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Role of Somatostatin in Migration of Hepatic Oval Cells in Injured Liver Model of Rats

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Title:
Role of Somatostatin in Migration of Hepatic Oval Cells in Injured Liver Model of Rats
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JUNG, YOUNGMI ( Author, Primary )
Copyright Date:
2008

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Antibodies ( jstor )
Cell growth ( jstor )
Cells ( jstor )
Cultured cells ( jstor )
Ellipses ( jstor )
Hepatocytes ( jstor )
Liver ( jstor )
Rats ( jstor )
Receptors ( jstor )
Stem cells ( jstor )

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University of Florida
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University of Florida
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Copyright Youngmi Jung. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
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8/31/2016

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ROLE OF SOMATOSTATIN IN MIGRAT ION OF HEPATIC OVAL CELLS IN INJURED LIVER MODEL OF RATS By YOUNGMI JUNG A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2006

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Copyright 2006 by Youngmi Jung

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This work is dedicated to my hus band, my son, and my loving parents

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iv ACKNOWLEDGMENTS I would like to express my indebtedness to Dr. Bryon E Petersen, whose direction, encouragement and support led me throughout my graduate school education. He has guided me to be a scientist. I express thanks to my committee members (Dr. Maria Grant, Dr. Chen Liu, Dr. Ammon Peck, and Dr. Lei Xiao) for their helpful suggestions and instructions during the research por tion of this study. I would like to thank all of the members of Dr. Peters enÂ’s laboratory, colleages and my friends: Dr. Seh-Hoon Oh, Dr. Thomas D. Shupe, Dr. Anna Piscag lia, Dr. Liya Pi, Dr. Rafal Witek, Dr. Jie Deng, Houda Darw iche, Susan Ellor, Jennifer Laplante, Donghang Zheng, and Alicia Brown. Special tha nks also go to Marda Jorgensen for all the help, suggestions, and instruct ion in histology techniques. Finally, I would like to show special gratitude to my husband, Byounghyok, my precious son, Minso, and my eternal s upporters, parents Won-Yeoung Jung, and NamYeon Cho.

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v TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................iv LIST OF FIGURES.........................................................................................................viii ABSTRACT....................................................................................................................... ix CHAPTER 1 INTRODUCTION........................................................................................................1 Liver Structure and Function........................................................................................1 Liver Regeneration.......................................................................................................3 Stem Cell...................................................................................................................... 6 Pluripotent Stem Cell............................................................................................7 Adult Stem Cell and Plasticity..............................................................................8 Hepatic Stem Cells.....................................................................................................10 Rodent Model of Oval Cell Induction.................................................................10 Characteristics of Oval Cells...............................................................................11 Origin of Oval cells.............................................................................................12 Oval Cell-mediated Liver Regeneration.....................................................................14 Inflammatory cytokines.......................................................................................14 Function of Hepatic Stellate Cells.......................................................................15 Stem Cell Factor..................................................................................................16 Interaction with the Extracellular Matrix............................................................17 Homing Capacity of Stem Cells.................................................................................18 Somatostatin and Its Receptors...................................................................................19 Somatostatin Receptor Subtypes.........................................................................20 Evolutionary Aspects...........................................................................................21 Ligand -Receptor Interactions.............................................................................22 SSTR Expression.................................................................................................23 Internalization, Desensitization and Oligomerization of SSTRs.........................25 Effects on Cell Proliferation................................................................................27 SSTR Signal Transduction.........................................................................................29 Study Design and Rationale........................................................................................32

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vi 2 A POTENTIAL ROLE OF SOMATOSTATIN AND ITS RECEPTOR SSTR4 IN THE MIGRATION OF HEPATIC OVAL CELLS...................................................34 Summary.....................................................................................................................34 Introduction.................................................................................................................35 Material and Methods.................................................................................................38 Animals................................................................................................................38 Rat Oval Cell Activation Protocol:......................................................................38 HOC Preparation.................................................................................................38 Immunohistochemistry........................................................................................39 RT-PCR and Real Time PCR..............................................................................39 Proliferation Assay..............................................................................................40 Migration Assay..................................................................................................40 Western Blot Assay.............................................................................................41 Statistical Analysis..............................................................................................42 Results........................................................................................................................ .42 Increased Expression of SST in the HOC Induction Model................................42 Chemotactic Role of SST for HOCs...................................................................43 Expression of SSTR4 in HOCs...........................................................................44 SST directing HOC Migration through SSTR4...................................................45 Discussion...................................................................................................................46 3 SOMATOSTATIN STIMULATES TH E MIGRATION OF HEPATIC OVAL CELLS WITHIN THE INJURED LI VER THROUGH PI3K PATHWAY..............58 Summary.....................................................................................................................58 Introduction.................................................................................................................59 Material and Methods.................................................................................................60 Animal.................................................................................................................60 HOC Preparation and Transplantation................................................................61 Immunohistochemistry........................................................................................61 Migration Assay..................................................................................................61 Stimulation and West ern Blot assay....................................................................62 Statistical analysis...............................................................................................62 Results........................................................................................................................ .63 SST analogue, TT232, Suppresses the Migration of HOC toward SST.............63 SST Affects HOC Homing through SSTR4, in vivo ...........................................64 SST Activates Intracellular PI3 kinase Pathway.................................................65 Discussion...................................................................................................................66 4 CHARACTERIZATION OF THY-1+ CELL IN VITRO CULTURE SYSTEM.......75 Summary.....................................................................................................................75 Introduction.................................................................................................................75 Isolation of Potential Liver Progenitor Cells.......................................................76 Oval Cell Lines....................................................................................................78 Material Methods........................................................................................................79

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vii Animal.................................................................................................................79 HOC Preparation and Culture.............................................................................79 Immunofluorescent Staining...............................................................................80 RT-PCR...............................................................................................................80 Results........................................................................................................................ .81 Discussion...................................................................................................................82 5 CONCLUSION...........................................................................................................86 LIST OF REFERENCES...................................................................................................89 BIOGRAPHICAL SKETCH...........................................................................................106

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viii LIST OF FIGURES Figure page 2-1. HOC induction in the 2AAF/PHx model...................................................................51 2-2. Increased SST expression in the HOC induction model............................................52 2-3. Immunohistochemical analysis for SST in the HOC induction model......................53 2-4. Potential role of SST as chemoattractant for HOCs...................................................54 2-5.Detection of SSTR4 in HOC -aided liver regeneration................................................55 2-6. Expression of SSTR4 by HOCs .................................................................................56 2-7. Effects of SST and SSTR4 on HOCs migration and actin rearangment....................57 3-1. Effect of SST analogues, TT232, on HOC migration................................................70 3-2. The plan for experiment was described......................................................................71 3-3. Suppression of SST/SSTR4 impairs the mi gration of HOC within the injured liver.72 3-4. SST/SSTR4-induced effects are mediated by PI3K pathway....................................73 3-5. PI3K plays a important role in SST-stimulated HOC migration................................74 4-1. Morphology of HOC on culture dish..........................................................................84 4-2. Albumin expression in HOC cultured on collagen-coated dish.................................85

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ix Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy ROLE OF SOMATOSTATIN IN MIGRAT ION OF HEPATIC OVAL CELLS IN INJURED LIVER MODEL OF RATS By Youngmi Jung August 2006 Chair: Bryon E. Petersen Major Department: Pathology, I mmunology and Laboratory Medicine Somatostatin (SST) is a regulatory pep tide that activates G protein-coupled receptors comprised of five members (SSTRs 1-5). Despite the broad use of SST and its analogs in clinical practice, the spectrum of SST activities has been incompletely defined. Recently, it has been demonstrat ed that SST can be a chemoattractant for hematopoietic precursor cells through somatostatin receptor type 2 (SSTR2). Since hepatic oval cells (HOCs) share common characte ristics with hematopoietic stem cells, we hypothesized that SST could act as a chemoattrac tant for HOCs by stimulating SSTRs. We showed an increased expression of SST in the 2-acetyl-aminofluorene (2AAF)/partial hepatectomy (PHx) HOC induc tion model, and this expression of SST was related to cell migration, but not prolif eration. SSTR4 was preferentially expressed by HOCs. SST stimulated rearrangement of actin filament in and migration of HOC through SSTR4. These results suggest a positive role for SST in the migration of HOCs, and that this effect is mediated through SSTR4.

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x We also investigated whether the effect of SST/SSTR4 on the migration of HOC is a functional consequence in HOC-mediated liver regeneration and what type of signaling molecules are associated with this chemotac tic action. The anti-migratory effect of TT232, which is a SST analogue with specific bi nding affinity for SSTR4, was determined in HOCs in vitro . Within cell transplantation model, a lower number of donor-derived cells was detected in TT232 treated animals, as compared to control animals. These data indicate that SST/SSTR4 appear to have an influence in the migration of HOC within the injured liver. Lastly, the activ ation of phosphatidylinositol-3-kinase (PI3K) was observed in HOC exposed to SST, and this activa tion was suppressed when the anti-SSTR4 antibody or TT232 was utilized. In addition, d ecreased motility of HOC by the treatment with a PI3K inhibitor revealed that PI3K was an essentia l signaling molecule in HOC migration. Our findings suggest that SST stim ulates the migration of HOC within the injured liver via SSTR4, and this action a ppears to be mediated by PI3K pathway.

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1 CHAPTER 1 INTRODUCTION Liver regeneration has evolved to protect animals in the wild from the catastrophic results of liver loss that can be caused by ingested toxins. The process has been recognized by scientists for many years and was even described by the ancient Greeks, who mentioned liver regeneration in the myth of Prometheus. Liver regeneration is a complex, evolutionarily conserved process that involves numerous hepatic cell types. In these processes, hepatocytes rapidly re-enter the cell cycles and di vide, although they are in the G (gap) 0 phase of the cell cycle and are norm ally quiescent. However, severe and chronic liver injury caused by drugs, viruses, and toxins impairs hepatocyte proliferation. When the ability of hepatocytes to divide and replace damaged tissue is compromised, a subpopulation of liver cells, hepatic stem cells, is introduced to proliferate and differentiate into hepatocytes and biliary epithelial cells. Stem cells are defined as cells capable of both self-renewal and multi-lineage differentiation. Also, stem cells have an ability to home to the appropriate site for their survival and/or proliferation. Homing is the first–and a fairly complex-process prior to cells’ proliferation. This chapter covers general aspects of the liver, such as f unction, anatomy, regeneration, and stem cell, specifically hepatic stem cells. Also, it re views chemokines and molecular mechanism affecting homing of hepatic stem cells. Liver Structure and Function The liver is the largest solid organ, we ights about 2–3% of body weight in human and 5% in mice.1 There has been much discussion a bout how to define the liver as a

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2 structural and functional unit. In its simplest formulation,2 the histological units of the liver are lobules, irregularly oriented struct ures which contain rows of hepatocytes . The lobules are demarcated by peripheral spaces (por tal triads) that have small branches of the portal vein, hepatic artery and bile ducts. In the portal spaces, the terminal segments of the biliary system connect with hepatocy tes in the liver parenchyma through the canals of Hering. The hepatocytes in the lobular plat es are separated by sinusoids and the plate extended from the portal spaces to the central vein located at the center of the lobular structure. Hepatocytes located at the periphery are referred to as periportal while more centrally located hepatocytes in the lobule are referred to as pericentral, perivenular or centrolobular.3 The functional units of liver are acinus. There are three acinar zones within the lobule. Zone 1 includes hepatocyte s that surround the portal triad; Zone 2 is composed of inter-zonal hepatocytes; Zone 3 consists of hepatocytes that surround the central vein. In this acinar structure, blood flows through sinusoids from Zone 1 to Zone 3, and the bile moves in the opposite direction fr om Zone 3 to Zone 1. It is believed that hepatocytes within different acinar zones have different functions and the hepatocytes within Zone 3 are characteri zed by their increased DNA conten t (4N to 16N) and largest size. Also, Zone 3 hepatocytes are found to be predominantly binucleated. Hepatocytes within Zone 1 are smaller and usually single nucleated (2N).4 The cell types of the liver that carried out most of the hepatic function are parenchymal cells, including hepatocytes a nd bile duct cell in hepatic plates. Nonparenchymal cells are lined in sinusoids and composed of sinusoidal endothelial cells, stellate cells, Kupffer cells, fibroblasts a nd leukocytes. The individual cell-cell contacts

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3 between each other in an intim ate relationship and with extracellular structure. These cells form a unique architectural arrangeme nt facilitating the function of the liver. The liver has many special functions First, the liver functions as a secretary organ, and is appropriately situated in the b ody to carry out its s ynthesis of protein, detoxification, metabolism, st orage of glycogen, and secretion of bile acid. Indeed, because of its numerous biochemical functions , the liver is considered the biochemical factory of the body. Second, the liver is organized strategically to coordinate its structure, including its blood circulation, with its functions. Liver has the structural and functional unit of the liver, called an acinus, and bile ca naliculi that transport bile produced by the liver cells. Third, the liver has a unique, dual blood supply, portal vein and hepatic artery. The portal vein carries oxygen rich blood, wh ile blood from hepatic artery nourishes the bile ducts and liver cells. They both jo in in tiny blood vessels called sinusoids.4 Liver Regeneration The liver regeneration is a well-known cap ability of the liver and occurs in response to an excessive metabolic worklo ad imposed by the body. However, we still do not fully understand what sorts of workloads, how metabolic work is translated into a growth signal, what starts the growth and st ops it, and what the growth effectors are? Therefore, the numerous studies for these questions are now being addressed in both in vitro and in vivo systems. The term “regeneration” is firmly embedde d in the literature, but is inaccurate in that the excised lobes do not regrow. Inst ead, the remaining lobes enlarge, undergoing what is more accurately termed a “comp ensatory hyperplasia”. The histological architecture is preserved throughout this process. In the norma l adult rat liver, hepatocytes are highly differen tiated and essentially all in a state of growth arrest, or G0.

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4 Hepatocytes are induced to ente r the cell cycle by the cell loss or functional inadequacy, for example due to surgical re section, infection, toxic or phys ical injury, or to metabolic imbalances caused by severe diabetes, to drasti c changes in nutrients, or to pregnancy in rats and mice where multiple fetuses occur in a small animal. When the excess metabolic workload is removed, the liver shrinks back to normal size by apoptosis. Partial hepatectomy (PHx) is widely used to induce liver regeneration, because it is reproducible with constant resu lts, well tolerated, delivers a quantifiable stimulus and is free of the side effects and damage to surviv ing cells associated with carbon tetrachloride (CCl4) or other commonly used toxic agents. Following the standard 70% hepatecptomy, the potentially interrelated growth and stress associated changes start almost immediately. The earliest documented events include monovalent cation fluxes, changes in intracellular pH, alterati ons in amino acid transport,5 and activation of certain liver function specific and immediate-early genes.6,7 Early events also involve activation of second messengers and signaling pathway and further changes in gene expression and attendant alteration in the bioc hemistry of the cells as they undergo the transition from G0 to the G1 phase of the cell cycl e an continue through G1 into the S (synthesis) phase. DNA (deoxyribonucleic acid) synthesis, ma rking the S phase of the cell cycle, begins at about 14 to 16 hour (hr) followi ng a partial hepatectom y in young adult rats, rises steeply to a sharp peak at 22 to 24 hr, then falls and c ontinues at a diminished level until the original liver complement is restor ed in a little more than a week. Mitosis follows DNA synthesis 6 to 8 hr later. DNA synthesis and mitosis are observed to occur first in hepatocytes in the periportal areas, subsequently spreadi ng centrally. During the first 72 hr, when the major cell division has occurred, about 80% of the new hepatocytes

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5 are formed in the periportal region of the li ver lobules. DNA synthe sis in ductal cells follows that in hepatocytes and peaks 12 to 24 hr later. 8,9The regenerative process is most rapid in weanling animals and slowest in old animals.10,11 In regenerating livers, DNA synthesis firs t appears periportally but progresses in time to involve the whole lobule with the possi ble exception of a very few cells close to the central vein.9 The fact that all hepatocytes have proliferative potential has been shown in animals given [3H] thymidine repeatedly for several days, during which essentially all of the hepatocytes become labeled.12 When rats are subjected to PHx at monthly intervals for a year, the liver regenerate repeatedly. A lthough at first the exis ting lobules enlarge, new lobules ultimately develop after additional hepatectomics.13 The rate at which regrowth occurs is proportional to the extent of liver loss. If the deficit is small, the liver regrows more slowly, despite its high growth potential.14 If the liver resection is increased beyond the usual 70% up to 80 to 90%, however, the animals are severely stressed and DNA synthesis is not further increased.15 The signals that initiate rege neration are clearly derived from extrahepatic sources, since they are transmitted by the blood. The si gnaling molecules are generally thought to be combinations of interacting hormones, gr owth factor, and cytokines, although direct action of nutrients or metabolites has not been ruled out. The complexity of this problem is compounded by the fact that the same growth factors can ac t both positively and negatively; transformi ng growth factor (TGF ), hepatocyte growth factor (HGF), and glucagons are well-known examples.12 Infusion of insulin and epidermal growth factor (EGF) individually into normal rats weak ly stimulates DNA synthesis, but when combined, the stimulus is substantial. Add ition of glucagons to this combination is

