u2009h after the onset of APR. An upregulation of transferrin, transferrin receptor 1 (TfR1), TfR2, ferritin-H, iron responsive element binding protein-1 (IRP1), IRP2 and divalent metal transporter gene expression was also found. Hemojuvelin (Hjv)-, ferroportin 1-, Dcytb-, hemochromatosis-gene- and hephaestin gene expression was downregulated. Hepcidin (Hepc) gene expression was not only detectable in extra-hepatic tissues such as heart, small intestine, colon, spleen and kidney but it was also upregulated under acute-phase conditions, with the Hjv gene being regulated antagonistically. Fpn-1 gene expression was downregulated significantly in heart, colon and spleen. Most of the genes of the known proteins involved in iron metabolism are expressed not only in the liver but also in extra-hepatic tissues. Under acute-phase conditions, acute-phase cytokines (eg IL-6) may modulate the gene expression of such proteins not only in the liver but also in other organs.

Thy-1 is an in vivo and in vitro marker of liver myofibroblasts

Thy-1, a glycophosphatidylinositol-linked glycoprotein of the outer membrane leaflet, has been described in myofibroblasts of several organs. Previous studies have shown that, in fetal liver, Thy-1 is expressed in a subpopulation of ductular/progenitor cells. The aim of this study has been to investigate whether the liver myofibroblasts belong to the Thy-1-positive subpopulation of the adult liver. The expression of Thy-1 has been studied in normal rat liver, in the rat liver regeneration model following 2-acetylaminofluorene treatment and partial hepatectomy (AAF/PH), and in isolated rat liver cells, at the mRNA and protein levels. In normal rat liver, Thy-1 is detected in sparse cells of the periportal area, whereas 7xa0days after PH in the AAF/PH model, a marked increase of the number of Thy-1-positive cells is detectable by immunohistochemistry. Comparative immunohistochemical analysis has revealed the co-localization of Thy-1 and smooth muscle actin, but not of Thy-1 and cytokeratin-19, both in normal rat liver and in the AAF/PH model. Investigation of isolated rat liver cell populations has confirmed that liver myofibroblasts are Thy-1-positive cells, whereas hepatocytes, hepatic stellate cells, and liver macrophages are not. Thy-1 is the first cell surface marker for identifying liver myofibroblasts in vivo and in vitro.

Single-Dose Gamma-Irradiation Induces Up-Regulation of Chemokine Gene Expression and Recruitment of Granulocytes into the Portal Area but Not into Other Regions of Rat Hepatic Tissue

Liver damage is a serious clinical complication of gamma-irradiation. We therefore exposed rats to single-dose gamma-irradiation (25 Gy) that was focused on the liver. Three to six hours after irradiation, an increased number of neutrophils (but not mononuclear phagocytes) was observed by immunohistochemistry to be attached to portal vessels between and around the portal (myo)fibroblasts (smooth muscle actin and Thy-1(+) cells). MCP-1/CCL2 staining was also detected in the portal vessel walls, including some cells of the portal area. CC-chemokine (MCP-1/CCL2 and MCP-3/CCL7) and CXC-chemokine (KC/CXCL1, MIP-2/CXCL2, and LIX/CXCL5) gene expression was significantly induced in total RNA from irradiated livers. In laser capture microdissected samples, an early (1 to 3 hours) up-regulation of CCL2, CXCL1, CXCL8, and CXCR2 gene expression was detected in the portal area but not in the parenchyma; with the exception of CXCL1 gene expression. In addition, treatment with an antibody against MCP-1/CCL2 before irradiation led to an increase in gene expression of interferon-gamma and IP-10/CXCL10 in liver tissue without influencing the recruitment of granulocytes. Indeed, the CCL2, CXCL1, CXCL2, and CXCL5 genes were strongly expressed and further up-regulated in liver (myo)fibroblasts after irradiation (8 Gy). Taken together, these results suggest that gamma-irradiation of the liver induces a transient accumulation of granulocytes within the portal area and that (myo)fibroblasts of the portal vessels may be one of the major sources of the chemokines involved in neutrophil recruitment. Moreover, inhibition of more than one chemokine (eg, CXCL1 and CXCL8) may be necessary to reduce leukocytes recruitment.

