Jacquelyn J. Maher, MD - US grants

Fibrosis of the hepatic subendothelial (Disse) space is a prominent component of the inflammation elicited by ethanol, and appears to correlate with clinically significant liver disease. The lesion consists of a collagen-rich new-matrix that replaces the low- density matrix normally present in this location. In recent and on-going studies from this laboratory, the lipocyte (fat-storing or Ito cell) has been examined in pure primary culture and found to be a major source of several matrix proteins including collagen, laminin and proteoglycans. In vivo, cells referred to as 'myofibroblasts' have been identified following chronic alcohol consumption and localized specifically to areas of fibrogenesis. Morphologic studies suggest that lipocytes and myofibriblasts represent the same cell in various stages of transition. The working hypothesis for the present studies is that ethanol 'activates' lipocytes, causing their conversion from resting, vitamin A-storing cells to matrix-secreting myofibroblasts. The pathway of lipocyte activation is the principal focus of the proposed research. Several possibilities will be explored, including stimulation by acetaldehyde or by soluble mediators of inflammation released as by-products of ethanol metabolism. The first series of experiments will establish wheter lipocyte activation occurs as a direct result of ethanol metabolism. This will be accomplished using a novel cell culture approach, in which hepatocytes and lipocytes are co-cultured as 'microorganoids' and incubated with varying concentrations of ethanol. Additional studies will examine whether lipocyte activation is indirect, involving compounds produced by neutrophils of Kupffer cells previously exposed to conditioned medium from ethanol-treated hepatocytes. Lipocyte activation will be judged with studies of ultrastructure as well as quantitative measurements of matrix protein secretion, matrix gene expression, and cell proliferation. Recently developed methods for evaluating matrix gene expression and for culture of individual parenchymal and non- parenchymal cell populations allow a direct approach to the pathogenesis of this common and important condition.

This is a request for an ADAMHA Research Scientist Development Award (RSDA). Three objectives are to be accomplished with this award: (1) expansion of an ongoing research program in alcoholic liver disease; (2) career development for the principal investigator in the areas of collaboration and mentoring; and (3) completion of experiments focusing on the pathophysiology of alcoholic liver injury and fibrosis. The applicant brings to the research program an in-depth understanding of hepatic fibrogenesis; dedication to the alcohol field is demonstrated by a clear and expanding focus on alcohol-induced liver disease. An RSDA will permit recruitment of young investigators to keep pace with new ideas. The research plan in this application addresses the mechanism underlying alcoholic liver fibrosis. As in all forms of fibrotic liver disease, lipocytes (hepatic stellate cells) play a prominent role; we hypothesize that in the setting of alcohol, lipocytes are activated to produce collagen by signals from an altered extracellular matrix. This postulate is based on observations that soluble mediators such as acetaldehyde, lipid aldehydes or TGFbeta, do not activate lipocytes in culture; cultured lipocytes activate readily, however, in response to changes in their surrounding extracellular matrix. This suggests that matrix alterations in vivo may lead to hepatic fibrosis. Inflammatory cells, particularly neutrophils, play an important role in our scheme of alcoholic liver fibrosis. Neutrophils migrate from the circulation to the liver by secreting extracellular matrix-degrading proteinases; we propose that in so doing, they provoke critical alterations in the hepatic perisinusoidal matrix that lead s to lipocyte activation. Neutrophil chemoattractants, produced by liver cells in the setting oc chronic ethanol ingestion, may contribute importantly to alcoholic fibrosis by stimulating neutrophil proteinase release. Interleukin-8 is a likely candidate, as preliminary studies implicate this chemokine in alcohol- induced liver injury. The studies proposed in this application fall logically into two categories. One focuses on the pathogenesis of "alcoholic hepatitis" (neutrophil recruitment to the alcoholic liver); the other addresses the consequences of inflammatory cell activation (manifest as proteinase secretion) on lipocyte behavior. Via an RSDA, the applicant hopes to expand and split these aims into two independent research projects. The ultimate goal is to build a competitive research group that performs high- quality alcohol-related research.

