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Cellular and molecular biology of Wnt signaling and versican expression in myofibroblast differentiation Carthy, Jonathon Morgan 2011

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CELLULAR AND MOLECULAR BIOLOGY OF WNT SIGNALING AND VERSICAN EXPRESSION IN MYOFIBROBLAST DIFFERENTIATION  by  Jonathon Morgan Carthy B.Sc., Queen’s University, 2004  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  DOCTOR OF PHILOSOPHY  in  The Faculty of Graduate Studies (Pathology and Laboratory Medicine)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  December 2011  © Jonathon Morgan Carthy, 2011  ABSTRACT Wound healing is a complex and dynamic process that restores tissue integrity after injury, but also contributes pathologically to the development of fibrosis. Growing evidence suggests a role for Wnt signaling during normal and aberrant wound healing. The proteoglycan versican is a target of Wnt signaling that is expressed following injury and accumulates pathologically in many chronic inflammatory conditions. In this dissertation, I hypothesized that Wnt signaling and its target versican are key regulators of mesenchymal cell phenotype. In Aim 1, I demonstrated that treatment of cultured fibroblasts with Wnt3a, a canonical Wnt ligand, stimulates the formation of a myofibroblast-like phenotype characterized by increased expression of smooth muscle αactin. These changes appear to be mediated by Wnt3a upregulating the expression of TGF-β and its associated signaling through SMAD2 in a β-catenin-dependent mechanism. In Aim 2, I show that Wnt3a alters the phenotype of vascular smooth muscle cells and stimulates the formation of a contractile and secretory phenotype in these cells that is associated with increased gap junction communication. Again, these changes occurred through a mechanism that was dependent on canonical Wnt signaling. In Aim 3, I explored the functional roles of versican by examining its expression following injury to cultures of valve myofibroblasts. My data indicate that versican is secreted as extracellular matrix following injury to valve cells, and suggests a role for the membrane receptor CD44 in organizing this provisional versican matrix. In Aim 4, I delved further into the functional roles of versican by expressing this proteoglycan in murine fibroblasts. In this aim I showed that versican expression promotes myofibroblast differentiation, and  ii  these changes appear to be mediated by activation of TGF-β signaling. Lastly, in Aim 5, I explored potential intracellular functions for versican, and provide evidence to suggest versican localizes to the nucleus in mesenchymal cells where it regulates the organization of the mitotic spindle during cell division. Collectively, these data suggest Wnt signaling and versican are key regulators of mesenchymal cell phenotype, and as such, are important mediators of a wound healing response.  iii  PREFACE This dissertation contains chapters which are based on published or submitted manuscripts:  A version of Chapter 2 has been published in the journal PLoS One [Carthy JM, Garmaroudi FS, Luo Z, McManus BM. Wnt3a induces myofibroblast differentiation by upregulating TGF-β signaling through SMAD2 in a β-catenin-dependent manner. PLoS One. 2011;6(5):e19809].  A version of Chapter 3 has been published in the journal Laboratory Investigation [Carthy JM, Luo Z, McManus BM. Wnt3a induces a contractile and secretory phenotype in vascular smooth muscle cells that is associated with increased gap junction communication. In press, October 2011].  A version of Chapter 4 has been published in the journal Cardiovascular Pathology [Carthy JM, Boroomand S, McManus BM. Versican and CD44 in in vitro valvular interstitial cell injury and repair. In press, May 2011]  A version of Chapter 5 is in preparation [Carthy JM, Boroomand S, Meredith A, Garmaroudi FS, Abrahahm T, Luo Z, Knight DA, McManus BM. Versican promotes myofibroblast differentiation in cultured fibroblasts].  iv  A version of Chapter 6 has been submitted and is in revision [Carthy JM, McManus BM. The intracellular localization of versican in vascular cells. Submitted April 26, 2011].  In all manuscripts, I was responsible for designing and performing the experiments, as well as writing the manuscripts. Co-authors assisted in intellectual guidance as well as technical aspects of data generation and analysis. Specifically, Ms. Zongshu Luo performed the qPCR reactions, Ms. Seti Boroomand isolated and cultured the human valve cells, and Dr. Thomas Abraham assisted me with the confocal microscope.  Ethics approval was obtained from the UBC/Providence Research Ethics Board, certificate numbers B05-0185, H05-50208, B09-0220, H09-01571.  v  TABLE OF CONTENTS ABSTRACT........................................................................................................................ ii PREFACE .......................................................................................................................... iv TABLE OF CONTENTS ................................................................................................... vi LIST OF FIGURES ............................................................................................................ x LIST OF ACRONYMS AND ABBREVIATIONS ......................................................... xii ACKNOWLEDGEMENTS ............................................................................................. xiv DEDICATION .................................................................................................................. xv CHAPTER 1. INTRODUCTION ....................................................................................... 1 1.1  Overview of wound healing ................................................................................. 1  1.2  Myofibroblasts ..................................................................................................... 2  1.3  The extracellular matrix ....................................................................................... 