CD109 Deficiency Promotes Skin Fibrosis in a Murine Model Liqin Xu Masters of Science Department of Surgery Division of Experimental Surgery McGill University Montreal, Quebec, Canada November 2018 A thesis submitted to the Faculty of Graduate Studies and Research at McGill University in partial fulfillment of the requirements of the degree of Master of Science in Experimental Surgery. © Copyright Liqin Xu, 2018. All rights reserved. 1 TABLE OF CONTENTS ABSTRACT ................................................................................................................................................. 5 Objectives................................................................................................................................................. 5 Hypothesis ................................................................................................................................................ 5 Methods .................................................................................................................................................... 5 Results ...................................................................................................................................................... 6 Conclusion ............................................................................................................................................... 6 RÉSUMÉ ..................................................................................................................................................... 7 Objectifs ................................................................................................................................................... 7 Hypothèse ................................................................................................................................................ 7 Méthodes .................................................................................................................................................. 7 Résultats ................................................................................................................................................... 8 Conclusion ............................................................................................................................................... 8 ACKNOWLEDGEMENTS ....................................................................................................................... 9 PREFACE .................................................................................................................................................. 10 CONTRIBUTIONS TO ORIGINAL KNOWLEDGE .......................................................................... 11 LIST OF ABBREVIATIONS .................................................................................................................. 13 GENERAL INTRODUCTION ................................................................................................................ 15 Skin Anatomy ........................................................................................................................................ 15 Normal Wound Healing ....................................................................................................................... 15 Fibroproliferative Skin Disorders ....................................................................................................... 17 TGF-β Signaling and Regulation ......................................................................................................... 18 TGF-β Co-Receptors ............................................................................................................................. 19 CD109 ..................................................................................................................................................... 20 Molecular Mediators of Fibrosis ......................................................................................................... 21 Objectives of Current Study ................................................................................................................ 21 MANUSCRIPT: ........................................................................................................................................ 22 BACKGROUND ....................................................................................................................................... 23 MATERIALS AND METHODS ............................................................................................................. 24 Generation of Mouse Colony ............................................................................................................... 24 Genotyping of CD109 Mice .................................................................................................................. 24 2 In Vivo Bleomycin Treatment .............................................................................................................. 24 Histology and Immunohistochemistry ................................................................................................ 25 Western Blot and Densitometry........................................................................................................... 25 Isolation of Dermal Fibroblasts ........................................................................................................... 26 Fibroblasts Cell Count and Proliferation ........................................................................................... 26 In Vitro Wound Healing Scratch Assay .............................................................................................. 27 Collagen Contraction Assay ................................................................................................................. 27 RESULTS .................................................................................................................................................. 28 Genotyping of CD109 Mouse Colony .................................................................................................. 28 Dermal Collagen Deposition and Density is Increased in CD109 KO Mice in Response to Bleomycin Injections ............................................................................................................................. 28 Dermal Fibronectin and CCN2 Production in Response to Bleomycin is Increased in CD109 KO Mice ........................................................................................................................................................ 28 CD109 KO Mice Display Enhanced α-SMA Expression ................................................................... 29 Bleomycin-Treated CD109 KO Mice Display Increased Phosphorylation of Smad1, Smad2, and Smad3 ..................................................................................................................................................... 29 CD109 KO Fibroblasts Display Accelerated Proliferation, Migration (Wound Closure), and Collagen Matrix Contraction ............................................................................................................... 30 DISCUSSION ............................................................................................................................................ 31 GENERAL DISCUSSION & CONCLUSION ....................................................................................... 34 REFERENCES .......................................................................................................................................... 37 FIGURES ................................................................................................................................................... 42 Fig. 1 Treatment Assignment for Bleomycin Injections in Mice. ..................................................... 42 Fig.2 Increased Dermal Type I Collagen Production, Deposition, and Density in CD109 KO Mice Skin Following Bleomycin Injections. ................................................................................................. 43 Fig.3 Bleomycin-injected CD109 KO mice display increased ECM production and myofibroblast differentiation. ....................................................................................................................................... 44 Fig.4 Increased Smad1, Smad2, and Smad3 Phosphorylation in CD109 KO Mice Skin Following Intradermal Bleomycin Injections. ...................................................................................................... 46 Fig.5 Cell Proliferation, and TGF-β-Induced Wound Closure and Contraction Rates are Significantly Increased in CD109 KO Mouse Fibroblasts, in vitro. .................................................. 47 TABLES ..................................................................................................................................................... 48 Table 1. Antibodies for Protein Detection .......................................................................................... 48 APPENDIX ................................................................................................................................................ 49 Supplementary Fig. 1 Genotyping of CD109 mouse colony. ............................................................. 49 3 Supplementary Fig. 2 Bleomycin injections increase dermal thickness in CD109 WT and KO mice. ......................................................................................................................................................