DESCRIPTION: (Adapted from Investigator's Abstract) Alcoholic hepatitis is a serious and potentially fatal complication of chronic alcohol abuse. The condition is marked by recruitment of inflammatory cells, in particular neutrophils, to the liver parenchyma. Once neutrophils invade the liver they can actively attack and destroy hepatocytes. The overall objectives of this research project are to determine how chronic alcohol consumption signal neutrophils to invade the liver, and how neutrophils and their chemoattractants cause liver damage. The main focus of the research will be on C-X-C chemokines. C-X-C chemokines are potent neutrophil chemoattractants and activators; when produced in the liver in vivo, they cause a severe neutrophilic hepatitis. In the setting of chronic ethanol abuse, C-X-C chemokines are induced in the liver and are believed to promote alcoholic hepatitis. However, the mechanism by which chronic ethanol consumption stimulates chemokine production is uncertain. Based on work to date, the investigators propose that ethanol alone is insufficient to induce hepatic chemokine production in vivo. The P.I. will explore the possibility that a cofactor, namely endotoxin, acts in conjunction with ethanol to stimulate C-X-C chemokine production by liver cells in intact rats. The investigators will address this question by administering low-dose endotoxin to ethanol-fed rats over a 4-wk interval, and by determining whether both insults induce C-X-C chemokines even if each alone does not. In separate studies they will investigate how C-X-C chemokines actually cause liver injury. Based on preliminary data, they propose that they induce liver damage by two independent mechanisms: one involving neutrophil recruitment and activation, and the other involving direct cytotoxicity toward hepatocytes. The investigators intend to explore this further by examining chemokine effects on liver cells in culture and in vivo. Cell culture studies will permit them to determine whether C-X-C chemokines cause death by apoptosis or necrosis, and whether they exert their effects intracellularly or by binding to cell-surface receptors. Studies in vivo will allow them to determine the extent to which neutrophils and direct cytotoxicity each contribute to chemokine-induced liver damage. Finally, they will investigate whether the adverse effects of chemokines are enhanced by chronic ethanol consumption.

DESCRIPTION (provided by applicant): Hepatic steatosis (fatty liver) progresses to steatohepatitis in only a small proportion of individuals. The risk factors that lead to progression are uncertain, but studies suggest that in some cases steatohepatitis is linked to a defect in hepatic triglyceride (TG) secretion. Hepatic TG secretion can be blocked in mice by feeding a diet devoid of methionine and choline (methionine-choline-deficient; MCD); when such a formula is administered together with excess sucrose, it provokes severe steatohepatitis. Preliminary data from our laboratory indicate that a sucrose-rich MCD formula causes hepatocellular apoptosis in vivo. This phenomenon is reminiscent of "lipoapoptosis" that has been described previously in other organs. In this application, we test the hypothesis that dietary sucrose provokes lipoapoptosis in susceptible mice and leads to steatohepatitis. Aim 1 is designed to confirm that lipoapoptosis takes place in the livers of MCD mice and document the importance of dietary sucrose to the pathogenesis of MCD steatohepatitis. Separate experiments will address whether dietary sucrose also induces lipoapoptosis in other mice that have related but independent defects in hepatic TG secretion (MTP-deficient; Scd-1-deficient). Aim 2 will investigate the importance of lipoapoptosis relative to lipid peroxidation in the pathogenesis of MCD steatohepatitis. Of particular interest is whether lipoapoptosis and lipid peroxidation are independent causes of liver injury or lipid peroxidation follows lipoapoptosis. Aim 3 concentrates on the mechanism by which saturated fatty acids cause hepatocyte apoptosis. Experiments will address whether fatty acid toxicity is due primarily to de novo synthesis of ceramide or to disruption of mitochondria with activation of an apoptosis cascade. Overall, the proposed work highlights lipoapoptosis as a potentially important cause of steatohepatitis in individuals with impaired hepatic TG secretion. Because lipoapoptosis can be regulated by dietary nutrients, some individuals at risk of steatohepatitis may be able to reduce their risk by modifying their diet.