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6 inhibitory, but when insulin is omitted glu cagons become weakly stimulatory. Similar effects are demonstrable in hepatocyte cultur es. Thus, it appears that hormone and growth factors interact in s ynergistic or antagonistic combinations, and the same substance may act positively or negatively, depending on the circumstances. After a long slow start, res earch on liver regeneration is gaining momentum at an accelerated rate. The framework, based on whole animal studies, is in place. Many of the parts that function within it are recognized and their potent ial roles are currently under intensive study at the molecular level. A lthough the complexities that are emerging may appear overwhelming, the technologies are at hand, and the pieces that fit into the puzzle are coming into sharper focus. The final step, assembly of the parts into the integrated whole that emcompasse s liver regeneration in vivo , is about to begin. Stem Cell No area of research since gene thera py has evoked so much enthusiasm and passionate debate as stem cell research. Torch as the cure for degenerative disease or the solution for the shortage of organ transplanta tion, stem cells start to unlock the mystery of human development and aging. Stem cells are unique cells. All stem cells can self-renew, proliferate indefinitely and differentiate into specialized tissues acco rding to the source of the stem cells. In general, stem cells are classified by the abil ity of differentiation. First, as a fertilized oocyte and a blastomere, totipotent stem cells can differentiate and generate a complete organism. Next, embryonic stem cells (ES) derived from the inner cell mass of a blastocyte. They are called as pluripotent st em cells, which retain the property of selfrenewal and the ability to differentiate into cells and tissue from all three germ layers. However, these cells cannot form tissues. Finally, multipotent stem cells are also

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7 undifferentiated cells found among di fferentiated cells in a tissu e or organ. They can selfrenew and differentiate to yield major special ized cell types of a tissue or organ. These stem cells persist into adu lthood and are responsible for the maintenance and regeneration of several organ systems. These tissue specific multipotent cells are defined as adult stem cells and called somatic stem cells. Pluripotent Stem Cell Debate over stem cells centered on the sour ce of the pluripotent stem cell. Stem cell isolation techniques are adopted from animal models that ha ve existed for more than 30 years.16,17 By extracting the inner cell mass from a mouse blastocyte, ES cells can be cultured and indefinitely maintained in vitro . The embryos from which human ES cells are derived are typically four or five day old hollow micr oscopic ball of cells called the blastocyte. The blastocyte incl udes three structures: the trophobl ast, which is the layer of cells that surrounds the blasto cyte; the blastocoel, which is the hollow cavity inside the blastocyte; and the inner cell mass, which is a group of an approximately 30 cells at one end of the blastocoel. The inne r cell mass is transferred into a plastic culture dish and subcultured during six months or more. The or iginal 30 cells of th e inner cell mass yield million of ES cells, which can proliferate in cell culture w ithout differentiating, and are refereed to as an ES cell line. From a therapeutic viewpoint the ES cell culture represents a potentially unlimited source for stem cell therapy. In an animal m odel for ParkinsonÂ’s diseases, transplantation of undifferentiated ES cells showed func tional improvement of motor asymmetry.18 However, the study of human ES cells ha s met with many obstacles on ethical and technical fronts. Human pluripotent cell line was obtained from two sources. Dr. ThomsonÂ’s group isolated human ES cell from unused embryos from in vitro fertilization

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8 cycles,19 and Dr. GearhartÂ’s group isolated human EG (embryo germ) cell from aborted fetuses.20 Both cell lines were donated for research purposes with informed consent of the donors and showed similar developmental potential.21 Because the embryo and unborn fetus are unable to grant consent for use, a dvocates have vowed to protect the rights of the human embryo and fetus.22 On the other hand, some i ndividuals argue that since multiple embryos are generated for a couple in the in vitro fertilization clinic and often are not all used they could be potentially useful for research purposes rather than destroying them or storing them indefinitely.23 In 2001, President Bush restricted research of human ES cell on 64 ES cell lines that existed before August 2001 by providing very strict criteria. The reality seemed much more restrictive because scientists complained that only a few of these lines ar e actually available and in some cases they are expensive or involve heavy in tellectual property entanglements. In addition, although the intent of the ES cell is to generate tissue/organ for transplantation, the success of cloning in animals such as Dolly has brought the fear of potential human cloning. With these mounting co ncerns over the use of ES and EG cells attention has been turned to adult stem cells with reexamination of their potential and plasticity. Adult Stem Cell and Plasticity Research on adult stem cell has generated a great deal of excitement. Scientists found adult stem cells in many more tissue than they once thought possible. This finding led scientist to ask whether adult stem cells could be used for transplants. In fact, the history of research on adult stem cells began about 50 years ago. In the 1960, the seminal work of Till and McCulloch 24 laid the foundation for the iden tification and enrichment of a population of bone marrow derived cells capab le of self-regenerati on and repletion of

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9 the entire hematopoietic cell line.25,26 After that, scientists have reported that adult stem cells occur in many tissues and that they en ter normal differentiati on pathways to form specialized cell types of the tissues in which th ey reside. Adult stem cells also exhibit the ability to form specialized cell types of other tissues, which is known as transdifferentiation or plasticity. Many experiments have challenged the long-st anding belief that adult stem cells or organ specific stem cells are li neage restricted. In other words, the theory that as cells become more differentiated, they lose plastic ity or the ability to differentiate may not hold true in all instances. The tracking of ce ll lineage with transgenic markers such as Lac-Z or green fluorescent protein (GFP), or with the Y chromosome suggest an unrealized potential for adult stem cell to cross tissue lines and transdifferentiate. Starting with these early experiments, other research es also found evidence suggesting that adult stem cells from various organs can contribute to the re generation of other, often dissimilar organs. In addition, a new prom ising type of adult stem cell, MAPC (mutipotent adult stem cell) was thought as versatile as ES cells without the risk of tumorgenicity and hence to have a great imp act on therapy for diseases of degenerative origin. However, many scientists ar gued that there was a lack of evidence demonstrating that adult stem cells can match the versat ility of ES cells derived from embryos. Andersen et al.27 suggested that traditional identi fication of lineage unrelated progeny by morphology, lineage specific antibodies or surface markers was not sufficient to demonstrate plasticity. Also the reproducibility of other tr ansdifferentiation results had

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10 been questioned.28,29 Moreover, reports of spontane ous cell fusion have cast further doubts on whether transdiffere ntiation actually occurs.30,31 Hepatic Stem Cells Over the years, substantial evidence has accumulated suggesting the existence of potential liver stem cells (LSCs) in the adu lt liver. In all cases, the putative LSCs were activated to proliferate and differentiate when the regenerative capacity of terminally differentiate hepatocytes was compromised. For that reason, LSCs were regarded as facultative stem cells or liver stem like cells.4,32-34 It would be almost impossible to assign a universal definition of LSCs. Following th e reasoning of Potten and Loeffler, it could be provided an updated definition of the ti ssue-specific LSC: (1) undifferentiated, (2) self-maintaining (self-renewing), (3) plurip otent and capable of producing multilineage (or at least bilineage) progeny, (4) capable of repopulating the liver, and (5) flexible in expressing these previous characteristic. The actual LSC will be the one that expresses all four characteristics. LSCs express one or more of the four char acteristics would be potential LSCs. Following this definition, a cl ear distinction among hepatic stem cells in embryonic, fetal, and adult liver can be ma de. The progeny of the potential LSCs are the well-described progenitor or oval cells in adult liver. Oval cells were first reported by Kinosita et al . 35 who observed small, ovoid cells in liver of rats exposed to the carcinogenic azo dye ‘Bu tter Yellow’. These cells were later termed oval cells because of their characteristic morphology; ovoid nucleus, small size (relative to hepatocytes) and high nuclear to cytoplasmic ratio.36 Oval cells are observed to proliferate in the periportal region of the liver and, as liver damage processes, they infiltrate into the parenchymal along th e bile canaliculi betw een the hepatic cords.4,37 Rodent Model of Oval Cell Induction

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11 Although many rodent models are currently used to study oval cell biology, most of the models of activation and pr oliferation of oval cells were developed in rats. Thus, the activation of oval cells was observed after tr eatment of rats with azo-dyes, feeding a choline-deficient diet supplemented with ethionine, feeding N-sfluorenyl-acetamide (2AAF) in combination with PHx, treatment w ith pyrrolizidine alkaloids in conjunction with PHx, after severe liver injury with D-galactosamine, or feeding rats a copperdeficient diet. Other murine-spe cific models were also descri bed: oval cells expansion in dipin-induced hepatocarcinogenesis,38 in phenbarbitalor co caine-induced periportal injury,39 and the 3, 5-diethoxycarbonly-1, 4-dyhydrocollidine (DDC) mo del of alcoholic liver diseases.40 Characteristics of Oval Cells Oval cells share some phenotypic characte ristics with hematopoietic progenitor cells, namely the receptor for stem cell factor (c-kit), and its ligand stem cell factor and the related proteins flt-3 and flt-3 ligand,41 considered to define early embryonic hematopoietic precursors.42 Expression of c-kit was also re ported in normal and pediatric human liver. However, c-kit-mediated signal transduction seems not to be essential for proliferation of oval cells in rodents, and it was reported rat ED (embryonic development) 13 fetal hepatoblasts do not express this receptor.43 Oval cells express CD34,44 a marker of early hematopoietic progenitor cells and va scular endothelium, implicated in signal transduction and cell adhesion. Thy-1 (C D90), found on the surface of many early hematopoietic progenitor cells, immature B ce lls, and T cells, is al so expressed in oval cells.45 Recently, it was also shown on the DDC model of mouse liv er injury that proliferating oval cells express A6 46 and another hematopoietic surface marker, Sca1, specific for hematopoietic stem cell (HSC).47

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12 Origin of Oval cells Two major views regarding the localization of oval cells in the quiescent liver have been developed over the years. The prevailing vi ew is that the potential hepatic stem cells are localized in the canals of Hering, the smalle st branches of the intr ahepatic biliary tree, lined by both hepatocytes and bile duct epithelial cell (BDEC).4,32,33,48 The second view is that hepatic stem cells are loca lized in the periduct ular/intraportal zone and that they are small nondescript cells that gi ve rise to progen itor cells of hepato cytes and BDEC and that the periductular progenito r cells may originate from an extrahepatic (bone marrow) source49. Petersen and coworkers50 transplanted syngenic bone marrow cells into lethally irradiated female animals treated with 2AAF and CCl4, which causes hepatic injury and impairment of hepatocytes proliferation, a nd found oval cells in the liver 9 days after injury and hepatocytes 4 days later. The fetal liver functions as a hepatic a nd hematopoietic organ. As stated earlier, hepatic oval cells express hematopoietic marker s not expresses in adul t liver. These data prompted Petersen et al 50 to study whether bone marrow prog enitors can give rise to oval cells in the liver. Lethally irradiated fema le rats were transplanted with genetically marked male bone marrow (CD23) and then su bjected to severe liver injury (2AAF/CCl4 protocol). The authors50 observed the appearance of dono r-derived hepatocytes in the liver of the injured animals. Theise et al.51 used a similar approach in mice without causing injury to the liver and sorting the CD34+linhematopoietic cell fraction. They found Y chromosome-positive hepatocytes with al bumin expression in the liver of female mice that originated from the donor bone marrow. To identify further the bone marrow cell capable of differentiating into hepatocytes, Lagasse et al.52 used purified HSCs (ckit+ Thy1low, lin-, and Sca-1+) and transplanted them into lethally irradiated FAH

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13 (fumarylacetate hydrolase)-/mice. Three weeks after cell transplantation, the animals were subjected to liver injury by removal of the drug NTBC, which is a pharmacological inhibitor of tyrosine catabolism upstream of FAH. Seven months later, numerous repopulating nodules of donor-derived FAH-positive hepatocytes were observed. Crossgender, or crossstrains, bone marrow and w hole liver transplants in mice have identified cells in the bone marrow that are capable of repopul ating the liver.50,51 These results are supported by human studies usi ng archival liver biopsy spec imens from recipients of cross-gender therapeutic bone marrow transp lants who later developed chronic liver damage due to recurrent disease. Analysis fo r the presence of the Y chromosome in cell of the liver biopsy specimens showed that bone marrow-derived cells give rise to hepatocytes alone, or both he patocytes and cholangiocytes53,54. However, there is disagreement on the extent of engraftment. Alison et al .55 reported a relatively low frequency of Y chromosome positive hepatocytes (0.5%-2%); whereas Theise et al.53 reported that, even in mild conditions, signi ficant engraftment (521% for hepatocytes and 4-18% for cholangiocytes) occurred; while in cases of severed injury, up to 64% of periportal hepatocytes and 38% of cholangiocytes were donor derived.53,54 Hence, hematopoietic cells are capable of migrat ion to the liver and differentiating into hepatocytes in rodents and humans; however, th e role of these cells in liver regeneration remains unclear. The origin of the hepatic oval cell remains a contentious issue. Nevertheless, it is increasingly apparent , as hypothesized by Sell,49 that three levels of proliferating cells are associated with the liver. These are: the ma ture adult hepatocytes; a tissue-determined stem cell, originating endogenous ly in adult liver in the terminal bile ducts; and a

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14 multipotent stem cell, that may be exogenous ly derived from circulating bone marrow stem cells, The direct lineage relationship that links bone marrow-derived hepatic stem cells and/or hepatic colony-forming-unit in culture (H-CFU-C)Â’s to oval cells has not been definitively proven. Oval Cell-mediated Liver Regeneration Regardless of the hepatic progenitor cell orig in, it is well established that oval cell proliferation occurs when the replicative capacity and function of hepatocytes are impaired. Oval cells are resistant to the e ffects of hepatotoxins/carcinogens, thus, they proliferate and migrate throughout damaged liv er lobules to replace lost parenchyma. Given the association between experimental models of hepa totoxicity and carcinogenesis, it is not surprising that oval cells have been identified in acute and chronic human liver pathologies including he patitis B virus/ C virus infecti on, hemachromatosis and alcoholic liver disease.56-60 An extensive inflammatory respons e is characteristic of many chronic liver diseases. This response is mediated th rough resident Kupffer, hepatic stellate cells and infiltrating inflammatory cells, which secrete chemokines, growth factors and cytokines in response to liver injury. As su ch, the histological changes that accompany oval cell proliferation pr ovide useful clues for identifying factors that mediate oval cell activation. Inflammatory cytokines Two important inflammatory cytokines rela ted with the regulati on of liver growth are tumornecrosis factor (TNF) and interle ukin-6 (IL-6). The majority of hepatic TNF produced during liver regeneration is K upffer cell derived. Inhibition of TNF by dexamethasone administration, or depleti on of Kupffer cells by treatment with gadolinium chloride prior to bile duct li gation completely ablates oval cell induction.61 In

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15 addition, TNF receptor 1 knockout mice have been shown to impaired oval cell proliferation.62 In contrast, there is conflicting evidence regarding the requirement for IL-6 in via oval cell –aided liver regeneration. IL-6 pr oduction following 2AAF/PHx is inhibited by dexamethasone with an accompanying reduction in oval cell numbers. This suggests its involvement in the activation of the oval cell.63 However, comparison of the cellular response to cocaine-induced peri portal injury in normal and IL-6-/mice demonstrated the increase in proliferation of periportal oval cells in IL-6-/mice to compensate for the decrease in restorative pro liferation of hepatocytes, b iliary epithelia and sinusoidal cells.39 Ten day after injury, the liver was complete ly repaired in all mice, indicating that IL-6 is not essential for oval ce ll proliferation. It is feasible that othe r members of the IL6 family, including leukemia inhibitory fact or (ILF) and/or oncostatin M (OSM), may compensated for the absence of IL-6 in these mice.64 Indeed, LIF is increased and remains elevated during oval cell induction, s uggesting that it may have a role in the expansion and differentiation of the oval cell compartments.65 OSM has recently been implicated in the maturation of fetal hepatocytes in vitro and in vivo ,66 and it may have a similar role in the hepatic differentiation of oval cells. Interferon (IFN) is another inflammatory cytokine considered to play an integral role in controlling stem cellmediated liver regeneration.67 Function of Hepatic Stellate Cells The concurrent proliferation of mesenchymal cells with oval cells was first reported by Popper et al . in 1957 68 and, using the 2AAF/Phx model, Evarts et al .69 showed that hepatic stellate cell proliferati on is closely associated with oval cell proliferation. Hepatic stellate cells express HGF, TGF, TGF, and acidic fibroblast growth factors (aFGF).