4  1.4  Extracellular matrix changes during wound healing ............................................ 5  1.5  Proteoglycans ....................................................................................................... 6  1.6  Versican ................................................................................................................ 7  1.6.1  Family and structure ..................................................................................... 7  1.6.2  Binding interactions and function ................................................................. 8  1.6.3  Regulation of versican expression .............................................................. 10  1.7  Wnt signaling ..................................................................................................... 10  1.8  Thesis overview.................................................................................................. 11  CHAPTER 2. WNT3A INDUCES MYOFIBROBLAST DIFFERENTIATION IN CULTURED FIBROBLASTS ......................................................................................... 17 2.1  Introduction ........................................................................................................ 17  2.2  Materials and methods ....................................................................................... 18  2.2.1  Cell culture .................................................................................................. 18  2.2.2  Cell proliferation and migration assays ...................................................... 18  2.2.3  Collagen gel contraction assay.................................................................... 19  2.2.4  Western blotting .......................................................................................... 19  2.2.5  Immunohistochemistry ............................................................................... 20  2.2.6  siRNA transfections .................................................................................... 21 vi  2.2.7 2.3  Statistical analysis ....................................................................................... 21  Results ................................................................................................................ 22  2.3.1  Wnt3a induces canonical Wnt signaling in mouse fibroblasts ................... 22  2.3.2  Wnt3a alters the morphology of mouse fibroblasts .................................... 22  2.3.3  Wnt3a inhibited fibroblast proliferation, but increased cell migration and contraction of collagen gels ........................................................................ 23  2.3.4  Wnt3a increases TGF-β expression, SMAD2 phosphorylation and smooth muscle α-actin expression ........................................................................... 23  2.3.5  Wnt3a-induced smooth muscle α-actin expression is dependent on βcatenin ......................................................................................................... 24  2.3.6  Wnt3a-induced change in cell phenotype is dependent on TGF-β expression ..................................................................................................................... 25  2.4  Discussion .......................................................................................................... 25  CHAPTER 3. WNT3A INDUCES A CONTRACTILE AND SECRETORY PHENOTYPE IN CULTURED VASCULAR SMOOTH MUSCLE CELLS ................. 39 3.1  Introduction ........................................................................................................ 39  3.2  Materials and methods ....................................................................................... 40  3.2.1  Cell culture .................................................................................................. 40  3.2.2  qPCR analysis ............................................................................................. 41  3.2.3  Cell proliferation ......................................................................................... 41  3.2.4  Western blotting .......................................................................................... 41  3.2.5  Collagen gel contraction assay.................................................................... 42  3.2.6  Immunohistochemistry ............................................................................... 43  3.2.7  Scrape loading dye transfer assay ............................................................... 44  3.2.8  Statistical analysis ....................................................................................... 44  3.3  Results ................................................................................................................ 44  3.3.1  Wnt3a activates canonical Wnt signaling in mouse vascular smooth muscle cells. ............................................................................................................ 44  3.3.2  Wnt3a alters the phenotype of vascular smooth muscle cells..................... 45  3.3.3  Wnt3a increases connexin 43 expression and intercellular communication in smooth muscle cells ................................................................................ 46 vii  3.3.4  Wnt3a-induced change in smooth muscle cell phenotype is dependent on canonical Wnt signaling .............................................................................. 47  3.4  Discussion .......................................................................................................... 48  CHAPTER 4. VERSICAN AND ITS MEMBRANE RECEPTOR CD44 ARE EXPRESSED FOLLOWING INJURY TO VALVE MYOFIBROBLASTS .................. 61 4.1  Introduction ........................................................................................................ 61  4.2  Materials and methods ....................................................................................... 63  4.2.1  Cell culture .................................................................................................. 