DESCRIPTION (provided by applicant) The Liver Center of the University of California, San Francisco is an inter-disciplinary consortium of basic and clinical scientists dedicated to understanding the biology and pathobiology of the liver and the treatment of liver diseases. From its inception in 1975, it has undergone continual evolution to meet current scientific challenges, with its Executive Committee monitoring research progress and creating initiatives to open up new areas. The main lines of Center research at present are represented by five inter-related themes: (1) Immunology; (2) Metabolism, Injury and Repair; (3) Genetics and Development; (4) Liver Cancer; and (5) Viral hepatitis. Three themes-Immunology, Genetics and Development, and Liver Cancer-have emerged during the current funding period through recruitment of new members and alliances with organized research units. A major initiative over the past two years has involved the UCSF Cancer Center and resulted in preparation of an application for an NCI SPORE in liver cancer. The Center supports four core facilities: (1) Cell and Tissue Biology; (2) Clinical and Translational Research; (3) Microscopy and Advanced Imaging; (4) Molecular Analysis. The four scientific Cores not only provide direct research support but are a venue for new collaborations and an entry point for new members. Finally, the Center fosters scientific exchange through a Visiting Scientist Program, a Seminar Series, and its Annual Scientific Retreat. In the 2000-01 academic year, these programs brought five leading researchers in basic biology or translational science whose work has particular relevance to the research themes of the Center. The Center continues to expand its support of member science through strategic partnerships, most recently with the Veterans Administration, the UCSF Cancer Center, and the graduate program in Biomedical Sciences. In addition to these affiliations, the Center enjoys close links with the UCSF programs in Immunology and Developmental Biology, respectively. Its funding base continues to grow, and support from the institution is sustained and strong. The opportunities in basic biology and in the application of basic science to clinical liver disease have never been greater, and the Center is poised to catalyze discovery in these areas.

DESCRIPTION (provided by applicant): This application is for renewal funding of research training in liver biology and disease at UCSF. The rationale is two-fold: (1) the field is growing rapidly and currently has insufficient numbers of well-trained investigators, and (2) the resources and environment at UCSF for training in liver research are exceptional. The program, which is based in the Division of Gastroenterology, provides two years of support to trainees who seek an intensive grounding towards a career as academic hepatologists. It is inter-disciplinary, encompassing the Department of Epidemiology and Biostatistics and the Liver Transplant Program for those undertaking clinical and outcomes research, and the UCSF Liver Center for those engaged in laboratory-based research. The mentor group comprises 13 faculty from the Departments of Medicine, Pathology, Biochemistry and Microbiology/Immunology. All have research programs in liver research, experience in training, and a record of working together. The program is overseen by a Steering Committee, which is responsible for screening applicants, tendering offers, and monitoring progress. Applications for admission will be solicited annually by national advertisement. The largest candidate pool will be M.D. applicants to the Investigator (research-intensive) track of the UCSF subspecialty program in Gastroenterology, but the process will be open to others including Ph.D.'s. The successful applicant will have a record of accomplishment in the form of publications and, in many cases, both M.D. and Ph.D. degrees. Applicants with a Ph.D. degree only must have a strong liver focus and orientation to translational research. The range of training within the program is broad, from clinical epidemiology, genetics, health outcomes and policy, to cell biology, organogenesis, immunology, host-pathogen interactions and fibrosis/carcinogenesis. Coincident with their research, trainees are provided formal instruction in basic and clinical sciences as part of the UCSF graduate programs in Clinical Research, Biomedical Sciences and Biological Sciences. A core curriculum encompassing ethical conduct of research and effective scientific communication is required of all students. Graduates of this research training in hepatology will be ready to assume a faculty-level position and to compete for individual extramural funding.

