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1 HEPATIC STELLATE CELLS INVOLVEMENT IN PROGENITOR CELL MEDIATED LIVER REGENERATION By DANA GABRIELA PINTILIE A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2009
2 2009 Dana Gabriela Pintilie
3 To my beloved family, the rock that steadies me
4 ACKNOWLEDGMENTS I thank my mentor Bryon E. Petersen for the scientific knowledge and ideals of perseverance towards understanding the unknown. I am also grateful to the other members of my committee, Drs. Maria Grant, Steven Ghivizzani, Edward Scott and Naohiro Terada for their continued support and scientific dialogue which helped me evolve into the scientist I am now. I would also like to extend my thanks to Drs. Sehhoon Oh, Thomas Shupe, Susan Ellor, Houda Darwiche, Anna Piscaglia, Jennifer Williams and Liya Pi for sh aring their research experience with me and for the friendship they have shown me throughout my time at the University of Florida. I cannot explain the importance of my family which never told me I could not achieve any goal toward which I set my mind. Their love and unconditional support kept me going through thick and thin. I can never tell them enough how much they mean to me.
5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ...................................................................................................... 4 LIST OF TABLES ................................................................................................................ 8 LIST OF FIGURES .............................................................................................................. 9 CHAPTER 1 INTRODUCTION ........................................................................................................ 20 1.1 Uncovering the Interactions between the Hepatic Stellate Cells and Oval Cells in Regenerating Liver ............................................................................... 20 2 BACKGROUND AND SIGNIFICANCE ...................................................................... 23 2.1 The Liver ............................................................................................................... 23 2.1.1 Liver Anatomy ........................................................................................... 23 18.104.22.168 Structure of the hepatic organ .................................................... 23 22.214.171.124 Microarchitecture of the liver ...................................................... 24 126.96.36.199.3 Biology and clinical significance of hepatic stellate cell ......... 28 2.1.2 Liver Physiology ........................................................................................ 33 188.8.131.52 Metabolic homeostasis ............................................................... 33 184.108.40.206 Storage ....................................................................................... 33 220.127.116.11 Blood re servoir ........................................................................... 34 18.104.22.168 Detoxification .............................................................................. 34 22.214.171.124 Liver endocrine functions ........................................................... 35 126.96.36.199 Liver exocrine function ............................................................... 35 188.8.131.52 Immune defense ......................................................................... 35 2.1.3 Liver Regeneration ................................................................................... 36 2.2 Stem Cells and Their Therapeutic Potential ........................................................ 38 2.3 Oval Cells Biology and Their Clinical Significance in Liver Regeneration .......... 41 2.3.1 Origin and Morphology of Oval Cells ....................................................... 41 2.3.2 Experimental Models and Mechanisms of Oval Cell Induction ............... 43 2.3.3 Oval Cell Plasticity .................................................................................... 46 2.3.4 Oval Cell and Hepatic Neoplasms ........................................................... 47 3 SPECIFIC AIMS .......................................................................................................... 56 4 METHODS AND MATERIALS ................................................................................... 60 4.1 Animal Treatments ............................................................................................... 60 4.1.1 Animals and Animal Housing Facilit ies .................................................... 61 4.1.2 Animal Sacrifice and Tissue Collection .................................................... 61 4.1.3 Oval Cell Induction in Rat ......................................................................... 62
6 184.108.40.206 2AAF pellet implantation ............................................................ 62 220.127.116.11 Two thirds partial hepatectomy .................................................. 63 4.1.4 2% L-cysteine Diet .................................................................................... 64 4.1.5 Animal Numbers ....................................................................................... 65 4.2 BrdU Analysis In Vitro Cell Response to L-cysteine ........................................ 65 4.2.1 Maintenance of WB -F344, HSC T6 Cells and Portal Fibroblasts in Cul ture ..................................................................................................... 65 4.2.2 Cell Synchronization and BrdU Treatment .............................................. 66 4.3 Histology and Immunohistochemistry .................................................................. 66 4.3.1 Hematoxylin and Eosin Staining of Paraffin Embedded Tissue ............. 66 4.3.2 Periodic Acid-Schiff Staining of Paraffin Embedded Tissue ................... 67 4.3.3 Immunohistochemistry ............................................................................. 67 18.104.22.168 Chromogen staining ................................................................... 67 22.214.171.124 Fluorescent staining ................................................................... 68 4.4 RNA Analysis Real Time Quantitative PCR ..................................................... 69 4.4.1 RNA Isolation ............................................................................................ 69 126.96.36.199 Tissue homogenization and phenol -chloroform phase separation .................................................................................. 69 188.8.131.52 Precipitation, washing and redissolving of RNA ........................ 70 184.108.40.206 Quantification of RNA by spectrophotometry ............................ 70 4.4.2 Real Time PCR (RT PCR) ........................................................................ 70 220.127.116.11 First -strand cDNA synthesis from total RNA using rt PCR ....... 70 18.104.22.168. PCR amplification of target cDNA ............................................. 71 22.214.171.124 Agarose gel electrophoresis ...................................................... 72 126.96.36.199 Real -Time PCR analysis of AFP levels ..................................... 72 4.5 Statistical Analysis ................................................................................................ 73 4.6 Solutions ............................................................................................................... 73 5 RESULTS .................................................................................................................... 78 5.1 Evaluation of in v itro Response to L -cysteine of Selected Hepatic Cell Populations ........................................................................................................ 79 5.2. Evaluation of in vivo Response of Hepatic Stellate Cells to L -cysteine Diet ..... 80 5.3. Evaluation of Cell Morphology in L -cysteine/2AAF/PHx Treated Rats .............. 82 5.4. Evaluation of Cell Proliferation in Regenerating Liver under L-cysteine Exposure ............................................................................................................ 85 5.5. AFP Expression in Periportal Regions of Regenerating Liver ........................... 86 5.6 Immunohistochemical Identification of Progenitor Cell Population ..................... 88 6 DISCUSSIONS AND FUTURE DIRECTIONS OF STUDY ..................................... 115 6.1 Interpretation of Results ..................................................................................... 115 6.2 Clinical Applications ............................................................................................ 121 6.3 Future Directions of Study .................................................................................. 122 7 SUMMARY AND CONCLUSIONS ........................................................................... 125
7 LIST OF REFERENCES ................................................................................................. 127 BIOGRAPHICAL SKETCH .............................................................................................. 138
8 LIST OF TABLES Table page 2 -1 Liver regeneration post 70% hepatectomy reflected by DNA synthesis in various resident hepatic cells; chosen time points indicate the number of days after the acute liver injury (adapted after Michalopoulos45). ........................ 55 2 -2 Molecular markers for several hepatic cell populations ........................................ 55 4 -1 Animals subjected to the stellate cell inhibition oval cell activation L cysteine/2-AAF/PHx protocol. ................................................................................ 77 4 -2 Antibodies and antigen retrieval methods used for immunohistochemistry ......... 77
9 LIST OF FIGURES Figure page 2 -1 Diagrams of liver blood supply and microarchitecture.. ........................................ 50 2 -2 Conceptual interpretations of the functional hepatic architecture. ....................... 50 2 -3 Histograms of Hematoxylin and Eosin (H&E) stained hepatic cell populations.. ........................................................................................................... 51 2 -4 Hepatic stellate cell: morphology and activation diagram.. ................................... 51 2 -5 Diagrammatic representation of the specific mechanisms differentiating compensatory regeneration from direct hepatic hyperplasia ................................ 52 2 -6 Diagrammatic representation of liver parenchyma growth after 70% partial hepatecomy in rat. .................................................................................................. 53 2 -7 Oval cell phenotype.. .............................................................................................. 53 2 -8 Oval cell plasticity. .................................................................................................. 54 4 -1 Diagrammatic representation of the 2% L-cysteine diet/2AAF/PHx experimental protocol ............................................................................................. 75 4 -2 Mechanism of action, metabolic activation and detoxification of 2AAF (2acetylaminofluorene). ............................................................................................. 75 4 -3 Proposed mechanisms for 2AAF inhibition of hepatic stellate cells. .................... 76 5 -1 L -cysteine effects on WB F344 ovall cell line. ....................................................... 89 5 -2 L -cysteine effects on portal myofibroblasts. .......................................................... 89 5 -3 L -cysteine effects on HSC T6 hepatic stellate cell line ......................................... 90 5 -4 C omparative image analysis of Brdu incorporation index in WB 344F oval cell line, PF portal fibroblasts and HSC T6 stellate cell line. ....................................... 90 5 -5 Comparative image analysis of desmin positive areas on liver sections from animals sacrificed 9 days post hepatectomy. ........................................................ 91 5 -6 Desmin immunostaining of normal rat liver. .......................................................... 92 5 -7 Desmin immunostaining of rat liver sections collected on day 9 post hepatectomy. .......................................................................................................... 93 5 -8 Comparative quantitative image analysis of desmin positive areas. .................... 94
10 5 -9 Hematoxylin and Eosin staining of normal rat liver ............................................... 95 5 -10 Comparative histological exam of H&E stained liver samples collected on day 9 post acute liver injury. ......................................................................................... 96 5 -11 Comparative histological exam of liver sections .................................................. 97 5 -12 Comparative histological exam of H&E stained liver samples collected on days 7 (A and B) and 11 (C and D) post acute liver injury. .................................. 98 5 -13 Comparative histological exam of H&E stained liver samples collected on days 15 (A and B) and 20 (C and D) post acute liver injury ................................. 99 5 -14 Comparative histological exam of Hematoxylin and Eosin stained control samples ............................................................................................................... 100 5 -15 Normal rat liver: the low levels of cytoplasmic glycogen are not visible by PAS staining. ........................................................................................................ 101 5 -16 Comparative histological exam of PAS stained liver samples collected on day 11 post acute liver injury ...................................................................................... 102 5 -17 Ki67 imm unostaining of normal rat liver ............................................................ 103 5 -18 Ki67 immunostaining of rat liver sections collected on day 9 post hepatectomy ......................................................................................................... 104 5 -19 Comparative quantitative image analysis of hepatic proliferation potential during compensatory hyperplasia. ....................................................................... 105 5 -20 D ouble IF for Ki67 and desmin on normal liver sections. ................................... 106 5 -21 Double IF for Ki67 and desmin on liver sections collected 9 days post acute hepatic injury. ....................................................................................................... 107 5 -22 AFP immunostaining of normal rat liver .............................................................. 108 5 -23 AFP immunostaining of rat liver sections collected on day 9 post hepatectomy. ........................................................................................................ 109 5 -24 Comparative quantitative image analysis of AFP po sitive cell presence. .......... 11 0 5 -25 Real time PCR analysis comparative expression of AFP ................................ 111 5 -26 OV6 immunostaining of normal rat liver .............................................................. 112
11 5 -27 OV6 immunostaining of rat liver sections collected on day 9 post hepatectomy ......................................................................................................... 113 5 -28 Comparative quantitative image analysis o f OV6 positive cell presence ........... 114
12 LIST OF ABBREVIATION S AA Allyl alcohol AAALAC American Association for the Accreditation of Laboratory Animal Care 2 -AAF 2 acetoaminofluorene AFP -fetoprotein Akt Protein kinases B family ALB Albumin AP-1 Activator protein 1 APS Ammonium persulfate bp Base pair BD bile duct branch BD1 Bile duct marker 1 BDS7 Bile duct specific marker 7 BrdU Bromodeoxyuridine C Cytoplasm CCC Cholangiocellular carcinoma CCl4 Carbon tetrachloride CDE choline deficient ethionine cDNA Complementary deoxyribonucleic acid Cdk2 Cyclin dependent kinase 2 Cdk4 Cyclin dependent kinase 4 CK Cytokeratin CK -19 Cytokeratin 19 CTGF Connective Tissue Growth Factor
13 DAB Diaminobenzidine DAPI 4',6-diamidino -2 phenylindole DAPM Methylene dianaline DEPC Diethyl pirocarbonate DHHS Department of Health and Human Services DMN Dimethylnitrosamine DNA Deoxyribonucleic acid DNase I Deoxyr ibonuclease I dNTP Deoxynucleotide triphosphate DTT Dithiothreitol ECM Extracellular matrix EDTA Ethylenediaminetetraacetic a cid EC Endothelial cell EGF Epidermal growth factor Egr1 Early g rowth r esponse p rotein 1 ERK Extracellular -signal regulated kinase ES Embryonic stem cells ET -1 Endothelin 1 F AK F ocal a dhesion k inase FBS Fetal bovine serum FDA Food and Drug Administration FGF Fibroblast growth factor FITC Fluorescein isothiocyanate
14 G0 Quiescent phase of cell cycle G1 Growth 1 phase of cell cycle G1/S cell cycle checkpoint between growth 1 and synthesis phases GAPDH Glyceraldehyde -3 p hosphate d ehydrogenase GFAP Glial fibrillary acidic protein GH -RH Growth hormone releasing hormone GGT Gamma glutamyl transpeptidase H Hepatocyte HA He patic artery branch HCC Hepatocellular carcinoma H&E Hematoxylin and eosin staining HES6 Hairy and enhancer of split 6 HGF Hepatocyte Growth Factor Hr Hour HSC Hematopoietic stem cells IACUC Institutional Animal Care and Use Committee IF Immunofluorescence IGF-2 Insulinlike growth factor 2 IHC Immunohistochemistry IL -1 Interleukin 1 i.p. Intraperitoneal JNK c -Jun N -terminal kinase M Membrane MAPK Mitogen activated protein kinase
15 MCP 1 Monocyte chemotactic protein 1 Me -DAB 3 methyl 4 -dimethylaminoazobenzene min Minute MMP Matrix metalloproteinase MnSOD Manganese superoxide dismutase MODS Multiple organ dysfunction syndrome mTOR M ammalian target of rapamycin n Number of animals included in an eperiment N Nucleus NFkB N uclear factor kappa -light -chain enhancer of activated B cells NIH National Institute of Health NK Natural killer cells N -OH 2AAF N hydroxyl 2 acetylaminofluorene NPC Non parenchymal cells NRL Normal rat liver Nt Nucleotide OC Oval cell OCT Optimal cutting temperature OD Optical density oligodTs oligodeoxythymidylic acid OLT Orthotopic liver transplant O/N Overnight p p value
16 PAS Periodic acid Schiff staining PBS Phosphate buffered saline PCR Polymerase chain reaction PDGF Platelet derived growth fa ctor PDGFR -b Platelet derived growth factor receptor beta PHx Partial hepatectomy PHS Public HealthServices PI3 -K Phosphatidylinositol 3 kinase PT Portal triad PV portal vein branch Ras Small GTPase involved in signal transduction Rb Retinoblastoma protein RBC Red blood cell RNA Ribonucleic acid mRNA Messenger RNA ROS Reactive oxygen species RPM Revolutions per minute RT Room temperature rt PCR R everse transcription polymerase chain reaction RT PCR Real time quatitative polymeras e chain reaction RT PCR Real Time PCR SC Stellate cell SCF Stem cell factor SDF1 Stromal derived factor 1
17 sec Second SHC Small hepatocyte-like cell -SMA Alpha smooth muscle actin Sp -1 Transcription factor SREBP1c Sterol regulatory element binding protein1c STAT3 Signal transducer and activator of transcription 3 TBE Tris -Borate -Edta Buffer TBS Tris -buffered saline TBST Tris buffered saline with 0.1% Tween TEMED Tetramethylethylenediamine TGF TGF Transforming TIMP Tissue -specific inhibitor of matrix metalloproteinases TNF Tumor necrosis factor v/v Volume per volume w/v Weight per volume
18 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy HEPATIC STELLATE CELLS INVOLVEMENT IN PROGENITOR MEDIATED LIVER REGENERATION By Dana Gabriela Pintilie December 2009 Chair: Bryon E. Peteresen Major: M edical Sciences The close association between hepatic stellate cells and oval cells has been observed during 2acetylaminofluorene/70% partial hepatectomy (2AAF/PHx) induced, progenitor mediated liver regeneration. Various interactions between these two cell populations have been proposed, including pro regeneration cytokine secretion and synthesis of a proliferation and differentiation conducive ECM (extracellular matrix). Previous studies conducted by our laboratory have demonstrated that suppression (transforming gr owth factor beta) mediated up-regulation of connective tissue growth factor ( CTGF ) by prostacyclin analogue iloprost resulted in a greatly diminished oval cell response to 2A AF/ PHx in rats. We hypothesized that this effect is due to decreased activation o f hepatic stellate cells. In order to test this hypothesis, we maintained rats on a diet supplemented with 2% L-cysteine as a means of inhibiting stellate cell activation during the oval cell response to 2AAF/PH x In vitro Bromodeoxyuridine (BrdU) incorp oration assay, reverse transcriptase polymerase chain reaction ( rt PCR ), real time polymerase chain reaction ( RT -PCR ), Immunofluorescent staining (IF), immunohistochemistry (IHC) and quantitative computer image analysis
19 were utilized to analyze the involvement of hepatic stellate cells in liver injury and oval cell activation. In vitro experiments demonstrated that L-cysteine did, indeed, prevent the activation of stellate cells while exerting no direct effect on oval cells. Desmin immunostaining of liver sections from 2AAF/PH x animals indicated that maintenance on the L-cysteine diet resulted in an 11.1-fold decrease in the number of activated stellate cells within the portal zones. The total number of cells proliferating in the portal zones of livers from animals treated with L-cysteine was drastically reduced. Further analyses demonstrated a four -fold decrease in the magnitude of the oval cell response in animals maintained on the L-cysteine diet as determined by immunostaining for both OV6 and AFP (alpha-fetoprotein) Global liver expression of AFP as measured by RT PCR was shown to be decreased 4.7-fold in the L-cysteine treated animals. These data indicate that the activation of hepatic stellate cells is required for an appropriate oval cell response t o 2AAF/PH x
20 CHAPTER 1 INTRODUCTION 1.1 Uncovering the Interactions between the Hepatic Stellate Cells and Oval Cells in Regenerating Liver The first scientific data which started to shed light on the regenerating potential of stem cells emerged in the second half of 20th century. Once the scientific world learned about the mechanisms of graft rejection, it became clear that stem cells have a real thera peutic potential. Further characterization led to discovery of the existing differences between the embryonic and adult stem cells and to the development of the first successful stem cell therapy. The clinical results quickly exceeded the initial expectati ons and encouraged the attempts to characterize, cells with stem potential isolated from various organs. The emerging gene therapy studies made stem cells the ideal candidates for correcting inborn errors of metabolism, whilst regenerating the diseased o rgan. The progresses registered in the stem cell research are encouraging signals for scientist who believe that in the near future novel stem cell therapies would be available for clinical trial. Due to their tremendous therapeutic potential, the discover y of stem cells has proven to be one of the most important scientific advances of the last century. Once the organ specific stem cells were identified and characterized from a morphological standpoint, the key to their therapeutical use became the underst anding of the complex molecular processes governing their functions, differentiation and interactions with a particular site of origin. It quickly became evident that a thorough characterization requires a lot of time and effort, due to the tremendous hete rogeneity of stem cell population and organ specific microenvironments. Several stem cell phenotypic markers have been proposed, but their use for cell labeling and lineage
21 tracing has its limitations. The alternative introduction of extraneous markers thr ough genetic manipulation could potentially interfere with the cells naturat fate. The isolation of stem cells takes them out of their natural environment and the chemical compunds able to manipulate their fate in vitro introduce variations in the cellular processes. Although imperfect, the current techniques have uncovered a considerable body of evidence which can provide a better understanding of stemness, differentiation, morphology, physiology and stem cell interactions with their environment. Currently scientists are striving to characterize the organ specific stem cells in relation with each step of their differentiation. In addition, effort is put in discovering the mechanisms responsible for maintaining the stemness versus promoting the different iation toward a specific fate. A detailed characterization of the the patways and their specific molecular signals is a mandatory step for developing stem cell therapies. Since its introduction in 1963, liver transplantation has become a widely accepted th erapy, able to increase the life expectancy of patients suffering of terminal hepatic failure. Unfortunately, the donor shortage has limited its patient accessibility. The discovery of liver specific oval stem cell gave hope to terminally ill patients in d ire need of a new liver. The perspective of an alternative stem cell therapy able to regenerate failing liver and correct the inborn metabolic diseases has accelerated the efforts toward understanding the hepatic stem cell. At this stage, the immediate goal is to expand the molecular characterization of various subpopulations in terms of stemness, differentiation fate, and in vitro manipulation toward a specific phenotype. Uncovering the mechanisms governing these processes would open the door to successful ly develop an alternative stem cell therapy.
