In the current study the thymidine kinase / ganciclovir-system was used to develop a prophylaxis in oder to prevent liver fibrogenesis. The intention was to eliminate activated hepatic stellate cells (HSC), those cells which became proliferative, fibrogenetic and contractile because of liver injury. Activated HSC are called myofibroblasts. They are mainly responsible for the expression of extracellular matrix protein leading to excessive scar formation and finally to cirrhosis, the end stage of fibrosis. Therefore the herpes simplex thymidinikinase gene was coupled to the human tissue inhibitor of metalloproteinases-1-promoter (h-TIMP-1-promoter), which is known as a strongly activated promoter within progressive liver fibrosis. Shuttle vector and recombinant replication-defective adonovirus was generated, containing the h-TIMP-1 / thymidine kinase transgene. After transfection of the plasmid into HSC and after infection of HSC with an adenovirus a strong expression of thymidine kinase-mRNA was observed in the cells. Furthermore protein synthesis of thymidinkinase and its correct enymatic activity was proven. The infected cells were able to modify the prodrugs aciclovir and ganciclovir into aciclovir- and ganciclovirtriphosphat. The toxicity of ganciclovir was determind via dilution series in cell culture. A successful treatment with the combination of thymidine kinase and ganciclovir in cells was observed and could be reproduced. The treatment caused cell death. It was shown that single components are not toxic to the cells. Furthermore, a Bystander Effect was observed. It became clear, that the activity of the h-TIMP-1-promoter is different depending on the celltype: under controll of the h-TIMP-1-promoter thymidine kinase was strongly expressed in HSC and less in myofibroblasts. To analyze the level of expression in quiescent HSC experiments were carried out in teflon tubes in order to to keep the cells as non-activated HSC. The results directed to the supposition that h-TIMP-1-promoter isnt active in quiescent HSC. The induced cell death was identified as apoptosis; necrosis could be excluded. For further animal experiments with rats a detailled plan was established. The effect of liver fibrosis as a consequence of bile duct ligation was qualified and quantified by monitoring the animals, analysing laboratory parameters and characterising tissue samples. It was not possible to make a certain conclusion about the effect of thymidine kinase / ganciclovir-system and the “bystander effect” in vivo.
LCN2 is a member of the lipocalin super-family with a diverse functionality, including transport of fatty acids and apoptosis induction.
Read moreDie Aktivierung der hepatischen Sternzellen (HSC) und ihre Transdifferenzierung in Myofibroblasten-ähnlichen Zellen (MFB) ist ein initialer Prozess der Leberfibrogenese. Ausgehend von einem Vitamin A speichernden Phänotyp entwickeln die Zellen dabei u.a. kontraktile Eigenschaften, die mit einer erhöhten Expression von Differenzierungsmarkern glatter Muskelzellen (SMC) einhergehen. Ein wesentlicher transkriptioneller Regulator dieser Gene ist der „Serum Response Factor“ (SRF), dessen Aktivität neben der Interaktion mit Kofaktoren auch durch repressiv wirkende Spleißvarianten kontrolliert wird. Das Ziel dieses Projektes war der Nachweis der SRF-Expression sowie seiner aus SMC bekannten Spleißvarianten in aktivierten HSC. Primäre Ratten-HSC wurden in einem in vitro-Transdifferenzierungsmodell zu verschiedenen Zeitpunkte lysiert und ihre RNA isoliert. Nach Umschreibung in cDNA wurde mittels spezifischer Primer die Expression von SRF, definierter SRF-Spleißvarianten sowie der SMC-Marker α-SMA und SM22α in einer semi-quantitativen PCR ermittelt und densiometrisch erfasst. Die SRF-Expression ist in frisch aktivierten HSC am stärksten und fällt zwischen dem 1. und 3. Kulturtag auf ca. 40% ab. Während der weiteren Kultivierung bis zum Tag 7 bleibt die Expression auf diesem Niveau, gefolgt von einem weiteren Abfall auf ca. 20% des Ausgangsniveaus in MFB. Dagegen steigt im gesamten Zeitraum die Expression der SMC-Marker konstant an. Wir konnten erstmals auch Spleißvarianten mit Deletionen der Exons 5, 4+5, bzw. 3–5 in HSC nachweisen, die im Verlauf des Transdifferenzierungs-prozesses eine jeweils ähnliche Expressionskurve wie die ungespleißte Variante aufweisen. Aus verschiedenen Muskelzelltypen ist bekannt, dass SRF-Spleißvarianten in unterschiedlichem Ausmaß negative Regulatoren der SRF-Aktivität sind. Inwieweit diese Varianten auch bei der Kontrolle glatter Muskelzellmarker in aktivierten HSC eine Rolle spielen konnte aufgrund ihres Expressionsmusters nicht geklärt werden. Die weitgehend parallele Expression aller Varianten im Laufe der Transdifferenzierung spricht eher für eine Feinregulation der SRF-Aktivität. Expressionskonstrukte der gefundenen Spleißvarianten können ihre individuelle Rolle in diesem Geschehen weiter aufdecken.
