Mark Zern - US grants

Long-term objective: to formulate a general model for the pathogenesis of hepatic fibrosis at the molecular level. Better understanding of changes in protein synthesis in cirrhotic liver will permit development of diagnostic methods to evaluate fibrogenesis in man. Specific aims: (1) To determine changes in hepatic protein synthesis caused by chronic ethanol administration to baboons which have the entire spectrum of alcoholic liver disease. (2) To evaluate the level of gene regulation responsible for differences in collagen mRNA in cirrhotic liver as compared to control liver. (3) To identify which cell(s) is primarily responsible for Type I collagen synthesis in alcoholic liver disease in baboons. (4) To investigate the influence of fibronectin synthesis as an initiator of fibrosis in liver. (5) To evaluate collagen synthesis by a noninvasive serum assay and correlate Type I collagen carboxy-terminal propeptide RIA with collagen mRNA sequence content infibrotic human liver biopsies. (6) To evaluate the possibility that a species' propensity for developing cirrhosis may result from a collagen gene polymorphism. (7) To determine whether any collagen gene polymorphism exists in man which may be related to development of cirrhosis in only a fraction of chronic ethanol users. Methods: cell-free protein synthesis, immunoprecipitation of protein products, "Northern" transfer and "Dot" blot hybridization will be employed to study the effect of chronic ethanol administration on RNA extracted from liver biopsy specimens in baboons. Wedge biopsy specimens from cirrhotic and control baboons will be studied by standard in vitro transcription assays to determine whether transcriptional regulation is responsible for changes in collagen mRNAs in fibrotic liver. In situ hybridization using a Type I procollagen cDNA probe, isolated hepatocytes and tissue sections from fibrotic and control liver will be used to localize the cell(s) which is primarily responsible for collagen synthesis. Investigation of collagen gene polymorphism will involve structural evaluation of Type I collagen genes using Southern blot hybridization and DNA sequencing of genomic DNA extracted from white blood cells of human and animal ethanol abusers. Health relatedness: determining a positive correlation between the collagen propeptide RIA and procollagen mRNA sequence content will help validate RIA as a non-invasive method for assessing prognosis in chronic liver disease. Identification of human collagen gene polymorphism may be useful in predicting which alcohol abusers are at a high risk to develop cirrhosis.

A successful approach to the therapy of hepatic injury and fibrosis should ideally involve the employment of inexpensive, non-toxic agents that will address rationally the pathophysiological processes that are responsible for the liver disease. At present no such agents are available in our standard therapeutic armamentarium. However, alternative therapeutic agents, in the form of herbal remedies, have been employed for generations in Asian countries in the treatment of liver diseases. Thus, our objective is to investigate the effectiveness of a number of herbal remedies, employing a rigorous scientific approach, to determine the relative effectiveness of these agents and the mechanisms by which they may be inhibiting liver injury and fibrosis. Specific Aims: 1) To determine the effectiveness of the herbal medicines in in vitro models of liver cell injury; and 2) to ascertain the effect of the herbal medicines on three models of liver injury and fibrosis. Methods: Hepatocytes and Kupffer cells from Osteogenic Disorder Shionogi (ODS) rats will be treated with ethanol, tert- butyl hydrogen peroxide (TBHP), or CC14 to characterize the injury and activation process. Mechanisms whereby herbal remedies inhibit this injury process will be explored by assays of lipid peroxidation, cytokine synthesis, tissue transglutaminase (tTG) expression, and NF-kappaB binding activity. Herbal remedies will also be employed to inhibit the injury and fibrosis induced by CC14 administration and bile duct ligation in in vivo systems, and the effectiveness of their action will be determined by histology and collagen protein determination, immunohistochemistry of tTG expression, and gel retardation assays of NF-kappaB activation. Health Relatedness: The successful use of one or more of the herbal agents in these models of liver injury and fibrogenesis will provide a rational basis for the use of these inexpensive, non-toxic agents in a series of clinical trials of liver disease in humans.