22 This project was designed to further understand the cell -cell interactions that g overn oval cell proliferation, migration and differentiation toward a hepatic lineage. Previous work ha s demonstrated the close p hysical proximity between the oval and stellate cells during liver regeneration. Stellate cells are known to be activated early after injury and subsequent changes in ECM (extracellular matrix) and cytokine levels have been observed. Through IHC, protein, and RNA analysis a correlation between the stellate cell activation and the specific profile of oval cell based liver regeneration was established. Chemical i nhibition of stellate cell activation both in vitro and in vivo resulted in an abnormal profile of the regenerative proces s, delayed and blunted, involving phenotypic changes of regenerating cells This project outlined the requirement of certain intercellular interactions for achieving the effective amplitude of oval cell response. Without a robust contribution from stellate cells oval cells proliferation was reduced and delayed, failing to migrate towards the centrolobular area, differentiating instead into small hepatocyte-like cells. This study only opens the door to a better understanding of th e intercellular interactions required by oval cell mediated liver regeneration. In addition, further studies would characterize the molecular and biochemical profile of the microenvironment created by the stellate oval cell interactions
23 CHAPTER 2 BACKGROUND AND SIGNI FICANCE 2.1 The Liver 2.1.1 Liver Anatomy 188.8.131.52 Structure of the hepatic organ Human liver is the largest parenchymal organ in adults, functions as an annex gland for the digestive tract and is the site of a vast number of metabolic processes. Weighing between 1400 and 1600g, it represents approximatively 2% of the total body weight. Rat liver, although very similar functionally, weighs 7 to 8g which accounts for a greater percent of the body weight1 (approximately 5%) The complex h epatic functions are possible in part, due to the particular dual blood supply of the liver. Almost 75% of the afferent circulation is supplied by the portal vein, carry ing venous poorly oxygenated blood loaded with nutrients absorbed in the gastro -intes tinal tract3. The remaning 25% of the afferent blood supply is provided by the hepatic artery which brings oxygenated blood, with poor nutrient content, from the celiac trunk. Once reaching porta hepatis these two main vessels subsequently divide into min or branches which ultimately reach the portal spaces. Both the portal and arterial blood enter the fenestrated sinusoids of the liver lobules, where an active exchange of metabolites takes place, mediated by the space of Disse (Figure 2-1) Sinusoidal blood converges toward the central venule of the liver lobule, to be further drained into the supralobular veins. It finally reaches the inferior vena cava, via the collecting and hepatic veins. Parallel to the blood vessels but opposing direction of flow, the biliary tree consists of excretory ducts that transport bile into the duodenum. The portal vein, hepatic artery and biliary tree form a central vascular bundle termed the
24 portal triad. The particular branching of hepatic vessels and biliary tree underlines the hepatic functional and surgical architecture. Human liver is formed by a right lobe (segments IVa, IVb, V, VI, VII and VIII), a left lobe (segments II and III), a quadrate lobe (segment IV), a caudate lobe (segment I), a caudate and a papill ary process4. The rat liver is composed of five lobes (right lateral, left lateral caudate and right medial incompletely separated from left medial) and a caudate process. Compared anatomy homologates left lateral lobe to human segments II, III, the right lateral lobe to segments VI, VII, the left medial to segments IV a, b the right medial lobe to segments V, VIII, whilst the caudate lobe together with the caudate process correspond to segment I5. 184.108.40.206 Microarchitecture of the liver There are three co nceptual interpretations of the functional hepatic architecture (Figure 22) : the classic hepatic lobule, based on endocrine function and structural parameters; the portal lobule, based on the exocrine function ( bile drainage pathway from adjacent lobules toward the same bile duct ); and the liver acinus concept, based on the gradient distribution of oxygen along the venous sinusoids in adjacent lobules2. The classic hepatic lobule is a hexagonal structure formed by cords of hepatocytes radiating from a cent ral venule ( which drains the sinusoids ) toward the portal triad. At the angles of the hexagon are situated t he portal triads : a bile duct together with terminal branches of portal vein and hepatic artery. A single portal triad forms the central axis of the portal lobule and drains bile from the surrounding hepatic parenchyma. The liver acinus concept is the most useful for understanding the liver regeneration patterns. It is defined as the parenchyma between two adjacent central veins, supplied by a termina l branch of hepatic artery. The flow of arterial and venous blood within the hepatic sinusoids creates gradients of oxygen and nutrients which
25 differentiate the three acinar zones of perfusion. Zone I, adjacent to the portal triad, is the richest in oxygen and nonmetabolized nutrients. Periportal hepatocytes specialize in glycogenolysis and gluconeogenesis and remove ammonia by producing urea. Zone III, closest to the central vein, is oxygenpoor, but rich in metabolism products. Centrolobular hepatocytes are active in glycolysis and glycogen synthesis and remove ammonia through glutamine synthesis. Zone II is intermediate in oxygen and nutrients. Within the liver acinus, blood flows through sinusoids from Zone I to Zone III, and the bile moves from Zone III to Zone I. Interestingly, hepatocytes within Zone III have an increased DNA (deoxyribonucleic acid) content (4N to 16N), predominant bi nucleation, large size and can undergo centrilobular necrosis. Conversely, hepatocytes within Zone I are smaller and us ually single nucleated (2N)6. Opposite to the blood, bile flows from the hepatocytes to the bile duct in the portal space. The smallest bile ducts lined by epithelium, named the canals of Hering, are situated at the periphery of the hepatic lobule. There are about 800 million cells in the liver. Hepatocytes, which represent 7090% of liver mass, are the functional exocrine and endocrine cells of the hepatic lobule. They have a life span of 180 400 days7 and most of them are able to sustain about 69 86 d oublings8, 9. Hepatocyte division results in compensatory hyperplasia, which restores the liver mass after injury. They form one cell thick anastomozing plates flanked by two sinusoidal spaces. The hepatocytes cord adjacent to the portal space forms the o uter limit of the hepatic lobule and is therefore named the limiting plate. Gap and tight junctions on lateral surfaces of adjacent hepatocyte membranes enable the
26 intercellular functional coupling in the plates and separate the basolateral from the apical membrane domain. The basolateral domain has abundant microvilli which protrude in the space of Disse and are actively involved in the exchange between hepatocytes and blood. The excess fluid in space of Disse is collected by the space of Mall, which is d rained by lymph vessels piercing the limiting plate. The basolateral domain is responsible of both absorption of blood -borne substances and secretion of plasma proteins synthesized by the hepatocyte. The apical domain is a membrane depression also lined b y microvilli. Occluding junctions connect the apical domains of adjacent hepatocytes sealing the bile canaliculus which drains the exocrine product of hepatocytes. The abundant rough endoplasmic reticulum of hepatocytes synthesizes plasma proteins (albumin fibrinogen, prothrombin, coagulation factors V, VII, IX), whilst the highly developed smooth endoplasmic reticulum is involved in cholesterol and bile salts synthesis, glucuronide conjugation of bilirubin, steroids and drugs, breakdown of glycogen into glucose, esterification of free fatty acids to triglycerides, removal of iodine from triiodothyronine and thyroxine hormones, detoxification of lipidsoluble drugs2. Associated with the hepatic microsomal membrane resides the cytochrome P450 (CYP 450) enzym atic complex which catalyzes a variety of reactions including monooxygenase reaction/hydroxylation, S oxydation, epoxidation, N dealkylation, O dealkylation. This family of hemeproteins is responsible for hepatic biotransformation and detoxification of a l arge number of exogenous and endogenous substances,
27 ranging from drugs and toxic compounds to bilirubin10, and also for cholesterol synthesis. Bile produced by hepatocytes is secreted into the intercellular spaces known as bile canaliculi, hence flows into cholangioles, thin intralobular ductules which drain into the terminal ductules named canals of Hering. The later are lined by squamos to cuboidal simple epithelium and exit the lobule through the limiting plate to open into the bile duct of the portal triad. The cuboidal epithelium lines all the intrahepatic bile ducts and expresses CK 19 (cytokeratin 19) marker. The liver sinusoids are lined by two cell types: fenestrated endothelium and Kupffer cells which are the liver resident macrophages. They lie in between or on top of endothelial cells but their cytoplasmic extensions allow direct cell -to -cell contact with the parenchymal hepatocytes. K upffer cells are specialized mainly in phagocytosis of hepatic cellular debris and blood borne pathogens but their morphology and biocapacity is variable, depending on their location. The periportal ones are larger and more active in phagocytosis, whereas the smaller centrolobular ones are more active in cytokine synthesis11. Also adherent to the sinusoid endothelium reside the pit cells, hepatic NK -cells cytotoxic for metastatic tumor cells that reach the liver via the portal venous system. Their functions are dependent on Kupffer cells which control their immunity, adherence, viability and cytotoxicity12 and together they are responsible of livers immune defense. Containing the largest population of macrophages in the body, liver is a potent modulator o f systemic immune response to severe infections13. Kupffer cells interact with neutrophils and T -cells being both antigen presenters and producers of TNF -
28 ( IL1 ( Interleukin 1) IL6 (Interleukin-6) and cytokines which induce the expression of acute phase proteins14. There is definite proof of Kupffer cells involvement in liver regeneration ( vide infra) helped by the bioactive mediators they express: TGF 1 2, 3), latent TGF protein 1, 215, TGF14), TIMP 1( Tissue -specific inhibitor of matrix metalloproteinase 1) 16 and TIMP -2 (Tissue -specific inhibitor of matrix metalloproteinase 2) 17. 220.127.116.11.3 Biology and clinical s ignificance of hepatic stellate cell Hepatic stellate cells, also known as the Ito cells, reside in the space of Disse in proximity to the hepatic sinusoids in close contact with hepatic regenerating cells18. They are in contact with the blood stream and regulate the sinusoid vascular tone19. Hepatic stellate cells are in direct contact with endothelial cells and interact with parenchymal cells via microspines extending from their cytoplasmic processes20. Stellate cells account for one-third of the nonpar enchymal cell population in normal liver21. They are spindle-shaped with oval or elongated nuclei and located in the space between the basolateral surface of hepatocytes and the ablumenual side of sinusoidal endothelial cells. Besides storing vitamin A, they are producing and secreting cytokines and fibers which form the hepatic ECM (extracellular matrix). Stellate cells can recruit inflammatory cells through expression of adhesion molecules, secretion of chemokines and cytokines and hence play regulatory r oles in liver inflammation. During tissue remodeling, quesicent stellate cells become activated and transdifferentiate into myofibroblast -like cells. They lose retinoids and become very proliferative, fibrogenic and contractile22. Activated stellate cells are also an important source of cytokines and synthesize a large amount of extracellular matrix components
29 including collagen, proteoglycan and adhesive glycoproteins. The production of ECM component is mediated through integrin -dependent signaling events including focal adhesion kinase, tyrosine kinase and ERK ( Extracellular activation. Transforming growth factor (TGF -SMA (Alpha smooth muscle actin) and adhesive receptors and the increasing synthesis of extracellular matrix molecules such as fibronectin. Normally, activated stellate cells ensure a finetuning of the woundhealing response according to the duration, the type and t he amount of damage, through extra cellular matrix production. However, overexpression of extracellular matrix components by these cells causes hepatic fibrosis. Stellate cells express some phenotypic markers such as desmin and alpha smooth muscle actin, b oth specific for activated stellate cells. Interestingly, they also express neuroendocrine markers including glial fibrillary acidic protein, neural cell adhesion molecule, the class VI intermediate filament protein nestin and synaptophysin23, 24. Such cha racteristics raise the question of a possible neural crest origin of stellate cells. These specialized hepatic pericytes of mesenchimal origin25 contain fat and retinyl palmitate in their cytoplasm, storing 80% of vitamin A body reserves. Under physiologic al conditions SCs (stellate cells) are quiescent, exhibiting a star like phenotype (Figure 23) Their activation is followed by proliferation and transdifferentiation into highly contractile myofibroblast like cells. Stellate cell activation is an essenti al step in both liver regeneration and in the initiation of liver fibrosis. The resulting myofibroblastic phenotype is represented by lipid droplets and vitamin A
30 reduction, increased collagen desmin -smooth muscle actin synthesis followed by actin c ytoskeleton reorganization and ECM deposition. Activated stellate cells proliferate and secrete cytokines and ECM proteins. They migrate to the liver injury site acting as specialized wound healing cells. Initially surrounding the hepatic necrotic areas, hepatic stellate cells appear enlarged; then they migrate amongst the oval cells and newly regenerated hepatocytes, emitting numerous extensions and preceding the migration of endothelial cells that will generate the sinusoidal walls. The stellate cell act ivation is a sequential process. It is initiated by transcriptional events, paracrine stimulation by Kupffer or other neighboring cells and early ECM changes. Subsequent cytokine secretion, receptor tyrosine kinase upregulation and accelerated ECM remodeli ng ensure its perpetuation. Once the healing is complete, the activation stops. It is not clear yet if its resolution is a process of reversion or if the activated hepatic stellate cells undergo apoptosis (Figure 24). Under pathological circumstances, th e activation process perpetuates and the increased deposition of collagen fibers and ECM within the space of Disse is followed by a loss of fenestrations and gaps between sinusoidal endothelial cells. As the fibrotic process advances, increasing numbers of hepatic stellate cells transdifferentiate into myofibroblasts, constricting the sinusoids lumen and increasing the vascular resistance inside the liver lobules. Together with the dissecting fibrous bands that distort the lobular architecture, the abnormal ly high resistance to blood flow in the hepatic sinusoids leads to portal hypertension, a complication of advanced cirrhosis19.
31 Almost 465 genes are highly expressed in activated stellate cells under various conditions26. Among the products synthesized by Ito cells, hepatocyte growth factor (HGF)27 and its receptors28, stem cell factor29 (SCF), platelet derived growth factor30 (PDGF), endothelin 1 (ET growth factor (CTGF) and its receptors, monoc yte chemotactic protein 1 (MCP1) are effectors known to be involved in autocrine, paracrine and endocrine interactions which result in liver healing and regeneration. TGF to latent TGF -d into the ECM where it is activated and exerts its matricrine action31. Many ECM components are secreted by Ito cells: collagen type 1, 3, 4, 14, and 1832, decorin and perlecan, fibronectin, dermatan and heparan sulfate, syndecan33, entactin, tenascin, l aminin and proteoglycans34. Hepatic stellate cells are also involved in a continuous process of ECM remodeling intermediated by their secretion products with antagonistic functions: matrixmetalloproteinases MMPs 1, 2, 3, 9, 10, 13, 1417, 35 and tissue spec ific inhibitor of matrix metalloproteinases TIMP 1, 236. These changes in protein expression are accompanied by a distinct phenotype and a specific migration pattern. Either hepatocyte, or stem/oval cell mediated, liver regeneration is always accompanied or preceded by stellate cell activation. Closely associated with stellate cells and surrounded by their processes, the oval stem cells seem to be nurtured by the non-parenchymal cells. Trough direct cell -to -cell interactions, growth factors and cytokine se cretion, stellate cells are thought to promote oval cell growth and differentiationetion 18.