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16 Interestingly, these cytokines have all b een identified in regenerating liver following PHx. Hepatic stellate cell-derived HGF may cause oval cell proliferation via the paracrine activation of HGF receptor, c-met.70 A marker increase in hepatic aFGF level has been reported at the peak of oval cell proliferation in the 2AAF/PHx model, and levels greatly exceeded those observed afte r PHx alone, suggesting a more prominent role for a FGF in oval cell-mediated regeneration.71 High level of TGFexpression are observed not only in hepatic st ellate cells, but also in oval cells in the 2AAF/PHx model,69,72 and TGFexpression is detected in foci of hepatocytes that appear to be oval cell-derived.57 In contrast, TGFis implicated as a negati ve regulator of oval cell activation. TGF1 expression on smooth muscle actin (SMA)-positive hepatic stellate cells coincides with oval cell proliferation in the 2AAF/PHx model and correlates with maximal oval cell apoptosis.73 As TGF1 is proposed to be a negative growth signal that controls liver size by the induc tion of apoptosis during comp ensatory hyperplasia, it is possible that TGF1 may assist in the remodeling of liver parenchyma during oval cellmediated liver regeneration by terminating oval cell activation.73 Stem Cell Factor Stem cell factor (SCF) and its receptor, c-ki t, play a fundamental role in survival, proliferation, differentiation, and migration of a variety of stem cells and may similarly affect oval cells. SCF is induced early in th e activation of oval cells by 2AAF/PHX, but this is not observed following PHx alone.41 Oval cell precursors express both SCF and ckit, suggesting that an autocrine mechanism may be involved, although the precise role of this receptor-ligand system in liver regene ration is unclear. Oval cell induction is significantly suppressed in Ws/Ws (white spotti ng on the skin) rats, in which the c-kit the c-kit receptor tyrosine kinase activity is severely impaired.74 Once oval cells appeared in

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17 Ws/Ws rats, expression of oval cell specific marker proteins and the proliferative response of these cells were similar to cont rol, implying that tyrosine kinase-mediating signal transduction is not esse ntial for the phenotype or proliferation of oval cells. It appears that the SCF/tyrosine kinase system plays a crucial role in oval cell appearance, either by regulating the number of hepatic progenitor cell within the liver or by committing hepatic progenitor cells to differentiation into oval cells. Interaction with the Extracellular Matrix The plasminogen activator and plasmin proteolytic cascades have an important function in stem cell-mediated regenerati on, as most regenerative responses are associated with changes in the extracellular matrix. The plasminogen activator/plasmin system is complex, involving many protei ns including urokine-type plasminogen activator (uPA), tissue type plasminogen activator (tPA), the uPA receptor (uPAR), and plasminogen activator inhibitor 1 (PAI-1).75 The upregulation of uPA mRNA accompanying oval cell proliferation has been reported, and infusion of uPA enhanced the mitogenic responses of cells located near bile ducts after the ad ministration of 2AAF. Expression of uPA and PAI-1 is upregulat ed in the 2AAF/PHx model of oval cell induction, and localized to the ductal structure formed by the oval cells.76 Urokinase-type plasminogen activator expression was also de tected in non-parenchymal cells along the hepatic sinusoids. In additi on to the significant remodeling and oval cell migration into the parenchyma that is occurring, this syst em may play a role in regulating several growth factors that are involved in oval ce ll regeneration. The expression of uPA, uPAR, and PAI-1 is upregulated in vivo and in vitro by a number of cytokines and growth factors, including HGF, EGF, and TGF. Additionally, HGF and TGFare both secreted as latent fa ctors and require activ ation by protesases.77-79

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18 A number of cytokines, growth factor, and chemokines released from Kupffer, hepatic stellate and oval cells act in concert to control oval cell proliferation and remodeling of the liver parenchyma. The identi fication of these factor that control this process, and clarification of their mechanism of action with respect to oval cell mediated liver regeneration will facilitate the establishment of in vivo models to investigate oval cell biology. Homing Capacity of Stem Cells The hallmark of stem cells is their mi gration and repopulatio n potential (selfrenewal and multi-lineage differentiation abiliti es). Interestingly, migratory capacity of stem cells in vitro correlates with their in vivo repopulation potentia l. Tissue damage leads to a dramatic increase in the leve l of secreted chemokines, cytokines, and proteolytic enzymes in many organs as part of the regeneration and repair process, which have profound impacts on stem cell migration and repopulation. For example, the stromal cell-derived factor 1 (SDF-1)/ CXC chemoki ne receptor 4 (CXCR4) pathway have been well known in homing of hematopoietic progenito r cell to BM or leuckocyte migration to tissue.80-82 However, there is relatively little information regarding the biological factors that influence hepatic oval cells, although these factors, which regulate the different hematopoietic lineages, are well characterized. Increasing evidence has emerged that gr owth factor and cytokines produced by Kupffer and hepatic stellate cells play a role in the prolifer ation of oval cells. In vitro experiments have shown that several interleu kins and chemokines are mitogens for oval cells.83 In oval cell mediated li ver regeneration, hepatic oval cells are observed in being scattered away from the porta l tract periphery deep into the lobular parenchyma. This topography of oval cell distributi on indicated that oval cells mi ght be mobile cells. Based

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19 on ultrastructure studies in rat liver, it has been proposed that oval cells migrate by amoeboid movement with the help of pseudopodia.59 Previous studies have shown a correlation between hepatic ova l cell proliferation and parenchymal inflammation. The finding that the inflammatory infiltrate influe nces the activation and localization of stem cells strongly suggested that one or more cytokines produced by the inflammatory infiltrate function as growth or chemotactic factors for the oval cells.59 The homing capacity of hepatic oval cells is a significant and in triguing aspect of stem cell biology. Therefore, the molecula r mechanisms regulating oval cell homing deserve more intensive study, especially given the importance of such homing in a variety of medical applications. Somatostatin and Its Receptors Somatostatin (SST) is an attractive regul atory hormone, which was first reported in 1973 as a hypothalamic hormone inhibiting gr owth hormone (GH) secretion. SST has two biological active forms, SST-14 and SST -28, which are generated as C-terminal product from proSST.84 SST-14 was originally described in the hypothalamus and the amino-terminally extended SST-28 discove red later on in the gut. SST-14 and SST-28 are predominantly expressed in neuron and s ecretary cells in the central and peripheral nervous systems, the gastrointestinal trac t, pancreas, pituitary , kidney, retina, and immune system.84 The major action of the neuropeptid e includes inhibition of hormone secretion from the pituitary, most notable that of GH,85 the pancreas, and other endocrine and exocrine secretion in a number of various organs. Also, SST controls the proliferation of normal and tumor cells.84 Additionally, it is invol ved in differentiation and migration of thymocytes.86 Recently, SST has been shown to exert a function as chemoattractant for immature ne uronal and hematopoietic cells.87,88

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20 These broad biological functions of SST ar e mediated by at l east five receptors subtypes, which all belong to the family of seven -helical transmembrane domain G protein -coupled receptors (GPCRs).89 Natural ligands of the different SST receptor subtypes are SST14 and SST28.89 Their identification in the early 1990s was a major step forward in elucidating the SST signaling sy stem and, during recent years, researchers were urged to find correlations between cloned and native rece ptors and to assign specific functions to individual receptor subtypes. This review will cover the latest advances that have been made to elucidate the somatostatinergic system with respect to SST receptor, receptor regulation, signal transduction. Somatostatin Receptor Subtypes In mammals all SST receptor (SSTR) s ubtypes, except, SSTR2A and SSTR2B, are encoded by separate genes on different chromosomes.84 In human, SSTR1-5 are encoded by five non-allelic genes on chromosomes 14, 17, 22, 20 and 16, respectively,84,89 and in rats, on chromosomes 6, 10, 7, 3 and 10, resp ectively. Both SSTR2 forms are derived from a single gene due to the pr esence of a crytic splice site.90 The genes of SSTR1, 3, 4, and 5 are not interrupted by introns in thei r protein coding regions. However, introns have been found in the 5Â’-untranslated regions of the SSTR2, 3, and 5 genes. All six SST receptor variants display the typical molecu lar architecture shared by GPCRs comprising seven putative transmembrane regions (T M), the conserved DRY motif at the cytoplasmic face of TM3, N-linked glycosyla tion sites in the N-terminal domain and, with the exception of SSTR3, putative palmitoylation motifs. Notably, a highly conserved sequences, YANSCANPI/VLY, in TM7 has been identified in SSTR subtypes, which is considered as a mammalian SSTR signature. Furthermore, all mammalian SSTR subtypes contain a consensus sequence (S/T)-X-Stop, in their C-terminus, where X can be any

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21 and is a large and hydrophobic amino acid residu e. Recent studies showed that this sequence is a potential PDZ domain-binding si te, which might be important for proteinprotein interactions between SST Rs and scaffolding proteins.91 Based on their sequence homology, the recep tor subtypes can be placed into two subgroups: SRIF1 and SRIF2 (somatotropin releas e inhibiting factor, which is a synonym for SST). The SRIF1 receptor group co mprises SSTR2, SSTR3, and SSTR5, while SSTR1 and SSTR4 are included in the SRIF 2 group. This classification is based on structural features and st rongly supported by their pha rmacological properties. Evolutionary Aspects According to the phylogenetic tree ba sed on the alignement of amino acid sequences of the known full-length SSTR in vertebrates, it might be proposed that the differentiation into SRIF1 and SRIF 2 receptor subgroups and the following differentiation into SSTR1 and SSTR4, and the two ancestral SSTRs (aSSTR3 and aSSTR2/5) happened before tetrapodes and tele osts split off, indicating a separation of the genes about 450 million years ago. This is consistent with the theory that SSTRs, like other GPCRs, arose very early during evolution.92 Furthermore, it might be suggested that an important regional DNAor genome dup lication event gave rise to two ancestral SSTR genes from which the groups of SR IF1 and SRIF2 receptors descended. An additional duplication event might have been responsible for the differentiation into SSTR1 and SSTR4 genes and two ancestral receptor genes. From aSSTR3 the present day SSTR3 genes evolved. The aSSTR2/5 gene split into ancestral SSTR3 and SSTR5 genes, from which the present fish and mammalian SSTR2 and SSTR5 gene developed. These data are consistent with the hypothesis that two to three partial or complete genome duplications have occurred dur ing vertebrate evolution.84 It is also very likely that the

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22 additional variants (A and B) of goldfi dh SSTR1, SSTR3 and SST R5 subtypes derived from polyploidization events during fish evol ution after fishes and tetrapodes separated, as the corresponding variants are closely re lated to each other. The two type one receptors share 98% identity in their am ino acid sequences, the SSTR5A and SSTR5B homologues exhibit an amino acid sequence identity of 85% and the goldfish SSTR5C has still about 69% similarity to goldfish SSTR5A. Both goldfish SSTR3 subtypes have a similarity of 65%.93 Furthermore, it is now well establishe d that most teleosts contains an excess of duplicated genes in comparison with mammals.94 Thus, it seems rather unlikely that the presence of these recep tor variants in goldfish point to the existence of a novel SSTR subtypes in mammals. However, furt her studies, for example the complete identification of the Fugu gemone and the pharmacological characterization of all fish receptors, are necessary to make a final stat ement about the representation of SSTRs in this species. Ligand -Receptor Interactions There are many structure-activity studies as well as investiga tions that involve receptor mutations from which conclusions about the ligand binding pocket and ligand receptor interactions were drawn. From these it appeared that the interaction of SST14 and SST28 with their receptors occurs, as in other peptides/receptor system, through amino acid residues located, deep within the lipid bilayer, most probably in a binding cleft involving resi dues of TM3 to TM7,95,96 and through amino acids in the extracellular loops.95 The formation of an ion pair bond betw een the critical K9 of SST14 (or the corresponding K in the analogues) and a cons erved aspartate residue in TM3 of the receptor likely represents an important early step in the binding of SST peptides to all SSTR subtypes.95

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23 Peptide-selective binding to SSTR subtypes is conferred by the extracellular loops and the upper parts of TM domains. Selectiv e binding of hexapeptide and octapeptide analogues to SSTR3 depends on regions about the second and-more im portantthe third extracellular loop and the adjacent TM6 and TM7 regions.96 Within these regions the precise interaction between the side chains of amino acid residues in the loops and the agonists determine the binding properties. Re placement of F (Phenylalanine) 265 located at the extracellular side in TM6 of SSTR5 by the corre sponding tyrosine of SSTR2 resulted in a mutant receptor w ith increased affinity for SST14.97 Greenwood et al .98 have reported that the second rather than the third extracellular loop play s a potential role for SST14 and SST28 binding to SSTR5. Similar e xperiments allowed to conclude that binding of SSTR1-selective peptides depe nd on amino acid residues in the second extracellular loop.99 Chen et a l. support a model in whic h the hydrophobic interaction of L (Leucine) 107 in TM2 with the SST anal ogues CH275 seems to be important for its specific binding to SSTR1. From these data it seems that ligand specificity depends on different regions in SSTRs in depe ndent of the investigated ligand. SSTR Expression The rationale for the therapeutic use of SST analogues is largely based on the expression of SSTRs in relevant normal and tumoural tissues.89 A high concentration of SSTRs can also be detected in the brain, and where primary SSTR1 and 2 seem to be involved in the inhibition of GH release.89 SSTR4 has recently been detected in rat hypothalamus, thus a role for this receptor subtype in the regula tion of GH secretion might be suggested. Tissues in the periphe ry that normally express SSTRs include gastrointestinal and pancreatic tissue. The pancreas is an important target of SST action, and all five SSTR subtypes have been detected in human islet cells, although in different

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24 amount. SSTR1, 2, and 5 are predominantly expressed in insulin-secreting human cells. In contrast to these receptors, SSTR3 and 4 were only poorly expressed or absent in specific cell types. In rodents, SSTR2 has been proposed to be the predominant SSTR subtype involved in the regulation of gluca gon release.These findings might be explained by species-specific differences. Most tumour tissues preferentially express SSTR2, less frequently SSTR1, 3, 5, and the SSTR4 is only rarely detected in tumour tissus.100 In human normal liver, none of receptor subtypes are detected; however all receptor subtypes, except for SSTR4, are expressed in hepatocellular carcinoma (HCC) in liver tissue and HCC cell line. However, the availa ble data on receptor expression in various tumours reveal major inconsistencies, possi bly owing to the small number of tumours examined so far, as well as the use of different detection methods, such as, receptor autoradiography, membrane-binding methods and immunohisochemistry using various antibodies and reverse transcriptasepolymerase chain reaction (RT-PCR).101-103 Although knowledge of the receptor distribution in normal and cancer tissues is of prime importance, inconsistent data on the abundance of individual receptor subtypes other than SSTR2 do not allow the therapeutic potential of receptor subtype-spe cific or universal analogues in oncological or certain endoc rine indications to be judged. These inconsistencies are shown in results from different studi es about rat liver. Reynaert et al .104 reported the presence of SSTR subtypes 1, 2, and 3 in ac tivated hepatic stellate cells in CCl4-treated rats, but not in normal rat liver. However, observation of SSTR3 mRNA in normal rat liver was provided by another study.105 Other experiments showed the expression of SSTR2, 3, and 5 by activated hepatic stellate cells in SD (Sprague Dawley)

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25 rat.106 One consistent thing observe d in these papers is absenc e of SSTR4 in normal adult rat liver. Thus, additional work on existing SST gene-deficient mice is required and in particular reports of new in vivo studies are eagerly awaited. Internalization, Desensitization and Oligomerization of SSTRs Receptor internalization is common among GPCRs. Accordingly, SSTRs can undergo ligand-induced interna lization depending on exposure time, ligand concentration and heterologous regulation th rough other signaling systems.101,107 SSTR subtypes SSTR2-5 are internalized more efficiently than SSTR1.107 The internalization of SSTRs, in particular of SSTR2, is be ing exploited therapeutically to induce the killing of tumour cells by receptor-targeted radiotherapy or chemotherapy. This strategy involves internalization of, for example, A uger-electron-emitting In-pentetreotide108 or SST analogues coupled to cytot oxis anticancer agents.109 Of potential releva nce for anticancer strategies is the observation that in AR42J tumour-bearing severe combined immunodeficient mice, the tumoral SSTR2 are downregulated following injection therapy with octreotide, whereas SSTR2 is upr egulated when octreotide is administered by continuous low-dose infusion.110 However, SSTR internalization might not be linked to desensitization of neuroendocrine tumours that have previ ously responded to SST analogue therapy.107 The loss of responsiveness of, for example, glucagonomas, insulinomas or carcinoid tumours after months or years of successful SST analogues therapy represents a major unmet medical need.111 However, there are numerous instan ces of lack of SST R desensitization, for example, in acromegalic patients treate d with octreotide for more than ten years.112

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26 Various families of GPCRs can apparent ly form oligomers when activated by ligand binding.113 SSTRs also exhibit SST-induced homo-heterodimerization, and thereby changes in their f unctional and binding properties.101,114,115 For example, heterodimerization of SSTR2A and SSTR3 result s in a protein complex that retains the binding properties of SSTR2 but loses affi nity for SSTR3-selectiv e ligands and SSTR3specific functions.116 By contrast, the SSTR2A and the related µ-opioid receptor heterodimerize in human embryonic kidney 293 cells without major changes in ligand binding and coupling properties ;however, the heterodimers show cross-phosphorylation and cross-desensitization on binding of SSTR2or µ –selec tive ligands, indicating that heterodimer formation might contri bute to the regulation of SSTRs.89 This could be physiologically relevant, as regional overlap in the expression of the SSTR2A and the µopiood receptor was reported in a region of the brain known as the peroaqueductal grey matter, pointing to a potentia l effect of SST on opioid-sens itive neurons mediated by the SSTR2A.89 Whether there is a synergistic action of SST and opioids on the same neuron remains to be determined. Following bindi ng to SST, human SSTR5 forms homodimers as well as heterodimers with human SSTR1.115 Moreover, Rocheville et al .114 found that human SSTR5 and dopamine receptors heterodime rize, which is consistent in view of their co-localization in the cerebral cortex, striatum and limbic system. The heterooligomer exhibits reciprocal induction of high-affinity binding by SST and dopamine, which translated to G-protein coupling and adenylyl cyclase regul ation. The role of heterodimerization is only beginning to be unrav eled, and it is possibl e that insight into SSTR oligomerization will have an impact on future drug design.113 But, it should be

