The Role of Flightless Protein in Hypertrophic Scarring and its Potential as a Target for a Novel Therapy. Alexander MacGregor Cameron University of Adelaide Faculty of Health Sciences School of Medicine Department of Surgery Supervisors: Professor Allison Cowin Professor Peter Anderson Regenerative Medicine Future Industries Institute Mawson Lakes September 2017 TABLE OF CONTENTS TITLE PAGE .................................................................................................................................. i TABLE OF CONTENTS .............................................................................................................. ii LIST OF FIGURES ..................................................................................................................... ix ABSTRACT ............................................................................................................................... xiii DECLARATION ......................................................................................................................... xv ACKNOWLEDGMENTS.......................................................................................................... xvi PUBLICATIONS ARISING FROM THIS THESIS .............................................................. xix NATIONAL AND INTERNATIONAL MEETINGS, SCIENTIFIC ABSTRACTS RESEARCH GRANTS AND AWARDS ARRISING FROM THIS THESIS ....................... xx CHAPTER ONE: LITERATURE REVIEW 1.1 Overview .............................................................................................................................. 2 1.2 Skin Biology ......................................................................................................................... 5 1.2.1 Epidermis ............................................................................................................ 6 1.2.2 Dermis ................................................................................................................... 9 1.3 Wound Healing ................................................................................................................ 10 1.3.1 Inflammatory Phase ..................................................................................... 10 1.3.2 Proliferative Phase ....................................................................................... 11 1.3.3 Remodelling Phase ........................................................................................ 12 ii 1.4 The Myofibroblast .......................................................................................................... 13 1.4.1 Myofibroblast origin .................................................................................... 14 1.4.2 Fibroblast-myofibroblast Differentiation and Tissue Stiffness….15 1.4.3 How do myofibroblasts sense mechanical stress? ............................. 16 1.4.4 The myofibroblast and fibroproliferative disease ............................. 17 1.5 The Cytoskeleton ...................................................................................................... 19 1.5.1 Actin dynamics during wound healing ................................................... 20 1.5.2 Actin cytoskeleton, cell motility and cell matrix ................................. 21 1.6 Flightless Protein ..................................................................................................... 23 1.6.1 The gelsolin family of actin remodelling proteins ............................. 31 1.6.2 Flightless I (Flii) protein .............................................................................. 26 1.6.3 Flightless I structures, expression and molecular functions………28 1.6.4 Flii in wound healing and tissue regeneration .................................... 33 1.7 Hypertrophic Scarring............................................................................................ 37 1.7.1 Defininig hypertrophic scarring ............................................................... 37 1.7.2 Epidemiology ................................................................................................... 39 1.7.3 Burn injury, hypertrophic scarring and current treatments………40 1.7.3.1 Burn injury ...................................................................................... 40 1.7.3.2 History of burn injury management ....................................... 41 1.7.3.3 Current management of hypertrophic scarring ................. 43 iii 1.7.4 Current understanding of the pathophysiology of hypertrophic scarring ....................................................................................................................... 51 1.7.4.1 Inflammation .................................................................................. 51 1.7.4.2 Extracellular matrix ..................................................................... 52 1.7.4.3 Keratinocyte fibroblast interaction ........................................ 54 1.7.4.4 Reversibility of fibrosis ............................................................... 55 1.8 Pre-clinical models of hypertrophic scarring ............................................... 55 1.9 Research Aims and Hypothesis ........................................................................... 64 CHAPTER TWO: MATERIALS AND METHODS 2.1 Materials ..................................................................................................................... 63 2.1.1 Molecular reagents ........................................................................................ 63 2.1.2 Immunohistochemical reagents ............................................................... 63 2.1.3 General reagents ............................................................................................ 66 2.1.4 Osmotic pumps ............................................................................................... 66 2.1.5 Human samples .............................................................................................. 67 2.1.6 Animal tissue ................................................................................................... 68 2.1.6.1 Flii+/- mouse generation .............................................................. 71 2.1.6.2 FliiTg/Tg mouse generation .......................................................... 72 2.1.7 Flii antibody generation .............................................................................. 73 2.2 Methods .......................................................................................................................74 2.2.1 A novel murine model of hypertorhic scarring using subcutaneous iv infusion of bleomycin .............................................................................................. 74 2.2.2 Murine surgery ............................................................................................... 75 2.2.3 Flii neutralizing antibody treatment ..................................................... 77 2.2.4 Sample processing, histology and immunohistochemistry ............ 77 2.2.5 Immunohistochemistry and image analysis......................................... 79 2.2.6 Cell lines and cell culture ............................................................................ 80 2.2.7 HFF isolation and culture ........................................................................... 81 2.2.8 Murine fibroblast isolation and culture ................................................. 81 2.2.9 Migration scratch assay ............................................................................... 82 2.2.10 WST-1 proliferation assay ....................................................................... 82 2.2.11 Immunocytochemistry, image analysis and fluorescence co- localization.................................................................................................................. 83 2.2.12 Protein extraction and quantification ................................................. 84 2.2.13 Western Blotting .......................................................................................... 85 2.3 Statistical analysis ........................................................................................................... 86 CHAPTER THREE: EXPRESSION OF FLII IN BURNS AND HYPERTROPHIC SCARS 3.1 Introduction ...................................................................................................................... 88 3.2 Results .................................................................................................................................90 3.3 Discussion ......................................................................................................................... 96 CHAPTER FOUR: A NOVEL MURINE MODEL OF HYPERTROPHIC SCARRING USING SUBCUTANEOUS INFUSION OF BLEOMYCIN v 4.1 Introduction ................................................................................................................... 101 4.2 Results .............................................................................................................................. 105 4.2.1 The bleomycin model produces tissue architecture that corresponds to hypertrophic scars .................................................................. 105 4.2.2 The bleomycin model displays histopathological features of hypertrophic scars ................................................................................................. 110 4.2.3 Collagen content and composition of bleomycin induced lesions is analogous to hypertrophic