32 ECM is composed of various macromolecules, including fibronectin, collagen, laminin, vitronectin, and proteoglycans. It functions as the physical support for epith elial and endothelial cells and modulates cell differentiation, migration, growth, and apoptosis. Liver ECM is distinct from other organs where ECM acts as a diffusion barrier between plasma and the epithelial cells and forms a basement membrane. Lacking a true basement membrane, the liver has ECM in the perisinusoidal space of Disse, which separates the basolateral domain of the hepatocyte membrane from the blood circulating in the endothelium lined hepatic sinusoid. The specific organization of the 0.2 proteins and other nutrients between circulating blood and hepatocytes. The ECM fibers of the perisinusoidal space are collagen (types I, III, and VI), laminin, entactin, perlecan, and the most abundant, insoluble fibronectin. Hepatocytes are the source of both soluble, circulating plasma fibronectin and insoluble, cellular, ECM form of fibronectin secreted during liver regeneration. Laminin, consisting of three distinct chains, is normally detected in the perisinusoidal space, large blood vessels, and biliary ducts. Although there are several lines of evidence suggesting that the laminin subunits are expressed in normal liver and regenerating liver, the exact composition of these l aminin subunits in normal liver and in regenerating liver remains unclear37. The ECM components interact with cells through cell surface heterodimer integrin receptors, a family of transmembrane proteins 37. During oval cell medi ated liver regeneration there is a constant remodeling of the ECM, which becomes more permissive for migration and facilitates cell to -cell
33 interactions. In this respect, ECM acts as a regulator of cell's dynamic behavior. ECM proteoglycans sequester a wid e range of growth factors which are released and activated after protease cleavage38. 2.1. 2 Liver Physiology 18.104.22.168 Metabolic h omeostasis Liver is an annex gland of the digestive tract It is also the site of multiple metabolic processes, thus playing a m ajor role in homeostasis. Being the main site of protein synthesis, it maintains the colloid osmotic pressure of the blood by producing the most abundant protein in the plasma, albumin. Other important plasma proteins such as lipoproteins, glycoproteins, prothrombin, fibrinogen and coagulation factors V, VII, IX, X, XI and the nonglobulins are synthesized by the liver. Thus, liver is the major regulator of coagulation process in the body. It also catabolizes the aminoacids by deamination an d conversion of resulting ammonia into urea39. Liver regulates the plasma levels of glucose: by converting it into its storage form, glycogen; thus reducing the glicemic level. When the energetic needs are increased, it cleaves the glycogen into glucose, i nducing a rise of glycemia ( vide infra ). 22.214.171.124 Storage Together with the muscular tissue, liver is the major deposit of glucose, stored in hepatocytes as glycogen. Whenever the energetic needs of human body increase, glycogen is cleaved and resulting gl ucose is oxidized to water, carbon dioxide and ATP. Also, the glucose released into the blood stream maintains the normal glycemic levels. Several vitamins are stored and converted into metabolically active forms in the liver. 80% of vitamin A reserves are in the stellate cells cytoplasmic lipid droplets. Vitamin B12, copper and iron reserves are also located in the liver. By storing and
34 releasing the iron in a metabolically active form, liver is an important regulator of its homeostasis. Because of its in volvement in iron metabolism, liver is the first organ affected in conditions associated with iron overloading. As part of the metabolic chain resulting in synthesis of biologically active vitamin D, liver plays an important role in rickets and osteomalaci a prevention. Vitamin D3 is converted into its circulating form 25 -hidroxy cholecalciferol, thus being available to the kidney for further transformation. Vitamin K absorbed in the colon is used by the liver for blood clotting factors synthesis, thus suppl ying the necessary proteins for a physiological coagulation cascade39. 126.96.36.199 Blood reservoir Hepatic particular capillary network and vascular supply associated with its large parenchymal mass make possible the storage of a large volume of blood. Liver has a dual afferent blood supply: 75% of hepatic blood comes from the portal vein, and 25% of it from hepatic artery. By receiving mixed (venous and arterial blood) the liver is able to store 25 30% of cardiac output and hold up to 1500 ml of blood39. 2. 1.2.4 Detoxification Acting as a waystation between the splanchnic and systemic circulation, liver receives xenobiotics which are detoxified by its cytochrome P 450 enzymes. The resulting water soluble products are excreted into urine by the kidney. Alcohol; dehydrogenase and isoforms of uridine diphosphoglucuronate glucuronosyltransferase (UGT) allow for the biotransfor mation of exogenous and endogenous toxic compounds. Conversion of ammonia into urea is such an example of endogenous waste product detoxification. Bilirubin degradation products and cholesterol are excreted through the bile into the small intestine.
35 2.1.2. 5 Liver endocrine functions Liver is considered a mixed gland. Its endocrine function cosists in synthesis and secretion of proteic products, as part of its metabolic homeostasis function. It has a significant impact on hormonal homeostasis by being part o f the metabolic pathways of degradation of hormones produced by other glands, such as estrogens, insulin and glucagon. It also controls the hormonal functions by regulating their secretion and activation3. Liver is an important source of GH RH (growth horm one releasing hormone) and converts thyroxine into triiodothyronine. 188.8.131.52 Liver exocrine function Liver is described as an annex gland of the GI tract due to its bile secretion. Collected by the intrahepatic biliary ducts, it is stored in the gallbladder and reaches the duodenum during the meals. Acting as a detergent, bile emulsifies the intestinal lipids and contributes to their absorption. Cholesterol secretion into the bile is its only mechanism of excretion in the human body. 184.108.40.206 I mmune defense Being a way station between the splanchnic and systemic circulation, liver receives, via the portal blood, toxins and pathogens which escape the enteric barrier into the bloodstream. Rich in Kupffer cells (resident macrophages) and pit cells (hepatic NK ly mphocytes) it is able to remove actively the antigens and act like a sieve for the portal blood. Kupffer cells are the hepatic resident macrophages responsible for phagocytosis of hepatocyte necrotic products and aged red blood cells. Pit cells, the Kupffe r cell dependent NK (Natural killer) cells phagocytose neoplastic cells coming to the liver from the digestive tract.
36 2.1.3 Liver Regeneration There are two distinct patterns of inducing hepatocyte proliferation: compensatory regeneration following liver mass loss and mitogen induced direct hyperplasia in the absence of significant cell loss40. Compensatory hyperplasia, generally and, in the absence of a liver blastema, improperly referred to as liver regeneration occurs after partial hepatectomy and hepat ocellular necrosis. Direct hyperplasia is a process of hepatocyte proliferation triggered by various chemicals which do not cause liver necrosis (Figure 2-5). Liver regeneration is the result of the interplay between two major sets of events: the adaptive changes elicited by the metabolic and vascular imbalance and the mitogenic changes which trigger the hepatocyte transition from a quiescent to a replicative state. This process requires the activity of multiple interacting pathways grouped into cytokines, growth factors and metabolic networks. Within minutes after a partial hepatectomy, hepatocytes exit G0 and enter G1. This so called priming phase governed predominantly by the cytokine network debuts with the enhanced expression of the early genes: c -fo s, c -jun proteins (components of AP 1, activation factor 1), c myc uclear factor kappa-light -chain enhancer of activated B cells ), Egr1 ( Early g rowth r esponse p rotein 1), STAT3 (Signal transducer and activator of transcription 3 ), HGF ( Hepatocyte Growth Factor ), TGFTransforming Growth ), IL 6 ( Interleukin -6) transcribed in the first 30 minutes 2 hours post injury. However, the cell cycle progression requires growth factors, responsible for overriding the G1/S c heckpoint (between growth 1 and synthesis phases of cell cycle). High levels of EGF ( Epidermal growth factor) and HGF ( Hepatocyte growth factor) are associated with increased expression of cyclins D, E, A. Phosphorylation of overly
37 expressed Rb ( Retinoblas toma protein) allows the formation of cdk4/cyclinD (Cyclin dependent kinase 4/cyclin D) and cdk2/cyclinE ((Cyclin dependent kinase 2/cyclin E) complexes. Although it is difficult to identify the mechanisms modulating the liver growth in accordance to the needs of the whole organism, it is generally accepted that the increased metabolic demands imposed on the liver remnants post injury are activating the DNA replication. Amino acids are known to activate the mTOR pathway responsible of sensing the nutrient and energy status, integrating growth factor signals and regulating the cell growth41. This metabolic regulation is also responsible for controlling the hepatocyte mediated liver regeneration. Compensatory hyperplasia is triggered by several damaging fact ors ranging from surgical resection to hepatotoxins and chronic viral infection. In response to the injury induced by these aggressors, the parenchymal loss is compensated by an active process of hepatocyte division. Despite their minimal replicative activ ity in the absence of injury, hepatocytes are able to restore the liver mass after hepatic necrosis. Figure 26 (adapted after the first depiction, by Higgins and Anderson, of the residual hepatic lobes growth after 70% partial hepatectomy43) shows the res toration of liver mass without the recovery of the original hepatic anatomy. Compensatory hyperplasia progresses in a centripetal direction inside the liver lobule, first hepatocytes to exit G0 (quiescent phase) and enter G1 (Growth 1 phase) being situated in the periportal areas. Mediated by cyclin D1 pathway activation44, 45, hepatocyte proliferation spreads gradually towards the centrolobular vein42, 46. According to Michalopoulos45 the DNA synthesis profile post 70% partial hepatectomy
38 has a camel back shape with an initial high peak after 1 day and a second small peak another day later. This particular pattern is caused by an initial hepatocyte proliferation in zones I and II, followed by non -parenchymal cells (NPC) and zone III hepatoc ytes division, as shown in Table 2 1. Whilst hepatocyte division is progressive, NPCs exhibit a simultaneous proliferation across the acinar zones. The original liver mass is usually restored within 2 weeks of the hepatectomy. Several hepatotoxins have been used to study the mechanisms of compensatory hyperplasia in the liver, amongst them CCl4 (carbon tetrachloride) and AA (allyl alcohol). Despite variations in necrotic area location47, 48, 49, and 50, in both animal models the hepatocytes are responsible for regeneration, without any contribution from other cell populations. It was later discovered that hepatocytes are not the only regenerating resource for damaged liver. When hepatocytes are severely impaired by pathogens and toxins, making impossible th eir division, a facultative regenerating cell population is activated. The hepatic stem cell compartment reacts to the inability of liver to recover its mass loss by multiplying in an amplification compartment represented by the oval cells. Although several data about oval cells origin have emerged lately, less in known about the molecular mechanisms which trigger oval cell proliferation and determine their lineage fate. 2.2 Stem Cells and Their Therapeutic Potential The interest in regeneration has been present for quite some time in the scientific world, triggered by observations made in freshwater hydra and axolotl which are able to regenerate parts of their bodies. Although few recordings from the 1908 Congress of Hematologic Society in Berlin51 have survived, we now know52 that Alexander Maximov heralded the existence of stem cells and even coined the term of stemzelle51. As a
39 result of his studies on blood cell lineages he hypothesized the existence of a pluripotent white blood cell, able to differen tiate into a different cell type51. The interest in stem cells soared after the devastating effects of atomic radiation on blood cells were discovered in the mid 20th century. The concept of stem cell therapy in the form of bone marrow transplantation date s from the same period, but the first results were utterly disappointing because of the little knowledge on immune compatibility. The stem cells were first identified in blood and characterized by James Till and Earnest McCulloch53, 54, work that has dubbed them the fathers of stem cell research. Discovering the proteins that enable stem cells to differentiate and mature, Till and McCulloch made possible the quantitative analysis of a single hematopoietic stem cell. Their researched revolutionized the success of bone marrow transplants. Further research55, 56 showed that a single cell type was able to repopulate bone marrow and set the basis for the current research boom in the field of stem cell therapy. Based on the emerging research, stem cells were de fined as multipotent cells able to self renew and differentiate into cells of various germ layers. They are located both in embryonic and adult tissues.Research data emerging in the early 1980s shed light on the clonogenicity and totipotential nature of t he embryonic stem cell and emphasized the differences between them and the adult stem cells. The only truly totipotent cells are found in the fertilized egg. To this date, many adult and embryonic stem cells were proven to be multipotent. However the differentiation capacity of stem cells is not fully described. Embryonic stem (ES) cells isolated from the inner cell mass of blastocysts58 60 are able to differentiate into cells originating from all three embryonic layers (ectoderm,
40 mesoderm and endoderm). Under specific culture conditions and in vivo ES were induced to differentiate into cells of mesoderma; origin: hematopoetic, hemangioblast, vascular, cardiac, skeletal muscle, chondrogenic, osteogenic, and adipogenic lineages6271. Endodermal lineages like pancreatic and hepatic cells were derived from ES. Ectodermal lieage differentiation of ES into neurons suggests the tremendous versatility and potential of stem cells for treating degenerative diseases, organ failure and inborn errors of metabolism. Unfor tunately, the ethicall issues and the formation of teratomas indicate that more research needs to be done in order to fully elucidate the nature of stem cells and pave the way for developing stem cell therapies. More data about the differential and self re newal capabilities, the nature of full lineage commitment of stem cells are needed. The discovery of murine embryonic stem cells represented a breakthrough in experimental medicine, offering the chance of isolating cells for replacement therapy. It became possible the development of a bone marrow transplantation protocol, which nowdays represents the greatest success of human stem cell therapy. Adult stem cells proved to have a great plasticity, too. They can be manipulated to differentiate into endodermal, mesodermal and ectodermal cell lines72 74, similar to ES. Reversible differentiation of neural stem cells into hematopoietic lineages was demonstrated75. Human adult bone marrow stem cells were manipulated to differentiate into hepatocytes76, 77 and endothelial cells82, proving for the first time the value of murine models in development of human adult stem cell therapies. In addition, bone marrow stem cells have been shown to participate in neural development83, 84. Further research has uncovered the cap acity of human bone marrow stem cells to differentiate into
41 neurons, intestine, pancreas, skin, skeletal and cardiac muscle cells in human recipients of bone marrow or organ transplants79, 80. Bone marrow mesenchymal cells have also been shown to be plurip otent81. These studies emphasize the broad differentiation spectrum adult stem cells possess. 2.3 Oval C ells B iology and T heir C linical Significance in L iver R egeneration 2. 3 .1 Origin and Morphology of Oval Cells Liver mass restoration post injury requires the coordinate interaction of several cell types. In addition to hepatocytes and non-parenchymal cells the liver contains stem cells which can generate a transit compartment of precursor cells. Named oval cells b y Farber85 who described them in 1956 these tissue specific, liver adult stem cells are able to give rise to both hepatocytes and cholangiocytes86. Embryologically, they are suggested to derive from the ventral foregut endoderm and form the main componen t of the liver primordium87. Initially thought to reside only in the canals of Hering, the traditional liver stem cell niche, there are several lines of evidence proving that bone marrow is an alternative source for the oval cells74,88. Alternative origins for oval cells were reported, starting with Petersen74 who has shown that some of them may actually arise from an extra hepatic source within the bone marrow. Several other investigators have confirmed that bone marrow derived cells possess the ability to produce functioning hepatocytes and bile ductular cells76, 88. Although a number of studies suggest that the oval cells reside in the hepatic parenchyma in close association with bile duct epithelium, the exact oval cell niche has yet to be characterized. Several recently published studies clearly demonstrate that stellate cells within the liver may, through a process of mesenchymal to epithelial transition, give rise to
42 hepatocytes89. It is possible that this phenomenon involves an intermediate cell t ype with oval cell properties. For the moment it is impossible to determine beyond any doubt if mesenchymal to epithelial transition contributes to the oval cell pool seen in the current experimental model s. However, this possibility deserves mention. It is believed that the oval cells constitute a heterogenic cell population which account for 1% 3% of the normal liver cell pool. Their morphological variability made Roskams et al .90 differentiate them into three categories: oval cells, intermediate bile duct -like cells and intermediate small hepatocyte-like cells. The last two populations were described as progeny of the first. Thought to be a reserve compartment activated only when hepatocytes fail to proliferate, the most common conditions involving oval ce lls in humans are: massive acute hemorrhagic necrosis, haemochromatosis, Wilson disease and chronic hepatitis C. During both acute necrosis and fibrotic reorganization stages of the disease a significant number of oval cells are present in the regeneration/proliferation areas of the human liver.Recent data suggest a correlation between the prevalence of oval cells and the fibrosis stage in pediatric patients with chronic hepatitis B91. Morphologically, the oval cell is small (5 id nucleus containing predominantly dense heterochromatin lining the nuclear envelope (Figure 27). The nucleus to cytoplasm ratio is very high compared to the hepatocyte and, in the electron microscopy the scanty cytoplasm appears slightly brighter due to the poorly developed organelles. The tonofilaments in the cytoplasm give them the flexibility and strength needed for their migration and attach on the desmosomes/macula adherens which connect their membranes to the neighboring hepatocytes and cholangiocy tes.