DESCRIPTION, OVERALL (provided by applicant): The Liver Center of the University of California, San Francisco (UCSF) is an interdisciplinary consortium of 82 basic and clinical scientists dedicated to understanding the biology and pathobiology of the liver and the treatment of liver diseases. Research by Center members is categorized under three basic biological themes: (1) Liver Injury and Repair, (2) Progenitor Cells, Growth and Development and (3) Hepatic Physiology and Metabolism. Across all themes, research spans bench to bedside, with approximately half of Center members being directly involved in clinical and translational studies. From 2008-2013 the Center will support three Biomedical Research Cores: (1) Cell Biology, (2) Liver Immunology & Cell Analysis and (3) Pathology. A Clinical Component, which offers biostatistical support to supplement the services offered by the UCSF Clinical and Translational Science Institute, is part of the Administrative Core. Also within the Administrative Core is the Tool & Technology Program; this is a user-defined or a la carte core designed to support member use of core services at UCSF not provided directly by the Center. The goals of the Center are to provide research support, technical training, a venue for new collaborations and an entry point for new members. The Center also fosters scientific exchange through an Enrichment Program that features seminars by local and visiting scientists, quarterly mini-symposia highlighting each research theme, and an annual 1-day symposium with a lively poster session. A vital component of the Center is its Pilot/Feasibility Program, which provides seed money to junior investigators and scientists new to investigative hepatology. This award program, like the cores, is an important recruitment vehicle. The Center continues to expand its support of member science through strategic partnerships with the Gladstone Institutes and affiliations, with other organized research units at UCSF. The funding base of the Center continues to grow, and support from the institution is sustained and strong. Opportunities for basic biologic discovery and the application of basic science to clinical liver disease are numerous; the Center is poised to catalyze achievements in these areas.

In fatty liver disease, the death of hepatocytes is an important occurrence because cell death can trigger downstream events such as hepatic inflammation and fibrosis. Certain fatty acids, particularly long-chain saturated species, kill hepatocytes directly and contribute to steatohepatitis in experimental animals. The mechanism by which saturated fatty acids kill cells is not uniform among all cell types and for hepatocytes the process is incompletely understood. The objective of this research plan is to clarify the cellular events involved in saturated fatty acid-induced hepatocyte [unreadable]lipotoxicity.[unreadable] Preliminary data indicate that saturated fatty acids block the mammalian target of rapamycin complex I (mTORCI) in hepatocytes and activate protein phosphatase 2A (PP2A). We propose that these two events are linked, because mTORCI typically maintains PP2A in an inactive state. Furthermore, we propose that events occurring downstream of PP2A activation, namely, suppression of the cyclin-dependent kinase inhibitor p27 and activation of cyclin-dependent kinases, are responsible for saturated fatty acid-induced cell death. We will address these hypotheses in four Specific Aims. In Aim 1 we will investigate how fatty acid-mediated inactivation of mTORC1 causes activation of PP2A, concentrating on the role mTOR in controlling the activity of α4, a negative regulator of PP2A. In Aim 2 we will test the hypothesis that fatty acid-mediated activation of PP2A prompts proteasomal degradation of p27 in hepatocytes, leading to cell death. In Aim 3 we will assess how p27 deficiency leads to cell death, focusing on cyclin-dependent kinases and their effects on mitochondrial integrity and autophagy. Finally, in Aim 4 we will assess whether the pathophysiologic pathway to lipotoxicity in cell culture is also operative in the liver in vivo, by modulating mTORC1, PP2A, p27 and cyclin-dependent kinases in a mouse model of SFA-mediated liver injury and determining their impact on liver-related outcome. The hope is that these studies will shed sufficient light on the pathogenesis of hepatocyte death in the setting of hepatic steatosis to suggest therapeutic options for patients with fatty liver disease.

PROJECT SUMMARY/ABSTRACT ? UCSF LIVER CENTER For the project summary/abstract please refer to the OVERALL/OVERVIEW section.

PROJECT SUMMARY/ABSTRACT ? UCSF LIVER CENTER For the project summary/abstract please refer to the OVERALL/OVERVIEW section.

