A NEW ROLE FOR TISSUE-TYPE PLASMINOGEN ACTIVATOR IN LIVER INJURY by Liang-I Kang B.S. in Biological Sciences, University of Delaware, 2006 Submitted to the Graduate Faculty of School of Medicine in partial fulfillment of the requirements for the degree of Doctor of Philosophy University of Pittsburgh 2013 i UNIVERSITY OF PITTSBURGH SCHOOL OF MEDICINE This dissertation was presented by Liang-I Kang It was defended on March 6, 2013 and approved by James L. Funderburgh, Ph.D., Professor, Departments of Ophthalmology and Cell Biology Youhua Liu, Ph.D., Professor, Department of Pathology George K. Michalopoulos, M.D., Ph.D., Professor and Chairman, Department of Pathology Donna Beer Stolz, Ph.D., Associate Professor, Department of Cell Biology and Physiology Committee Chair: Charleen T. Chu, M.D., Ph.D., Professor, Department of Pathology Dissertation Advisor: Wendy M. Mars, Ph.D., Associate Professor, Department of Pathology ii Copyright © by Liang-I Kang 2013 iii A NEW ROLE FOR TISSUE-TYPE PLASMINOGEN ACTIVATOR IN LIVER INJURY Liang-I Kang, Ph.D. University of Pittsburgh, 2013 Chronic liver disease is increasing in prevalence worldwide; however, few medical therapies are available to treat liver cirrhosis and failure. Hepatic stellate cell (HSC) activation and trans- differentiation into myofibroblast-like (MFB-like) cells is a key process in liver injury and fibrogenesis. Greater understanding of the role of matrix regulating proteases, such as the plasminogen activators, in HSC activation could provide new therapeutic targets for treating chronic liver disease. Mice lacking plasminogen activators exhibit delay in liver repair; however, their exact functions after liver injury remain unclear. Recent studies in kidney demonstrate that low density lipoprotein receptor-related protein 1 (LRP1)-dependent signaling by tissue-type plasminogen activator (t-PA) is an essential regulator of the myofibroblast phenotype after injury. This study investigated the role of t-PA and LRP1 in HSC activation and in vivo liver injury. We find that, in contrast to kidney fibroblasts, exogenous t-PA antagonizes activation of primary and immortalized HSCs in vitro. Similar to kidney, these effects are independent of the proteolytic function of t-PA and require phosphorylation of LRP1. Antagonism of LRP1 or PI3K/Akt signaling pathways is able to prevent t-PA-mediated decreases in α-SMA. During recovery following acute liver injury, mice lacking t-PA (globally) or LRP1 (conditionally on HSCs) retain higher densities of the α-SMA+ MFB-like cell population compared to control mice. These differences are seen at time points that correspond to the appearance of co- iv localization between p-LRP1 and α-SMA, as well as t-PA immunolocalization at sites of α- SMA-positive cells. Additionally, t-PA may regulate macrophage phenotype and drug metabolism, as t-PA null mice exhibit increased macrophage accumulation and lack of normal compensatory down-regulation of a key metabolic enzyme after acute injury. Finally, more collagen I deposition remains in the livers of t-PA null mice up to two weeks after cessation of chronic liver injury, suggesting a decreased rate of matrix turnover. These data reveal that t-PA has multiple functions in liver repair and is able to affect the phenotype of several cell types, in addition to its classical plasminogen activating role. Further preclinical studies are needed to evaluate the clinical potential of using t-PA as a treatment for chronic liver injury and fibrosis. v TABLE OF CONTENTS PREFACE ................................................................................................................................. XIII 1.0 GENERAL INTRODUCTION ................................................................................... 1 1.1 THE LIVER AND ITS REGENERATION AFTER INJURY........................ 1 1.1.1 Cell types of the liver ....................................................................................... 2 1.1.2 Regeneration models ....................................................................................... 5 1.1.3 Cellular sources for repopulation in liver regeneration ............................... 7 1.1.3.1 Resident liver cells ................................................................................. 7 1.1.3.2 Bone marrow derived cells ................................................................. 10 1.1.3.3 Oval/Progenitor cells and trans-differentiation ............................... 10 1.2 LIVER INJURY AND FIBROSIS ................................................................... 12 1.2.1 Hepatic fibrogenesis ...................................................................................... 12 1.2.2 Challenges to treating hepatic cirrhosis ...................................................... 15 1.3 PLASMINOGEN ACTIVATORS ................................................................... 16 1.3.1 Structure, function, and clearance of plasminogen activators .................. 16 1.3.2 Plasminogen activators in liver homeostasis ............................................... 19 1.3.3 Plasminogen activators in liver injury and fibrosis .................................... 20 1.4 LOW DENSITY LIPOPROTEIN RECEPTOR-RELATED PROTEIN 1 (LRP1) 22 vi 1.4.1 General structure and function .................................................................... 22 1.4.2 t-PA and LRP1 in kidney fibrosis ................................................................ 