Hepatische Sternzellen (HSC) sind die entscheidenden zellulären Effektoren bei der Leberfibrogenese, die nach Aktivierung durch TGF-β1 zu proliferativen, extrazelluläre Matrix bildenden Myofibroblasten (MFB) transdifferenzieren. In experimentellen Fibrosemodellen kann die Expression von Fibrosemarkern nach adenoviralem Gentransfer durch systemische Blockade von TGF-β1 oder seiner intrazellulären Signaltransduktion reduziert werden. Für eine nebenwirkungsfreie Intervention der Leberfibrogenese durch TGF-β-Antagonisten ist es allerdings sinnvoll die Transgen-Expression lokal auf HSC/MFB und zeitlich auf die Krankheitsausprägung zu begrenzen. Wir untersuchen deshalb die Möglichkeiten über transkriptionelles targeting eine HSC/MFB-spezifische Expressionsregulation in der Leber zu vermitteln. Hierzu nutzen wir Promotorfragmente des kodierenden Gens für das LIM-Domänen-Protein CRP2 (CSRP2), das endogen in der Leber ausschließlich in aktivierten HSC exprimiert wird.Das GFP-Reportergen wurde unter transkriptionelle Kontrolle unterschiedlich langer Promotorfragmente (0,8 kbp und 4,2 kbp) sowie des konstitutiven CMV-Promotors gesetzt und in Adenoviren (Ad5) verpackt. Die Infektion kultivierter HSC/MFB und Hepatozyten zeigte, dass beide Promotorfragmente die Genexpression in aktivierten HSC effizient steuern. In in vivo-Experimenten mit Ad5-CMV-GFP infizierten Ratten fand sich zudem als reglementierender Faktor die biologische Qualität und Reinheit der eingesetzten Adenoviren. Weiterhin wird das Ausmaß der Aufnahme adenoviraler Partikel durch Leberzellen stark durch die Applikationsmethode bestimmt. So führte die systemische Infektion über die Schwanzvene im Vergleich zur lokalen Infektion über die Pfortader prinzipiell zu einer homogeneren Verteilung der Adenoviren. Somit bietet der adenovirale Transfer CSRP2-Promotor gesteuerter TGF-β-Antagonisten einen Ansatz zur Zelltyp-spezifischen Blockade der Transdifferenzierungsprozesse in HSC.
Read moreAims: The first pathological implication of chronic toxic liver injury including alcoholism is subsequent cell damage or cell death which results in transdifferentiation of HSC into the myofibroblast phenotype. Activated hepatic stellate cells (HSC) accelerate production of ECM, TIMPs, cytokines, and several MMPs promoting hepatic fibrogenesis. TIMP–1 was found to suppress apoptosis in activated HSC1. Our approach was to antagonize TIMP–1 by proteolytically inactive MMP–9 mutants in order to induce HSC apoptosis and thus to reduce hepatic fibrogenesis.
Read moreAims: TGF-β1 causes excessive extracellular matrix deposition in the setting of fibrogenic lesions. In the liver, BMP-7 is an antifibrotic ligand abrogating profibrogenic TGF-β1 responses [1]. However, the exact molecular details regulating this antagonism are unknown. We employed a model system (L6E9 myoblasts) which shows a myofibroblastic phenotype upon treatment with TGF-β1 [2] resembling that of the transition of hepatic stellate cell (HSC) into myofibroblast-like cells (MFB). Previously, we have already shown that TGF-β1 responses are counteracted by BMP-7 in (L6E9-) myoblasts [3].
Read moreUNLABELLED: Peripheral CD8 T-cell tolerance can be generated outside lymphatic tissue in the liver, but the course of events leading to tolerogenic interaction of hepatic cell populations with circulating T-cells remain largely undefined. Here we demonstrate that preferential uptake of systemically circulating antigen by murine liver sinusoidal endothelial cells (LSECs), and not by other antigen-presenting cells in the liver or spleen, leads to cross-presentation on major histocompatibility complex (MHC) I molecules, which causes rapid antigen-specific naïve CD8 T-cell retention in the liver but not in other organs. Using bone-marrow chimeras and a novel transgenic mouse model (Tie2-H-2K(b) mice) with endothelial cell-specific MHC I expression, we provide evidence that cross-presentation by organ-resident and radiation-resistant LSECs in vivo was both essential and sufficient to cause antigen-specific retention of naïve CD8 T-cells under noninflammatory conditions. This was followed by sustained CD8 T-cell proliferation and expansion in vivo, but ultimately led to the development of T-cell tolerance. CONCLUSION: Our results show that cross-presentation of circulating antigens by LSECs caused antigen-specific retention of naïve CD8 T-cells and identify antigen-specific T-cell adhesion as the first step in the induction of T-cell tolerance.
Read moreBackground/Aims: Platelet-derived growth factor (PDGF)-BB and transforming growth factor (TGF)-β1 are important profibrogenic cytokines involved in liver fibrogenesis. They trigger the activation of hepatic stellate cells (HSC) and the synthesis of extracellular matrix (ECM) components, respectively. The pivotal role of both cytokines has been figured out by using antagonistic strategies [1]. It has been previously shown that PDGF-BB may exponentiate profibrogenic TGF-β signaling through a MAP Kinase (MAPK)-mediated modulation of Smad activity [2].