43 This morphology corresponds mostly to the type I oval cell described by Roskams. The intermediate small hepatocyte-like cells are twice bigger than the progenitors, but half the diameter of a mature hepatocyte. Their nuclei, similar to the mature hepatocytes, have less heterochromatin and occasionally nucleoli were reported. Mitochondria, peroxisomes and rough endoplasmic reticulum are visible in the less dense cytoplasm as visible in the electron micrograms published by Sobaniec -Lotowska91. The inter mediary bile duct -like cells have a rounder nucleus, more organelles and tonofilaments in the cytoplasm and microvilli at the apical pole. The small hepatocytelike cells are intermediary in size between oval cells and hepatocytes, have a higher nucleus to cytoplasm ratio due to a larger volume of basophilic cytoplasm. They are very active metabolically and numerous vacuoles filled with lipids are present in the cytoplasm. Oval cells express different combinations of phenotypic markers from both the hepatoc yte and biliary lineage. Similar to cholangiocytes in their cytoskeletal proteins (CK-19) and isoenzymatic profile (gammaglutamyl transpeptidase GGT), they also express alpha feto protein (AFP), OV6, OC2, A6 antigens92. Classical hematopoietic markers Thy 1, c kit (SCF receptor) and CD34 expressed on the surface of oval cells strongly support their bone marrow origin. Table 2 -2 shows a comparative expression of cell markers by various hepatic cell types. 2.3.2 Experimental Models and Mechanisms of Oval Cell Induction Several models of oval cell induction have been developed in rodents, such as choline deficient/ethionine supplemented diet, 2acetylaminofluorine/70% partial hepatectomy, galactosamine and dipin injection. They all have in common the associati on of liver injury with impairment of hepatocyte division, creating a
44 pathophysiological mechanism similar to the severe acute or chronic viral, toxic or drug induced liver pathology in humans. There are some differences though, regarding the onset, phenot ype and location of the regenerating cells. Activated oval cells observed in the above mentioned murine models migrate from the periportal region towards the centrolobular zone, forming primitive ductular structures with poorly defined lumena93. The origin of oval cells remains unclear. According to some reports94 which analyze their involvement in periportal repair, some data suggest that the oval cell niche is located in the periportal area. Although the oval cell presence has not been observed in the reg enerative response to PHx or CCl4 injury, prior administration of 2AAF induces their proliferation in the periportal areas. 2AAF is converted into its N hydroxylated active metabolite by the cytochrome P 450 enzymes in hepatocyte endoplasmic reticulum. After being translocated into the nucleus 2AAF metabolites form voluminous adducts with DNA, rendering it unable to be approached by the transcription apparatus. As a result, cyclin D1 synthesis is inhibited and the hepatocytes are arrested in the cell cyc le at the G1/S checkpoint. Once the hepatocyte proliferation comes to a halt, injured liver mobilizes the alternative regeneration mechanisms resulting in oval cell activation. As with allyl alcohol induced injury, oval cell proliferation in these models begins in the periportal region, followed by migration deeper into the acinar zone I as regeneration progresses. Depending on the site of injury, the migration may stop either in acinar zone II, or in the pericentral area. 2AAF/PHx model, developed after the Solt Farber 1976 protocol95 elicits a vigorous oval cell response which is associated with oval cell activation in the periportal
45 area where progenitor cells are proliferating18, 96, 97. The concomitant activation and close proximity between oval and s tellate cells during the stem cell mediated regeneration led to the hypothesis that perisinusoidal stellate cells may control the developmental fate of the progenitor cells. The potential regulatory mechanisms are either direct by secreting growth factors, such as hepatocyte growth factor (HGF) and transforming growth factors (TGF), or indirect via urokinase induced extracellular matrix (ECM) remodeling98. Other studies emphasized the self reliance of oval cells, able to synthesize TGF growth factor (FGF), or IGF -2 (insulin -like growth factor 2). All these factors are responsible for autocrine regulation of their differentiation fate6. To which extent are oval cells dependent on other resident cell population for creating the cytokine an d growth factors microenvironment conducive to a certain lineage commitment remains to be found. The proposed sequence of events for oval cell activation includes an initial immune response with a significant increase of cytokines secretion. These signals promote the infiltration of the damaged area by Kupffer and stellate cells which further produce cytokines and growth factors. If the hepatocyte regeneration is impaired, resident oval stem cells located in the canals of Hering are activated. Hepatic progenitor cells may also be recruited from the bone marrow by chemoattractants, such as SCF (stem cel l factor) and SDF 1 (stromal derived factor 1) and further infiltrate the liver lobules in response to the cytokines produced by Kupffer and stellate (Ito) cells. TNF (tumor necrosis factor) stimulates oval cell proliferation. HGF, EGF and TGF t he plasminogen activators responsible for TGF trigger apoptotic pathways in oval cells, resulting in cessation of oval cell mediated liver
46 regeneration. The delicate balance between the various cytokines and growth fact ors at a definite time point is believed to dictate the amplitude and the duration of the oval cell response. 2.3.3 Oval Cell Plasticity Although the oval cell potential to differentiate into hepatocytes was suggested over seventy years ago99, it took alm ost two decades before the first supporting data emerged. Later research confirmed the hepatocyte differentiation and revealed a second fate commitment towards bile duct cells100 103. Initially the bipotentiality was attributed to the heterogeneity of ov al population, but the increasingly consistent findings led to a consensus over the capacity of oval cells to differentiate into both hepatocytes and cholangiocytes. More recent data105, 106 have shown the oval cell potential to differentiate into several cell lines, ranging from hepatocytes and cholangiocytes, to intestinal and pancreatic acinus epithelium, or neural cells (Figure 28). It was also reported that hepatic oval cells express not only AFP (indicating their non-terminally differentiated status) but also hematopoietic cell markers like Sca 1108 for mice and Thy 1107 in rats. These data suggest that both oval cells and HSCs might share, to a certain extent, a common developmental pathway. An alternative explanation might be that they share a comm on stem progenitor located in the bone marrow. In this situation, the actual tissue specific environment might be the trigger which induces the development of oval cells towards hepatic parenchymatous cells. This interpretation is supported by the well est ablished fact that the particular developmental program stem cells engage to reach a certain differentiation fate is dictated by the specific regulation of their gene expression. Various liver specific extracellular signals are believed to interfere with expression of
47 genes responsible for stemness, self renewal and differentiation. Describing these signals and their specific signaling pathways is the main goal for researchers striving to establish novel cell therapies. The roles of stellate and Kupffer cel ls are currently evaluated as part of cell -to -cell interactions able to influence oval cells fate. 2.3.4 Oval Cell and Hepatic Neoplasms From a pathophysiological standpoint, the neoplasm inducing mechanism is considered to trigger either the dedifferenti ation of a terminally differentiated cell, or the maturation arrest of a non-fully differentiated one. Since liver is known to be populated essentially by two distinct cellular pools, the unipotential hepatocytes and the multipotential oval cell, it is reasonable to assume that both major populations can become the origin of neoplastic tumors. The ongoing debate regarding the carcinogenetic potential of oval cells was triggered by the fact that the non-terminally differentiated state of the oval cell is ass ociated with their capacity of undergoing a large number of cell divisions. Since the likelihood of a preneoplastic mutation is proportional to the number of mitoses, oval cells became a candidate for being the cell of origin for neoplastic tumors. Early s tudies seemed to support this hypothesis43, demonstrating that hyperplasia of small round cells, somewhat similar phenotypically to the oval cells is associated with subsequent development of hepatocellular carcinomas. Since the morphology was the only proof invoked by the authors, it is hard to affirm beyond any doubt the oval cell nature of all hepatocellular carcinomas. More recent data showed that the transitional duct cell, progeny of oval cell, might be implicated in initiation and progression of liver carcinogenesis110. One study111 examining the cellular markers of primary HCC (hepatocellular carcinoma) tumors demonstrated that oval cells, hepatocyte nodules and malignant areas express both oval cell and hepatocyte
48 antigens, suggesting a precursor relationship between oval cells and HCC111. Subsequent examination of alternative oval cell markers in different models of HCC112, 113 found these markers in HCC nodules. The current histological grading, considers that the degree of differentiation of a ma lignant tumor is dictated by the differentiation status of the cell of origin. This would suggest that the highly differentiated hepatic tumors may not originate in an oval cell. There are published reports109 documenting the existence of cancers with high er differentiated status, like hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCC) which originate in hepatocytes. A study examining HCC tumors developed in patients with hepatitis B found oval cells expressing both hepatocyte and bile duct marke rs114, 115. Although these data seem to support the hypothesis of oval cell origin of HCC, they are not able to eliminate the possibility of hepatocyte dedifferentiation as origin of the neoplastic process. Proponents of both theories had to face criticis m. Although it is assumed that various cell types are able to resist to a different extent to mutations and epigenetic transformations, it is not known yet if the oval cell is less resistant to carcinogens than the fully differentiated cells. For these reasons, it is hard to believe that the oval cell is the only cell type with carcinogenetic potential in the liver, but it is more probable to assume that at least some of the cancers originate in hepatocytes and cholangiocytes. Whether the presence of cells with oval cell like phenotype is due to the dedifferentiation of mature hepatocytes undergoing the carcinogenic process, or they are real oval cells engaged on a procarcinogenic pathway is not known. Experiments using oval and epithelial liver cells expo sed to various carcinogens showed a chemical
49 dependent phenotype in the preneoplastic and neoplastic cells116119. It was also impossible to replicate in vitro the exame sequence of events happening in vivo during the carcinogenetic process. Considering th ese conflicting data, until further proof, it is safe to conclude that any cell type in the liver, including oval cells, has the potential to undergo the neoplastic cascade.
50 B Figure 21. Diagrams of liver blood supply and microarchitecture. A) Repr esentation of hepatic biliary flow and blood supply (flow direction indicated by arrows), B) Portal triad and intralobular blood flow (adapted after Kierszembaum2). Figure 22. Conceptual interpretations of the functional hepatic architecture (adapted after Kierszenbaum 20022). I acinar zone I, II acinar zone II, III acinar zone III, dotted triangle contour of a portal lobule, dotted hexagon contour of a hepatic (classic) hepatic lobule, dotted diamond contour of the liver acinus, color code: blue represents veins red represents arteri es green represents bile ducts
51 A B Figure 23. Histograms of Hematoxylin and Eosin (H&E) stained hepatic cell populations. A) Hepatocytes, Kupffer cells and the space of Disse, B) The intimat e relationship between hepatic stellate cells and hepatocytes. A B Figure 24. Hepatic stellate cell: morphology and activation diagram. A) Representation of hepatic stellate cell activation (adapted after www.medscape.com), B) Electron microscopy image of a quiescent hepatic stellate cell in the space of Disse (adapted after Kierszembaum2; a hepatocyte, b space of Disse, c lipid droplets containing vitamin A).
52 Figure 25. Diagrammatic representation of the specific mechanisms differentiating compensatory regeneration from direct hepatic hyperplasia (adapted after Columbano40).
53 Figure 26. Diagrammatic representation of liver parenchyma growth after 70% partial hepatecomy in rat; compensatory hyperplasia is respomsible for restoring the ori ginal hepatic mass (adapted after Higgins and Anderson 193143). A B Figure 27. Oval cell phenotype: A) Light microscopy of Hematoxylin and Eosin (H&E) stained image of small ovoid cells with high nucleus/cytoplasm ratio (black arrow heads indicate the o val cells radiating from the periportal area into acinar zone I; magnification 40x). B) Electron microscopy histogram of an oval cell (OC), adjacent to a hepatocyte (H): N nucleus, M membrane, C cytoplasm with scarce organelles, white arrowhead indic ating the oval cell membrane and black arrowheads pointing to the desmosome junctions between the oval cell and the neighboring hepatocyte..
54 Figure 28. Oval cell plasticity: diagrammatic representation of hepatic progenitor cells differentiation fate under various experimental conditions, depicting their transformation into various normal cell lines, but also into hepatocellular and cholangiocarcinomas (modified after Williams 2007104).
55 Table 2 1. Liver regeneration post 70% hepatectomy reflected by DNA synthesis in various resident hepatic cells; chosen time points indicate the number of days after the acute liver injury (adapted after Michalopoulos45). Cell type Regeneration onset Pea k of regeneration End of regeneration Duration of regenerationr Hepatocyte Within 12 hrs posthepatectomy Beginning of 2nd day post hepatectomy End of 4 th day posthepatectomy 4 days Cholangiocytes End of 1 st day posthepatectomy 2 nd day post hepatectomy End of 4 th day posthepatectomy 3 days Nonparenchymal (Kupffer and Ito cell) Beginning of 2 nd day post hepatectomy End of 2 nd day post hepatectomy End of 10 th day post hepatectomy 9 days Sinusoid endothelial cells During the 2 nd day post hepatectomy End of 4 th day post hepatectomy End of 10 th day post hepatectomy 8.5 days Table 2 2 Molecular markers for several hepatic cell populations Marker Oval cell Hepatocyte Cholangiocyte Stellate cell AFP + ALB + A6 + BD1 + BDS7 + c kit + CK7 + CK8 + CK18 + CK19 + CD34 + Desmin + GFAP + GGT + + HES6 + OC2 + + OC3 + + OV1 + + OV6 + + Thy 1 +
56 CHAPTER 3 SPECIFIC AIMS The extraordinary regenerative capacity of the liver makes it one of the most resourceful viscera, able to restore its original mass during the next two weeks post injury. The first line of regeneration employed is represented by mature hepatocytes divisio n, an ongoing process during the lifetime. Certain pathological contexts, such as massive acute hepatic necrosis and chronic long term injury result in impairment of hepatocyte division. Under these circumstances the liver stem cell compartment takes over, acting as a backup regenerative system. The progenitor dependent compensatory hyperplasia takes place in the amplification compartment, composed of multiplying stem cells progeny known as oval stem cells and located traditionally in the canals of Herin g120. Recent studies proved the bone marrow origin of at least one subpopulation of oval cells107. Their progenitor phenotype exhibits transitional features between hepatocytes and cholangiocytes and expresses markers evoking their bone marrow ancestry. Si milar to the hepatocyte dependent compensatory hyperplasia, stem cell mediated liver regeneration is an orchestrated response induced by specific local and systemic stimuli which elicit sequential changes in gene expression, followed by growth factor production, cellular activation, proliferation, migration and differentiation. The first and foremost question concerning oval cells is which signals are involved in oval cell activation and which cellular interactions are responsible for these signals? It is g enerally accepted that the different hepatic cell types influence the fate of oval cells by cell -to -cell interactions and also by creating an extracellular environment conducive for regeneration. The overarching question/hypothesis is i n which interactions and to
57 what extent are hepatic stellate cells involved during oval cell activation, engraftment, and differentiation? The overall project goal is to explore the effects of stellate cell inhibition on oval cell activation, proliferation and differentiation. In order to test this hypothesis, two specific aims were designed: Specific aim I: Test the hypothesis that stellate cells play a necessary role in facilitating oval cell proliferation in the liver. Several studies have shown that hepatic stellate cells are important in the early stages of oval cell proliferation121, 122. The complex interactions between these cell types are mediated both by cytokines and extracellular matrix proteins. Oval cells express many receptors for stellate cells pr oduced growth factors which may trigger regulatory molecular pathways for liver stem cell activation, growth and development. As a major source of ECM proteins, the stellate cells are involved in the recently described matricrine regulation123. They also r emodel the extracellular environment, indirectly influencing the migration and the differentiating fate of oval cells. We hypothesize that stellate cell activation and proliferation is required for the oval cell mediated liver regeneration, being a major player in the complex cellular interactions which result in oval cell activation. To test the complex role of stellate cells we will utilize the well characterized 2acetylaminoflourene/partial hepatectomy (2AAF/PHx) acute hepatic injury model associated wi th the L -Cysteine diet, a proven stellate cell inhibitor124. The specificity of L -cysteine inhibition was determined by in vitro treatment of stellate cells, portal fibroblasts and oval cells. Changes of oval cell activation were analysed for corresponding time points during the study. Specific a im II: To test the hypothesis that in the absence of a robust stellate cell activation the oval cell activation is delayed. Liver regeneration is a very complex
58 process involving several cell -to -cell interactions. O ne of the histological features of diseased liver is the numeric imbalance between the hepatic cell types. In order to circumvent this issue and increase the chances of success for cell therapy, it might be necessary to inject more than one cell type. A po tential mixture between oval cells and other hepatic cells (most likely stellate and Kupffer cells) might be necessary. Since the recruitment of monocytes is persisting to a certain extent in the ailing liver, it is possible to stimulate their hepatic homi ng by injecting monocyte specifi c chemoattractants. This chemoki ne mediated recruitment is not operating in the case of stellate cells and it might be necessary to isolate, expand and reinject them together with the oval cells. The exact speed of oval cell mediated liver regeneration is crucial for survival of patients with terminal hepatic failure. If too slow, the irreversible hepatic encephalopathy, coupled to the associated multiorgan dysfunction syndrome (MODS) could lead to a fatal outcome, despite of a promising onset of oval cell proliferation. For these reasons, even a delay in oval cell activation might prevent a successful outcome of oval cell therapy. There are several redundant mechanisms in the body and specifically in the liver. To determine i f other hepatic cell types are compensating for the lack of adequate stellate cell contribution to oval cell mediated liver regeneration, the effects of stellate cell inhibition was analyz ed quantitavely and also in terms of timing of the regenerative resp onse. A general evaluation of regeneration was done comparatively for animals kept on the inhibiting diet comparative to control animals during the whole time course of the experiment. Samples collected at corresponding time points were immunostained for m arkers of stellate and oval cell activation and the positivity was assessed by quantitative analysis. Expression of AFP gene which is a hallmark for the regeneration
59 amplitude was assessed by real time PCR. A clearer picture of amplitude and timing of live r regeneration in the absence of robust stellate cell contribution emerged.
60 CHAPTER 4 METHODS AND MATERIAL S 4.1 Animal T reatments In order to test our hypothesis we combined an experimental model of oval cell activation with the stellate cell inhi bition. The 2AAF/PH x protocol (a modern modification of the original Solt and Farber technique95) was utilized to induce the progenitor mediated liver regeneration, explore and characterize oval cell activation, proliferation and differentiation under vari ous extraneous factors introduced in the experimental system. This well established model shown in Figure 4 1 makes possible the isolation of oval cells necessary for in vivo studies, but more importantly, overcomes the limitations of cell culture systems. Although informative, the in vitro studies can provide data limited to the proliferation and differentiation status of oval cell, but are not able to reproduce the complex molecular interactions which take place in the liver lobule. Our animal model reproduces in rats the pathological circumstance s present in the liver of human patients suffering of chronic hepatic conditions which exhaust or inhibit the na tural proliferative capacity of hepatocytes. 2AAF is a mitogen known to be metabolized by the cy tochrome P 450 enxymes into its bgiologically active, N -OH derivatives. After entering the nucleus 2AAF metabolites form voluminous adducts with DNA, rendering it unable to be approached by the transcription apparatus. This results in cyclin D1 and E inhib ition, followed by G1/S check point cell cycle arrest, as shown in Figure 42 The stellate cell inhibition was performed subjecting the laboratory animals to a diet supplemented with 2% L-cysteine, a non essential amino acid. There are several proposed me chanisms for L-cysteine inhibition of stellate cells, as shown in Figure 4 -3. Either by ROS (reactive oxygen species) inhibition of cyclin D1
61 mu rine models of liver fibrosis124. 4.1.1 Animals and Animal Housing Facilities For this study we used Fisher 344 male r ats obtained from Charles River Laboratories, Inc. (Wilmington, MA). Animals were housed in the Animal Care Services Facility in the Medic al Science and Communicore Buildings During the whole study, constant veterinary care was provided by the animal care facility: Animals were checked regularly and assistance with euthanasia decisions was provided. The animal protocols used were approved by the Univers ity of Florida IACUC (Institutional Animal Care and Use Committee). The animal care program at University of Florida is accredited by AAALAC (American Association for the Accreditation of Laboratory Ani mal Care ). University of Florida meets National Institutes of Health standards as described in DHHS publication #NIH 86-23 T he PHS (Public Health Science) Policy on Humane Care and Use of Laboratory Animals by Awardee Institutions and the National Institute of Health Principles for the Utilization and Care of Vertebrate Animals Used in Testing, Research and Training are mandatory for the animal protocols at the University of Florida. 4.1.2 Animal Sacrifice and Tissue Collection At determined tim e points a number of three animals were sacrificed for tissue collection. Each animal w as euthanized by injection of 150 mg/kg Nembutal Sodium Solution (OVATION Pharmaceuticals, Inc., Deerfield, IL) according to Euthanasia D Special protocol. This procedure conforms to the recommendations of American Veterinary Medical Association and the Guide for the Use and Care of Laboratory Animals (US Department of Health and Human Services/NIH Publication #86-23).
