Bryon E. Petersen - US grants

Activation, proliferation and differentiation of a distinct phenotype of stem cells, called pancreatic oval cells, are observed after pancreatic injuries. Under certain physiological conditions, oval cells can act as bipotential progenitors of the different types of epithelial cells of the pancreas, the ducts, acinar and islet cells. Because pancreatic oval cells and hepatic oval cells share an uncanny number of similarities in the conditions that cause their activation, some have considered these pancreatic and hepatic oval cells to be one in the same cell type. The hepatic oval cells have been usually thought to be the progeny of a hepatic stem cell, native to the liver. Recently, however, we as well as others have obtained clear evidence that in the rat hepatic oval cells, or at least a fraction of them, can be derive from a precursor cell of bone marrow origin. Perhaps, the pancreatic oval cells, which lie in the ductular region and gives rise to the different epithelial cell types may in fact be derived from an extra-pancreatic source (i.e. bone marrow). Having been the first to show that oval cells can be derived from an extra-hepatic source we now have the technology to answer the question of cell of origin through the use of sex mismatched bone marrow transplants. The goals of this project are to identify and phenotypically characterize the bone marrow-associated cell that can act as a progenitor of pancreatic oval cells. Towards achievement of these goals, we will perform a series of studies aimed at to test the hypothesis that bone marrow derived stem cells participate in the regeneration process in the injured rat pancreas. (Specific Aim I); and to establish whether the CD- 34+ or CD-34- sub-population of bone marrow cells contains the oval cell progenitor and produces the highest percentage of bone marrow derived pancreatic cells (Specific Aim II). It is anticipated, with confidence, that performance of the proposed studies will yield new and significant data about the overall biology of pancreatic oval cells, and the basic phenotype and properties of their bone marrow precursor. The same data could potentially provide a valuable resource for future tissue engineering and cell therapy technique interventions in pancreatic regeneration.

DESCRIPTION (provided by applicant): Activation, proliferation and differentiation of a distinct phenotype of hepatic cells, called oval cells, are observed after severe hepatic injuries, especially when proliferation of hepatocytes is inhibited. Under those conditions, oval cells can act as bipotential progenitors of the two types of epithelial cells of the liver, the hepatocytes and bileductular cells. Oval cells have been usually thought to be the progeny of a hepatic stem cell, native to the liver. However, we have obtained clear evidence that in the rat, hepatic oval cells, or at the least a fraction of them, can derive from a precursor cell of bone marrow origin. The goals of this project are therefore to determine how bone marrow stem cell emigration to the liver is regulated, what local hepatic environment promotes engraftment of bone marrow cells as an oval cell population, and what bone marrow-derived cell population can best act as a progenitor of hepatic oval cells. Towards achievement of these goals, we will perform a series of studies aimed at increasing the efficiency of bone marrow cell recruitment to test the hypothesis that the type of injury (periportal vs. pericentral) and chemokine (CC vs. CXC) response being invoked determines the efficiency with which bone-marrow stem/precursor cells engraft in an injured liver in both rat and mouse models (Specific Aim I); to determine whether the homing chemokine SDF- 1 and its receptor CXCR4 play a role in directing the bone marrow precursor cell to the liver (Specific Aim Il); and to establish whether the CD-34+ or CD-34- sub-population of bone marrow cells contains the oval cell progenitor and produces the higher percentage of bone marrow derived hepatocytes (SpecificAim III). It is anticipated that performance of the proposed studies will yield new and significant data about the overall mechanisms for recruitment of hepatic oval cells, and the basic phenotype and properties of their bone marrow precursor. These data will be critical for developing novel therapeutic strategies for the treatment of liver disease.

DESCRIPTION (provided by applicant): The liver has an enormous capacity to regenerate, as demonstrated by the 2/3 partial hepatectomy model in rodents. In addition, the liver has a stem cell compartment acting as a backup regenerative system. Activation of the stem cell compartment takes place when hepatocytes are functionally compromised, are unable to divide, or both. In stem cell-aided liver regeneration, progeny of the stem cells multiply in an amplification compartment composed of hepatic oval cells. Several studies have shown that bone marrow cells can differentiate into hepatocytes, and we have also shown that bone marrow (BM) cells are able to produce hepatic oval cells. The foremost questions are: what molecular mechanisms are involved in oval cell physiology, and can these pathways be manipulated to enhance their therapeutic value in treating liver disorders? The experiments described within this proposal are designed to address the above stated questions. We will pursue the following specific aims: Specific Aim 1: We hypothesize that activation of the JAK2 and MAPK signal transduction pathways by G-CSF interaction with G-CSF receptor on the cell membrane enhances both proliferation and migration of liver oval cells. Specific aim 2: We hypothesize that activation of the MEK and PI3K signal transduction pathways following SDF-1 binding to CXCR4 receptor on the cell membrane enhances both proliferation and migration of liver oval cells. Specific aim 3: We hypothesize that modulation of the oval cell phenotype by G-CSF and SDF-1 will positively affect engraftment and expansion of compensatory oval cells into mouse liver afflicted with a genetic disorder, resulting in a measurable enhancement of liver function. It is anticipated that the proposed studies will yield new and significant data about the mechanisms of governing the bone marrow contribution to liver regeneration and signals involved in oval cell activation, proliferation and differentiation.