Agrin, a novel basement membrane component in human and rat liver, accumulates in cirrhosis and hepatocellular carcinoma

Agrin is a multifunctional heparan sulfate proteoglycan originally discovered in the neuromuscular junctions and later observed in numerous other localizations. The presence of agrin in the liver, either healthy or diseased, has formerly not been reported. We detected agrin in minor amounts in the basement membranes of blood vessels and bile ducts in the healthy liver. The proliferation of bile ductules and the formation of new septal blood vessels in liver cirrhosis, as well as neoangiogenesis in the hepatocellular carcinoma (HCC) result in a dramatic increase in the quantity of agrin. Vascular and peribiliary basement membranes were strongly immunopositive for agrin in 29/29 human liver specimens with cirrhosis and HCC. However, sinusoidal walls of regenerative nodules in the cirrhotic liver consistently remained negative. Given the selectivity of agrin for tumor microvessels, agrin immunohistochemistry may prove helpful in recognizing malignant transformation in cirrhotic livers. Similar immunohistochemical observations were made on the liver of rats exposed to a combined cirrhosis/HCC induction treatment. In both human and rats, agrin probably originates from activated myofibroblasts, vascular smooth muscle cells and biliary epithelial cells. Increased agrin expression in human specimens, in the liver of 4/4 treated rats, as well as in isolated rat liver mesenchymal cells was verified by quantitative RT-PCR. Considering that agrin binds various growth factors, and it directly interacts with cell membrane receptors such as αv-integrins, we hypothesize a stimulatory role for agrin in neoangiogenic processes such as tumor vascularization, and a supportive role in bile ductule proliferation.

Thy-1 is expressed in myofibroblasts but not found in hepatic stellate cells following liver injury.

Thy-1 (CD90) is an adhesion molecule induced in fibroblast populations associated with wound healing and fibrosis. In this study the question whether Thy-1-gene-expression can be induced in hepatic stellate cells (HSC) in vivo, under conditions of liver injury or liver regeneration was addressed. Acute and chronic rat liver injury was induced by the administration of CCl4. For comparison, cirrhotic human liver, and rat 67% partial hepatectomy (PH) was studied as well. Thy-1-gene-expression was examined also in isolated human liver myofibroblasts. Thy-1-mRNA expression was significantly upregulated in chronic liver injury. Thy-1+ cells were detected in the periportal area of rat liver specimens in normal-, injured- and regenerative-conditions. In chronic human and rat liver injury, Thy-1+ cells were located predominantly in scar tissue. In the pericentral necrotic zone after CCl4-treatment, no induction of Thy-1 was found. Gremlin and Thy-1 showed comparable localization in the periportal areas. Thy-1 was not detected in either normal or capillarized sinusoids, in isolated rat HSC, and was neither inducible by inflammatory cytokines in isolated HSC, nor upregulated in treated myofibroblasts. Based upon these data Thy-1 is not a marker of “activated” sinusoidal HSC, but it is a marker of “activated” (myo)fibroblasts found in portal areas and in scar tissue.