63  4.2.2  In vitro scratch injury model ....................................................................... 64  4.2.3  Immunohistochemistry ............................................................................... 64  4.2.4  Collagen gel contraction assay.................................................................... 65  4.2.5  Statistics ...................................................................................................... 66  4.3  Results ................................................................................................................ 66  4.3.1  Phenotypic analysis of valvular interstitial cells ......................................... 66  4.3.2  Versican immunostaining in valvular interstitial cells................................ 66  4.3.3  CD44 immunostaining in valvular interstitial cells .................................... 67  4.3.4  Blocking CD44 disrupts pericellular matrix organization and stress fibre formation in valvular interstitial cells ......................................................... 68  4.3.5  Blocking CD44 inhibits valvular interstitial cell-mediated contraction of type I collagen gels ..................................................................................... 69  4.4  Discussion .......................................................................................................... 70  CHAPTER 5. VERSICAN EXPRESSION INDUCES A MYOFIBROBLAST-LIKE PHENOTYPE IN CULTURED FIBROBLASTS ............................................................ 83 5.1  Introduction ........................................................................................................ 83  5.2  Materials and methods ....................................................................................... 84  5.2.1  Cell culture .................................................................................................. 84  5.2.2  Creation of the versican V1 cell line........................................................... 84  5.2.3  Western blotting .......................................................................................... 85  5.2.4  Immunohistochemistry and confocal microscopy ...................................... 86  5.2.5  Cell proliferation, migration and adhesion assays ...................................... 87  5.2.6  Collagen gel contraction assay.................................................................... 87 viii  5.2.7  qPCR analysis ............................................................................................. 88  5.2.8  Statistical analysis ....................................................................................... 88  5.3  Results ................................................................................................................ 89  5.3.1  Expression and secretion of human versican in mouse fibroblasts............. 89  5.3.2  Versican alters the phenotype of cultured fibroblasts ................................. 90  5.3.3  Versican induces a myofibroblast-like phenotype in cultured fibroblasts .. 90  5.3.4  Versican mediates increased TGF-β signaling in cultured fibroblasts ....... 92  5.4  Discussion .......................................................................................................... 92  CHAPTER 6. THE INTRACELLULAR LOCALIZATION OF VERSICAN IN MESENCHYMAL CELLS ............................................................................................ 104 6.1  Introduction ...................................................................................................... 104  6.2  Materials and methods ..................................................................................... 105  6.2.1  Reagents .................................................................................................... 105  6.2.2  Cell culture ................................................................................................ 106  6.2.3  Immunohistochemistry ............................................................................. 107  6.2.4  Western blotting ........................................................................................ 107  6.2.5  siRNA transfections .................................................................................. 108  6.3  Results .............................................................................................................. 108  6.3.1  Intracellular localization of versican ......................................................... 108  6.3.2  Versican localizes to the nucleolus in non-dividing vascular smooth muscle cells ........................................................................................................... 109  6.3.3  Association of intracellular versican with mitosis .................................... 110  6.3.4  Versican localizes to polar ends of the mitotic spindle and is required for normal spindle organization ...................................................................... 110  6.4  Discussion ........................................................................................................ 111  CHAPTER 7. CONCLUSIONS AND FUTURE DIRECTIONS .................................. 123 REFERENCES ............................................................................................................... 128  ix  LIST OF FIGURES Figure 1. Phases of wound healing. .................................................................................. 13 Figure 2. Structure of the versican isoforms. .................................................................... 14 Figure 3. The interaction of versican with its binding partners. ....................................... 15 Figure 4. Overview of canonical Wnt signaling. .............................................................. 16 Figure 5. Wnt3a induces canonical Wnt signaling in mouse fibroblasts. ......................... 