62 T issue samples were excised from various organs ( br ain, heart, lung, liver, pancreas, spleen, kidney and intestine ) and collected for embedding in OCT compound, paraffin embedding, RNA and protein isolation. Tissue collected for cryosections was immersed in Tissue -Tek OCT Compound (Sakura Finetek USA Inc Torrance, CA) snap frozen in a 2methylbutane histobath and stored at 80C until sectioning. Organ samples for paraffin embedding were fixed in 10% Neutral Buffered Formalin (Richard -Allan Scientific, Kalamazoo, MI) O/N (over night) After being transfer red in PBS (phosphate buffered saline) the following day, the collected tissues were submitted to the University of Florida Molecular Pathology Core Facility for paraffin embedding. For histological analysis all samples used in our study were cut in to 5 thick sections Samples used for protein and RNA extraction were snap frozen in a 2methylbutane histobath and stored at -80C until isolation was performed. 4.1.3 Oval Cell Induction in Rat 220.127.116.11 2AAF pellet implantation 2 -Acetylaminofluorene (2AAF) is the DNA adducts forming mitogen used in our protocol to selectively inhibit hepatocyte proliferation. Its inhibitory activity on cyclins D1 and E associated with the formation of voluminous DNA adducts block s the cell cycle at the G1/S checkpoint125, a s shown in Figure 42. It has been used for quite some time, administered by gavage to rats undergoing the 2AAF/PHx protocol. It has become commercially available in intraperitoneal slow release pellet (70mg/pellet over a 28 day release, Innovative Researc h of America Inc., Sarasota, FL, USA) In this form insures the quick absorption of an efficient dose of 2.5 mg/day. The pellet is inserted seven days before the partial hepatectomy through a 0.5 cm long incision made in the left hind quadrant of the abdominal cavity Prior to the
63 surgery, the animals w ere anesthetized with isofl u orane. The abdomen was shaved and scrubbed in a centrifygal pattern three times with ethanol and three times with Betadine (Purdue Pharma L.P., Stamford, CT). After drapping the abdominal wall with a Steri Drape (3M, St. Paul, MN) a 0.5 cm long incision was made in the lower left quadrant of the abdomen ensuring a convenient distance from the liver M idline incision s are discouradged, due to the post operatory fibrosis resulting i n adhesions between the pellet and the hepatic tissue. The incision sectioned the skin, subcutaneous tissue, muscle and parietal peritoneum, allowing the pellet insertion into the abdominal cavity. The muscle was then sutured with 3 -0 Vicryl (Ethicon, Inc. Cornelia, GA) and t he skin was closed with the Autoclip Wound Closing System (Braintree Scientific Inc Braintree, MA). Post operatory the animals were housed in warmed cage s and monitored every six hours until complete recovery. Ten days later t he staples were removed. The potential complications, hemorrhage, dehydration and hypothermia, were absent. 18.104.22.168 Two -thirds partial hepatectomy To induce compensatory hyperplasia, 70% partial hepatectomy is performed according to the protocol described by Higgins and Anderson43. Left lateral, left medial and right medial lobes of rat liver were removed. Prior to the surgery, the animals w ere anesthetized with isofl u orane. The abdomen was shaved and scrubbed in a centrifygal pattern three times with ethanol and three times with Betadine (Purdue Pharma L.P., Stamford, CT). The xyphoid process is palpated and after drapping the abdominal wall with a Steri -Drape (3M, St. Paul, MN) a 1.5 cm long midline incision was made. Skin, subcutaneous tissue and muscle ar e sectioned and the xyphoid process was removed. The three lobes were extruded through the incision and were tied off with silk. After
64 exposure, the lobes were removed and the stump hemostasis was performed. After carefully examining the abdominal cavity f or signs of persisting hemorrhage, the abdominal wall was closed. Any post operatory bleeding is a fatal complication for animals. The muscle is sutured with 3 -0 Vicryl (Ethicon Inc. San Angelo, TX ). Autoclip Wound Closing System (Braintree Scientific, Inc .) was used for skin closure. Staples were removed ten days later. After surgery the animals were housed in warmed cages and monitored for post operatory complications until complete recovery. Dehydration, bleeding and hypothermia were not present. Animals were sacrificed at days 3, 5, 7, 9, 11, 13, 15, 17, and 20 days post PH x 4.1.4 2% L cysteine D iet The 6 week old Fisher 344 male rats (Charles River Laboratories, Wilmington MA) were maintained on standard laboratory chow supplemented with 2% L -cysteine (Dyets Inc. Betlehem PA) for the duration of the experiment, according to a protocol established by Horie124. The diet was custom made by the manufacturer using standard rat food supplemented with laboratory purity standard L-cysteine (MP Biomedicals LLC Cleveland OH) To identify any potential repulsion to the diet, which might incur unwanted consequences on the weight gain and introduce variables into the experiment, the animal reaction and feeding habits were monitored and their weight was measured daily. Since there were no differences in terms of food repulsion reactions and weight gain between the animals kept on diet and the control group kept on standard rat food, L-cysteine diet was offered ad libitum to the whole group of animals included in the experiment.
65 4.1.5 Animal Numbers Several animal groups were included in this study. Besides normal liver, diet, 2AAF, diet/2AAF, diet/PHx controls, for each time point of animal sacrifice in the Lcysteine/2A AF/PHx group there was a corresponding 2AAF/PHx control. As table 4-1 shows, a nimals were sacrificed on days 1, 3, 5, 7, 9, 11, 13, 15, and 20 days after PH x. Animal numbers at the various time points and the experimental protocol they were subjected are s hown in Table 41. All animals included in this study survived the experimental protocols performed on them. 4.2 BrdU Analysis In Vitro Cell Response to L -cysteine 4.2.1 Maintenance of WB -F344, HSC T6 Cells and Portal Fibroblasts in Culture WB -F344 oval stem cell line, graciously provided by Dr. William Coleman w as cultured in chamberslides with RPMI 1640 medium (Mediatech Inc., Herndon VA) supplemented with 10% fetal bovine serum (Sigma Aldrich, St. Louis MO) 1 0 IU Penicillin/ml and 10 streptomyc in (Mediatech Inc., Herndon VA) in a 37C humidified incubator containing 5% CO2 and 95% air The culture medium was changed every other day. Primary isolated portal fibroblasts kindly provided by Dr. Rebecca Wells were maintained in chamberslides on DMEM medium (Hyclone Lab Inc., Logan UT) supplemented with 10% fetal bovine serum (Sigma Aldrich, St. Louis MO), 1 0 IU Penicillin/ml and streptomycin (Mediatech Inc., Herndon VA) at 37C, 5% CO2 humidified incubator. Culture medium was replaced every other day. HSC T6 hepatic stellate cell line, kind gift from Dr. Robert Friedman, was cultured in chamberslides on DMEM F12 50/50 medium (Hyclone Lab Inc., Logan UT) supplemented with 10% fetal bovine serum (Sigma Aldrich, St. Louis MO) 1 0 IU Penicillin/ml and 10
66 streptomycin (Mediatech Inc., Herndon VA) in a 37C humidified incubator containing 5% CO2 and 95% air The culture medium was changed every other day. 4.2.2 Cell Synchronization and Br dU Treatment When each cell culture achieved 30% confluence, they were synchronized with 0.4 g/ml Demecolci ne (Sigma Aldrich, St. Louis MO), an inhibitor of mitotic spindle formation added to their respective culture media. When cell cycle was blocked in mitosis after 24 hours of exposure, demecolcine was washed and 100 M L -cysteine (MP Biomedicals LLC, Cleveland OH) was added to the culture media, thus inhibiting the hepatic stellate cell activation. After a three day exposure to L-cysteine, the chamber slides were treated with 10 M bromodeoxyuridine (BrdU) (Sigma Aldrich St. Louis MO) and fixed in 4% paraformaldehyde. 4.3 Histology and Immunohistochemistry 4. 3 .1 Hematoxylin and Eosin Staining of Paraffin E mbedded T issue 5 m thick t issue sections were cut and placed in a 42C water bath. After being mounted to a Superfrost Plus slide (Thermo Fisher Scientific Inc. Waltham, MA) the sections were air dried overnight ( O/N ) at room temperature ( RT ). The sections were de paraffin ized by immersing them in xylene for 2 x 5 minutes. Further immersion in several baths of ethanol rehydrated the sections: 100% e thanol 2 x 2min, 95% e thanol 2 x 1min, and distilled H2O for 1min. Nucleic acids were stained with h ematoxylin 7211 (Richard -Al lan Scientific Kalamazoo MI ) for 2min and rinsed with H2O for 2 x 1min. Consecutive baths of c larifier 1 (Richard -Allan Scientific Kalamazoo MI ) for 1min, distilled H2O for 1min, b luing r eagent (RichardAllan Scientific Kalamazoo MI ) for 1min, distilled H2O for 1min, and 80% e thanol for 1min intensified the h ematoxylin color. For cytoplasm staining was used e osin Y (Richard -Allan Scientific Kalamazoo MI ) for 1min
67 30 sec. Several baths of 2 x 1min 95% ethanol, 2 x 1min 100% ethanol, and 3 x 1min xylene de hydrated the sections before coverslipping with Cytoseal XYL (Richard-Allan Scientific Kalamazoo MI ). 4.3.2 Periodic Acid Schiff Staining of Paraffin E mbedded T issue 5 m thick t issue sections cut and mounted to a Superfrost Plus slide (Thermo Fisher Scientific Inc. Waltham, MA) air dried O/N at RT were deparaffinized and rehydrated according to the same protocol described above. They were immersed 5 min in Periodic Acid solution (Richard -Allan Scientific Kalamazoo MI ), rinsed in distilled H2O, stained with Schiff Reagent (Richard -Allan Scientific Kalamazoo MI ) for 15 min and rinsed for 10 min in lukewarm tap H2O. After 1 min hematoxylin staining, 30 sec rinsing in distilled H2O, bluing for 1 min, rinsing for 30 sec in distilled H2O, the sections were dehydrated 2x1 min in 100% alcohol. Sections cleared in 3x1 min baths of clearing reagent were mounted with with Cytoseal XYL (Richard-Allan Scientific Kalamazoo, MI ). 4.3.3 Immunohistochemistry 22.214.171.124 Chromogen staining P araffin and frozen sections w ere incubated O/N at 4C for primary antibody and 30 min for secondary antibodies then stained using Vector ABC, DAB (Diaminobenzidine) (Vector Laboratories Burlingame, CA) as per manufacturers instructions. DAB slides were counterstained with Vector Hematoxylin QS (Vector Laboratories Burlingame, CA) and mounted with Cytoseal XYL (Richard-Allan Scientific Kalamazoo, MI). Aditional antigen retrieval methods are mentioned, together with the antibodies, in Table 4-2.
68 126.96.36.199 Fluorescent staining 5 m thick f ro zen sections were cut and placed on Superfrost Plus (Thermo Fisher Scientific Inc. Waltham, MA). The s ections were air dried for 5 min at RT and fixed for 10 min in ice cold methanol After 5 min incubation in TBS plus 0.1% Tween (TBS -T) and 20 min serum block the slides were incubated with the primary antibody for 1 hr at RT or O/N at 4C. Slides were then washed for 5min in 1X TBS -T at RT and incubated with a fluorochrome labeled secondary antibody for 30 min. After being washed again for 5 min in TBS -T at RT the slides were coverslipped with Vectashield Mounting Media with DAPI (Vector Laboratories). 5 m thick paraffin sections were cut and placed on Superfrost Plus slides (Thermo Fisher Scientific Inc. Waltham, MA). Two xylene baths of 5 min each were used for deparafinization. The slides were then rehydrated by immersing in successive baths of 100% ethanol (2 x2 min), 95% ethanol (3 min), 70% ethanol (1 min) and H2O (2 x 1 min). After antigen retrieval at 95C using Dako Target Retrieval 1X (Dako, Car pinteria, CA see table 4-2 for details) and serum block, the slides were exposed O/N to a mixture of rabbit anti rat Ki67 antibody (Novocastra Leica, Bannockburn, IL) and mouse antihuman desmin clone D33 (Dako, Carpinteria, CA). The slides were washed in 1X TBST (Tris buffered saline with 0.1% Tween), incubated with fluorochrome conjugated secondary antibodies for 30 min at RT. After washing they were coverslipped with Vectashield Mounting Media with DAPI ( 4 6 diamidino -2 phenylindole ), (Vector Laboratories Burlingame, CA ). Fluorescence was observed and photographed with a fluorescent microscope : BX51 Olympus Fluorescent microscope fitted with cubes for FITC (Fluorescein isothiocyanate) Texas Red, DAPI and dual pass FITC/Texas Red, an d Optromic Digita l Cemera with Image Pro 3.1 Software and
69 Magnafire 3.1. Computer image analyses of immunostained sections w as performed using Aperio ScanScope Image Analysis Platform (Aperio, Vista CA) and MetaMorph software (MDS Technologies, Concord ON, Canada) for hist ological evaluation and quantitation. 4.4 RNA Analysis Real Time Quantitative PCR The mRNA (messenger RNA) levels were assessed by two -step quantitative real -time PCR (Polymerase chain reaction) reaction, using a DNA Engine Opticon 2 Continuously Fluorescence Detector (MJ Research Inc Waltham MA). Total RNA was extracted using the RNA Bee isolation kit (Tell -Test Inc. Friendswood TX), treated with Dnase I (Deoxyribonuclease I) (Ambion, Austin, TX) and reverse transcribed with the Superscript III First Strand Synthesis System for rt PCR (Reverse transcription polymerase chain reaction), (Invitrogen, Carlsbad, CA). Amplification was performed on a customized RT2Profiler PCR Array plate for the gen es of interest (SA Biosciences, Frederick MD) using iQ SYBR Green Supermix (Bio -Rad Laboratories, Hercules CA). 4.4.1 RNA Isolation 188.8.131.52 Tissue homogenization and phenol -chloroform phase separation 50 mg liver tissue samples kept on ice were homogeni zed in 1ml of RNABee Reagent (Tel -Test, Inc. Friendswood, TX). Then 0.2 ml of chloroform per 1ml of RNABee Reagent was added and the tubes shaked for 30 s ec and incubated on ice for 15 min. The samples were then centrifuged at 12, 000 x g for 15 min at 4C and, as a result, the lysate separated into a lower blue, phenolchloroform phase, a white opaque interphase, and a colorless upper aqueous phase. RNA remains exclusively in the aqueous phase which makes for approximately 50% of the mixture volume.