DESCRIPTION (provided by applicant): Treatment of liver disease with orthotopic liver transplantation (OLT) carries considerable morbidity and mortality. Moreover, due to organ shortages, thousands of people die each year without getting transplanted. Therefore, safer and more convenient alternative therapies will benefit many people requiring liver transplantation. An approach that might address this problem is the development of a proliferative cell line that expresses liver-specific genes which could be employed for cell transplantation or for a bioartificial liver. Developing such a line from monkey embryonic stem cells (ESC) would provide a cell line valuable for pharmacology and toxicology studies, as well as establishing an approach that could be employed in human cells. Specific Aims: 1) to develop and characterize ESC derived from rhesus monkeys; 2) to determine conditions for directing the cells into a hepatocyte lineage, and to assess the effects of ethanol administration on the differentiation process; 3) to elucidate the effects of ethanol on the ESC-derived cells' ability to repopulate the liver; 4) to delineate the therapeutic utility of the cells in an in vivo model of liver injury; and 5) to assess the effectiveness of gene transfection protocols in treating liver injury induced by ethanol in in vitro and in vivo systems. Methods: Initial experiments will include the development and characterization of ESC and their differentiation towards a hepatocyte lineage. Single cell clones will be developed in an attempt to establish uniform lines with high levels of liver-specific function. The effects of ethanol will be determined on the ESC-derived cells by monitoring liver-specific function, growth curves, and oncogenic potential in in vitro systems. The effects of ethanol on the ability of the cells to engraft, proliferate, and function in vivo will also be assessed. Liposomes or viral vectors will be employed to deliver extracellular superoxide dismutase or catalase to the ESC-derived cells in an attempt to inhibit ethanolinduced injury. Health Relatedness: If the studies are successfully undertaken, it will provide for the development of an unlimited source of differentiated primate hepatocytes that can be used for toxicology and pharmacology studies, and provide the basis for the establishment of similar lines from human cells which could then be employed in liver cell transplantation studies in man.

[unreadable] DESCRIPTION (provided by applicant): Treatment of liver disease with orthotopic liver transplantation (OLT) carries considerable morbidity and mortality. Moreover, due to organ shortages, thousands of people die each year without getting transplanted. Therefore, safer and more convenient alternative therapies will benefit many people requiring liver transplantation. An approach that might address this problem is the development of a proliferative cell line that expresses liver-specific genes which could be employed for cell transplantation or for a bioartificial liver. Developing such a line from human embryonic stem cells (ESC) would provide cells valuable for pharmacology and toxicology studies, as well as establishing an approach that could be employed in human cells. Specific Aims: 1) to determine conditions for directing the ESC to differentiate into hepatocytes in vitro, and to characterize the differentiated cells; and 2) to establish the in vivo potential of human ESC. Methods: A variety of conditions will be empirically assayed to delineate the most effective approach to differentiate the embryonic cells along a hepatocyte lineage. The NIH code number of the cells that are being used is WA01. To delineate mechanisms aimed at inducing either proliferation or hepatocellular gene expression in the cells, specific manipulations will be utilized with assays of liver-specific gene products and growth factor responsiveness. To determine whether the cells can engraft, survive and proliferate after transplantation, studies will be conducted in immunodeficient NOD-SCID mouse models. Health Relatedness: If the studies are successfully undertaken, it will provide for the development of an unlimited source of differentiated human hepatocyte-like cells that can be employed in cell-based therapeutics or in pharmacology studies. [unreadable] [unreadable] [unreadable]

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Embryonic stem cells (ESC) hold great potential for the treatment of liver diseases. The goals of these studies focused on methods to differentiate rhesus ESC to a hepatocyte lineage.

DESCRIPTION (provided by applicant): Treatment of liver disease with orthotopic liver transplantation (OLT) carries considerable morbidity and mortality. Moreover, due to organ shortages, thousands of people die each year without getting transplanted. Therefore, safer and more convenient alternative therapies will benefit many people requiring liver transplantation. An approach that might address this problem is the development of a proliferative cell line that expresses liver-specific genes which could be employed for cell transplantation or for a bioartificial liver. Developing such a line from human embryonic stem cells (hESC) would also provide cells valuable for pharmacology and toxicology studies. Specific Aims: 1) to determine conditions for directing the hESC to differentiate into hepatocytes in vitro;2) to characterize the differentiated hESC;3) to establish the in vivo potential of hESC in NOD-SCID mice;and 4) to establish the in vivo potential or hESC in nonhuman primates. Methods: A variety of conditions will be empirically assayed to delineate the most effective approach to differentiate the hESC along a hepatocyte lineage. This will include empiric studies of optimizing media, extracellular matrix, and growth factors. The purity of the cells will be enhanced with transduction of liver-specific lent virus vectors. The purified cells will be characterized with assays of liver-specific gene products, growth factor responsiveness, and potential oncogenicity. To determine whether the cells can engraft, proliferate, and function after transplantation, studies will be conducted in immunodeficient NOD- SCID mouse models of liver cell injury and in nonhuman primates. Innovative imaging approaches will be utilized to assess the viability and proliferation of the cells over time in vivo. Health Relatedness: If the studies are successfully undertaken, it will provide for the development of an unlimited source of differentiated human hepatocytes that can be used for toxicology and pharmacology studies, and can be employed in cell-based therapeutics in man.