PROJECT SUMMARY/ABSTRACT ? UCSF LIVER CENTER The Liver Center of the University of California, San Francisco (UCSF) is an interdisciplinary consortium of 59 basic and clinical scientists dedicated to understanding liver biology and contributing new knowledge about the pathophysiology and treatment of liver diseases. The overarching theme of the center is ?translating science to benefit patients with liver disease.? The Center's research base is made up of 40 individuals: these investigators receive $21 million dollars in liver-related research funds annually (direct costs). The research base of the Center is evenly split between practicing physicians and non-physicians. Their research is categorized into three focus areas. One focus area is entitled Liver Injury, Repair & Transplantation: this group includes scientists who study liver immunology as well as clinician-investigators who study the epidemiology, management and outcomes of chronic liver disease. Second is Liver Cell Biology, Plasticity & Transformation: this area is home to scientists who study cellular responses to liver injury such as toxicity and fibrosis, as well as cell growth in contexts ranging from developmental biology to cancer. Third is Hepatic Metabolism & Metabolic Derangements: members in this category study basic aspects of nutrient and xenobiotic metabolism as well as the mechanisms, diagnosis and outcomes of fatty liver disease. Across all focus areas, research spans bench to bedside. More than 75% of Center members perform research directly with human subjects or human materials. A major goal of the Liver Center is to promote research excellence and accelerate research progress in hepatology. As it continues in 2018-2023, the Center will accomplish this goal by supporting three Biomedical Research Cores: (1) Cell Biology, (2) Liver Immunology & Cell Analysis and (3) Pathology & Imaging. The Center also operates a separate Clinical Component, which offers biostatistics services and is being expanded to include support for biospecimen procurement, storage and retrieval. A second goal of the Liver Center is to foster a vibrant community of liver researchers at UCSF. In this case the strategy will be to stimulate scientific exchange through an Enrichment Program that features seminars by local and visiting scientists, mini-symposia highlighting specific member constituencies, and an annual 1-day symposium featuring the progress of the entire group. Throughout the year the Center's Administrative Core will maintain member connections and keep them informed of new developments through newsletters and a comprehensive website. A third goal of the Center is to sustain a trajectory of growth in liver research by developing promising new investigators. Several means will be used to achieve this end. First, the Center will continue its Pilot/Feasibility Program, which provides seed money to junior investigators and scientists new to investigative hepatology. This award program is an important recruitment vehicle and gives priority to junior scientists. Second, the Center will continue a popular mini-symposium dedicated to junior faculty and trainees, providing a forum for open discussion and offering advice to those in transition to independent careers. Third, through personal mentorship from the Internal Executive Committee with grant applications and priority access to research support, the Center will aim to provide every benefit possible to those at the beginning of their careers. The Center is fortunate to have strong support from the university leadership to continue its mission. It also has solid partnerships with 2 other NIDDK P30 Centers at UCSF (Diabetes Research Center, Nutrition & Obesity Research Center). With renewed focus after a restructuring effort in 2013-2018, the Center is positioned to innovate and make important contributions to basic and clinical science as it relates to liver disease.

PROJECT SUMMARY/ABSTRACT Nonalcoholic fatty liver disease (NAFLD) and the related disorder nonalcoholic steatohepatitis (NASH) are now the most prevalent liver diseases in the world. Research into the pathophysiology of NAFLD/NASH has uncovered mechanisms by which lipids can accumulate in liver cells and cause liver injury ? but has also revealed the tremendous complexity of the disease and its variability among global populations. Mouse models of NAFLD/NASH, despite their many benefits, fall short of replicating the human disease. For this reason there is an important need to study the pathogenesis of NAFLD/NASH directly in humans. The goal of this exploratory/developmental research project is to use human induced pluripotent stem cells (iPSC) to promote better understanding of NAFLD/NASH. Our proposed strategy is to take human iPSC, generated from 48 patients with biopsy-proven NAFLD/NASH and 19 healthy controls, and study them in culture after differentiating them to iPSC-derived hepatocytes (iPSC-heps). At the core of the research plan is the hypothesis that certain critical features of human NAFLD/NASH are hepatocyte-autonomous. In other words, we believe iPSC-heps from NAFLD/NASH patients will exhibit phenotypic differences from control iPSC-heps that underlie their predisposition to injury and disease. We will test this hypothesis by completing several Specific Aims. In Aim 1 we will determine whether iPSC-heps from NAFLD/NASH patients are distinguishable from iPSC-heps from healthy subjects at baseline, without any exogenous stimulus. This will reveal whether liver cells from NAFLD/NASH patients exhibit an inherent predisposition to disease that manifests without provocation. In Aim 2 we will assess whether iPSC-heps from NAFLD/NASH patients are more sensitive to metabolic stresses than iPSC-heps from healthy subjects. Together, Aims 1 and 2 will establish whether iPSC-heps hold promise as a means for expanding knowledge about a complex human liver disease. While conducting studies on the existing collection of iPSC we will pursue a 3rd Aim to expand our cohort, by collecting clinical information and blood from additional NAFLD/NASH patients and healthy controls. We will store PBMC from these individuals with the longer term goal of broadening our iPSC research to larger numbers of subjects. This research project promises to provide valuable information about NAFLD/NASH pathogenesis with direct relevance to NAFLD/NASH patients. Importantly, success with these studies will also prove that research based on iPSC-heps can extend beyond the investigation of individuals with monogenic liver diseases to populations of patients with a complex disease. Positive results will set the stage for more in- depth study of human NAFLD/NASH in family kindreds and/or other populations based on genotype, gender, ethnicity and geography. Overall, the proposed work has the potential to answer important basic research questions about NAFLD/NASH by focusing directly in human subjects through the power of iPSC.