24 1.4.3 Role of LRP1 in smooth muscle cells ........................................................... 25 2.0 TISSUE-TYPE PLASMINOGEN ACTIVATOR DOWN-REGULATES HEPATIC STELLATE CELL ACTIVATION THROUGH LDLR-RELATED PROTEIN 1 IN RATS AND MICE .............................................................................................................. 27 2.1 INTRODUCTION ............................................................................................. 27 2.2 EXPERIMENTAL PROCEDURES ................................................................ 29 2.2.1 Reagents and Antibodies ............................................................................... 29 2.2.2 Rat hepatic stellate cell isolation and cell culture ....................................... 29 2.2.3 MTT and [3H]Thymidine Incorporation Assays ........................................ 30 2.2.4 SDS-PAGE and Western Blot ...................................................................... 31 2.2.5 Immunoprecipitation..................................................................................... 32 2.2.6 Reverse Transcription and Polymerase Chain Reaction ........................... 32 2.2.7 Gene Array Analysis ..................................................................................... 33 2.2.8 Carbon tetrachloride acute injury and resolution experiments ................ 34 2.2.9 Immunohistochemical staining and fluorescent labeling ........................... 34 2.2.10 Image Capture and Analysis ....................................................................... 35 2.2.11 Statistical Analysis ........................................................................................ 36 2.2.12 Animal use approval .................................................................................... 36 2.3 RESULTS ........................................................................................................... 37 2.3.1 Exogenous t-PA decreases markers of HSC activation in primary and transformed cells ........................................................................................................ 37 vii 2.3.2 Global gene expression changes after t-PA treatment of HSC-T6 cells ... 39 2.3.3 The effects of t-PA on HSC activation are dependent on LRP1-mediated signaling ...................................................................................................................... 39 2.3.4 p-LRP1 and t-PA co-localization with α-SMA precedes resolution of injury in WT mice ...................................................................................................... 43 2.3.5 t-PA- and LRP1-deficient mice retain more α-SMA-positive cells after injury 46 2.4 DISCUSSION ..................................................................................................... 50 3.0 FURTHER INSIGHTS INTO TISSUE-TYPE PLASMINOGEN ACTIVATOR FUNCTION IN LIVER AND LIVER INJURY ....................................................................... 54 3.1 INTRODUCTION ............................................................................................. 54 3.2 EXPERIMENTAL PROCEDURES ................................................................ 55 3.2.1 Carbon tetrachloride chronic injury and resolution .................................. 55 3.2.2 SDS-PAGE and western blot from liver tissue ........................................... 56 3.2.3 Immunohistochemical staining and image capture .................................... 57 3.3 RESULTS ........................................................................................................... 58 3.3.1 Increased macrophage populations in t-PA null mice after injury........... 58 3.3.2 Cytochrome P450 2E1 expression in WT and t-PA knockout mice.......... 60 3.3.3 Chronic injury studies in WT and t-PA deficient mice .............................. 64 3.4 DISCUSSION ..................................................................................................... 66 4.0 GENERAL REMARKS ............................................................................................ 68 4.1 SUMMARY ........................................................................................................ 68 4.2 DISCUSSION AND FUTURE DIRECTIONS ............................................... 69 viii 4.2.1 Dissection of t-PA and LRP1 signaling partners in HSCs ......................... 69 4.2.2 t-PA and LRP1 kinetics and distribution after injury ............................... 72 4.2.3 Evaluation of observed differences in t-PA null mice ................................ 74 4.2.4 Follow-up studies in LRP1 conditional null mice ....................................... 76 4.2.5 Evaluation the efficacy of t-PA treatment in chronic liver injury ............ 77 APPENDIX A .............................................................................................................................. 79 APPENDIX B .............................................................................................................................. 97 BIBLIOGRAPHY ....................................................................................................................... 98 ix LIST OF TABLES Table 1. Fold changes in selected genes in HSC-T6 cells after treatment with t-PA. .................. 79 Table 2. Absolute changes in selected genes in HSC-T6 cells after treatment with t-PA. ........... 84 x