Altered regulation of Prox1-gene-expression in liver tumors

BackgroundProspero-related homeobox 1 (Prox1) transcription factor was described as a tumor-suppressor gene in liver tumors. In contrast, Prox1 knock out in murine embryos drastically reduces proliferation of hepatoblasts.MethodsWe have studied the expression of Prox1 in normal liver, liver cirrhosis and peritumoral liver samples in comparison to hepatocellular (HCC) and cholangiocellular carcinoma (CCC) at mRNA, protein and functional levels.ResultsProx1 was found in hepatocytes of normal liver, while normal bile duct epithelial cells were negative. However, Prox1+ cells, which co-expressed biliary epithelial makers and showed ductular morphology, could be detected within fibrotic septa of cirrhotic livers, and in both HCC and CCC. Two Prox1 mRNA isoforms (2.9 kb and 7.9 kb) were identified with a prevalence of the longer isoform in several HCC samples and the shorter in most CCC samples. Evidence was provided that Myc-associated zinc finger protein (MAZ) might significantly contribute to the gene expression of Prox1 in HCC, while neo-expression of Prox1 in CCC remains to be resolved. A point mutation in the prospero domain of Prox1 was found in one HCC sample.ConclusionOur study shows dysregulation of Prox1 in liver cirrhosis, HCC and CCC, such as neo-expression in cells with biliary epithelial phenotype in liver cirrhosis, and in CCC. Altered Prox1 mRNA expression is partly regulated by MAZ, and mutation of the prospero domain in HCC indicates an involvement for Prox1 during tumor progression.

Expression of stem cell factor and its receptor c-Kit during the development of intrahepatic cholangiocarcinoma.

Stem cell factor (SCF) and its receptor, c-Kit, constitute an important signal transduction system with proliferative and anti-apoptotic functions. Besides regulating hemopoietic stem cell proliferation and liver regeneration, it has been implicated in the regulation of human malignancies. However, the cellular expression of the SCF–c-Kit gene system in the liver during cholangiocarcinogenesis has not been studied to date. The protein- and mRNA-expression levels of SCF and c-Kit genes were examined in normal rat liver, in isolated normal rat liver cells and in a thioacetamide-induced rat model of intrahepatic cholangiocarcinoma (CC). Immunohistochemical analysis of the normal liver showed that SCF is expressed in the wall of the hepatic artery and in some cells, which were located along the sinusoids, although it was absent from hepatocytes and biliary epithelial cells. The mRNA analysis of isolated normal liver cell populations revealed a co-expression of SCF- and c-Kit-mRNA in sinusoidal endothelial cells and in Kupffer cells, whereas passaged and cultured liver myofibroblasts (MFs) expressed only SCF. Low levels of the SCF- and c-Kit-mRNA expression could be detected in isolated hepatocytes of the normal liver. Immunohistochemical analysis of the CC tissue showed SCF positivity in proliferating biliary cells (CK-19+), in macrophages (ED-1+) and in MFs (α-smooth-muscle-actin, α-SMA+) of the tumoral microenvironment. c-Kit-positivity could be detected on hepatocytes of the regenerating nodules and on the proliferating bile ducts of CC. Compared with the normal liver tissue, SCF-mRNA from the CC tissue was upregulated up to 20-fold, whereas c-Kit-mRNA was upregulated up to fivefold. These data indicate that several cell populations may become able to express SCF and/or c-Kit during cholangiocarcinogenesis. Therefore, the SCF–c-Kit system may contribute to tumor development, for instance, by inducing proliferation of hepatocytes and of biliary cells and by acting as a surviving factor for CC cells.

Inflammation and repair in viral hepatitis C.

Hepatitis C viral infection (HCV) results in liver damage leading to inflammation and fibrosis of the liver and increasing rates of hepatic decompensation and hepatocellular carcinoma (HCC). However, the host’s immune response and viral determinants of liver disease progression are poorly understood. This review will address the determinants of liver injury in chronic HCV infection and the risk factors leading to rapid disease progression. We aim to better understand the factors that distinguish a relatively benign course of HCV from one with progression to cirrhosis. We will accomplish this task by discussion of three topics: (1) the role of cytokines in the adaptive immune response against the HCV infection; (2) the progression of fibrosis; and (3) the risk factors of co-morbidity with alcohol and human immunodeficiency virus (HIV) in HCV-infected individuals. Despite recent improvements in treating HCV infection using pegylated interferon alpha (PEGIFN-α) and ribavirin, about half of individuals infected with some genotypes, for example genotypes 1 and 4, will not respond to treatment or cannot be treated because of contraindications. This review will also aim to describe the importance of IFN-α-based therapies in HCV infection, ways of monitoring them, and associated complications.