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27 noted that so far results on SSTR oligomer ization have mostly been generated using transfected cells expressing high numbers of certain SSTR subtypes.101 Effects on Cell Proliferation A large number of human tumour express SSTRs, such as adenomas causing acromegaly or carcinoid tumours of th e gut. Stable SST analogues and SST radiopharmaceuticals, mainly de signed for SSTR2 activation, have been widely used for tumour treatment and diagnosis.84 However, although a successf ul long-term therapy of acromegaly patients is possible, patients sufferi ng from other tumour types usually escape from SST analogues therapy after several mont hs. The tumours then continue to grow and hormone hypersecretion starts again.84 The reasons for this are still not known and new tools need to be developed for a more effective treatment of cancer and neuroendocrine disorders. Thus it is of particular clinical interest to elucidate the fundermental molecular mechanisms invol ved in SSTR-mediated control of cell proliferation. Therefore, in ma ny studies proliferative, as well as antiproliferative, effects of SST mediated by different SSTR subtypes ha ve been investigated for distinct cell types. For example, Rosskopf et al .117 proposed a SST growth factor-like activity on human B lymphocytes, mediated via SST R2A involving the activation of PLC (phospholipases C). Other inves tigators reported pr oliferative effects on transfected CHO (Chinese hamster ovary) cells induced by th e activation of SSTR4 or SSTR2B, which a sustained extracellular signal-re gulated kinase (ERK) activity.118-120 However, stimulation of SSTR4 also resulted in a phosphorylation of the transcription factor STAT3,119 which in addition has been shown to be critical for the pro liferative effect of SSTR4.119,121 In contrast to its proliferative eff ects, SSTR4 has been shown to mediate

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28 antiprolifertive effects via the stimulation of p38 that re sults in an activation of the cyclin-dependent protein kinase inhibitor p21cip1/WAF1.120,121 Today, the modulation of phosphotyrosin e phosphatase (PTPase)s activity, has been proposed to play an important role in the inhibition of cell pr oliferation mediated by SST.122 Buscail et al.122 showed that antiproliferation observed after SSTR2 stimulation in transfected CHO cells is coupled to the activ ation of a PTPase and that, in contrast to SSTR2, SSTR5 mediates a cessation of cell growth through coupling to the PLC/inositol phospholipids/Ca2+ inhibitory pathway. In addition, it was suggested that SST modulated cell proliferation and mitogen-activated prot ein kinase (MAPK) signaling pathway were negatively controlled via SSTR5 involv ing a cGMP (cyclic guanosine monophosphate)dependent pathway in CHO cells.123 However, Sharma et al.124 reported a PTP-mediated antiproliferative action of SSTR5 in the same cell type, involving the induction of the retinoblastoma tumour suppressor protein (R b) and p21, but also suggested the possible existence of a PIP (phosphatidylinos itol phosphate)-independent pathway. Antiproliferative effects of SST can al so be a consequence of apoptosis.84 In this case, SSTR2 caused apoptosis via two diffe rent mechanism: first through the downregulation of mitochondrial Bcl-2 protei n expression and second via upregulating expression of death receptors belonging to th e TNF family. In addition, it has been shown that SSTR2 inhibits cancer cell proliferation via the induction of an autocrine/paracrine SST loop.84 SST inhibits prolifera tion of various malignat hematopoietic cell types.87 However, SST did not exerted any signifi cant antiprolifertive effects on normal hematopoietic stem cell.87 Interestingly, SST has been shown to exert a function of

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29 chemoattractant for hematopoi etic stem cell via SSTR2.87 Also, this chemotactic effect of SST has been observed in primitive neuronal cells.88 In conclusion, the observation that SSTR 2 and SSTR4 are able to mediate opposing effects on cell proliferation a nd that stimulation of the ER K pathway can be involved in mitogenic but also anti-mitogenic processes demonstrates once more the complexity of the involved signal transduction machinery. The activity in vitro data regarding the coupling of SST to PTPase deliver a rather co mplicated picture, from which it is hard to refine a clear statement about the interacti on of single SSTRs with a specific PTP. In general, it can be said that di rect antiprolifertive effects of SST are mainly mediated via a transduction pathway involving the coupling of SSTs to PTPases. SSTR1, 2, 4, and 5 mediate in vitro direct cytostatic effects and SSTR 2 and 3 may be responsible for the apoptotic effects of SST. Nonetheless, as in the case of signal tran sduction, it has to be kept in mind that most data results from in vitro experiments using di fferent cell lines and experimental settings and furt her studies are necessary to investigate whether signaling cascades stimulate by SSTR subtypes in vitro are also activated in vivo . SSTR Signal Transduction Signaling pathways coupled to the diff erent SSTR subtypes have been studied extensively. SST binding to SSTRs in native membranes results in the modulation of a wide range of second messenge r systems via the stimulati on of different types of pertussis toxin (PTX)-sensitive and insensitive GTP (guanosine triphosphate)-binding protein.95 The diversity of the transduction pathways reflects the pleiotropic actions of the receptors. The presence of multiple SSTR subt ypes in many tissues and cell lines made it difficult to assign particul ar transduction pathway to single receptors. Thus, scientists tried to overcome this problem by investig ating recombinant receptors expressed in

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30 appropriate cell lines and through the us e of SSTR subtype-selective agonist and antagonists. Binding of SST and SST analogues to SST Rs induces G-protein activation and signalling through various pathways. As a c onsequence, the activit ies of several key enzymes, including adenylyl cyclase, PT Pases and MAPK are modulated along with changes in the intracellular level of calciu m and potassium ions. Accordingly, calcium and potassium channels in addition to the sodium-proton antiporte r respond to SSTR activation.95 Which types of signalling prevails in certain cells depends on the tissuespecific distribution of ligands, SSTR intern alization, desensitizat ion and /or receptor crosstalk.95 SSTR stimulation is coupled to the resp ective intracellular signalling pathways through activation of specific G-protein, including pertussis-toxin-sensitive G i and G 0, as well as pertussis-toxin-insensitive G q, G 14, and G 16 proteins.95,117 The interaction of certain G-proteins and SSTR s ubtypes depends on factors such as their tissue-specific expression; for example, G 0 is found in neuronal cells, but is absent in B lymphocytes, whereas both cell types express the SSTR2.117 All known human SSTRs can inhibit adenyl yly cyclase and hence decrease cyclic AMP (adenosine monophosphate) level.89 This PTX-sensitive action affects various downstream elements, in particular protein ki nase A. The latter, in turn, acts as an activator of a cAMP (cyclic adenosin e monophosphate)-response-element-binding protein, as shown in GH4 cells expressing SSTR2A.125 MAPK signalling cascades is also modulated by SST.84 Several investigators proposed that SSTR-coupled inhibition 126-128 or activation129 of different MAPK cascade

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31 members has been mediated by PTPase. However, in CHO cells expressing SSTR5, inhibition of MAPK activity was rela ted to a cGMP-dependent pathway.123 Sellers118 proposed a SSTR4-mediated acute activati on of MAPK involving Ras and src and a sustained activation of MAPK-dependent on PKC that could be activated by phosphoinositide-3 kinase (PI3K). PI3K has been shown to play a fundamental role for the stimulation of MAPK signa lling cascade through human SSTR4130 or rat SSTR2B, respectively.120 Furthermore, recent studies have s hown that SSTR2 and SSTR4 can also coupled to an alternative MAPK cascades, thereby causing a prolonged activation of p38 that culminates in the induction of the cell cycle inhibitor, p21cip1.120,121 In most cells, Ca2+ signalling is downregulated by SSTR activation owing to the inhibition of calcium channels and intracellular Ca2+ release or the activation of K+ channels, which results in membrane hyperpolarization.81 Accordingly, all humans SSTR subtypes can be coupled to various PLC isoforms.131 In certain systems, however, SSTR activity increases the enzymatic activity of PLC 2 and PLC 3, for example, and hence the intracellular levels of IP3 (phospha tidylinositol triphosphate) and Ca2+.117,132 Finally, it has been shown that SST can couple to Na+-H+ exchanger, which plays a role in cell adhesion, migr ation and proliferation.84,93 Our current understanding of subtype-s elective signaling reflects a rather complicated picture. It is mostly based on experiments using receptors from different species expressed in different cell types. A nother level of complexity may be added by the observation that receptor coupling to a gi ven pathway may be strongly influenced by the ligand used.84 Therefore, the yet available data should be interpreted with caution.

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32 Study Design and Rationale The liver has an enormous capacity to re generate, as demonstrated by the 70% PHx model in rodents. In addition, the liver ha s a stem cell compartment acting as a backup regenerative system. Activation of the stem cell compartment occur when the 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 the so-called oval “stem” cells.58 There is a substantia l body of evidence to suggest that oval cells are i nvolved in liver regeneration, as they differentiated into hepayocyte and biliary cells.58 We have shown that bone marrow cells are able to produce hepatic oval cells with the capacity to repopulate the liver,50,51,53 while others have only shown the end product of hepato cytes and bile duct epithelial cells.55 Regardless of their origins, it has been show n that numerous cytokine, growth factor, and chemokines mediate oval cellaided liver regeneration.133 However, there is relatively little information regulating ova l cell activation, whereas the biological factors regulating the different hematopoietic lineag es are well characterized. Increasing evidence suggests that the neur oendocrine system influences blood cell development and function.87 SST is a pleiotropic neur opeptide, exerting a variety function in central nerve system (CNS) and peripheral tissues.84,89 Also, SST-producing cells are present at the interface between bone and bone marrow, a location where the most primitive hematopoietic cell reside.87 And there, SST have been shown to exert chemotactic function for these cell types via SSTR2.87 Thereby, this novel finding with SST and its receptor suggested that this signalling system might be important for hematopoietic stem cell development a nd migration. Based on common origin and characteristic between hematopoietic stem cell and hepatic oval cells, we hypothesized

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33 that SST might be involved in oval cell activation, especially homing. To prove our hypothesis, we investigated whether SST is associated with 2 AAF/PHx HOC induction model. Since all known functi on of SST is mediated by it s specific receptors, we postulated that the chemotactic role of SST might be mediated by certain types of receptor. We examined receptor subtypes prof iles expressed by HOCs and investigated if certain type of receptor was involved in the effects of SST on HOCs. Finally we examined whether the effect of SST/SST R4 for oval cell migration is functional consequence and what kind of effectors molecu les are related to the chemotactic activity of SST. PI3K pathway was investigated in order to identify whether this pathway is related to SST-stimulated HOC migration, because PI3K has been shown to be an essential molecules in cell migration. The overarching question is which systemic signals are responsible for determining the magnitude and efficiency of oval cell activ ation within this type of hepatic repair? The functions of SST in these processes are i nvestigated in this study. This may lead to better understanding of liver rege neration processes by HOCs.

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34 CHAPTER 2 A POTENTIAL ROLE OF SOMATOSTATIN AND ITS RECEPTOR SSTR4 IN THE MIGRATION OF HE PATIC OVAL CELLS Summary Somatostatin (SST) is a regulatory pep tide that activates G protein-coupled receptors comprised of five members (SSTR 1-5). Despite the broad use of SST and its analogs in clinical practice, the spectrum of SST activities has been incompletely defined. Recently, it has been demonstrat ed that SST can be a chemoattractant for hematopoietic precursor cells. Since hepatic oval cells (HOCs) share common characteristics with hematopoietic stem cells, we hypothesized that SST could act as a chemoattractant for HOCs by stimulating somatostatin receptors (SSTRs). Reverse transcriptase-polymerase chain reactions (RT-PCR) and Western blot assays revealed an increased expression of SST in the 2-acetyl-aminofluorene (2AAF)/ partial hepatectomy (PHx) HOC induction model. Immunohistochemical staining showed the expression of SST in 2AAF/PHxtreated rat liver, as compared to normal liver. Proliferation and migration assays demonstrated that the increase of SST is re lated to migration of HOCs, but not their proliferation. RT-PCR and quantitative real-time PCR showed that SSTR4 was preferentially expressed by HOCs. Western blot assay a nd immunohistochemical staining confirmed the expression of SSTR4 by HOCs. In addition, pretreatment with anti-SSTR4 antibody cultures resulted in a dr amatic reduction of cell migra tion as compared to that of control. Lastly, SST stimulat ed the rearrangement of actin filaments in HOCs, while

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35 HOCs treated with anti-SSTR4 antibody failed to do so. These results suggest a positive role for SST in the migration of HOCs, and that this effect is mediated through SSTR4. Introduction Hepatic oval cells (HOCs) are known to participate in liver regeneration under certain conditions, and are implicated in he patic carcinogenesis. When liver damage is severe, and the ability of hepatocytes to divide and replace damaged tissue is compromised, HOCs are induced to proliferate.134 Morphologically, ova l cells are small in size (approximately 10 m in diameter), with a large nuc lear to cytoplasm ratio, and an oval shaped nucleus.4 Proliferating oval cells in both th e rat and murine models appear to radiate from the periportal region, forming primitive ductular structures with poorly defined lumena.40 They are similar to bile ductular epithelial cells in their distinct isoenzyme profile, expressing certain kera tin markers (e.g. CK19), and gamma-glutamyl transpeptidase (GGT). HOCs also express high levels of alpha-fetopr otein (AFP) as well as hematopoietic stem cell markers (i.e. Thy-1, CD34 and c-Kit).44,45 In addition, several monoclonal antibodies, such as OV6, OC.2 and BD1, have been developed to aid in their identification.135-137 These markers may be used fo r isolating HOCs by fluorescence activated cell sorting (FACS) or ma gnetic activated cell sorting (MACS).45,55 Petersen et al (1998)45 showed that by using Thy-1 in conj unction with FACS sorting, a 95-97% enriched population of oval cells could be obtained. Though oval cells do not normally participate in the regenerative response to PHx or CCl4 injury, they can be made to do so by s uppressing mature hepatocyte proliferation. Administration of 2-AAF prior to and dur ing hepatic injury induced by PHx or CCl4 will block the proliferation of hepatocytes by inte rfering with the cyclin D1 pathway. Oval

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36 cell proliferation can thus be induced in th ese otherwise non-oval cell aided regenerating models.138,139 In HOC-mediated liver regeneratio n, HOCs arise from the portal tract periphery and migrate deep in to the lobular parenchyma. Th is HOC distribution suggests that HOCs may be mobile. Based on ultrastr uctural studies in rat liver, it has been proposed that HOCs migrate by amoeboid mo vement with the help of pseudopodia.59 Trafficking, mobilization, and homing of stem cells are multifactorial processes that are regulated not only by adhesion molecules and cy tokines, but also by chemotactic factors that direct transendothelial migration.140 Somatostatin (SST) is a unique regulato ry hormone, which was first reported by Brazeau et al . 1973141 as a hypothalamic hormone i nhibiting growth hormone (GH) secretion. SST has two biologically activ e forms, SST-14 and SST-28, which are generated as C-terminal produc ts from pro-SST. SST-14 was originally described in the hypothalamus, and the amino-terminally ex tended SST-28 was disc overed later in the gut.84 SST-14 and SST-28 are predominantly expre ssed in neurons and secretory cells in the central and peripheral ner vous systems, gastrointestinal tract, pancreas, pituitary, kidney, retina, and immune system.142 The major action of this neuropeptide includes inhibition of hormone secretion from the pi tuitary, the pancreas, as well as other endocrine and exocrine secretion in a number of various organs.85 SST has also been shown to control the prolifera tion of normal and tumor cells.89 In addition, it is involved in the differentiation and migration of thymocytes.86 These broad biological functions are mediated by five receptor subtypes, all of which belong to the seven -helical transmembrane domain G protein -coupled receptor (GPR) family. Somatostatin receptors (SSTRs) are widely distributed throughout many tissues and show different

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37 functions in various cell and tissue types.89,143 However, studies on receptor expression in various tumors reveal incons istencies, which are also obs erved in rat liver studies.104,105 Reynaert et al .104 reported the presence of SSTR1, 2, a nd 3 in activated hepatic stellate cells in CCl4-treated rats, but not in normal rat liver. However, Bruno et al . 105 provides the observation of SSTR3 mRNA in normal ra t liver. Another study shows the expression of SSTR2, 3, and 5 by activated stella te cells in Sprague Dawley rats.106 Therefore, inconsistent data on individual receptor subtyp es have lessened the therapeutic potential of receptor subtype-specific or universal analogues in oncolog ical and certain endocrine disorders. Despite the broad use of SST and its analogs in clinical practice, the spectrum of SST activities has been incompletely defined. Recent evidence has emerged that neuroendocrine-like SST-producing cells are present at the interface between bone and bone marrow, a location where the most primitive hematopoietic cells reside.144 SST can act as a chemoattractant for primitive hemat opoietic progenitor cells, which is mediated exclusively via SSTR2.87 Given that HOCs share co mmon characteristics with hematopoietic stem cells,133 these novel findings led to the hypothesis that SST could possibly influence the migration of HOCs. In the present study, the 2-acetylaminofluorene (2AAF) followed by partial hepatectomy (PHx) model for HOC activation was utilized in order to show the effect of SST on the migration of HOCs. SST and the SSTR1-5 expression patterns were examined via molecular and biochemical tec hniques. Also, the chemotactic ability of SST for HOCs was investigated. The current study demonstrates that SST induces HOC migration via SSTR4.