70 184.108.40.206 Precipitation, washing and redissolving of RNA The aqueous phase is carefully aspirated and transferred to a fresh tube. Aspiration of interphase would result in RNA contamination and compromise its quality. RNA is precipitated from the aqueous phase by adding 600 for each 1ml of RNABee Reagent used for the initial homogenization. The samples were then incubated at RT for 10 min and centrifuged at 12, 000 x g for 5 min at 4C. The RNA precipitate s as a white gel atinous pellet on the side o f the tube. The supernatant was discarded, the pellet was resuspended and vortexed in 1 ml 75% ethanol, then centrifuged for 5 min at 7 500 x g at 4C For a better quality of extracted RNA, the washing step was repeated. The supernata nt was decanted and the RNA pellet was air dried for 510 min, avoiding it being dryed completely, as this would greatly decrease its solubility. The RNA was dissolved in R N ase -free water and stored at 80C. 220.127.116.11 Quantification of RNA by spectrophotome try The RNA was diluted by mixing sample with -treated H2O (Diethyl Pyrocarbonate-treated H2O ). For this solution, t h e absorbance at OD (Optical density) of 260 n m and 280 n m was measured by a spectrophotometer. RNA purity was dete rmined based on an OD 260/280 ratio and found between 1 .6 1.9. The concentration of RNA was determined using the following formula: 260 40 ng/ dilution factor of 100 4.4.2 Real Time PCR (RT PCR ) 18.104.22.168 First -strand cDNA synthesis from total RNA using rt PCR First strand cDNA was synthesized usin g SuperScript III First -Strand Synthesis System (Invitrogen Carlsbad, CA ) according to manufacturers instructions:
71 A volume of solution containing 5 g RNA was mixed with 1l 10 mM dNTPs (Deoxynucleotide triphosphate), 1l oligodTs (oligodeoxythymidylic acid) and DEPC H2O was added up to 10 ml. After a short spin, the mixture was incubated at 65C and then kept on ice for 1 min. In the meantime the cDNA (Complementary DNA) synthesis mix was prepared by mixing: 2l 10X RT buffer B, 4l 25 mM MgCl2, 2l 0.1M DTT (Dithiothreitol), 1l Rnase OUT (40 U/l) and 1l Superscript III RT (200 U/l). 10l of cDNA synth esis mix was added to each RNA/primer mixture, mixed, briefly centrifugated and incubated for 50 min at 50 C The reaction was terminated at 85C for 5 min and then was chilled on ice. After a brief centrifugation, 1 l of Rnase H was added to each tube and incubated for 20 min at 37C 22.214.171.124. PCR amplification of target cDNA cDNA was amplified using GAPDH ( Glyceraldehyde -3 -Phosphate Dehydrogenase ) primers as a loading control for the RT PCR reaction. The following reaction mixture prepared on ice was used: 2 l 10X PCR buffer B, 0.6 l 25mM MgCl2, 0.4 l 10mM dNTP mix, 1 l GAPDH forward primer, 1 l GAPDH reverse primer, 13.81 l H2O, 1 l cDNA and The GAPDH primers used for the reaction had an annealing temperature of 58C and produced a 577 bp cDNA: Forward primer TGA GGG AGA TGC TCA GTG TT Reverse primer ATC ACT GCC ACT CAG AAG AC
72 T aq p olymerase was added immediately before samples were placed in a thermocycler. The PCR reaction was run as follows : 1. 94C for 10min 2. 31 cycles of 94C for 30 sec Annealing temp 58 C for 30 sec 72C for 30 sec 3. 72C for 10min 4. 4 C indefinitely 126.96.36.199 Agarose gel electrophoresis The 0.7% w/v (wheight per volume) agarose gel for cDNA electrophoresis was prepared by microwaving agarose with 30 ml 0.5 X TBE. When the agarose was completely dissolved, 0.001% v/v (volume per volume) ethidium bromide was added. Then, the gel was poured into a gel p oring apparatus and, after inserting the well comb, it was left to solidify. The comb was removed before placing the gel pouring apparatus in the gel electrophoresis chamber. 0.5X TBE was added to cover the gel and the wells were loaded with a mixture of 0 .5 a garose gel loading buffer 3.5 i -Q H2O and 1 each cDNA sample. One well loaded with an appropriate base pair ladder was used to identify the cDNA of interest. The gel was run at 90 110 volts until the desired separation of b ands was achieved. Pictures of cDNA bands were taken with a GelDoc XR (Bio-Rad Hercules, CA) apparatus 188.8.131.52 Real -Time PCR analysis of AFP levels To accurately assess the variations of AFP message levels after L -cysteine exposure at day 9 post hepatectomy in animals subjected to 2AAF/PHx protocol, Real Time PCR quantitative analysis was performed using an RT2ProfilerTM PCR array produced by SA Biosciences (Frederick, MD). The PCR microarray plate used had
73 primers for betaactin, rat genomic DNA contamina tion, AFP, RT PCR control and rt PCR control. The amplification conditions were: 1. 94C for 10min 2. 40 cycles of 9 5 C for 15 sec 55C for 30 sec 72C for 30 sec 3. 72C for 10min 4. 4 C indefinitely The comparative Ct threshold cycle method was used to assess the expression level, actin mRNA expression. 4.5 Statistical A nalysis Values were expressed as mean +/ standard deviation (SD). Statistical significance was determined by ANOVA, and student t test performed in Microsoft Excel. p v alues <0.05 were considered statistically significant. 4.6 Solutions 10X Agarose Gel Loading Buffer 1. 15.0 mg bromophenol blue 2. 15.0 mg xylene cyanol 3. 8.0 g sucrose 4. Milli -Q H2O qs to 10 ml 10X PBS (Phosphate buffered saline) 1. 80.0 g NaCl 2. 2.0 g KCl 3. 11.5 g Na2HPO4 7H2O 4. 2.0g KH2PO4 5. Milli -Q H2O qs to 1 L 5X TBE (Tris -Borate -EDTA Buffer ) 1. 54 g Tris base. 2. 22.5 g Boric acid 3. 4.7 g EDTA (Ethylenediaminetetraacetic Acid), 4. Milli -Q H2O qs to 1 L
74 10X TBS 1. 80.0 g NaCl 2. 2.0 g KCl 3. 30.0 g Tris base 4. 800 ml H20 5. Milli -Q H2O qs to 1L Adjust pH to 7.4 using 1M HCl DEPC water 1. Diethylpirocarbonate 1ml 2. Milli -Q H2O 999ml After being mixed well and let set at RT for 1 hour, it was autoclaved at 121C for 60 min and let cool at RT before use.
75 Figure 41. Diagrammatic representation of the 2% L-cysteine diet/2AAF/PHx experimental protocol combining chemical inhibiti on of hepatic stellate cells and oval cell activation. Figure 42. Mechanism of action, metabolic activation and detoxification of 2AAF (2 acetylaminofluorene), N -OH 2AAF (N hydroxyl 2AAF), G1/S checkpoint between G1 and S phase of cell cycle.
76 Figure 43. Proposed mechanisms for 2AAF inhibition of hepatic stellate cells: glutathione mediated, ROS mediated G1/S cell cycle arrest, PDGFR beta downregulation with proliferation inhibiting effects; ROS reactive oxygen species, MnSOD manganese superoxide dismutase, NFkB Nuclear factor kappa-light -chain enhancer of activated B cells, AP 1 activator protein 1, Sp1 transcription factor, PDGFR platelet derived growth factor receptor beta, Ras small GTPase involved in signal transduction, MAPK Mitogenactivated protein kinase, JNK c -Jun N -terminal kinase, FAK focal adhesion kinase, PI3-K Phosphatidylinositol 3 kinase, Akt protein kinases B family.
77 Table 4 1. Animals subjected to the stellate cell inhibition oval cell activation Lcysteine/2-AAF/PH x protocol. Time point Number of animals Experimental protocol Control normal 3 No experimental procedure performed Control diet 3 L cysteine diet alone Control 2AAF 3 2AAF ip implanted Control diet/ 2AAF 3 L cysteine diet, 2AAF ip implanted Control diet /PH x 3 L cysteine diet, 70% PH Control 2 AAF/PH x 3 per time point 2AAFip implanted, 70% PH Diet/2AAF/PHx day 1 3 L cysteine, 2AAF ip implanted, 70% PH Diet/2AAF/PHx day 3 3 L cysteine, 2AAF ip implanted, 70% PH Diet/2AAF/PHx day 5 3 L cysteine, 2AAF ip implanted, 70% PH Diet/2AAF/PHx day 7 3 L cysteine, 2AAF ip implanted, 70% PH Diet/2AAF/PHx day 9 3 L cysteine, 2AAF ip implanted, 70% PH Diet/2AAF/PHx day11 3 L cysteine, 2AAF ip implanted, 70% PH Diet/2AAF/PHx day13 3 L cysteine, 2AAF ip implanted, 70% PH Diet/2AAF/PHx day15 3 L cysteine, 2AAF ip implanted, 70% PH Diet/2AAF/PHx day20 3 L cysteine, 2AAF ip implanted, 70% PH Table 4 2 Antibodies and antigen retrieval methods used for i mmunohistochemistry Protein Animal Retrieval Concentration Manufacturer Cat. # BrdU mouse 10mM Na citrate pH6 1:50 Dako M0744 Ki67 mouse 10mM Na citrate pH6 1:300 BD Pharmingen 556003 AFP rabbit Trilogy 1:600 Dako A0008 OV6 mouse N/A 1:150 Dr. Stuart Sell laboratory N/A Desmin Dako Target Retrieval 1x 1:50 Dako M0760 Ki67 Rabbit Dako Target retrieval 1:2000 Novocastra Leica NCL Ki67p
78 CHAPTER 5 RESULTS Previous studies conducted by our laboratory have demonstrated that chemical suppression of transforming growth factor regulation of connective tissue growth factor (CTGF) resulted in a greatly diminished oval cell response to 2 acetylaminofluorene/partial hepatectomy (2AAF/PH x) in rats. We hypothesized that this effect is due to decreased activation of hepatic stellate cells. In order to test this hypothesis, we maintained rats on a diet supplemented with 2% Lcysteine as a means of inhibiting stellate cell activation during the oval cell response to 2AAF/PH x. In vitro exp eriments demonstrate that L-cysteine did, indeed, inhibit the activation of stellate cells Desmin immunostaining of liver sections from 2AAF/PH x animals indicated that maintenance on the L-cysteine diet resulted in an 11.1fold decrease in the number of a ctivated stellate cell s within the periportal zones. No direct effects on oval cells, regarding the proliferation index or phenotypical chages were observed. The total number of cells proliferating in the periportal zones of livers from animals treated wit h L -cys teine was drastically reduced. Further quantitative analyses using immunostaining fo r both OV6 and AFP demonstrated a greater than four -fold decrease in the magnitude of the oval cell response in animals maintained on the Lcysteine diet Global liv er expression of AFP as measured by real time PCR was shown to be decreased 4.7-fold in t he L -cysteine treated animals. These data indicate that the activation of hepatic stellate cells is required for an appropriate oval cell response to 2AAF/PH x
79 5.1 Eva luation of in vi tro Response to L cysteine of Selected Hepatic Cell Populations Since the direct effects of L -cysteine on hepatic stem cells hasnt been explored yet, before starting the in vivo model we evaluated its in vitro effects on the oval cells, q uiescent stellate cells and portal fibroblasts. WB -F344 cells t he only oval cell line currently available, were maintained on RPMI -1640 medium Primary isolated portal fibroblasts are a hepatic population rich in myofibroblasts exhibiting a phenotype very similar to the activated stellate cells HSC T6 cells are the only available quiescent hepatic stellate cell line and were maintained under the same conditions in DMEM F12 50/50 medium. When the desired confluence was achieved, the cells were synchronized with Demecolcine and then treated with L-cysteine for three days The choice of L -cysteine was dictated by its proven capacity of inhibiting the hepatic stellate cell activation both in vivo and in vitro in murine liver fibrosis models. The cells undergoing the S-phase were identified by BrdU incorporation into newly synthesized DNA and immunostaining with anti BrdU antibodies Less is known about the L-cysteine effects on oval cells. In order to exclude a potential direct interference w ith their phenotype and proliferation attributable to Lcysteine, t he hepatic progenitor cell line WB F344 was cultured both with and without L cysteine in the culture medium. We werent able to identify any visible changes in the WB F344 morphology under any of these circumstances examined (Figure 51 A and B). We determined that the BrdU incorporation index was similar for both L -cysteine treated and untreated cells. Figure 5 -4 suggests that L -cysteine ha s no effect on the proliferation rate of these cell s
8 0 We next examined primary portal myo fibroblast cultures (Figures 5 -2 A and B), as well as the hepatic stellate cell line HSC -T6 (Figures 5 -3 A and B). In contrast to the progenitor cell line, both of the mesenchymal cell cultures demonstrated a significa nt reduction in proliferation rates when culture media was supplemented with 100 M L cysteine. Its worth mentioning that HSC T6 cells are non activated stellate cells, whilst portal fibroblasts are terminally differentiated mesenchymal cells thought to d erive, at least in part, from activated stellate cells. Quantitative image analysis was performed in order to determine the BrdU incorporation index which reflects the comparative proliferative capacity of the examined hepatic cell populations. A 3.56-fold decrease in BrdU incorporation for HSC T6 and a 5.6-fold reduction for portal fibroblasts were observed (Figure 5 4 ). Taken together, quantitative image analysis data suggest that L -cysteine acts selectively on the mesenchymal cell populations examined. L -cysteine appears to be a selective in vitro inhibitor of hepatic mesenchymal populations examined. 5.2. Evaluation of in vivo Response of Hepatic Stellate Cells to L -cysteine Diet Desmin immunofluorescent staining was performed on liver samples collected 9 days after acute injury during the 2AAF/P Hx protocol as a preliminary step in assessing the stellate cell activation in vivo The deliberate choice of day 9 post hepatectomy as a reference time point in our study is dictated by the peak of oval cell acti vation observed constantly at this particular time in the animals subjected to the 2AAF/PHx protocol. We have observed a global reduction of desmin expression in animals exposed to Lcysteine (Figure 5 5). Since tissue morphology is not visible on IF (Immu nofluorescence) labeled cryosections, it was difficult at this stage to discern which cells are responsible for the noted difference in hepatic desmin expression. Further
81 immunostaining of paraffin sections was performed in order to reveal the phenotype of desmin positive cells. Normal liver tissue expresses desmin in endothelial cells, myofibroblast -like cells which surround the arteries in the portal space and in activated stellate cells scatered throughout the hepatic parenchyma, as Figure 5-6 shows. In the areas located outside the periportal zones, the presence of desmin positive cells is very seldom. Under pathological circumstances generated by any form of liver injury, desmin positive cells are present in higher numbers in close proximity to the nec rotic cells, being observed among the early post injury changes128. As a consequence of the hepatectomy injury in our model, numerous desmin positive hepatic stellate cells are present in the acinar zone one, in close proximity to the small oval -shaped cells which are the regenerating phenotype in the 2AAF/PHx model. Interestingly, the radiating pattern of oval cell migration toward the centrolobular area is followed by the stellate cells which seem to extend projections around the progenitors, as Figure 57 A) and B) shows. When L-cysteine exposure was added, a considerable lower number of desmin positive stellate cells were present in the periportal areas. Not restricted to the perivascular structures, these cells are in higher number than in the normal l iver and are also spatially associated with the same oval shaped phenotype, as seen in Figure 5 7 C) and D). For a more accurate assessment of the stellate cell activation status, a quantitative computer image analysis was performed. A significant 11.1-fo ld reduction in hepatic pericyte presence on the entire liver section was observed when the animals were exposed to L-cysteine, as opposed to the situation when only the oval cell activation
82 protocol was performed (Figure 58). Taken together, these data s uggest that L cysteine has an inhibitory effect on the hepatic stellate cell population in vivo 5.3. Evaluation of C ell M orphology in Lcysteine /2AAF/PHx Treated Rats Histological characterization of liver regeneration in the 2AAF/PH x model for rat oval cell activation demonstrated the expected robust proliferation of smaller oval shaped cells emanating from the portal zone ( Figure 5 -10 A and B ). These cells were not present in untreated rat liver ( Figure 59 A and B ). In animals that were maint ained on t he 2% L -cysteine diet the small oval cell response in the portal zone remained quite modest ( Figure 5 10 C and D ). The disparity between the amplitude of the small oval cell response in the two groups is most evident on days 9 post partial hepatectomy. This time point is known to coincide with the peak of oval cell proliferation following 2AAF/PHx in rats. Aside from the reduced oval cell presence in L-cysteine treated animals, there is also a notable difference in cell morphology. In the L-cysteine treated group, the cells tended to be rounded nuclei and basophilic or vacuolar cytoplasm (Figure 5 10 C and D) The comparative analysis of liver morphology for the two animal groups studied was further performed for all time points. The histological exam of liver samples from animals kept on hepatic stellate cell inhibitory diet revealed a delayed onset of oval cell response after 2 -AAF/PHx injury. By day 3 post hepatectomy ver y few portal spaces had isolated cells (1-2/field) with morphology suggestive for the oval cell phenotype (Figure 511 B). The control group which was submitted only to the 2 AAF/PHx treatment displayed by day 3 a robust onset of progenitor -like oval cell presence in the peri portal spaces (Figure 5-11 A), spreading into the acinar zone I by day 5 (Figure 5-
83 11 C). The oval cell response remains very modest during the first week in the diet group (Figure 5-11 D) and the individual cell morphology observed in both groups is suggestive for oval cells in Roskams90 classification (Figures 511 and 512 A and B). The disparity between the amplitude of the regenerative response in the two groups persists on days 9 and 11, the peak of oval cell activation in 2 -AAF/P Hx model, as shown in Figures 510 and 512 B and D. The newly observed cells on day 9 sections are more numerous by day 11, as seen in Figure 512 D). Their morphology is closer in appearance to the small hepatocyte-like phenotype, named by Roskams50 intermediate hepatocyte-like cells and is the center of a controversy over being considered an intermediary differentiating stage of oval cells or a distinct immature progenitor population, as Best and Coleman126 suggest. Unfortunately, because of the lack of reliable markers, it is hard to discern at this stage if they are indeed the progenitor subset described by the above mentioned authors, or oval cells which alr eady made the commitment toward the hepatocyte lineage. In the end of the second week and during the third week post hepatectomy the oval cell response gradually decreases in the 2-AAF/PHx group (Figure 5 -13 A and C). In these animals, by day 20 most of the oval cells have undergone differentiation and only few cells with morphology suggestive for t he oval cell phenotype are visible in the periportal space (Figure 5-13. C). The regenerating response is still vigorous in the Lcysteine exposed group and interestingly, most of the cells exhibiting small hepatocyte like phenotype start having clear vacu oles in their cytoplasm (Figure 5-13 B and D). The presence of vacuoles is indicative of intense metabolic activity and metabolite storage in these small hepatocyte-like cells. They appear clear on Hematoxylin and Eosin stained
84 sections suggesting that the y might hold a lipidic material. In the end of the third week posthepatectomy almost the entire zone I of the acinus is occupied by cells with small hepatocyte like morphology and clear vacuoles in the cytoplasm, but small islands of oval cells are still v isible among the small hepatocyte-like cells (Figure 513 D). Although L -cysteine and 2AAF by themselves could be incriminated for inducing this particular phenotype, our control images show that neither L -cysteine alone (Figure 5 -14 D), nor in combination with 2AAF (Figure 5-14 B), administered to animals kept under identical conditions induce clear vacuole formation in hepatocytes cytoplasm. The presence of these vacuoles could be the result of either increased liponeogenesis from glucose, or a form of lipid storage. Periodic acid Schiff (PAS) staining was performed in order to identify if glycogen, the storage form of glucose is present, as a substrate for increased liponeogenesis in small hepatocyte like cells. Normal liver has few glycogen in the peri portal area, due to the acinar zone I hepatocytes propensity to glycogenolysis, as Figure 5 15 shows. Day 11 post hepatectomy was chosen as timepoint for this staining, due to the presence at this stage of a larger number of small hepatocyte like cells. O n images from animals subjected only to the 2AAF/PHx protocol, we havent noticed glycogen inside the cytoplasm of oval cells or periportal hepatocytes, as expected (Figure 516 A and B). Interestingly, the animals kept on diet had glycogen only in the cyt oplasm of zone III mature hepatocytes, but not in the vacuolated small hepatocyte like cells (Figure 5-16 C and D). It is possible that the content of cytoplamic vacuoles in transitional hepatocytes to be of lipid origin. PAS staining results suggest that lipid presence might be the result of increased storage and not of active liponeogenesis from glucose.
