ABSTRACT
In contrast to AAV, Simian Virus 40 (rSV40) not inducing neutralizing antibodies (NAbs) allowing re-treatment seems a promising vector for neonatal treatment of inherited liver disorders. Several studies have reported efficacy of rSV40 in animal models for inherited liver diseases. In all studies the ubiquitous endogenous early promoter controlled transgene expression establishing expression in all transduced tissues. Restricting this expression to the target tissues reduces the risk of immune response to the therapeutic gene. In this study a liver specific rSV40 vector was generated by inserting a hepatocyte specific promoter. This increased the specificity of the expression of hUGT1A1 in vitro. However, in vivo the efficacy of rSV40 appeared too low to demonstrate tissue specificity while increasing the vector dose was not possible because of toxicity. In contrast to earlier studies, neutralizing antibodies were induced. Overall, the lack of a platform to produce high titered and pure rSV40 particles and the induction of NAbs, renders it a poor candidate for in vivo gene therapy.
Subject(s)
Glucuronosyltransferase/genetics , Hyperbilirubinemia, Hereditary/pathology , Simian virus 40/genetics , Animals , Antibodies, Neutralizing/genetics , Antibodies, Neutralizing/metabolism , Cell Line, Tumor , Genetic Therapy/methods , Genetic Vectors/genetics , Genetic Vectors/metabolism , Glucuronosyltransferase/deficiency , Glucuronosyltransferase/metabolism , Humans , Hyperbilirubinemia, Hereditary/genetics , Liver/metabolism , Mice , Mice, Knockout , Promoter Regions, Genetic , Tissue Distribution , Transcriptional ActivationABSTRACT
Adamant progression of chronic cholangiopathies towards cirrhosis and limited therapeutic options leave a liver transplantation the only effective treatment. Insulin-like growth factor 1 (IGF1) effectively blocks fibrosis in acute models of liver damage in mice, and a phase I clinical trial suggested an improved liver function. IGF1 targets the biliary epithelium, but its potential benefit in chronic cholangiopathies has not been studied. To investigate the possible therapeutic effect of increased IGF1 expression, we crossed Abcb4(-/-) mice (a model for chronic cholangiopathy), with transgenic animals that overexpress IGF1. The effect on disease progression was studied in the resulting IGF1-overexpressing Abcb4(-/-) mice, and compared to that of Abcb4(-/-) littermates. The specificity of this effect was further studied in an acute model of fibrosis. The overexpression of IGF1 in transgenic Abcb4(-/-) mice resulted in stimulation of fibrogenic processes - as shown by increased expression of Tgfß, and collagens 1, 3 and 4, and confirmed by Sirius red staining and hydroxyproline measurements. Excessive extracellular matrix deposition was favored by raise in Timp1 and Timp2, while a reduction of tPA expression indicated lower tissue remodeling. These effects were accompanied by an increase in expression of inflammation markers like Tnfα, and higher presence of infiltrating macrophages. Finally, increased number of Ck19-expressing cells indicated proliferation of biliary epithelium. In contrast to liver fibrosis associated with hepatocellular damage, IGF1 overexpression does not inhibit liver fibrogenesis in chronic cholangiopathy.
Subject(s)
ATP Binding Cassette Transporter, Subfamily B , Cell Proliferation , Cholestasis/metabolism , Epithelium/metabolism , Gene Expression Regulation , Insulin-Like Growth Factor I/biosynthesis , Animals , Cell Line , Cholestasis/genetics , Cholestasis/pathology , Chronic Disease , Collagen/biosynthesis , Collagen/genetics , Epithelium/pathology , Fibrosis/genetics , Fibrosis/metabolism , Fibrosis/pathology , Mice , Mice, Knockout , Tissue Inhibitor of Metalloproteinase-1/biosynthesis , Tissue Inhibitor of Metalloproteinase-1/genetics , Tissue Plasminogen Activator/biosynthesis , Tissue Plasminogen Activator/genetics , Transforming Growth Factor beta/biosynthesis , Transforming Growth Factor beta/genetics , ATP-Binding Cassette Sub-Family B Member 4ABSTRACT
A comprehensive understanding of the mechanisms that underlie hepatic differentiation inside a bioartificial liver (BAL) device is obtained when functional, histological, and gene expression analyses can be combined. We therefore developed a novel cell-sampling technique that enabled us to analyze adherent hepatocytes inside a BAL device during a 5-day culture period, without the necessity of terminating the culture. Biochemical data showed that hepatocyte-specific functions were relatively stable, despite an increase in glycolytic activity. Quantitative reverse transcriptase polymerase chain reaction analysis of hepatic genes cytochrome p450 3A29, albumin, glutamine synthetase, alpha-1 antitrypsin, and carbamoyl-phosphate synthetase, but also de-differentiation marker pi-class glutathione S transferase showed stable messenger ribonucleic acid (mRNA) levels from day 1 to 5. In contrast, mRNA levels of alpha-fetoprotein, pro- and anti-apoptotic genes Bax-alpha and Bcl-X(L), metabolic genes lactate dehydrogenase and uncoupling protein 2, and cytoskeleton genes alpha- and beta-tubulin and beta-actin increased in 5 days. Histological analysis revealed viable tissue-like structures with adaptation to the in vitro environment. We conclude that hepatocytes show a tendency for de-differentiation shortly after seeding but thereafter remain acceptably differentiated during 5 days of culture. Furthermore, partly impaired mitochondrial function is suggestive for local hypoxic regions and may trigger the observed metabolic changes. Anti-apoptotic activity seems to balance pro-apoptotic activity. This new cell-sampling technique facilitates the analysis of dynamic processes of hepatocyte culture inside a BAL.