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38 Material and Methods Animals Male F344 rats (age 6-8 weeks, weight 130-150g) were purchased from Charles River Laboratories and maintained on standa rd laboratory chow and daily cycles of alternating 12 hours of light and dark. They were used at approximately 8-10 weeks of age and 150-180g weight. All animal work was conduced under protocols approved by IACUC at the University of Florida. Rat Oval Cell Activation Protocol: The basic design of the 2-AAF/injury mode ls have been described previously by Petersen et al .45 Briefly, 2-AAF pellets (70 mg/28 day release, 2.5 mg/day) were inserted subcutaneously 7 days prior to surgical res ection of the hepatic mass (PHx injury) this follows protocols similar to those described by Novikoff et al .145 and Hixson et al . 136 Normal rat liver was used as a time ze ro-control. PHx (70%) were performed as described by Higgins and Anderson .146 HOC Preparation To isolate HOCs, the 2AAF/PHx oval cell activation model was used.147 The liver was harvested at 11 days post PHx and cells were isolated via standard two-step collagenase perfusion. Obtained cells were gradient centrifuged at 500 g to isolate hepatocytes. The non-parenchymal cell (NPC) fraction containing the HOCs was collected at 1000 g. Isolated cells were in cubated with anti-Thy-1 FITC conjugated antibody followed by anti-FITC-microbeads. After incubation, cells were positively selected using MACS sort. Cell viability wa s determined to be >90% as established by trypan blue exclusion.148 After isolation, HOCs were re-s uspended in IscoveÂ’s Modified

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39 DulbeccoÂ’s Medium (IMDM, purchased from GIBCO, Grand Island, NY) (10%FBS, 1% insulin, 1X antibiotic-antimycotic) for experiments.8 Immunohistochemistry BrdU (Dakocytomatin, Carpinteria, CA) staining was conducted as described by Sum et a l.149 All immunostaining was performed on HOCs, cytocentrifuged HOCs, or frozen liver sections using standard st aining protocols. Samples were fixed and permeabilized, saturated, and processed for immunostaining with primary antibodies. Anti-SST (Santa Cruz Biotech., Santa Cr uz, CA), CD45 (Becton Dickinson), Thy-1 (Becton Dickinson), OV6 (a kind gift from Dr. Stewart Sell), SSTR4 (Santa Cruz Biotech.), F-actin (Sigma-Aldrich Corp. St. Louis, MO) antibodies were used in this procedure. Vector ABC kit (Vector Laboratories, Burlingame, CA) and DAB reagent (Dako Comp.) were employed in the detec tion procedure. For double immunofluorescent staining, Texas red anti-goat IgG (Vector Laboratories) and Fluor anti-mouse IgG (Vector Laboratories) were used as secondary antibody. RT-PCR and Real Time PCR RT-PCR and real time PCR were performed as described by Bar et al. 150 Oligonucleotide primers specific for SST a nd SSTRs were designed with Primer 3 software (http://frodo.wi.mit.edu/cgi-bin/ primer3/primer3_www.cgi) and GenBank entries: SST (308 bp) 5Â’-TGG CAG AAC TGC TGT CTG AG-3Â’ forward, 5Â’-TAA CGC AGG GTC TAG TTG AGC-3Â’reverse, SSTR1 (365 bp) 5Â’-CAC GCA CCG CAG CCA ACA-3Â’ forward, 5Â’-GGA AG C CGT AAG AGG ATG GGG TT-3Â’ reverse, SSTR2 (376 bp) 5Â’-ATC ATC AAG GTG AAG TCC TCT3Â’ forward, 5Â’-GGG TCT CCG TGG TCT CAT T-3Â’ reverse, SSTR3 (329 bp) 5 Â’-GGG GAG TTT CAG AAA GCA AT-3Â’ forward, 5Â’-TTG GGC AAG TCA CTT CTC TC-3Â’ reve rse, SSTR4 (364 bp) 5Â’-TCG TGG GGG

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40 TGA GGC GT AG-3’ forward, 5’-CAT AGA GAA TCG GGT TGG CAC AG-3’ reverse, SSTR5 (388 bp) 5’-CAC GGA TGT CCA GGA GGG-3’ forward, 5’-GTA GAG CAG GGG GTT GGC ACA-3’ reve rse. The following primers were used for real time PCR : SSTR4 (145bp). 5’-ATG TGT CCC TC TCC TCA GC-3’ and 5’-TCT TCC TCA GCA CCT CCA GT-3’. These sequences for SSTR4 were obtained from Bar et al .150 All PCR products were directly sequenced for ge netic confirmation using an AmpliTaq cycle sequencing kit (PerkinElmer, Boston, MA). Proliferation Assay Prior to cells being placed in culture, trypan blue exclusion assay was performed. At the time of plating cell viability was meas ured at greater that 95%. Cells were seeded in 6-well plate (4.5 X 104 cell/well) and grown in IMDM supplemented with 10%FBS. After 48 h, the medium was replaced with serum-free IMDM for 16 h. The cells were subsequently cultured with 0.5% BSA (bovine serum albumin)-containing medium, or medium with addition of 10% FBS or SST at a concentration of 100 nM. Cells were counted after 1, 2, and 3 days, and trypan blue dye exclusion was used as an indicator of cell viability. All experiments were perf ormed three times to ensure statistical significance. Migration Assay Migration was assessed in Transwell culture dishes with 5µm pore filters (Transwell, 6.5mm diameter, 24 -well cell clusters; Coring In corporated Costar, Coring, NY) that were precoated overnight at 4 oC with 0.001% collagen. Cells (7.5 104) were suspended in IMDM (10%FBS, 1% insulin), and were allowed to attach overnight. Unadherent cells were removed from the top of the transwell chambe r and attached cells were re-fed in the migration buffer (IMDM). At this time the motility assay was initiated

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41 by transferring the entire tran swell chamber to new cluster plate well containing various doses of SST-14 (synthesized by the Interdis ciplinary Center for Biotechnology Research at the University of Florida) (1, 10, 100,1000 nM) in migration buffer. After determining the optimal concentration of SST thr ough preliminary studies, migration buffer containing 100 nM SST was placed in the lowe r chamber and plates were maintained at 37oC, 5% CO2 for either 4 or 6 hours. In some expe riments, cells were pretreated for 30 minutes with anti-SSTR4 antibody (5 and 10 µg As a negative control, SST was either not added to the lower chamber or a dded to both lower and upper chambers. At the end of the experiment, cells were fi xed and stained as described by Stolz et al .151 Cells that had migrated to the bottom of the transw ell filter were enumer ated by counting each transwell chamber, at X4 ma gnification. Each migration a ssay was performed a minimum of three-times. Western Blot Assay Whole liver tissue or HOCs were homogeni zed in triton lysis buffer (20mM Tris, pH7.4, 137mM NaCl, 10% glycerol, 1% Triton X-100, 2 mM EDTA, 1mM PMSF, 10mM NaF, 5µg/ml aprotinin, 20µM Leupept in, and 1mM Sodium ortho-vanadate) and centrifuged at 10,000 g for 15 minutes. Protein co ncentrations were measured using the Lowry assay. Immunoblotting was performed using 1: 500 anti-SST (sc13099, Santa Cruz Biotech.), 1: 150 anti-SSTR4 (sc11620), and 1: 5000 antiactin (ab6276-1000, abcom. Stockholm, Sweden) antibodies. Immunocomplexes were detected with horseradish-peroxidase conjugated secondary antibodies. Membranes were developed by chemoluminescence (ECL; Amersham Bioscience, Buckinghamshire, England).

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42 Statistical Analysis All results are expressed as the mean ±SD. Statistical differences were determined by Student’s t-test. P values of <0.05 were considered to statistically significance. Results Increased Expression of SST in the HOC Induction Model To examine whether SST is involved in liver regeneration by HOCs, the 2AAF/PHx model was employed. In this model, HOC proliferation peaks on approximately day 9 and continues through day 13.1 Small cells with a large nuclear to cytoplasm ratio are observed in 2AAF/PHx-treat ed rat liver, radiating from the periportal region (Figure 2-1A). OV6, a known HOC ma rker, confirmed these cells as HOCs (Figure 2-1B). In normal rat liver, OV6 was de tected in ductal cells (Figure 2-1D). Figure 2-1C shows a negative control in which anti -mouse IgG was used in place of the primary antibody. These data confirm that the 2AAF/PHx protocol for rat HOC s is reliable and reproducible. Utilizing RT-PCR an alysis with specific primers for rat SST detects newly synthesized message from the SST gene (F igure 2-2A). Western blot assay for SST showed that SST was initially expressed at negligible levels and increased when HOC proliferation increased, peaking at approxi mately day 11 of HOC activation (Figure 22B). Immunofluorescent staining revealed fe w SST positive cells within normal or PHx treated liver (Figure 2-3A-C). However, the number of positive cells significantly increased within the 2AAF/PHx model. SST was expressed on CD45+ cells early in the HOC activation process (day 5) (Figure 23D-F, and J), whereas by day 13 of HOC activation, SST expression was localized to the OV6 positive cells (Figure 2-3G-I and K). These observations were visible only in th e HOC activation model. This specific and

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43 increased expression of SST in HOCmediat ed liver regeneration suggested a potential role of SST in HOC activation. Chemotactic Role of SST for HOCs. SST is a pleiotropic peptide mediating inhi bitory functions in various secretory and proliferative processes as well as a stimulatory role in cell proliferation.89,117,130 These broad effects are cell type-specific. Thus, th e effect of SST on HOC proliferation was investigated. Figure 2-4A shows that there is a significant increase in the number of HOCs after stimulation with 10% FBS, reaching up to 7.4x104 cells at day 3. However, SST-stimulated HOCs show only slight proliferation (4.5x104 cells at day 3), which is similar to the level of prolifer ation seen in th e control (3.8x104 at day 3). In this assay, 0.5% BSA was employed in order to inhi bit cell death. Therefore, HOCs in SSTcontaining or control medium had a normal morphology and excluded trypan blue up to the end of the experiment. To confirm this re sult, BrdU incorporati on assay was utilized. The number of BrdU-positive cells was consid erably different among the FBS, SST, and media alone (control) cultures. 44% of the cells cultured in 10% FBS were positive for BrdU, while only 14% of cells cultured in SST-containing medium were BrdU-positive (Figure 2-4B). There was no significant differe nce in the number of BrDu-positive cells between the SST-including medium (SST ) and untreated medium (control). These results demonstrated that SST did not aff ect the proliferation of HOCs. Recently, SST has been shown to function as a chemoattractant for immature neuronal and hematopoietic cells.87,88 Hence, this study was designed to determine whether SST might be involved in migration of HOCs. In the migration assay, when SST was added to the medium in the lower chamber, HOCs crossed the filter in a dosedependent fashion. In the presence of 100 nM SST, the number of migrating HOCs

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44 showed a significant peak, a threefold incr ease on average (Figure 2-4C). However, HOCs showed limited mobility in the absence of SST or in the pr esence of SST in both lower and upper chamber. Migration assa y using the transwell chamber clearly demonstrated that HOCs migrated along the SST gradient, suggesting that increased expression of SST might be involved in HOC migration. Expression of SSTR4 in HOCs All of the known functions of SST are mediated by five receptor subtypes. Therefore, it was hypothesized that the effect of SST in HOC migration might be mediated by a certain type of receptor. In order to determine receptor subtype expression profiles by HOCs, RT-PCR for each SSTR s ubtype was performed. SSTR1, 2, 4, and 5 were up-regulated in HOCs, compared to the expression of these recep tors in hepatocytes and normal liver. However, it was found that SSTR4 was the only subtype expressed exclusively by HOCs (Figure 2-5A , asterisks). All other recept or subtypes were found in normal liver and hepatocytes, as well as HOCs. Thus, SSTR4 was thought to have a specific function in HOCs. Real-time PCR wa s conducted to obtain quantitative data for SSTR4 expression in the HOC induction model. Gene expression for SSTR4 determined by real-time PCR confirmed that expressi on of SSTR4 significantly increased during HOC activation, peaking around day 7 post PHx, whereas normal liver was devoid of any such expression (Figure 2-5B). In additi on, immunohistochemical staining showed the SSTR4 protein within the HOC population area (Figure 2-5C). Also, Western blot assay of protein extract from Thy1 sorted HOCs showed a band co rresponding to glycosylated SSTR4 (70kDa), while this band was not det ected in normal liver (Figure 2-6A). Double immunofluorescent staining on purified and cyto centrifuged HOCs showed that a portion of the Thy-1-positive and OV6-positive popul ations express SSTR4 protein (Figure 2-

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45 6B). Considering the rare detection of SST R4 in the normal liver, the significant expression of SSTR4 in HOCs suggests a spec ific function for SSTR4 in these cells SST directing HOC Migration through SSTR4 SST and SSTR4 are expressed in the HOC induction model, and SST acts as chemoattractant for HOCs in migration assa ys. These results suggest that SST might exert a chemotactic effect on HOCs via SSTR4. To test this possibili ty, migration assays using transwell chambers were performe d on HOCs pretreated with anti-SSTR4 antibody. This antibody was raised against th e N-terminus of mouse SSTR4 and crossreacts with SSTR4 of rat. As shown in Fi gure 2-7A, the mobility of HOCs was abolished by adding 5 or 10 µg anti-SSTR4 antibody before SST stimulation, as compared to the un-inhibited migration of HOCs toward SST. Hence, the migratory effect of SST on HOCs appears to be related to SSTR4. Dynamic changes in actin filaments are rela ted to various cellular processes, such as cell motility, cell cycle control, cellular structure, and cell signaling.152 The rearranged actin filaments form discrete structures, such as stress fibers, la mellipodia, filopodia, and membrane ruffles, at the edges of cell membrane. These structures are essential for cell migration.152 Therefore, additional studies were incorporated to determine whether SST/SSTR4 would induce rearrangement of ac tin filaments in HOCs. HOCs cultured in IMDM without SST showed evenly distri buted actin filaments throughout the cell (Figure 2-7B, left image). In contrast , treatment with 100 nM SST induced the distribution of actin filaments into the cell me mbrane to form cell mobility structures at the leading edges of the cells (Figure 2-7B , middle image). These mobility structures were not detected in HOC treated with SST along with the anti-SSTR4 antibody (Figure

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46 2-7B, right image). These data show that the rearrangement of actin filaments in HOCs occurs following stimulation of SSTR4 by SST. Discussion SST is a regulatory peptide with a wide variety of functions, mainly linked to the neuroendocrine and immune systems.95 Thus far, SST has been found to act predominantly as an inhibitor of secretory and proliferative responses. However, recent studies have shown that SST can act as a chemoattractant for primitive hematopoietic progenitor cells.87 There is increasing evidence revealing common characteristics between HOCs and hematopoietic stem cells.45,47,153 The novel function for SST to act as a chemoattractant for stem cells led us to propose that SST could affect HOC migration. The focus of these studies was to determin e whether or not ther e was ligand/receptor interaction during oval cell aided regeneration. In order to achieve our goal we chose to use the time of maximum SST protein expres sion, which was determined to D11 postPHx, which is clearly shown via Western analysis (Figur e 2-2B). To confirm the expression if SSTR4 was also present at D11 immunohistochemistry (Figure 3-5C) and Western analysis (Figure 2-6A ) were performed. This also indicates that the ligand is present within the liver as the same time the that receptor is present on the HOCs. We also show that SST stimulates cell migra tion and cytoskeletal rearrangement, and the effects of SST appear to be medi ated by the receptor SSTR4 sub-type. Previous studies have shown a corre lation between HOC proliferation and inflammation within the liver parenchyma.59 The present study show s that expression of SST by CD45 positive immune cells occurs ea rly in HOC activation and is followed by expression in OV6 positive oval cells. In normal or PHx-(alone) -treated liver, very little SST expression was observed (Figure 2-3A-C). The finding that the inflammatory cells

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47 affect the activation and local ization of stem cells strongl y suggests that one or more cytokines produced during inflammation functio n as growth or chemotactic factors for the HOCs.59 Accordingly, this may suggest that CD45+ cells express SST in response to tissue damage, leading to recruitment of HOCs to the site of injury. These newly recruited HOCs appear to also express SST, amplify the activation signal, bringing additional HOCs to mediate liver regeneration. In the migration assay, four different concentrations (1, 10, 100, and 1000 nM) of SST were tested. A bell-shape dose response wa s observed (Figure 2-4C). This pattern is often observed in G-coupled receptor-mediate d responses and is thought to occur via desensitization.87 Based upon these results, a concentra tion of 100 nM of SST was chosen as the optimal concentrati on for these studies. In fou r-hour migration assay, HOCs migrated with a significant chemotactic res ponse. However, this response of HOCs slightly decreased following six-hour incuba tion. There could be several factors to explain this. First, SST is an unstable peptide which could degrade and lose its effectiveness. Second, because of it small size, SST could diffuse between the two chambers, allowing the cells access without migration. The chemotactic response of HOCs to the SST gradient led us to predict that HOCs might express one or more of the particul ar receptor subtypes responsible for the chemotactic properties of SST. The biological effects of SST are mediated by SSTRs that are highly cell-specific. Phys iological responses vary with the expression of individual receptor subtypes which are functionally couple d to the effectors of signal transduction, resulting in physiological impact.143 Within the context of this study, SSTR4 was uniquely expressed by HOCs within the liver. The expression of SSTR4 in rat liver has

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48 not previously been observed. Consequentl y, detection of SSTR4 in the HOC population during liver regeneration suppor ts the hypothesis that SSTR4 is involved in the action of SST as a chemoattractant. This notion is furt her supported by the fact that SST fails to stimulate HOC migration when SSTR4 is blocked (Figure 2-7A). Another possible explanation for the lack of migration within the transwell studies could be from an apoptotic effect brought on by an tibody exposure. It has been reported that SST can act as an antiproliferative agent, however, thes e studies were conducte d on cancer cell lines (MCF-7 and CHO-K1) and it has been shown that these cells can be coaxed into apoptosis via the SHP-1/Caspase 8 pathway.154 Sharma et al (1996)155 showed that of all the different receptor sub-types, the only one capable of inducing apoptosis was SSTR3. In addition, two independ ent studies, Anderson et al (2001)121 and Sellers et al (1997)119 showed that only SSTR4 was capable of induci ng a proliferative effect in cells via the phosphorylation of STAT3. To date there is no data showing an apopt otic effect through SSTR4. It should also be pointed out that in Figure 2-7B we show oval cells untreated (left panel), treated with SST (middle panel) and SST/anti body (right panel) and stained for actin. In untreated cells the actin stress fibers are visible uniformly distributed throughout the cell. When SST is given to the cell culture these actin fibers are lost and focal adhesion points are seen along the edge of the cell membrane. However, when the cells exposed to SST plus antibody the actin fi bers are intact and th e cell appear to be normal. It is also worth pointing out that no irregular/picnotic nuclei we seen following SSTR4 antibody treatment. This further indi cates that the anti body treatment did not induce apoptosis in the migration assay.