85 5.4. Evaluation of Cell Proliferation in Regenerating Liver under L-cysteine Exposure We next sought to determine the proliferation status of the cells within the periportal zones of livers from 2AAF/PH x treated animals both with, and without the Lcysteine diet. Immunostaining for Ki67 nuclear antigen was performed on samples collected on day 9 following PH x Ki67 identifies all cells that have exited t he quiescent state, G0, and have entered the cell cycle Hepatocytes are the cell population which normally proliferates after hepatic injury. In the absence of any lesion, hepatocytes only divide to replace the aged cells which suffer apoptosis. Thus, i n the normal liver very few hepatocytes mainly localized in the periportal zone proliferate, as Figure 5 17 shows A large number of proliferating cells were present in the peri portal zones of rat liver subjected to the 2AAF/PH x protocol as seen in Figur e 5 -18 A) and B) Considerably fewer cells were Ki67 positive on the sections collected from animals kept on L-cysteine diet (Figure 5-18 C) and D). The quantitative image analysis performed on these sections showed a 3.96 fold reduction in the number of p roliferating cells i n the peri portal zones of the liver from rats maintained on the L-cysteine diet as seen in F ig ure 5 19. These data suggest that L cysteine exposure is associated with reduced cell proliferation in periportal ar eas The y also indicate a significant reduction of oval cell contribution to hepatic mass recovery. A double IF (immunofluorescent) staining was performed to investigate if the reduced proliferation correlates with low levels of stellate cell activation in the L-cysteine treated liver. Sections were stained for Ki67 and desmin and in normal liver very few hepatocytes, mainly located in the limiting plate, are proliferating (Figure 520). Desmin is an intermediary filament which is present in the activated stellate cells and the
86 va scular wall. In the normal liver is very hard to identify the activated stellate cells located in the normal hepatic parenchyma because of their scarcity, but the myofibroblasts, their active form, are present around the portal triad, as shown in Figure 5 -20. The liver exposed to 2AAF and PHx has a very robust population of cells with ovoid nuclei positive for Ki67. Numerous desmin positive activated stellate cells are seen in the periportal areas, in close proximity to the Ki67 positive cells. The stellat e cell projections are wrapped around the proliferating oval cells, as seen in Figure 521 A and B. In contrast, on sections collected from animals maintained on L-cysteine diet considerably fewer Ki67 positive proliferating oval cells are present. The des min presence is reduced too, confirming the effectiveness of L-cysteine inhibition of stellate cell activation. The spatial distribution of Ki67 and desmin positive cells is similar to the pattern observed in the 2AAF/PHx group (Figure 521 C and D). Taken together, these results suggest that the observed reduction in the oval cell proliferation is associated with decreased stellate cell activation induced by L -cysteine diet. 5.5. AFP Expression in Periportal Regions of Regenerating Liver Alpha -feto protein (AFP) is a fetal globulin found in adult liver only in hepatocellular carcinoma, rarely in cholangiocarcinoma, but constantly in the oval stem cells. Known as a marker of their nonterminally differentiated status, it is currently used t o identify the oval cell population. As expected, on the normal liver sections used as controls no AFP positive cells were apparent as Figure 5 22 shows Under normal circumstances very few oval cells are present in the canals of Hering and they are rarely seen on light mi croscopy sections.
87 Sections collected from animals subjected to the 2AAF/PHx protocol alone, or in association with L -cysteine diet were examined for AFP positive cells. As expected, the portal zones of livers from 2AAF/PH x treated animals contained a lar ge population of AFP positive cells ( Figure 5-23 A and B ). They exhibit the characteristic radiary migration from the periportal space towards the centrolobular vein. The phenotype of these positive cells correlates with Roskams description of oval cells, having an oval shaped nucleus and reduced nucleus to cytoplasm ratio. A sensibly lower presence of AFP positive cells was observed on the sections from L cysteine treated group. Some cells exhibited oval cell phenotype. It is worth noting though, that a greater percentage of the cells within the portal zones of livers from animals maintained on the L-cysteine diet appeared to be transitional hepatocytes (small hepatocyte like cells). Morphologically, they are larger than the average diameter of an oval cel l (5 -10 m) but smaller than a mature hepatocyte (2030 m). On Hematoxylin and Eosin stained sections their cytoplasm is basophilic (Figures 512 D and 5 -13 B) and they express weak ly AFP as shown in Figure 5 -23 C) and D) Quantitative image analysis o f scanned liver samples revealed that in the a nimals exposed to L-cysteine there was present a 4.3-fold decrease in AFP positive areas (Figure 5 24). Global AFP expression in the livers of animals subjected to 2AAF/PH x was determined by quantitative real -time PCR (Figure 5 25). AFP message was measured relative to the normal liver and normalized to bet a actin. Animals that were exposed to L -cysteine demonstrated a 4.7-fold decrease in total liver expression of AFP as
88 compared to animals that were fed the n ormal diet. These results suggest a significant reduction of oval cell contribution to hepatic mass recovery. 5.6 Immunohistochemical Identification of Progenitor Cell Population An alternative oval cell marker was used to confirm the findings of the AFP immunostaining. OV6 is a well characterized marker for oval cells and bile duct cells binding to both CK 14 and CK 19 (cytokeratins 14 and 19) present in the oval cells. In normal liver, only the bile ducts within the portal triad were positive for OV6, d ue to their characteristic expression of CK 19 in the cytoskeleton (Figure s 5 26 ) and to a reduced presence of oval cells under physiological circumstances Cryosections collected from animals subjected to 2AAF/PH x contained a large population of OV6 posi tive cells within the periportal zone that radiated out toward the central vein (Figure 5 27 A and B ), which correlates with AFP findings This is consistent with a normal oval cell response at day 9 foll owing PHx in the 2AAF/PHx model. In contrast to this the liver sections from animals maintained on the L -cysteine diet displayed a very modest oval cell response at day 9 following PH x (Figure 5 27 C and D ), mostly confined to the periportal areas The reduced contribution of oval cells to the regeneration process is confirmed by the OV6 immunostaining. Since cell morphology is not so well conserved on cryosections, we scanned the slides for quantitative image analysis of the OV6 positive cells on our samples. Computer image analysis of scanned slides confirmed the 3.5-fold disparity in the magnitude of oval cell response in animals that were fed the normal rat food, as compared to animals that were administered L-cysteine ( Figure 528). Taken togeth er, all the se observations strongly suggest that the L -cysteine associated to the 2AAF/PHx model of hepatic injury induces a diminished and delayed regenerative response in rat liver.
89 A B Figure 51. L-cysteine effects on WB F344 ovall cell line. Representative pictures of BrdU stained chamber slides taken during an experiment done in triplicate. A) L -cysteine untreated control vs. B) 100 M L -cysteine exposed cells, showing no significant changes in morphology and BrdU uptake. A B Figure 52. L-cysteine effects on portal myofibroblasts. Representative pictures of BrdU stained chamber slides taken during an experiment done in triplicate. A) L cysteine untreated control vs. B) 100 M L -cysteine exposed cells displaying a marked reduction in BrdU i ncorporation.
90 A B Figure 53. L-cysteine effects on HSC T6 hepatic stellate cell line. Representative pictur es of BrdU stained chamber slides taken during an experiment done in triplicate. A) L -cysteine untreated control vs. B) 100 M L -cysteine exposed cells showing reduced number of cells undergoing S phase Figure 54. Comparative image analysis of Brdu incorporation index in WB 344F oval cell line, PF portal fibroblasts and HSC T6 stellate cell line; white columns L cysteine exposed cells, black columns controls maintained on regular culture media. Data represent the mean +/ SD of three independent experiments, p<0.05.
91 A B C D Figure 55. Comparative image analysis of desmin positive areas on liver sections from animals sac rificed 9 days post hepatectomy; r epresentative pictures of desmin immunofluoresce stained cryosections were taken during an experiment done in triplicate. A) L -cysteine exposed animals (n = 3) 20X magnification B) control animals (n = 3) subjected only to the 2AAF/PHx protocol, 20X magnification C) same group (n = 3) as A) 40X magnification and D) same group (n = 3) as B) 40X magnification; PT portal triad.
92 A B Figure 56. Desmin imm unostaining of normal rat liver; representative pictures taken during an experiment done in triplicate Samples collected from three different animals (n = 3) were used each time. A) 20X magnification, B) 40X magnification showing positive vascular walls and rare hepatic stellate cells/myofibroblasts in the periportal areas. HA hepat ic artery branch, PV portal vein branch, BD bile duct branch, SC stellate cell, EC endothelial cell.
93 A B C D Figure 57. Desmin immunostaining of rat liver sections collected on day 9 post hepatectomy from animals (n = 3) exposed to L -cyst eine C) 20X magnification, D) 40X magnification, as opposed to 2AAF/PHx treated rats (n = 3) A) 20X magnification, D) 40X magnification, showing positive vascular walls and reduced hepatic stellate cells in the diet exposed animals. Representative picture s were taken during an experiment done in triplicate. HA hepatic artery branch, PV portal vein branch, BD bile duct branch, HSC hepatic stellate cell, EC endothelial cell.
94 Figure 58. Comparative quantitative image analysis of desmin positiv e areas reflecting decreased activated stellate cell presence on sections from animals exposed to L -cysteine diet : N normal rat liver, 9AP samples collected on day 9 post acute liver injury in animals under 2AAF/PHx protocol, 9DAP liver sections from animals kept on L-cysteine diet associated to 2AAF/PHx (timepoint day 9 post hepatectomy). Data represent the mean +/ SD of three independent experiments, p<0.05.
95 A B Figure 59. Hematoxylin and Eosin staining of normal rat liver shows that the per iportal areas are devoid of oval cell presence. Representative pictures were taken during an experiment done in triplicate, using sections collected from three different animals (n = 3) each time. A) 20X magnification, B) 40X magnification. HA hepatic artery branch, PV portal vein branch, BD bile duct branch, H hepatocyte, RBC red blood cell.
96 A B C D Figure 510. Comparative histol ogical exam of H&E stained liver samples collected on day 9 post acute liver injury from: animals (n = 3) on 2AAF/PHx protocol A) 20X magnification, B) 40X magnification and from rats (n = 3) maintained on L -cysteine diet and 2AAF/PHx C) 20X magn ification, D) 40X magnification. Representative pictures were taken during an experiment done in triplicate; HA hepat ic artery branch, PV portal vein branch, BD bile duct branch OC oval cell, SHL small hepatocyte-like cell.
97 A B C D Figure 511. Comparative histologic al exam of liver s ections ; H&E staining of samples collected on days 3 (A and B) and 5 (C and D) post acute liver injury: from animals (n = 3) on 2AAF/PHx protocol (panels A and C) and from rats 9n = 3) maintained on L-cysteine diet and 2AAF/PHx (panels B and D). 20X magnification. Representative pictur es were taken during an experiment done in triplicate and samples collected from three different animals (n = 3) were examined each time. OV oval cell, HA hepatic artery branch, BD bile duct branch, PV portal vein branch.
98 A B C D Figure 512. Comparative histologic al exam of H& E stained liver samples collected on days 7 (A and B) and 11 (C and D) post acute liver injury: from animals (n = 3) on 2AAF/PHx protocol (panels A and C) and from rats (n = 3) maintained on L-cysteine diet and 2AAF/PHx (panels B and D). 20X magnification. Representative pictur es were taken during an experiment done in triplicate. SHL small hepatocyte-like cell, OV oval cell, HA hepatic artery branch, BD bile duct branch, PV portal vein branch.
99 A B C D Figure 513. Comparative histological exam of H&E stained liver samples collected on days 15 (A and B) and 20 (C and D) post acute liver injury: from animals (n = 3) on 2AAF/PH protocol (panels A and C) and from rats (n = 3) maintained on L -cysteine diet and 2AAF/PH (panels B ad D). 20X magnif ication. Representative pictur es were taken during an experiment done in triplicate. HA hepatic artery branch, PV portal vein branch, BD bile duct branch, SHL small hepatocyte-like cell starting to form vacuoles in the cytoplasm, OC oval cell.
100 A B C D Figure 514. Comparative histological exam of Hematoxylin and Eosin stained control samples A) normal liver, B) L -cysteine diet and 2AAF, C) 2AAF alone, D) Lcysteine diet alone. Representative pictur es were taken during an experiment done in triplicate. Sections from three animals in each group (n = 3) were examined. HA hepatic artery branch, PV portal vein branch, BD bile duct branch, 20x magnification.
101 A B Figure 515. Normal rat liver: the low levels of cytoplasmic glycogen are not visible by PAS staining as distinct cytoplasmic granules in periportal hepatocytes. A) 20X magnification, B) 40X magnification. Representative pictures were taken during an experiment done in triplicate and sections from three different animals (n = 3) were examined each time. HA hepatic artery branch, PV portal vein branch, BD bile duct branch.
102 A B C D Figure 516. Comparative histologic al exam of PAS stained liver samples collected on day 11 post acute liver injury from animals (n = 3) exposed to 2AAF/PHx protocol (panels A and B) and rats (n = 3) kept on L-cysteine diet (panels C and D). 20X magnification (panels A and C) and 40X magnification (panels B and D). Representative pictur es of glycogen loaded hepatocytes (pink) were taken during an experiment done in triplicate. HA hepatic artery branch, PV portal vein branch, BD bile duct branch, arrowheads indicate PAS positive mature hepatocytes.
103 A B Figure 5 17. Ki67 immunostaining of normal rat liver ; representative pictur es were taken during an experiment done in triplicate and tissue samples collected from three animals (n = 3) were examined each time. A) 20X magnification, B) 40X magnification showing very few, periportally scattered hepatocytes which exit G0 and enter the cell cycle. HA hepatic artery branch, PV portal vein branch, BD bile duct branch, arrowheads indicate hepatocytes undergoing the cell cycle.
104 A B C D Figure 518. Ki67 immunostaining of rat liver sections collected on day 9 post hepatectomy from control animals (n = 3) undergoing 2AAF/PHx protocol A) 20X magnification and B) 40X magnification and same time point rats (n = 3) exposed to L-cysteine and 2AAF/PHx oval cell a ctivation C) 20X magnification D) 40X magnification. Representative pictures were taken during an experiment done in triplicate. HA hepatic artery branch, PV portal vein branch, BD bile duct branch, arrowheads indicate Ki67 positive nuclei.
105 Figu re 519. Comparative quantitative image analysis of hepatic proliferation potential during compensatory hyperplasia; Ki67 immunostaining of liver sections collected from normal rats (n = 3) N and, on day 9 post hepatectomy, from control animals (n = 3) undergoing 2AAF/PHx protoco l 9AP and rats (n = 3) exposed to L-cysteine associated with 2AAF/PHx oval cell activation 9DAP. Data represent the mean +/ SD of three independent experiments, p<0.05 and ** p<0.005.
106 A B Figure 520. Double IF for Ki6 7 and desmin on normal liver sections showing very few hepatocytes located in the limiting plate which exit G0 and enter the cell cycle. Representative pictur es were taken during an experiment done in triplicate, using liver samples from three animals (n = 3) each time. A) 20X magnification, B) 40X magnification. HA hepatic artery branch, PV portal vein branch, SV small vessels, branches of hepatic artery arrowheads indicate hepatocytes undergoing the cell cycle (nuclai stained green with FITC fluorescein isothiocyanate) and arrows indicate desmin positive myofibroblasts and endothelial cells in the vascular wall (stained red with Texas red).
107 A B C D Figure 521. Double IF for Ki67 and desmin on liver sections collected 9 days post acute hepatic injury.from animals (n = 3) exposed to the 2AAF/PHx protocol A) 20X magnification, B) 40X magnification and from animals (n = 3) kept on L -cysteine diet as sociated to the 2AAF/PHx protocol C) 20X magnification, D) 40X magnification. Representative pictur es were taken during an experiment done in triplicate. HA hepatic artery branch, PV portal vein branch, arrowheads indicate hepatocytes undergoing the cell cycle (nuclei stained green with FITC) and arrows indicate desmin positive activated stellate cells and endothelial cells (stained red with Texas red).
108 A B Figure 522. AFP imm unostaining of normal rat liver; representative pictur es were taken during an experiment done in triplicate and samples from three different animals (n = 3) were examined each time. A) 20X magnification, B) 40X magnification showing the absence of AFP positive cells in animals with normal hepatocyte mediated regeneration.
109 A B C D Figure 523. AFP immunostaining of rat liver sections collected on day 9 post hepatectomy from 2AAF/PHx treated rats (A 20X magnification, B 40X magnification), as opposed to animals exposed to L-cysteineand 2AAF/PHx (C 20X magnification, D 40X magnification), showing reduced numbers of oval cells and few small hepatocyte-like cells in the diet exposed animals. Arrows indicate AFP positive oval cells. Representative pictur es were taken during an experiment done in trip licate and samples from three different animals (n = 3) were examined each time.
110 Figure 524. Comparative quantitative image analysis of AFP positive cell presence reflected by immunostaining. Liver sections were collected from: normal rats (n = 3) N; on day 9 post hepatectomy, from control animals (n = 3) undergoing 2AAF/PH protocol 9AP; at the same timepoint, rats (n = 3) exposed to L-cysteine and 2AAF/PHx oval cell activation protocol 9DAP. Data represent the mean +/ SD of three independent experiments, p<0.05.
111 Figure 525. Real time PCR analysis comparative expression of AFP reflecting the regeneration process on samples collected on day 9 post hepatectomy from animals (n = 3) subjected to the 2AAF/PHx protocol (9AP), respectively ra ts (n = 3) treated with L-cysteine during the same protocol (9DAP); values normalized to beta actin and normal liver tissue (N) expression of AFP; GAPDH was used as a loading control. Data represent the mean +/ SD of three independent experiments, p<0.05.