PROJECT SUMMARY/ABSTRACT ? UCSF LIVER CENTER The Liver Center of the University of California, San Francisco (UCSF) is an interdisciplinary consortium of 59 basic and clinical scientists dedicated to understanding liver biology and contributing new knowledge about the pathophysiology and treatment of liver diseases. The overarching theme of the center is ?translating science to benefit patients with liver disease.? The Center's research base is made up of 40 individuals: these investigators receive $21 million dollars in liver-related research funds annually (direct costs). The research base of the Center is evenly split between practicing physicians and non-physicians. Their research is categorized into three focus areas. One focus area is entitled Liver Injury, Repair & Transplantation: this group includes scientists who study liver immunology as well as clinician-investigators who study the epidemiology, management and outcomes of chronic liver disease. Second is Liver Cell Biology, Plasticity & Transformation: this area is home to scientists who study cellular responses to liver injury such as toxicity and fibrosis, as well as cell growth in contexts ranging from developmental biology to cancer. Third is Hepatic Metabolism & Metabolic Derangements: members in this category study basic aspects of nutrient and xenobiotic metabolism as well as the mechanisms, diagnosis and outcomes of fatty liver disease. Across all focus areas, research spans bench to bedside. More than 75% of Center members perform research directly with human subjects or human materials. A major goal of the Liver Center is to promote research excellence and accelerate research progress in hepatology. As it continues in 2018-2023, the Center will accomplish this goal by supporting three Biomedical Research Cores: (1) Cell Biology, (2) Liver Immunology & Cell Analysis and (3) Pathology & Imaging. The Center also operates a separate Clinical Component, which offers biostatistics services and is being expanded to include support for biospecimen procurement, storage and retrieval. A second goal of the Liver Center is to foster a vibrant community of liver researchers at UCSF. In this case the strategy will be to stimulate scientific exchange through an Enrichment Program that features seminars by local and visiting scientists, mini-symposia highlighting specific member constituencies, and an annual 1-day symposium featuring the progress of the entire group. Throughout the year the Center's Administrative Core will maintain member connections and keep them informed of new developments through newsletters and a comprehensive website. A third goal of the Center is to sustain a trajectory of growth in liver research by developing promising new investigators. Several means will be used to achieve this end. First, the Center will continue its Pilot/Feasibility Program, which provides seed money to junior investigators and scientists new to investigative hepatology. This award program is an important recruitment vehicle and gives priority to junior scientists. Second, the Center will continue a popular mini-symposium dedicated to junior faculty and trainees, providing a forum for open discussion and offering advice to those in transition to independent careers. Third, through personal mentorship from the Internal Executive Committee with grant applications and priority access to research support, the Center will aim to provide every benefit possible to those at the beginning of their careers. The Center is fortunate to have strong support from the university leadership to continue its mission. It also has solid partnerships with 2 other NIDDK P30 Centers at UCSF (Diabetes Research Center, Nutrition & Obesity Research Center). With renewed focus after a restructuring effort in 2013-2018, the Center is positioned to innovate and make important contributions to basic and clinical science as it relates to liver disease.