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49 The activation of ligand induces SSTR di merization which alters the functional properties of the receptors, such as, ligand binding affinity, agonist-induced receptor internalization, and up-regulation.115 Increased expression of SSTR1, 2, and 5 were observed in this study (Figure 2-5A). Hence, it is possible that the dimerization of SSTR4 with another SSTR isotype may affect migra tion or other responses of HOCs. Although a role of SSTR4 in HOC migration was demons trated in this study, further studies on the interaction among SSTRs and/or the effect of other re ceptor subtypes in HOC are required. Because SSTR5 was so strongly up-re gulated in oval ce ll activation it would seem logical to focus the next set of experi ments on this receptor subtype to determine if there is a role for this receptor subtyp e in oval cell activation and or migration. This study demonstrates that SST induces th e reorganization of actin filaments. In order for cells to migrate, F-actin requires rearrangement. Consequently, the cells can form mobility structures and move along th eir pathway to engraftment. With the rearrangement seen within this study, it woul d appear that SST is required for this rearrangement. It was also demonstrated that the effect of SST in actin rearrangement was abolished by anti-SSTR4 antibody. Thes e data indicate that SST/SSTR4 might stimulate an upstream signal to rearrange act in filaments needed for motility. The SSTRs are coupled to Gi-protein, which has been reported to be required for homing of hematopoietic stem cells.80 This receptor class can be c oupled to the activation of PI3 kinase, the downstream effectors of which, Cd c42 and Rac1, have been implicated in the formation of mobility structures.156,157 Therefore, it is conc eivable that SST would mediate the migration of HOCs through the SSTR4-coulped Gi-protein/PI3K/Rac signaling pathway, but this remains to be tested.

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50 Stem cells “home” or migrate to appropr iate sites where they exert unique functions, such as self-renewal and mu lti-lineage differentia tion. The molecular mechanisms regulating stem cell homing require more study, especially given the importance of such homing in a variety of medical applications. A new function for SST and SSTR4 in HOC migration is presented in th e current study. This may lead to a better understanding of HOC movement within the injured liver. However, more work is required to fully understand the signi ficance of the present findings.

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51 Figure 2-1. HOC induction in the 2AAF/PHx model. (A) Hematoxylin-eosin staining at day 11 liver section of 2AAF/PHx–tr eated rats (X20, inserted image X40, arrows indicate small HOCs). (B) I mmunohistochemical staining for OV6 at day 11 liver section of 2AAF/PHx –tr eated rats. Brown color indicates OV6positive cells (X20). (C) Liver section from the same animal as A and B stained by anti-mouse immunoglobulin G se rving as a negativ e control (X20). (D) Liver section from normal liver tissue (X20). Data shown represents one of three experiments with similar results.

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52 A B Figure 2-2. Increased SST expression in th e HOC induction model. (A) RT-PCR of SST in 2AAF/PHx (D 9), Thy-1 sorted HOCs (OC), normal hepatocytes (HEP), and normal liver (NL). GAPDH was used as an internal control. Data shown represents one of three experiments w ith similar results. (B) Western blot analysis for SST in liver tissue obtained from 2AAF/PHx treated rats. Protein extracted from the brain was employed as positive control and actin as an internal control. Data shown represents one of three experiments with similar results.

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53 Figure 2-3. Immunohistochemical analysis fo r SST in the HOC induction model. (A) Normal liver, (B) PHx-treated liver ( 12 hr post PHx), and (C) PHx-treated liver (24 hr post PHx) were stained w ith SST (Texas red). Representative slides were viewed at X20 magnifi cation. DAPI (blue) was employed for nuclear staining. (D-F) Double immunoflo rescent staining for SST (D; Texas red), CD45 (E; FITC), and merged imag e (F) of D & E in 2AAF/PHx treated rats (Day 5, X10). (G-I) Double immunoflorescent staining for SST (G; Texas red), OV6 (H; FITC), and merged image (I) of G & H in 2AAF/PHx treated rats (Day 13, X10). (J) Magnified imag e from squares in figure F. (K) Magnified image from squares in figure I. (Co-localized cells appear as yellow to orange. Arrows indicate co-l ocalization with SST and CD45 (J) or OV-6 (K). Original magnifications of J and K are X10 and X20, respectively. Data shown represents one of thr ee experiments with similar results .

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54 C Figure 2-4. Potential role of SST as chemoa ttractant for HOCs. (A) Effects of SST on HOC proliferation. HOCs were incuba ted in 10 % FBS supplemented IMDM medium ( , FBS), or serum free IMDM medi um containing 0.5% BSA with SST at 100nM ( , SST), or without SST ( , control) for the indicated times. These medium were changed every day. Each point represents the mean ± SD, n=3 of independent experiments. (* P<0.001) (B) BrdU incorporation assay. Cells were seeded in 6-well plate (4.5 X 104 cell/well) and grown in IMDM supplemented with 10%FBS. After 48 h, the medium was replaced with serum-free IMDM for 16 h. The cells were subsequently cultured with 0.5% BSA-containing medium, or medium with addition of 10% FBS or SST at a concentration of 100 nM. After 1hour incubation, 100 µM of BrdU was added. Cells were fixed after 24 hours. BrdU-positive cells are presented as percentage of total cell number. Da ta shown represents one of three experiments with similar results (*P<0.01). (C) Migration assay using Transwell chamber. HOCs were seeded on the top of the chamber with 1, 10, 100, or 1000 nM of SST protein placed in the bottom chamber or 100 nM of SST protein place in both upper and lower chamber. Controls were used with no SST protein in either chamber. Data represents mean value ± SD of three independent experiments. Data were normalized for each independent experiment with respect to cont rol migration (*P<0.05, **P<0.01, relative chemotactic index vs control).

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55 C Figure 2-5.Detection of SSTR4 in HOC-aided liver regeneration. (A) RT-PCR analysis of SSTR subtypes in HOCs, normal hepatocytes (HEP), and normal liver (NL). Asterisks indicate no band for SSTR4 in HEP sample. GAPDH was used as internal control. Data shown represents one of three experiments with similar results. (B) Relative quan tification of SSTR4 in HOC induction model using real-time PCR. Data represen ts the mean value ± SD (C) Immunohistochemical staining for SSTR 4 at day 9 liver section of HOC induction model (Original magnification was taken by using a X40 objective). Anti-goat immunoglobulin (IgG) was employe d in staining liver section from the 2AAF/PHx treated rats, in order to serve as negative c ontrol. Data shown represents one of three experi ments with similar results.

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56 A B Figure 2-6. Expression of SSTR4 by HOCs . (A) Western blot analysis for SSTR4 in Thy1 sorted cells from 2AAF/PHx-treated ra ts and total liver tissue from normal rats. Data shown represents one of thr ee experiments with similar results. (B) Double immunofluorescent staining for SST R4 (Texas red) and Thy-1 or OV6 (FITC) on cytocentrifuged preparations of Thy-1 sorted cells from 2AAF/PHs-treated rats (Magnification of the small images is X40. Original magnification of the merge images is X40). In merged image, SSTR4 are shown as yellow to orange. Data shown represents one of three experiments with similar results.

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57 A B Figure 2-7. Effects of SST and SSTR4 on HOCs migration and actin rearangment (A) Stimulation of HOCs migration by SST and SSTR4. HOCs were subjected to chemotaxis assays with 100 nM SST ( SST) or without SST (Con). Cells were treated with anti-SSTR4 Antibody (5 and 10 µg ) and cultured in 100 nM SST– containing medium for 4 hours (5Ab & SST and 10Ab & SST). Addition of anti-SSTR4 antibody sign ificantly reduced the chemotactic response. Data were normalized for each independent experiment with respect to control migration (*P<0.01, relative chemotactic index vs control). Data represents mean value ± SD of th ree independent experiments. (B) Rearrangement of actin filament in HOCs through SST and SSTR4. HOCs were incubated in IMDM medium cont aining no addition (control), 100 nM SST-containing medium for 4 hours (SST). Cells were pretreated with antiSSTR4 Antibody and then incubated in 100 nM SST– containing medium for 4 hours (Anti-SSTR4 Ab & SST). (all images X40) Data shown represents one of three experiments with similar results.

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58 CHAPTER 3 SOMATOSTATIN STIMULATES THE MI GRATION OF HEPATIC OVAL CELLS WITHIN THE INJURED LIVER THROUGH PI3K PATHWAY Summary Somatostatin (SST) is a pleiotropic peptid e, exerting a variety of effects through its receptors. Recently, SST has been shown to act as a chemoattractant for hematopoietic progenitor cells via receptor type 2 (SSTR2 ). Also, SST was shown to stimulate the migration of hepatic oval cells (HOC) thr ough its receptor SSTR4. However, it remains to be elucidated whether the effect of SST/SSTR4 on the migration of HOC is of functional consequence in HOC-aided liver regeneration. Also the type of signaling molecule associated with this chemotactic ac tion remains to be determined. In order to investigate the function of SST/SSTR4 in HOC within the damaged rat liver, the effects of TT232, an SST analogue having specific bi nding activity to SSTR4, was tested in HOC in vitro . Signaling molecules mediating this action of SST/SSTR4 were examined. In the current studies, the anti-migratory action of TT-232 was determined in HOC. In cell transplantation experiment s, a lower number of donor-deriv ed cells was detected in TT232 treated animals, as compared to control animals. Activation of phosphatidylinositol-3-kinase (PI3K) was obser ved in HOC exposed to SST, and this activation was suppressed by either the anti -SSTR4 antibody or TT232-pretreatment. In addition, decreased motility of HOC by treatmen t with a PI3K inhibi tor revealed that PI3K is an essential signaling molecule in HOC migration. In conclusion, our findings

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59 suggest that SST stimulates the migration of HOC within the injured liver via SSTR4, and this action appears to be mediated by PI3K pathway. Introduction Hepatic oval cells (HOC) are activated to proliferate and differentiate when the regenerative capacity of terminally di fferentiated hepatocytes is compromised.158 In HOC-mediated liver regeneration, tissue damage l eads to a dramatic increase in the level of secreted chemokines, cytokines, and pr oteolytic enzymes which impact on stem cell migration and repopulation.59 Stem cell factor (SCF), hepatocytes growth factor (HGF), granulocyte colony-stimulatung f actor (GCSF) and stromal cell-derived factor-1 (SDF-1) are well known factors that cont rol the migration of stem cells.80-82,159,160 However, there is relatively little information regarding the biological factors that influence HOC, although factors which regulate the differ ent hematopoietic lineages, are well characterized. Somatostatin (SST) is a plei otropic hormone, exerting variety of effects within the body, such as a control for hormone secretion and influence in the proliferation, motility and development of a wide variety of cells.89 85,86 In addition, SST has been shown to function as a chemoattractant for hematopoi etic progenitor cells, HOC, and immature neuron.87,88,158 Physiological effects of somatostat in are mediated through a family of seven transmembrane spanning G-protein coupled receptors.89 Increasing evidence has been reported that the distant effects on ce ll response elicited by the individual receptor types correlated with activation of th e various intracellular signaling pathways.95 The phosphatidylinositol-3-kinase (PI3K) signaling pathway is crucial for many aspects of cell growth and survival.156,157 PI3K is a key signaling molecule in the regulation of cell migration and invasion in response to biological factors.157 Akt is an

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60 essential downstream protein of PI3K and is involved in a variety of biological functions including angiogenesis, glycoge n synthesis, gene expression , inhibition of apoptosis, cell cycle arrest, endocytosis , vesicular trafficking, and cell transformation.157 p21 activated kinase 1 (PAK1) has been identified as a downstream molecule of activated Cdc21/Rac or Akt. PAK activity is regulated by different classes of membrane receptors including Gprotein coupled receptors, tyrosine kinase receptors, and cytokine receptors.156 Activation of PAK1 has been shown to induce formation of motility structures,156,161 although it remains unclear how signaling molecules are involved in the effects of PAK on forming these structures. Thus, components of the PI3K pathway, including the key effector, Akt and PAK1 are important for controlling the surv ival and proliferation of stem cells in a similar way to their role in mature cell system.162 In addition, the PI3K system has recently been shown to be an essential step in mediating the migration of stem cell.156,157 In the current study, whether SST has a role in the migration of HOC through SSTR4 in vivo was examined. Also, the signaling molecules regulating HOC migration within SST/SSTR4 were investigated. The da ta demonstrate that SST/SSTR4 stimulate HOC migration within the inju red liver and, these effects appear to be mediated by the intracellular PI3K signaling pathway. Material and Methods Animal Dipetidyl peptidase IV deficient (DPPIV-) female F344 breeding animals were inbred-house and maintained on standard labora tory chow with daily cycles of alternating 12 hours of light and dark. They were used at approximately 8-10 weeks of age and 150180g weight. Normal male DPPIV+ F344 rats (age 8-10 week s, weight 180-220g) were purchased from Charles River Laboratories and were used as donor animals for all

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61 transplantation studies. All animal work was conduced under protocols approved by the IACUC at the University of Florida. HOC Preparation and Transplantation The standard protocol for oval cell ac tivation, 2-acetly-aminofluorene (2AAF)/ partial hepatecomy (PHx), was used and HOC were isolated as described by Jung et al .158 For transplantation, DPPIVfemale recipients were treat ed with monocrotaline (MCT) (two I.P. injection) /PHx in accordance with previously published work .163 Donor HOC (2.4 X 105 cells/rat) from DPPIV+ males were injected intras plenically. Donor HOC were pre-treated with 5 µM of TT232 ( a generous gift from Biostatin, Budapest, Hungary) for 15min, and then transplanted into receipen t animals (treated gr oup). The treated group was subsequently injected intravenously w ith 5 µg/kg concentration of TT232 on a daily basis. Animals were sacrificed on day 13 and 24 post transplantation for tissues collection and further examination. Immunohistochemistry DPPIV staining was performed as described by Dabeva et al.164 TUNEL staining was performed as the manufacter’s instruc tions (BD Biosciences, Mountain view, CA USA). Brieftly, cells were s eeded in 6-well plate (4.5 X 104 cell/well) and grown in IMDM supplemented with 10 %FBS at 37oC, 5% CO2. After 48 hr, the medium was replaced with serum-free IMDM for 16 h. Th e cells were subsequently cultured with medium alone, or medium with addition of TT232 at a concentration of 1, 5, 10, 25, 50 µM for 24hours. Migration Assay Migration assays were conducte d as described by Jung et al158 in transwell culture plates. The motility assay was initiated by tr ansferring the entire transwell chamber to

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62 new cluster plate well contai ning 100nM of SST-14 (synthesized in ICBR, UF) or TT232 (1 and 5µM) in migration buffer. Plates were maintained for 4 hour. In some experiments, cells were pretreated for 30 minutes with 5 µM TT232. As a negative control, cells were incubated without SST or TT232. At the end of the experiment, cells were fixed and stained as described by Stolz et al.151 Cells that had migrated to the bottom of the transwell filter were enumerated by counting each transwell chamber, at X4 magnification. Each migration assay was performed a minimum of three-times. Stimulation and We stern Blot assay Quiescent HOC were incubated for 10, 30, and 60 minutes at 37°C in the presence of 100 nM SST. In the inhibition experiment , HOC were pretreated with 5 µM TT232, 5 µg/ml anti-SSTR4 antibody, 20 µM LY 294002 (LC lab., Woburn, MA) or 0.08% DMSO for 30 minutes, and then incubated with SST at the indicated times, followed by PBS washing. HOC were homogenized in Triton Lysis Buffer (20 mM Tris, pH7.4, 137 mM NaCl, 10% glycerol, 1% Trit on X-100, 2 mM EDTA, 1 mM PMSF, 10 mM NaF, 5 µg/ml aprotinin, 20 µM Leupeptin, and 1 mM Sodi um ortho-vanadate) and centrifugated at 10,000 g for 15 minutes. Protein concentrations were measured using the Lowry assay. Immunoblotting was performed using anti-pAKT (Cell signaling tech., Denvers, MA), anti-AKT(Cell signaling tech.), anti-pAP K1(Cell signaling tech.), anti-PAK1(Cell signaling tech.), antibodies. Membranes we re developed by chemoluminescence (ECL; Amersham Pharmacia). Statistical analysis All results are expressed as the mean ±SD. Statistical differences were determined by Student’s t-test. P values of <0.05 were considered statistically significance.