Read moreHintergrund: CC binding protein 2 (CCBP2) Ist ein kürzlich identifizierter Chemokinrezeptor, welcher keine intrazelluläre Signaltransduktion aufweist und daher als Scavenger-Rezeptor angesehen wird. CCBP2 reguliert Entzündungsprozesse, indem es Chemokine während einer Entzündungsreaktion bindet und deren Abbau einleitet. Das Ziel dieser Studie war die Expression von CCBP2 im murinen Modell und bei Patienten mit HCV Infektion zu analysieren und zu überprüfen, ob genetische Variationen von CCBP2 einen Einfluss auf die Entzündungsreaktion bei HCV haben.
Read moreAims: Platelet-derived growth factor (PDGF) plays an important role in liver fibrogenesis, whereby PDGF-B and –D act as potent mitogens in culture-activated hepatic stellate cells (HSC) (1, 2). Induction of PDGFR-beta in HSC has been well documented in single dose CCL4 induced acute liver injury and in early bile duct ligated models in vivo (3). Of the newly discovered Isoforms PDGF-C and -D, only PDGF-D shows significant upregulation in BDL rat model (2). We further investigated the expression of PDGF isoforms and their receptors in chronic liver injury through long term repeated dosing of CCl4 in rats.
Read morePleiotrophin (PTN) is a heparin-binding protein and a growth factor with a broad spectrum of biological activities very similar to the Fibroblast Growth Factors (FGFs). FGFs and PTN promote the growth, survival, and migration of various cells, and play many roles in epithelial mesenchymal interactions during organogenesis. Particulary, expression of PTN has been implicated in the development and tissue repair of the liver. PTN interacts with several receptor proteins which may transduce PTN signaling in a cell type specific way. As PTN receptor has been proposed syndecan-3, receptor-tyrosine phosphatase (RPTP)zeta, anaplastic lymphoma kinase (ALK) and the Wnt co-receptors LRP5/6. Syndecan-3 and RPTPzeta have been proposed to be the receptor of PTN-induced neurite outgrowth, migration and survival of embryonic neurons. In this study we analysed the expression of PTN, the expression of the receptors ALK and LRP5/6 in rat liver cells, in activated rat hepatic stellate cells and in human and rat myofibroblasts. Ligand and receptor expression was investigated at the mRNA level using reverse transcription polymerase chain reaction. All analysed hepatic nonparenchymal cells including stellate cells, sinusoidal endothelial cells and Kupffer cells demonstrate PTN-, ALK- and LRP5/6- mRNA expression. Hepatocytes did not express the anaplastic lymphoma kinase. Interestingly, since ALK and PTN are implicated in tumor formation, ALK expression was also not detected in the hepatoma cell line HepG2. Since PTN prevents apoptosis in serum-starved NIH3T3 fibroblasts via activation of ALK at concentrations much lower than those needed to induce cell growth, the function of PTN in the diseased liver may mainly be its anti-apoptotic signaling in HSCs.
Read moreBackground/Aims: Despite tremendous efforts in disclosing the pathophysiological and epidemiological factors that are associated with liver fibrogenesis, non-invasive diagnostic opportunities to estimate the clinical outcome or progression of liver fibrogenesis are limited [1]. Presently, approximately 0.6% of the population in Germany are infected and more than 170 million people worldwide are estimated to suffer from chronic hepatitis C (HCV) infection causing 300.000 deaths annually [2]. Therefore, there is an absolute demand on high-throughput methodologies allowing the reasonable and reliable assessment of the severity and/or progression of hepatic fibrogenesis. MALDI-TOF (matrix assisted laser desorption ionization time-of-flight) mass spectrometry (MS) is a sensitive method to screen for protein profiles in samples. Following MS analysis spectra obtained are analyzed for sample-class specific but differential expression (pattern profiling). Mathematical models obtained are suitable for classification of further unknown samples.
Read moreThere are currently no approved antifibrotic therapies for liver cirrhosis. We used vitamin A-coupled liposomes to deliver small interfering RNA (siRNA) against gp46, the rat homolog of human heat shock protein 47, to hepatic stellate cells. Our approach exploits the key roles of these cells in both fibrogenesis as well as uptake and storage of vitamin A. Five treatments with the siRNA-bearing vitamin A-coupled liposomes almost completely resolved liver fibrosis and prolonged survival in rats with otherwise lethal dimethylnitrosamine-induced liver cirrhosis in a dose- and duration-dependent manner. Rescue was not related to off-target effects or associated with recruitment of innate immunity. Receptor-specific siRNA delivery was similarly effective in suppressing collagen secretion and treating fibrosis induced by CCl4 or bile duct ligation. The efficacy of the approach using both acute and chronic models of liver fibrosis suggests its therapeutic potential for reversing human liver cirrhosis.
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