DESCRIPTION (provided by applicant): The liver has an enormous capacity to regenerate, as demonstrated by the 2/3 partial hepatectomy model in rodents. A stem cell compartment within the liver is activated when mature hepatocytes are unable to perform their role in this process. The overarching question of this research proposal is;which systemic signals regulate stem cell mediated liver regeneration and what molecular mechanisms underlie this regulation? The proposed research will identify the role and mechanism of action for specific factors involved in the activation, trafficking, expansion and differentiation of liver stem cells. Previous experiments have implicated connective tissue growth factor (CTGF), Notch-1, Wnt1, Somatostatin (SST), insulin-like growth factor binding protein 3 (IGFBP3) as playing key-roles in the liver stem cell response to 2- acetylaminofluorene/partial hepatectomy (2AAF/PH) liver injury in rats. All of these factors have a relationship to TGFb. We will characterize these relationships in three specific aims. Specific aim 1 will test the hypothesis that the TGF2/CTGF axis mediates the synthesis of a fibronectin rich provisional extracellular matrix (ECM) by activated portal fibroblasts that is required for the expansion of the liver stem cell population following 2-AAF/PH in rats. The main goal of this aim is to determine the role of TGFb and CTGF in forming the appropriate extracellular microenvironment for liver stem cell proliferation and migration. Specific aim 2 will test the hypothesis that IGFBP3 and SST mediate the trafficking of liver stem cells within the liver, and that SST potentiates the up-regulation of CTGF during the oval cell response to 2AAF/PH in rats. The role of these factors in directing the migration of liver stem cells and the interaction of these pathways with TGF2 signaling will be elucidated. Specific aim 3 will test the hypothesis that Notch/Jagged and Wnt/Frizzled signaling play a required role in lineage selection during liver stem cell differentiation. Each of these pathways is influenced by TGFb, and the integration of these signals appears to determine the phenotype of the differentiated stem cell. The data generated by these studies will provide a more complete understanding of the regulation of liver stem cells. This knowledge will help to guide the development of strategies for the therapeutic use of stem cell transplants for the treatment of liver disease. PUBLIC HEALTH RELEVANCE: Only 5,000 livers suitable for transplant become available each year, while 18,000 patients await these organs. Clearly, an alternative to whole organ transplant is needed. The studies proposed within this application are designed to identify mechanisms that regulate repair of the liver by stem cells. These mechanisms may be targeted therapeutically to bolster stem cell transplants into diseased organs offering an alternative to whole organ transplant in many patients.

ABSTRACT: Liver fibrosis and its advanced form, cirrhosis, can occur in virtually all types of chronic liver disease (CLD) and are major health problems due to their high mortality rates and predisposition to cause liver failure, portal hypertension, and hepatocellular carcinoma (HCC). According to the American Liver Foundation, 5.5 million Americans are currently afflicted with CLD or cirrhosis, and the National Institutes of Health (NIH) reports cirrhosis as the 12th leading cause of death due to disease in America. The long-term goal of this proposal is to understand mechanisms underlying hepatic reparative processes and to discover novel therapy targets for boosting repair and reducing fibrosis. Connective tissue growth factor (CTGF) is a secreted matricellular protein in the Cyr61/CTGF/Nov (CCN) protein family that acts as an extracellular modifier through binding to growth factors, receptors, and the extracellular matrix (ECM). It is transcriptionally activated by transforming growth factor (TGF)-? and the transcriptional co-activator Yes-associated protein (YAP). Overexpression of CTGF has been identified as a hallmark of fibrotic disorders. Our studies have demonstrated the importance of CTGF in hepatic progenitor cell (HPC) response and fibrosis in animal models. In addition, we have found that another negative regulator, miR-133b, targets the 3' UTR of Ctgf mRNA and exhibits anti-fibrotic potential. The hypothesis underlying this proposal is that CTGF promotes HPC mediated fibrotic response through binding to key regulators, whereas CTGF downregulation negatively affects this process. This hypothesis will be tested utilizing our recently developed rat models in the following two specific Aims. AIM I: Determining if deletion of the Ctgf gene by CRISPR/Cas9 mitigates the fibrotic response in alcohol induced rat liver injury models: Transgenic animals deficient for CTGF production will be subjected to chemically induced liver damage. The necessity of CTGF for initiation and progression of liver fibrosis will be evaluated. AIM II: Determining whether miR-133b inhibits HSC activation and subsequent liver fibrosis. The expression pattern of miR-133b during HSC activation and liver fibrosis will be examined. Systemic delivery of miR-133b using an AAV delivery system will be applied in vivo to alcohol damaged rat livers to assess the anti-fibrotic potential of miR-133b. This study will provide new mechanistic insights into the modulation of fibrosis, aimed at the discovery of novel therapeutic strategies and new molecular targets to enhance repair mechanisms and reduce liver fibrosis.