112 A B Figure 526. OV6 immunostaining of normal rat liver showing the presence of positive bile duct cells in animals with normal hepatocyte mediated regeneration. Representative pictur es were taken during an experiment done in triplicate. Samples col lected from three different animals (n = 3) were analyzed each time. A) 20X magnification, B) 40X magnification,
113 A B C D Figure 527. OV6 immunostaining of rat liver sections collected on day 9 post hepatectomy from: 2AAF/PH treated animals (A 20X magnification, B 40X magnification) as opposed to rats exposed to L-cysteine and 2AAF/PHx (C 20X magnification, D 40X magnification), showing reduced numbers of oval cells in the diet exposed animals. Representative pictur es were taken during an e xperiment done in triplicate. Samples collected from three different animals (n = 3) were analyzed each time. Arrowheads indicate clusters of OV6 positive oval cells.
114 Figure 528. Comparative quantitative image analysis of OV6 positive cell presence reflected by immunostaining. Liver sections were collected from normal rats (n = 3) N and, on day 9 post hepatectomy, from control animals (n = 3) undergoing 2AAF/PHx protocol 9AP, and rats (n = 3) exposed to Lcysteine and 2AAF/PHx oval cell activation 9DAP. Data represent the mean +/ SD of three independent experiments, p<0.05.
115 CHAPTER 6 DISCUSSIONS AND FUTURE DIRECTIONS OF STUDY 6.1 Interpretation of Results Progenitor cell mediated liver regeneration is an alternative compensatory hyperplasia, able to restore hepatic mass when hepatocyte proliferation is severely impaired by massive liver necrosis or chronic cirrhogenic conditions127. It involv es sequential waves of cytokine secretion and remodeling of the extracellular matrix. These two process es are intimately coupled, as the matrix can liberate chemical signals when degraded, and concentrate chemical signals132 that bind to th e matrix within specific regions38, 133. Thus ECM functions as a primary reservoir of biologically active molecules in the liver132. Hepatic stellate cells are the resident hepatic pericyte responsible for secreting cytokines and growth factors, but also for matrix remodeling. During hepatic regeneration the presence of increased numbers of hepatic stellate cells was noted in close proximity to the oval cells and direct contact between stellate cell projection and oval cell membranes was reported18. Activated hepatic stellate cells are the main source of MMPs and TIMPS that participate in matrix remodeling and release of bo und cytokines17, 128. Matrix remodeling results in the establishment of a unique microenvironment, conducive for the proliferation and migration of cells within the regenerating zone. This renders the activation of hepatic stellate cells a n important compo nent of progenitor cell mediated regeneration process. Previous research conducted by our group demonstrated that during progenitor cell mediated liver regeneration, a fibronectin rich provisional matrix is synthesized in the peri portal zone132. We feel t hat it is likely that this provisional matrix contributes to the oval cell response, acting as a required substrate on which oval cells may proliferate
116 and providing binding sites for signaling molecules that regulate oval cell phenotype. One such example is CTGF which binds to the fibronectin rich provisional matrix of the periportal zone, where it is concentrated and made available to the oval cells which are known to express CTGF receptors38. Given these complex direct and indirect interactions between s tellate and oval cells during regeneration, it is of great interest to understand to which extent is the regeneration process affected in the absence of stellate cell contribution. Knowing that parenchymal mass recovery in a timely manner is crucial for maintaining a normal hepatic function, we decided to inhibit the stellate cell activation. Oval cells and stellate cells are among the first cells to enter the cell cycle following injury in the 2AAF/PH x progenitor activation protocol128. L ess is known regar ding the exact temporal relationship between oval and stellate cell activation in the regenerating liver. It is for this reason that we chose to begin the L-cysteine diet well before initiation of the 2AAF/PH x protocol (Figure 4 1). The daily food intake and the body weight of all animals were monitored for the duration of the study in order to identify any potential effects attributable to the diet, and no significant differences were seen between animals fed the experimental and control diets Before es tablishing the animal model we tested the inhibitory effects of L-cysteine in vitro Our stud y demonstrated that L -cysteine per se does not directly affect WB F344 oval cell proliferation in culture; nor does it induce any alterations of their phenotype. We opted to use two different mesenchymal cell populations for this assay. HSC T6 cells are the only available quiescent stellate cell line and they are demonstrated to maintain their non activated status for a minimum of one week in culture. The second
117 po pulation examined, improperly named portal fibroblasts is composed mostly of myofibroblast cells believed to result from the activation of stellate cells. They exhibit the contractile properties necessary to modulate the blood influx into the hepatic lobul e, one of the main functional characteristics of the activated stellate cell. Either in activated (portal fibroblasts) or quiescent state (HSC T6 stellate cells), both mesenchymal cells included in this study were sensitive to L-cysteine inhibiting effects Their reduced proliferation was reflected by a sensibly diminished BrdU index. Interestingly, portal myofibroblasts have proved to be more sensitive to the inhibiting effects of L -cysteine, exhibiting a 5.6-fold reduction of their proliferation index, as opposed to 3.56-fold reduction in HSC T6 cells. This observation correlates with previous reports about Lcysteine reversing chemically induced fibrosis in DMN (Dimethylnitrosamine) fed rats, by directly inhibiting the proliferation of activated hepatic s tellate cells124. The hepatocyte proliferation inhibitor used in our study ( 2 -AAF ) is activated and detoxified by the liver through rounds of hydroxylation and conjugation129 which lead to renal excretion of water -soluble derivatives130, 131. Being a precursor for glutathione synthesis, it is reasonable to consider the possibility that L-cysteine could increase the rate of 2AAF detoxification. This would potentially lead to incomplete suppression of hepatocyte proliferation in the 2AAF/PH x model. However, examination of Ki67 stained liver sections from the L-cysteine treated group showed no signs of mature hepatocyte proliferation, the only Ki67 positive cells exhibiting oval cell and small hepatocyte like phenotype (Figure s 5 14, 515). This would seem to exclude differential 2AAF metabolism as a complicating factor in these studies. This finding is explained in part by glutathiones lack of effectiveness in competing with macromolecules for trapping the
118 reactive metabolites of 2AAF, and also by its inabil ity to interfere with DNA adduct formation135. Since the ECM fibers, enzymes and growth factors are secreted in the extracellular environment, the most used marker for oval cell activation is desmin, an intermediate filament wich is present in the activated Ito cells cytoskeleton. Our immunostaining for desmin showed that the presence of activated stellate cells is significantly reduced during L-cysteine exposure, although their distribution in the vicinity of oval cells is conserved. No changes have been noticed in the characteristic pattern of stellate cell extensions wrapping around the regenerating cells. It is reported that due to the direct membrane contacts Ito cells are able to exert their nourishing function during oval cell proliferation18. The parallel reduction of stellate and oval cell presence in the regenerating liver (Figure 521) seems to be associated with a direct inhibition of their activation and not with an anomaly of migration or disruption of cell -to cell interactions. The human conditions associated with oval cell activation are characterized by exhaustion of hepatocyte function and proliferation capacity. The resulting hepatic failure would potentially benefit from an oval stem cell therapy able to recover the liver function before the onset of end-stage MODS ( multiple organ dysfunction syndrome) For these reasons, any delay in the oval cell mediated liver regeneration can have fatal consequences. Comparative monitoring of oval cell activation revealed differences in regards to the onset and progression of liver regeneration. When stellate cells are inhibited the onset of regeneration is delayed. Only 12 cells/field exhibit morphology suggestive for the oval cell phenotype 3 days post hepatectomy (Figure 5-11 B) and a
119 robust oval c ell response onset is observed only in the end of the first week (Figure 5-12 B). In contrast, the group exposed only to 2AAF/PHx exhibits a robust onset of regeneration starting with day 3 post acute injury (Figure 5-11A). This disparity between the respo nses at corresponding time points is persistent during the whole course of hepatic regeneration in our model. The hepatic mass would eventually be restored, but not exclusively by oval cell contribution. Once the oval cell differentiation is complete and t he 2AAF release is over, the resulting hepatocytes would take over and start divide. However, it would take considerably more time for the liver mass to be restored. A combined oval and stellate cell therapy might accelerate the regeneration and overcome t he time constraints imposed by the onset of fatal MODS Another interesting feature of the L-cysteine treated livers is the apparent accumulation of small hepatocyte-like cells, also known as transitional hepatocytes (Figure 5 12 D ). These cells are morphologically similar to hepatocytes, but are much smaller and weakly express AFP. They have been observed in retrorsine hepatic injury models and are considered by some authors an intermediary stage in normal differentiatiation towards hepatocyte, whilst others see them as an independent progenitor cell126. In our model, we speculate that in the absence of an adequate stellate support, oval cells might take two pathways. If the differentiaton cues are missing, they might f ail to complete the differentiation pr ogram and the intermediary hepatocytes we have seen are a developmental arrested phenotype. Conversely, if during regeneration the coupling between cytokine mediated priming phase and growth factor promoted proliferation fails, oval cells might start diffe rentiation towards hepatocytes earlier than expected and the small hepatocyte like cells are just a normal
120 differentiating phenotype. This would suggest that inhibition of stellate cell activation following 2AAF/PH x not only affects oval cell proliferation but also oval cell differentiation. Once again, this would most likely result from the lack of an appropriate microenvironment within the regenerating zone. One recently published stud y demonstrate s that stellate cells within the liver may, through a pro cess of mesenchymal to epithelial transition, give rise to hepatocytes89. It is possible that this phenomenon involves an intermediate cell type with oval cell properties. It is impossible though, to determine if decreased mesenchymal to epithelial transit ion contributes to the reduction in oval cell proliferation seen in our model. However, this possibility deserves mention. Two distinct cellular markers, AFP and OV6, were used to identify the oval cell population in our study. Quantitative image analyses and real time PCR analysis showed a disparity in oval cell presence at corresponding time points between the two animal groups. Overall, the blunted oval cell response in animals fed the L-cysteine diet is likely due to a combination of the loss of a major cytokine and growth factor source (i.e. activated stellate cells), coupled with the loss of an appropriate microenvironment for expansion of the oval cell population (i.e. the fibronectin rich provisional matrix). The end result of L-cysteine treatment is a delayed restoration of liver mass following the 2AAF/PH x protocol Because the injury does eventually resolve in the L-cysteine treated group, redundant pathways for the regulation of oval cell phenotyp e likely exist. However, the significant reduction in oval cells seen at what would normally be considered the time of maximal oval cell proliferation proves, for the first time, the critical role of stellate cells in oval cell mediated liver regeneration.
121 6.2 Clinical A pplications Terminal cirrhosis is a medical challenge in the sense that to this date it has no effective cure and irreversibly progresses to decompensated liver failure. A fairly significant number of hepatic conditions progress toward cirr hosis. Despite their various pathophysiological mechanisms ranging from autoimmune disorders like primary billiary cirrhosis to genetic mutations (seen in Wilson disease or hemochromatosis) and chronic hepatitis C infection they are all accompanied by persistent activation of stellate cells which results in cirrhosis. In an ideal outcome, the oval cell hepatic regeneration which takes place in the cirrhotic nodules would restore the liver mass and the fibrosis would decrease to a point where the neovasc ularization would reestablish functional connections with the indemn hepatocytes inside the nodules. The common denominator for these ideal outcome is the hepatic stellate cell which is able to influence the amplitude of regeneration and synthesize the fib rotic tracts in cirrhotic liver. The more we know about stellate cells involvement and interactions with the other resident cell populations, the more chances of developing a strategy for dissociating these two functions and inhibiting the fibrogenic outc ome. Due to their remarkable capacity of proliferation, pluripotency and engraftment in various tissues, stem cells have shown great promise as a potential candidate for cell therapy. Ideally, stem cells isolated from an individual would be expanded in vit ro, primed towards a certain differentiation path and reinjected into the desired organ. It is known that hepatocyte transplantation is followed only by transient engraftment, thus limiting its potential of being used in cell therapy. Researchers and clini cians turned to exploring the oval cells potential of being used as an alternative cell therapy. Since at
122 least one subpopulation of oval cells is bone marrow derived, there is a chance of isolating them from blood. In various experimental models oval cells have been implanted into mammalian liver and subsequent presence of donor derived tissue has been demonstrated. Over the years the engraftment potential of oval cell has been thoroughly documented. Unfortunately, very few hepatocytes derived from donor oval cells have been identified in the recipient liver. Increasing the numbers of donor derived cells might be achieved by priming the local environment to become more conducive for oval cell proliferation, migration and differentiation. Perhaps chemicall y induced, a transient activation of stellate cells might be the boost that injected oval cells need to proliferate in higher numbers. Or, injecting oval and stellate cells concomitantly would result in a better outcome in terms of graft size. Using cell therapy as an alternative for transplantation in patients with cirrhosis requires a very thorough characterization of the interactions between the oval and stellate cells. The hyperactivated stellate cells, already existing in the cirrhotic liver at the moment of progenitor cell injection might induce a hyperproliferation of progenitors, or conversely, might keep them in a nondifferentiated state. The prior administration of L -cysteine, can inhibit stellate cell proliferation and reduce the fibrotic tracts to an extent which might allow the vessel formation and migration of oval cells into the adjacent areas of hepatic lobules. 6.3 Future Directions of Study This present research opens the door for two main directions of study which would shed more light on the complex interactions between the hepatic stellate cells and oval cells in the liver. One direction might be the characterization of the hepatic extracellular
123 matrix in terms of the delicate balance between MMPs and TIMPs during the L-cysteine inhibiti on of stellate cells. It is reasonable to assume that certain variations in MMPs expression would result in matrix degradation which would favor deposition of new fibers produced by stellate cells. ECM profile is very important at each step of oval cell mi gration from the periportal space towards the pericentral zone, ensuring the adequate supply with regenerative cells at the site of injury. Also, MMP mediated cleavage liberates and activates growth factors necessary for oval cell proliferation and differentiation. During liver regeneration, after the priming step, the recovery is not possible without an adequate supply of growth factors produced by existing hepatocytes, Kupffer cells and hepatic stellate cells. Some of these necessary growth factors are pr oduced in an inactive form and are bound to the ECM. Without the MMP degradation, they would never be available for the matricrine regulation of liver regeneration. An alternative avenue would be represented by the characterization of cytokine production o f stellate cells when their activation is inhibited by L -cysteine. It is possible that the delayed regenerative response is at least in part the consequence of a reduced cytokine and growth factor stimulation by the stellate cells. Corroborated with the st udy of ECM profile under L-cysteine exposure this new research avenue would shed light on the significant contributors to oval cell proliferation, migration and differentiation. An unexpected finding in our study was the presence of small hepatocyte like c ells at time points during regeneration when they are usually absent in the 2AAF/PHx model. Whether they are the result of an accelerated differentiation of the oval cells, a differentiation arrested cell, or are an alternative progenitor recruited when th e oval cell
124 response is weak it is still to be found. Exploring this new regenerative cell player in the context of stellate cell inhibition represents a new direction for future research.
125 CHAPTER 7 SUMMARY AND CONCLUSI ONS The aim of this study was to gain a better insight into the cellular interactions between the stellate cells and the oval cells in the liver regeneration process by chemically blocking the Ito cell activation In summary, our results suggest that L cysteine, the non essential amino acid used for this purpose, is an effective inhibitor of hepatic stellate cells in our model of liver regeneration. T he blunted oval cell response in animals fed the L-cysteine diet result s in a significant delay of the regeneration process fo llowing 2AAF/PH x T he significant reduction in oval cells seen at corresponding time points throughout the oval cell mediated regeneration proves the critical role of stellate cells in oval cell mediated liver regeneration. We believe that by influenci ng the proliferation rate of oval cells and, as other studies have shown38, changing the ECM profile in the regenerating liver, the stellate cells do play a necessary role in facilitating oval cell proliferation in the liver. Our data also indicate that wh en the stellate cells are not able to contribute effectively to the oval cell mediated regeneration, the liver mass recovery takes place at a considerably lower pace. Compared to the 2AAF/PHx regeneration model, the regeneration at identical time points po st acute injury is reduced and the whole process is delayed. The presence of small hepatocyte like cells is a novel finding. It indicates the existence of redundant pathways which are activated in the injured liver when the oval cell mediated regeneration is blunted. The transitional small hepatocyte-like cells might also be the result of premature or arrested differentiation of oval cells in the absence of an adequate support from the inhibited stellate cells.
126 Chemical i nhibition of stellate cell activati on resulted in an abnormal profile of the rege nerative proces s, delayed and blunted, involving phenotypic changes of regenerating cells Considering all our findings, this study concludes that the activation of hepatic stellate cells is required for an appropriate oval cell response to 2AAF/PHx hepatic injury
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138 BIOGRAPHICAL SKETCH Dana Gabriela Pintilie was born in Suceava, Romania. She attended George Bacovia High School, graduating the Mathematics and Physics program in 1986. She obtained a medical doctor degree from the University of Medicine and Pharmacy Gr. T. Popa Iasi in 1992. Deciding to further her education, she enrolled in the graduate program at the same university and, under the mentorship of Dr. Mircea Chiriac she obtained a PhD in anatomy in 2003, with the thesis A telomic and systemic interpretation of the celial trunk. Searching for an opportunity to learn new molecular te chniques and better prepare for the complex approach modern research requires, Dana decided to come to the United States and enroll in the Interdisciplinary Program in Biomedical Sciences at the University of Florida, beginning the fall of 2005. After four years and a half of diligent work under the mentorship of Dr. Bryon Petersen, Dana will receive her doctorate in molecular cell biology at the University of Florida College of Medicine. Her project consisted in elucidating the effects of stellate cell inh ibition on oval cell proliferation. Her scholastic accomplishments include being an Alumni fellow at the University o f Florida during her Ph.D. studies She received the Outstanding International Student Award 2009 from the University of Florida College of Medicine.