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63 Results SST analogue, TT232, Suppresses the Migration of HOC toward SST Several SST analogues have been developed for the purpose of clinical applications. TT232 is a stable SST analogue with the highest binding affinity for SSTR4.89,165 TT-232 has been shown to exert an antip roliferative effect on several tumor cell lines and animal mode ls by inducing apoptosis.166 This effect has been shown to be very specific and is based on the concentra tion used. In these experiments, the higher doses are required for inducing a poptosis, and this type of cell death is independent from SSTRs.167,168 As specific SST analogue for SSTR4, the lower TT232 dose below 10 µM was tested,89 and has been shown to have no effect on cell proliferation.169 To clarify dose-dependent function of TT232 for HOC, I examined apoptotic cell death by TT232 at different concentrations. Approxi mately 35% and 5 % of apoptot ic cells were detected in HOC incubated with 50 µM and 25 µM of TT2 32, respectively. However, below 2% of apoptotic cells were observed in HOC treat ed with these concen tration (1, 5, 10 µM) (Figure 3-1A). These numbers of apoptotic ce lls was similar to level of apoptotic cells observed in control (0 µM of TT232).These data demonstrated that TT232 induced HOC apoptosis in dose-dependent manner, not promote HOC apoptosis at a lower concentration (< 10 µM). Our previous study revealed that the SST/SSTR4 pathway was involved in the migratory response of HOC.158 We examined the effect of TT232 on HOC motility. HOC were incubated with TT232 and tested their migratory capacity. When 1 or 5 µ M of TT232 was placed in the lower chamber, HOC failed to migrate toward TT232, (Figure 3-1B). However, pretreatment of HOC w ith TT232 abrogated the SST-mediated motility to the level of the control, whereas SSTstimulated HOC showed approximately a two

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64 and half-fold increase in motility toward SST (Figure 3-1C). These results indicated that TT232 suppressed the chemotactic action of SST on HOC migration. SST Affects HOC Homing through SSTR4, in vivo To examine whether the effect of SST/ SSTR4 on HOC migration was a functional consequence in HOC-mediated liver regenera tion, I designed the cell transplantation model as described in Figure 3-2. In this model, DPPIV+ HOC were transplanted into DPPIVfemale rats which were pretreated with MCT/PHx. The reaction product of DPPIV appears as diffused orange staini ng of the bile canaliculi membranes of hepatocytes. There is no-s taining observed in DPPIVanimals, so the transplanted cells can be easily distinguishe d from recipient cells.170 MCT is taken up by liver, metabolized, and subsequently inhibits endoge nous hepatocyte regeneration.163 PHx was given to create liver injury, thus pr oviding a suitable environment fo r repopulation of the liver by transplanted donor cells.171 The inhibitory function of TT232 on the chemotactic action of SST was expected to suppress motility of HOCs in transplantation model. Immunohistochemical staining for DPPIV positive cells was not detect ed in either the treated or control groups at day 13 post transplantation (data not shown). However, DPPIV+ cells were observed in recipient liver sections at 24 days post transplantation (Figure 3-3A). As expecte d, a high number of DPPIV+ cells and clusters were observed in control rat livers, whereas a si gnificantly lower number of DPPIV+ cells and cluster were observed in TT232-treated livers. It is possible that daily injection of TT232 could induce apoptosis in transplant ed cell, and for that reason, a lower number of donor cells could be observed in TT232-treated group. To te st this possibility, TUNEL assay on liver section of recipient group wa s conducted (Figure 3-3B). Apoptotic cells were rarely detected in both groups (day 13 and 24 post tr ansplantation), confirming that there were

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65 no apoptosis induced by TT232. These results demonstrated that suppression of SST/SSTR4 by TT232 decreased mobility of HOC, indicating that SST/SSTR4 might influence HOC homing to the damaged liver. SST Activates Intracellular PI3 kinase Pathway. In recent studies, the PI3K pathway ha s been shown to be required in cell migration.156,157 The activation of Akt and PAK1 was examined, in order to identify whether SST activated PI3K signaling system in HOC. Akt is a known PI3K effector, and PAK1 is a known Akt or direct PI3K effector.156 SST stimulated activation of Akt after 30 minutes and PAK1 after 60 minut es, whereas SST-induced phosphorylation of Akt and PAK1 were suppressed by an anti -SSTR4 antibody (Figure 3-4A & B). Also, TT232 decreased the activation of Akt and P AK1 (Figure 3-4C & D). These inhibitory effects of anti-SSTR4 antibody and TT232 on SST-mediated phosphorylation of Akt and PAK1 confirmed that TT232 impeded signaling event elicited by SST, in similar manner with anti-SSTR4 antibody. The activation of Akt and PAK1 suggested that PI3K signal pathway might be involved in stimulation of HOC via the SST/SSTR4 pathway. In order to investigate whether PI3K is essential for cell migration, we employed PI3K inhibitor, LY294002, in a migration assa y of HOC with SST. Western blot assay showed that LY 293002 significantly blocked the action of PI3K on Akt and PAK1 even in the presence of SST. DMSO, a solvent for LY 294002, did not affect Akt and PAK1 activation (Figure 3-5A). Mi gration assay revealed that pretreatment of LY 294002 abrogated HOC migration toward SST, whereas SST increased the numbers of migrating HOC, approximately a threefold increase (Fi gure 3-5B). Our result suggested that intracellular PI3K pathway might be an esse ntial step for HOC migration mediated by SST.

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66 Discussion After a cytotoxic therapy and growth factors administration, stem cells are mobilized to the location of in jury, but still retain the abil ity to selectively repopulate a tissue system. Trafficking, mobilization, and homing of stem cells is a multifactorial process that is regulated by adhesion molecu les and cytokines as well as chemotactic factors.140 In hematopoietic stem cells, recent studies have provided evidence that several chemotactic factors, particularly the SD F-1 and its receptor chemokine receptor 4 (CXCR4), are involved in re gulation of mobilization and hom ing of hematopoietic stem cells.81,172 Thus, homing is the hall mark of hematopoietic stem cells, for which mechanism and cytokines are vigorously stud ied and well characterized. However, there is relatively little informati on regarding those that influen ce oval cells homing. In this context, a major challenge is represented by the identification of the chemotactic function of SST/SSTR4 on HOC migration in vivo and the mechanism induci ng this effect of SST. In this study, we investig ated the effect of TT232 on HOC and found that apoptosis brought on by TT232 was dose-dependent with higher doses (> 25 µM) carrying apoptosis. TT232 did not promote apoptosis, but abrogated HOC migration mediated by SST at a low dose of TT232 which was repres ented as appropriate concentration for SSTR4 binding.89 These inhibitory effects of TT232 for SST/SSTR4 also were proven in functional consequences in HOC migration, showing that blockage of SSTR4 decreased motility of HOC within the damaged liver. In signaling mechanism, inhibitory effects of TT232 was similar to that of anti-SST R4 antibody on suppressing SST-induced activation of Akt and PAK1 (Figure 3-4), confirming that TT232 bind to SSTR4, which then blocks the signaling pathway triggered by SST.

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67 In the HOC-transplantation experiment, th e data indicates a significant difference of DPPIV+ cell and cluster number between the control animals and TT232-treated animals. A few DPPIV+ cells in TT232-treated group are lik ely to result from incomplete blockage of SSTR4 by TT232. Although it is al ready verified that TT232 specifically bind to SSTR4, blockage of receptor subtypes by TT232 is dependent on applied concentration of TT232.89 Following the guideline described by Weckbecker, et al ,89 I utilized different concentrations to bloc k the function of SSTR4 and injected low concentration which would neither affect ot her receptor subtypes nor induce apoptosis. However, concentration of a drug is change d in inner body by certain factors and TT232 has been shown to be effective up to four hours post injection, according experimental report provided by the Biostatin. Considering these points, it appear s that TT232 did not inhibit completely the interac tion of SST and SSTR4 in this procedure. Also, it should be considered that oligomerization between SSTR4 and SSTR2 can occur.173 SSTR2 is the major receptor for SST and a cellular inter action between SSTR4 and SSTR2 can affect functions of individual receptors.174 Thus, there is a possibility that SSTR2 can compensate for loss of function of SSTR4. Although this action was not investigated, it remains a possibility. Recently, two studies have provided evidence that SDF-1/CXCR4 might be involved in HOC-aided li ver regeneration and HOC migration.175,176 Accordingly, it is conceivable that SST ma y make synergetic effects on migration of HOC with SDF-1. When the response of SSTR4 on HOC to SST was blocked, cells escaping from the suppressive effect of TT232 or having decreased motility induced by SSTR4-blockage may respond to SDF-1 and migrate to the damaged liver. However, it still remains unclear how SST interacts with SDF-1.

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68 Following the full ligand-dependent tyrosi ne phosphorylation, SSTR4 expressed on HOC was able to activate the PI3K pathwa y. SSTR4 belongs to the family of seven helical transmembrane domain G protein -c oupled receptors (GPRs), which are coupled to heterotrimeric G-proteins.89 This receptor can be linked to the activation of PI3K which is directly stim ulated by the G-protein subunits released from -subunit when the latter is activated by the binding GTP.156 The downstream effector, PI3K, has been implicated in the several cell types of migration.156,157,177 The motility of HOC has been shown to be abrogated by anti-SSTR4 antibody and TT232 in our previous and present study, respectively.158 Western blot assays within the cu rrent system have showed that the activity of PI3K was suppressed by anti-SSTR4 antibody or TT232. These results suggest that PI3K is an essential molecules in HOC migration when SST/SSTR4 are involved. Migration assay with a PI3K inhibitor added evidence that SST stimulated HOC migraton through a PI3K-dependent way. Since the anti-proliferative e ffect of TT232 in cancer ce ll lines has been shown to be induced through p38 169, we examined whether anti-proliferation via these molecule or cell cycle arrest though p21159 caused anti-migratory results in HOC. During the experiments, the expression of p21 and p38 were not observed (data not shown). In the current study, we found that trea ting cells with SST resulted in Akt activation, peaking at 30 minutes and subs equently PAK1 activation, peaking at 60 minutes. Although PI3K also was shown to induce directly the activation of PAK1,156 the sequential activation of Akt and PAK1 sugge sts a potential signal transduction pathway, PI3K/Akt/PAK1, within HOC migration stimulated by SST/SSTR4. In addition, antiSSTR4 antibody inhibited the activation of PAK1, while TT232 seemed to delay it,

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69 although these differences were subtle. It is assumed that anti-SSTR4 antibody binds more predominantly to SSTR4 than SST does, thereby it blocked SSTR4 response to SST, whereas TT232 inactivated SSTR4-coupl ed signaling molecule, PI3K. However, both consequently induced inac tivation of PI3K, and finally abrogated motility of HOC toward SST. In conclusion, our findings suggest that SST could stim ulate liver regeneration by mobilization, migration, and in corporation of HOC via SSTR4, and this effect appears to be mediated by intracellular PI3K pathway. However, more detailed studies about relationship with other chemokines will be ne cessary to clarify mechanism of homing.

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70 Figure 3-1. Effect of SST an alogues, TT232, on HOC migration . (A) TUENL assay was conducted in order to examine apoptotic cells. A higher number of apoptotic cells are observed in HOC-treated with high dose (50µM) of TT232, compared with other treated groups. Da ta shown represents one of three experiments with similar results. (B) Migration assay with TT232 on HOC. HOC were seeded on the top of the chamber with 1 and 5 µM of TT232 placed in the bottom chamber. Controls were used with no TT232 in either chamber. (C) HOC were subjected to chemotaxis assays with 100 nM SST (SST). Cells were treated with 5 µM TT232 for 30 minutes and cultured in 100 nM SST– containing medium for 4 hours (TT/SST). Controls were used with neither TT232 nor SST. Data represents mean value ± SD of three independent experiments. Data were normalized for each independent experiment with respect to control migration. (*P<0.01, relative chemotactic index vs control).

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71 Figure 3-2. The plan for experi ment was described. The DPPIVfemale rats received two doses of 30mg/kg MCT and PHx. HOC were isolated from DDPIV+ rats which were treated with 2AAF/PHx. HOCs for treated group was pretreated with TT232 (5 µM) for 15 minute and tr ansplanted into spleen of DPPIVfemale rats at 7 day post PHx. Treated group was injected intravenously with 3 µg/kg concentration of TT232 on daily base. Both groups were killed at 13 and 24 days post transplantation.

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72 Figure 3-3. Suppression of SST/ SSTR4 impairs the migration of HOC within the injured liver. (A) DPPIV staining for transplanted cell in DPPIV-/rats at 24 day post transplantation. Control (a) and treated animals (b). (Arrows indicate DPPIV+ cells. Representative slide were viewed as X20, and insert viewed at X40 magnification). (c) The number of DPPIV+ cells and clusters (B) Apoptosis assay on recipient liver section us ing TUNEL. (a & b) Day 13 post transplantation (a; control, b; trea ted animal). (c & d) Day 24 post transplantation (c; control, d; treated animal). (A rrows indicate apoptotic cells) PI (red) was employed for nuclear staining. Representative slide were viewed as X10, and insert viewed originally at X20 magnification.

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73 Figure 3-4. SST/SSTR4-induced effect s are mediated by PI3K pathway . (A) SST induced phosphorylation of Akt and PAK1 with a maximum effect at 30 and 60 minutes, respectively. SST-induced activation of Akt and PAK1 was suppressed by 5µg/ml auti-SSTR4 anti body. (B) Densitometry analysis to show the relative activation of Akt and PAK1. Histogram function of Adobo photoshop was used to approximate density of each band. (C) The effects of SST on phosphorylations of Akt and P AK1 were decreased by 5µM TT232. (D) Densitometry analysis to show th e relative activation of Akt and PAK1. Histogram function of Adobo photoshop was used to approximate density of each band. Figure 4A &C represent one of three experiments with similar results. Data shown in figure 4B & D represent mean value ± SD of three independent experiments. Data were normalized for each independent experiment with respect to control band density

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74 pAkt Akt DMSO None LY SST pPAK1 PAK1 pAkt Akt DMSO None LY SST pPAK1 PAK1A Figure 3-5. PI3K plays a important ro le in SST-stimulated HOC migration. (A) HOCs were pretreated with PI3K inhib itor (LY194002), DMSO (solvent for LY 294002), and none-added medium for 30 minut es. And then, these cells were stimulated with 100nM SST. Western bl ot analysis for Akt and PAK1 was conducted in order to test whethe r PI3K was blocked by LY295002. Data shown represents one of three experiment s with similar results. (B) HOC were subjected to chemotaxis assays with 100nM SST only (SST), and SST after 30 minutes pretreatment with PI3K i nhibitor (LY/SST). Control was used without LY and SST (con). Data represents mean value ± SD of three independent experiments. Data were normalized for each independent experiment with respect to control migration. (*P<0.01, relative chemotactic index vs control). .