Prospero-related homeobox 1 (Prox1) is a stable hepatocyte marker during liver development, injury and regeneration, and is absent from "oval cells"

The aim of this study was to analyse the changes of Prospero-related homeobox 1 (Prox1) gene expression in rat liver under different experimental conditions of liver injury, regeneration and acute phase reaction, and to correlate it with that of markers for hepatoblasts, hepatocytes, cholangiocytes and oval cells. Gene expression was studied at RNA level by RT-PCR, and at protein level by immunohistochemistry. At embryonal stage of rat liver development (embryonal days (ED) 14–16) hepatoblasts were found to be Prox1+/Cytokeratin (CK) 19+ and α-fetoprotein (AFP)+, at this stage Prox1−/CK19+/AFP- small cells (early cholangiocytes?) were identified. In fetal liver (ED 18–22) hepatoblasts were Prox1+/CK19−/AFP+. CK7+ cholangiocytes were detected at this stage, and they were Prox1−/AFP−. In the adult liver hepatocytes were Prox1+/CK19−/CK7−/AFP−, cholangiocytes were CK19+ and/or CK7+ and AFP−/Prox1−. In models of liver damage and regeneration Prox1 remained a stable marker of hepatocytes. After 2-acetyl-aminofluorene treatment with partial hepatectomy (AAF/PH) the amount of Prox1 specific transcripts was low in the liver, when CK19 and AFP gene expression was high, and at no time point AFP+/CK19+ “oval cells” were found to be Prox1+. However, a few Prox1+/CK19+ and a few Prox1+/CK7+ cells were identified in the liver of AAF/PH-animals, which may represent precursors of hepatocytes, or a precancerous state.

Expression and regulation of the insulin-like growth factor axis components in rat liver myofibroblasts

Apart from hepatic stellate cells (HSC), liver myofibroblasts (MF) represent a second mesenchymal cell population involved in hepatic fibrogenesis. The IGF system including the insulin‐like growth factors I and II (IGF‐I, ‐II), their receptors (IGF‐I receptor, IGF‐IR; IGF‐II/mannose 6‐phosphate receptor, IGF‐II/M6‐PR), and six high affinity IGF binding proteins (IGFBPs) participate in the regulation of growth and differentiation of cells of the fibroblast lineage, possibly contributing to the fibrogenic process. The aim of this work was to study the expression and regulation of the IGF axis components in rat liver MF. Methods: Cultures of MF from passages 1 to 4 (P1–4) were studied. IGFBP secretion was analyzed by [125I]‐IGF‐I ligand and immunoblotting. IGF‐I, IGF‐IR, IGF‐II/M6‐PR, and IGFBP messenger RNA (mRNA) expression was assessed by Northern blot hybridization. DNA synthesis was evaluated by 5‐bromo‐2′‐deoxyuridine (BrdU) incorporation assay. Results: MF from P1 to 4 constitutively expressed mRNA transcripts specific for IGF‐I, IGF‐IR, and IGF‐II/M6‐PR. In MF, biosynthesis of IGFBP‐3 and ‐2 was observed that was stimulated by IGF‐I, insulin, and transforming growth factor β (TGF‐β), whereas platelet‐derived growth factor (PDGF‐BB) revealed inhibitory effects. IGF‐I and to a lesser extent insulin increased DNA synthesis of MF. Simultaneous addition of recombinant human IGFBP‐2 or ‐3 with IGF‐I diminished the mitogenic effect of IGF‐I on MF whereas preincubation of MF with IGFBP‐2 or ‐3 further potentiated the IGF‐I stimulated DNA synthesis. In conclusion, the present study demonstrates that the IGF axis may play a role in the regulation of MF proliferation in vitro which might be relevant in vivo for the process of fibrogenesis during acute and chronic liver injury.