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75 CHAPTER 4 CHARACTERIZATION OF THY-1+ CELL IN VITRO CULTURE SYSTEM Summary Hepatic oval cells (HOCs), capable of matu ring into hepatocytes and biliary cells, are hypothesized to be involved in all fo rms of liver regeneration and may prove clinically useful at reconstituting damaged livers. A HOC population from young adult rat liver tissue has been isolated and charac terized to establish validity for our studies. HOCs were induced by 2AAF/PHx protocol, is olated using modifica tion of a two-stage liver perfusion technique followed by low speed centrifugation. Cellular analysis by fluorescent microscopy and RT-PCR demonstrat ed that HOC cultured on collagen-coated dish over 6 weeks phenotypically and genetically differentiated into hepatocytes, whereas cells cultured on none-coated dish maintained their properties. Thes e data suggest that HOC can differentiate into hepatocytes in vitro and HOCs cultured on none-coated dish within 4 weeks can be used in our model for HOC transplantation, migration and molecular approaches. Introduction A unique marker for oval cells in the adu lt liver has not been assigned, and because there cell are under constant rene wal, unlike epithelial cells of the intestine or the skin, they escape detection in the quiescent liver. For this reason, potential stem cells in the adult liver have not yet been isolated co mpletely. Also, culture methods maintaining properties of oval cells remain unclear. In this chapter, the method of isolating oval cells

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76 and hepatic stem cell lines are reviewed. Also the characteristics of Thy-1+ oval cells in vitro culture system we re investigated. Isolation of Potential Liver Progenitor Cells Attempts aimed at purifying fetal liver hepa toblasts, considered to be fetal liver progenitor cells, have been more successf ul. Three major approaches have been developed. The first protocol entails hepatoblasts fetal li ver cells by “panning” of the fetal liver cell suspension with a re d blood cell antibody, followed by panning of macrophages and endothelial, myeloid, and lymphoid cell with OX-43 and OX-44 antibodies. This procedure resulted in s a six fold enrichment of hepatoblasts.178 Magnetic cell sorting (MACS), depletion of fetal liv er cell with OX-43 and OX-44 antibodies with parallel enrichment for T hy-1+ and CK (cytokeratin)-18+ cells, was also used successfully.153 A negative selection with CD (clust er of differentia tion) 45 (leukocyte common antigen) and TER119 (an antibody reco gnizes a molecules associated with glycophorin A, expressed on all erythroid ce lls) results in enri chment of hepatic progenitor cells from murine fetal liver.179 The second approach utilized antibodie s raised against cell surface antigens expressed on hepatoblasts and combined with fluorescence activated cell sorting (FACS) or MACS. Suzuki et al .43used 6 and 1 intergrin subunits that are receptors for laminin and are highly expressed in fetal liver to enri ch the primary murine fetal liver isolates in hepatocytes and cholangiocyte progenitor cells . Another cell membra ne protein used for isolation of fetal hepatoblasts was the cel l-cell adhesion glycoprotein E-cadherin, highly expressed in epithelial tissues, includi ng fetal liver. Using MACS and monoclonal antibodies against the extracellular domain of E-cadherin, Shiojiri180 and coworker isolated fetal liver progenito r cells from ED 13.5 mouse liver with 90% purity and 40%

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77 yield. After transplantation into the splee n, only hepatocytic proge ny were observed in the liver. The third approach was enrichment of hu man hepatoblast using hematopoietic cell surface markers. It was reported that 0.9% of th e cells in human liver at 14 to 22 weeks of gestation are CD34+.181 Three to 8% of the CD34+ cells co-expressed cytokeratin CAM 5.2.182 Another hematopoietic marker used for enrichment of hepatic fetal progenitor cells is the surface protein Thy1 (CD90) . It was shown that 1% of all Thy1+ cells coexpress the epithelial marker CK18.153 Miyajima and coworkers183 developed a method based on an innovative idea to search for cell surface antigens expressed in mouse fetal hepatic cells, applying the si gnal sequence trap method. Thes e investigators identified such a gene, Dlk (Pref-1) of unknown func tion, expressed in ED10.5 embryos and in mouse fetal liver until ED16.5. FACS-isolate d Dlk+ cells are hepatoblast and not hematopoietic cells; they express albumin and AFP ( -fetoprotein); and a single cell is capable of differentiating into albumin-, CK19-, and albumin/CK19-positive cells, showing that the Dlk positive cells are bipotential. However, in vivo , after transplantation into the spleen, only hepatocytic progeny were observed. In addition, oval cells have been isolated fr om the liver of adult rats in which these cells have been induced in response to hepatocarcinogenesis. When hepatocytes proliferation is suppressed by hepatic inju ry, expansion of oval cell compartment is resulted. 45 Oval cells express liver progenitor ce ll markers, such as Thy-1, AFP, GGT, CK19, OC.2 and OV6.32,45,184 These markers are used in identification, ch aracterization, and isolation. When the Thy-1 antibody is used as a new marker for the detection of oval cells, a highly pure population can be obtained. The flow cytometry has established a

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78 method to isolate a 90-97% pure Thy-1+ oval cell population.45 Utilizing cell-sorting techniques in combination with th e Thy-1 antibody will facilitate both in vivo and in vitro studies of hepatic oval cells. Oval Cell Lines Liver epithelial cell lines ar e considered the cell cultu re counterpart of liver stem/progenitor cells.184 Several laboratories have succeeded in isolating clonogenic epithelial cell lines from normal liver and from the liver after hepatocarcinogenic regimens. In summary, liver epithelial cell lines possess the following characteristics: (1) They express phenotypic properties of undiffere ntiated fetal hepatoblast/oval cell. (2) When exposed to appropriate conditions in vitro, the cell initiates differentiation and acquires some hepatocytic or biliary epithelial characteristics. LE (liver epithelial)/2 and LE/6 oval cell lines isolated by centrifugal elutri ation from the liver of rats fed a cholinedeficient diet differentiate into hepatocytes in a three-dimensional culture system composed of a collagen gel and a feeder layer.185 Oval cell lines obtained from allyl alcohol-treated rats, propagated in feeder layers, express he matopoietic stem cell markers (c-kit, CD34) and early hepatocytic mark ers (AFP, albumin, and CK14). Upon removal of the feeder layer, the cells begins to express mature hepatocytic markers (H4 antigen and CYP1AII (cytochrome P450 1AII) ) or acquire a bile duct-like structure, if plated on Matrigel.186 (3) Liver epithelial cells differentiate in vivo into hepatocytes. The cells from one normal liver epithelial cell line, BAG-2WB, transplanted into the liver of syngeneic animals became incorporated into the hepati c plate of the host parenchyma and acquired hepatocytes-specific gene expression.187,188 The advantage of the epithelial cells is that they can be easily propagated and immort alized spontaneously in culture.

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79 However, these cell lines are immortali zed by certain types of mutations, which lead to problems in validity of our approac h. Also, primary oval cells show slow growth, short life, and liable contamination. Most of a ll, they differentiate into hepatocytes at a certain time point, and long-term culture of primary oval cells is impossible. Therefore, we characterized Thy-1+ oval cell culture system to show the validity of the approach. Accordingly, this procedure was needed in our further studies, in order to establish experimental condition and practice in vitro prior to in vivo . Material Methods Animal Dipetidyl peptidase IV deficient (DPPIV-) female F344 breeding animals, which were originally obtained from the Albert Eins tein College of Medicine, were a generous gift from Dr. Sanjeev Gupta. These anim als were in-house bred and maintained on standard laboratory chow and daily cycles of alternating 12 hours of light and dark. They were used at approximately 8-10 weeks of age and 150-180g weight. Normal male DPPIV+ F344 rats (age 8-10 weeks, weight 180-220g) were purchased from Charles River Laboratories and were used as donor an imals. All animal work was conduced under protocols approved by IACUC at th e University of Florida. HOC Preparation and Culture To isolate HOCs, 2AAF/PHx oval cell activation model was used.147 The liver was harvested at 11 days post PHx and cells were isolated via standard two-step collagenase perfusion. Obtained cells were gradient centrifuged at 50 g to isolate hepatocytes. Then, fraction of non-parenchymal cell (NPC)s containing HOCs was collected at 100 g. Obtained fraction of cells was incubated with Thy-1 FITC conjugated antibody (Becton Dickinson, Flanklin Lakes, NJ) and then w ith anti-FITC-microbeads (Miltenyi Biotec

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80 GmbH, Auburn, CA). After inc ubation, cell suspension is lo aded on a magnetic column which is placed in the magnetic field of a MACS Separator. The magnetically labeled cells are retained in the co lumn while the unlabeled cells run through. After removal of the column from the magnetic field, the magneti cally retained cells can be eluted as the positively selected cell fraction. Thy-1+oval cell population obtain ed from MACS shows about 90% purity148 and cell viability was determined by Trypan blue exclusion. After isolation, HOCs were resuspended in Isc ove’s Modified Dulbecco’s Medium (IMDM) (10%FBS, 1% insulin, 1X antibiotic-antim ycotic) on the collagen (0.001%)-coated or non-coated bent tissue culture flask. Additiona lly, we added 4 different types of growth factor (SCF, IL-6, IL-3, FLT ( fms -like tyrosine kinase); e ach 10ng/ml, except for IL-6 20ng/ml), to maintain state of HOCs. Immunofluorescent Staining Immunostainings were performed on cyto centrifuged HOCs using standard staining protocols. Samples were fixed and permeabilized, saturated, and processed for immunostaining with anti-albumin (Dako Comp ., Santa Cruz, CA). Texas red anti-goat IgG (Vector Laboratories) wa s used as secondary antibody. Vector ABC kit (Vector Laboratories, Burlingame, CA) were employed in blocking procedure. RT-PCR For the Reverse transcriptase polymerase chain reactions (RT-PCR) analysis, total RNA (ribonucleic acid) was is olated from the normal liv er, oval cell cultured on collagen-coated or non-coated tissue flask by RNeasy kit (Qiagen, Valencia, CA). Total of 2 µg RNA was used for each cDNA synthesi s. RT-PCR were performed as previously described by Bar et al .150 Following primers for albumin were used: 5’GCTACGGCACAGTGCTTG-3’ (sense strand), 5’-CAGGATTGCAGACAGATAGTC-

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81 3Â’ (antisense strand), which delineated a 260-bp product. The resulting RT-PCR products were amplified and subjected to electrophores is in 1.2% agarose gel and stained with ethidium bromide. The purified PCR produc ts were directly sequenced using an AmpliTaq cycle sequencing kit (Perkin-El mer Setus, Branchburg, NJ) for genetic confirmation. Results The same number of freshly isolated Thy-1+ HOCs was placed in collagen-coated and non-coated bent culture flask. After four weeks, the normal dish was colonized with HOCs (Figure 4-1A&B) which showed mor phology of original HOC before placing. However, HOCs on collagen-coated tissue flask were observed having different morphology from it of HOCs on normal tissue flask (Figure 4-1C-F). HOCs on collagencoated culture tissue flask seemed to be differentiated into small hepatocytes morphologically. In order to examine whether mo lecular properties of these cells changed to hepatocytes, immunofluorescent staini ng and RT-PCR for albumin were conducted. As shown in figure 4-2A, a portion of HOCs on collagen-coated tissue flask expressed albumin, whereas albumin positive cells were not detected in HOCs cultured in normal tissue flask (data not shown). RT-PCR re sult showed that HOCs cultured on collagencoated tissue flask expressed albumin, while the message of albumin was not observed in HOCs on non-coated tissue flask (Figure 42B). When HOCs on non-coated tissue flask were cultured over 4 months, these cells lo st their properties which were checked by CK19-, OV6-, AFP-, and interestingly SSTR4 (data not shown). Th ese results indicated that collagen and long-term culture might i nduce differentiation of HOCs. Therefore, we will conduct in vitro experiment only in short-term (within 6weeks) cultured HOCs and on non-collagen treated normal dish for validity of our technique.

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82 Discussion HOCs have been considered facultive bipotential precursor cells that can differentiate into hepatocytes and bilila ry epithelial lineages depending on the environmental conditions or regulatory factors.134 Identification and isolation of liver stem cells may ultimately allow a large number of autologous hepatocytes suitable for transplantation to be grown in vitro from a small liver biopsy. However, the lack of specific markers hampers the identification of HOCs. Although liver explant culture, an attractive method to expand hematopoietic stem cells, has been used to isolate HOCs, no pure population of HOCs had yet not been obtai ned from adult livers. In addition, it is difficult to maintain undifferentiated status, de fine the time point of differentiation, and expand primary HOCs in vitro . Therefore, this study aims to define culture condition for HOCs. Extracellular matrices (ECMs), which can insoluble macromol ecular substances consisting of proteins and carbohydrates, fix a nd support cells as physical anchorages. In the liver, the ECMs produced by stellate cells regulate the proliferation and differentiation of hepatocytes. 189 Type I fibrillar collagen is very abundant and is widely distributed around the vascular spaces and G lissonÂ’s capsule, and within the space of Disse in contact with hepatocytes and sinus oidal endothelial cells . This provides an appropriate microenvironment for hepatic differentiation.189 Several in vitro studies reported that cloned derived from oval cells were shown to differentiated into mature hepatocytes or to form duct-lik e structures in collagen gel culture when coclutured with fibroblasts as feeder cells or when supplie d with growth factors. These observations suggest that biodegradable co llagen matrices regulate th e differentiation of HOCs by providing an appropriate microenvironment. In this study, we established HOCs in short-

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83 term culture and characterized their phenotype in vitro . Our data demonstrated that collagen matrix facilitated differentiation of HOCs, which lost the expression of HOC markers and gained the phenotypic and molecu lar characteristics of hepatocytes. In addition, three independent experiments sugge sted the transition time points for HOC differentiation, forming the basis for the use of HOCs in our in vitro system. In the absence of the appropriate oval cell lines, these preliminary experiments may allow us to get the stabilized data from our approach.

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84 Figure 4-1. Morphology of HOC on culture di sh (A & B) HOC cultured on non-coated culture tissue flask. (C-F) HOC culture d on collagen-coated culture tissue flask. Photos were taken at 30da ys after being seeded freshly.

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85 Figure 4-2. Albumin expressi on in HOC cultured on collag en-coated dish. (A) HOC cultured on collagen-coated tissue flask were cytospinned at 40 days post placing and a part of these cells expresse d albumin.( Representative slide were viewed as X20 original magnification) (B) In RT-PCR, only HOCs culture on collagen-coated tissue flask showed messa ge for albumin . NL (normal liver), OV (hepatic oval cells cultured on noncoated culture tissue flask) DOC (hepatic oval cells culture on coll agen-coated culture tissue flask) .

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86 CHAPTER 5 CONCLUSION Hepatic oval cells (HOCs) are activated to proliferate and differentiate when the regenerative capacity of terminally differ entiated hepatocytes are compromised. In oval cell mediated liver regeneration, tissue damage l eads to a dramatic increase in the level of secreted chemokines, cytokines, and proteoly tic enzymes in many organs as part of the regeneration and repair process, which ha ve profound impacts on stem cell migration and repopulation. SST is a regulatory peptide that activates G-protein-coupled receptors of a family comprising five members (SSTR1-5). Despite the broad use of SST and its analogs in clinical practice, the spectrum of SST activities has been incompletely defined. Recently, it has been demonstrat ed that SST functions as a chemoattractant for immature neuronal and hematopoietic cells. Hence, we proposed to test the hypothesis that SST influenced on the homing of hepatic oval cells under pathological conditions, based on the evidence that hepatic oval cells, acting as liver stem cells, share several characteristics with hematopoietic stem cells Therefore, the main purpose of this research was to identify a potential role of SST and ascer tain its mechanism on hepatic oval cell homing. To accomplish this, we utilized the rat 2 AAF/PHx model of ova l cell activation and examined SST and SSTR1-5. RT-PCR and West ern blot assay revealed an increased expression of SST in 2AAF/PHx HOC induc tion model. Immunohistochemical staining showed the expression of SST in 2AAF/PHx-t reated rat liver, as compared to normal liver. Proliferation and migrati on assays demonstrated that th e increase of SST is related to migration of HOCs, but not their proliferation. RT-PCR and quantitative real-time

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87 PCR showed that SSTR4 was preferentially expressed by HOCs. Western blot assay and immunohistochemical staining confirmed the expression of SSTR4 on HOCs. In addition, pretreatment with anti-SSTR4 antibody cultures resulted in a dramatic reduction of cell migration as compared to that of control. Lastly, SST stimulated the rearrangement of actin filaments in HOCs, while HOCs treate d with anti-SSTR4 antibody failed to do so. These results suggest a positive role for SST in the migration of HOCs, and that this effect is mediated through SSTR4. In the next experiments, we investigated whether the effect of SST/SSTR4 on the migration of HOC is a functional consequen ce in oval cells-mediated liver regeneration and what type of signaling mo lecules are associated with this chemotactic action. TUNEL assay and migration assay showed that TT 232, which is SST analogue with specific binding affinity for SSTR4, did not induce cell apoptosis, but abrogate the motility of HOC by SST. In cell transplantation model to examine the potential role of SST and SSTR4 in HOC migration in vivo , a lower number of donor-deriv ed cells was detected in the TT232 treated animals, as compared to the number of donor-derived cells in the control animals. Activation of phosphatidyli nositol-3-kinase (PI3K) was observed in HOCs exposed to SST, and this activation was suppressed by eith er the anti-SSTR4 antibody or TT232-pretreatment. In addition, decreased motility of HOC by the treatment with a PI3K inhibitor revealed that PI3K might be an e ssential signaling molecule in HOC migration by SST. Our findings suggest that SST and SSTR4 stimulate the migration of HOC within the injured liver, a nd this action appears to be mediated through the PI3K pathway.

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88 Stem cells “home” or migrate to appropr iate sites where they exert unique functions, such as self-renewal and mu lti-lineage differentia tion. The molecular mechanisms regulating stem cell homing require more study, especially given the importance of such homing in a variety of me dical applications. Our findings suggest that SST and SSTR4 could stimulate liver re generation by mobilization, migration, and incorporation of hepatic oval cells. This may lead to a be tter understanding of HOC movement within the injured liver. However, more work is required to fully understand the significance of the present findings.

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106 BIOGRAPHICAL SKETCH Youngmi Jung was born in Seoul, South Korea. She attended Ewha Womans University from March 1992 to February 1997. During undergraduate study, she received the Korean Leader Scholarship. At junior grade, she participated in Korean Biology Conference for undergraduate students. After obt aining her Bachelor of Science degree in science of education (biology), she worked as a research assist ant at Seoul National University, College of Medicine, where sh e continued her education from 1997 to 1999. Miss Jung obtained her Master of Medici ne degree in pathology in March 1999. In 2002, Miss Jung was accepted to the Interdisciplinary Program at the UF, College of Medicine, where in May 2006 she received the degree of Doctor of Philosophy.