30 Figure 6. Wnt3a induces a spindle-like morphology with increased stress fibre formation after 72 hours of treatment. ............................................................................................... 31 Figure 7. Wnt3a inhibits cell proliferation, but increases cell migration and contraction after 72 hour treatment. ..................................................................................................... 33 Figure 8. Wnt3a increases TGF-β expression, SMAD2 phosphorylation and smooth muscle α-actin expression. ................................................................................................ 35 Figure 9. Wnt3a-induced change in cell phenotype is dependent on β-catenin. .............. 36 Figure 10. Wnt3a-induced change in cell phenotype is dependent on TGF-β expression. ........................................................................................................................ 38 Figure 11. Wnt3a activates canonical Wnt signaling in vascular smooth muscle cells. ... 53 Figure 12. Wnt3a alters the phenotype of vascular smooth muscle cells. ........................ 54 Figure 13. Wnt3a increases contractile properties of vascular smooth muscle cells. ....... 55 Figure 14. Wnt3a inceases collagen synthesis and protease expression in smooth muscle cells. .................................................................................................................................. 56 Figure 15. Wnt3a increases connexin 43 expression in smooth muscle cells. ................. 57 Figure 16. Wnt3a increases gap junction communication in vascular smooth muscle cells. .................................................................................................................................. 58 Figure 17. Wnt3a-mediated change in smooth muscle cell phenotype and connexin ..... 43 expression is dependent on canonical Wnt signaling. ...................................................... 59 Figure 18. Cultured valvular interstitial cells display a myofibroblast-like phenotype. ... 75 Figure 19. Versican expression in cardiac valvular interstitial cells. ............................... 76 Figure 20. Versican expression during injury and repair in cardiac valvular interstitial cells. .................................................................................................................................. 77 Figure 21. Expression of CD44 in cardiac valvular interstitial cells. ............................... 78 x  Figure 22. Expression of CD44 during injury and repair in cardiac valvular interstitial cells. .................................................................................................................................. 79 Figure 23. Blocking CD44 inhibits pericellular matrix organization and stress fibre formation in valvular interstitial cells. .............................................................................. 80 Figure 24. Blocking CD44 inhibits valvular interstitial cell-mediated contraction of type I collagen gels...................................................................................................................... 81 Figure 25. Blocking CD44 inhibits stress fibre formation and versican deposition into the ECM in valvular interstitial cells cultured in collagen gels. ............................................. 82 Figure 26. Versican expression in murine fibroblasts. ..................................................... 97 Figure 27. Versican increases N-cadherin expression. ..................................................... 98 Figure 28. Versican alters the function of fibroblasts. ...................................................... 99 Figure 29. Versican increases smooth muscle α-actin expression. ................................. 100 Figure 30. Versican increases fibroblast-mediated contraction of a collagen lattice. .... 101 Figure 31. Versican increases TGF-β signaling in cultured fibroblasts.......................... 103 Figure 32. Versican expression in vascular smooth muscle cells. .................................. 116 Figure 33. Versican localizes to the nucleus in vascular smooth muscle cells. .............. 117 Figure 34. Colocalization of versican and nucleolin confirms nucleolar localization of versican in smooth muscle cells. ..................................................................................... 118 Figure 35. Intracellular versican is associated with mitosis. .......................................... 120 Figure 36. Versican localizes to polar ends of the mitotic spindle. ................................ 121 Figure 37. Versican knockdown alters mitotic spindle organization. ............................. 122 Figure 38. Hypothetical framework. ............................................................................... 127  xi  LIST OF ACRONYMS AND ABBREVIATIONS ADAMTS  A disintegrin and metalloproteinase with thrombospondin motifs  APC  Adenomatous polyposis coli  BSA  Bovine serum albumin  CS  Chondroitin sulphate  CBP  Complement binding protein  CRD  Carbohydrate recognition domain  DAPI  4',6-diamidino-2-phenylindole  DKK1  Dickkopf-related protein 1  DMEM  Dulbecco modified Eagle’s minimal essential medium  ECM  Extracellular matrix  EDA  Extra domain A  EGF  Epidermal growth factor  EGFR  Epidermal growth factor receptor  FAK  Focal adhesion kinase  FBS  Fetal bovine serum  FGF  Fibroblast growth factor  GAG  Glycosaminoglycan  GSK  Glycogen synthase kinase  IL  Interleukin  LEF  Lymphoid enhancer binding factor  LDL  Low density lipoprotein  LRP  Lipoprotein receptor-related protein  MAPK  Mitogen-activated protein kinase  MMP  Matrix metalloproteinase  PBS  Phosphate buffered saline  PARP  Poly (ADP-ribose) polymerase  PDGF  Platelet-derived growth factor  PSGL-1  P-selectin glycoprotein ligand-1  RHAMM  Receptor for hyaluronan-mediated motility xii  SDS-PAGE  Sodium dodecyl sulfate polyacrylamide gel electrophoresis  SOD  Superoxide dismutase  TBS  Tris buffered saline  TBST  Tris buffered saline and tween  TCF  T-cell factor  TGF  Transforming growth factor  TIMP  Tissue inhibitor of metalloproteinases  VEGF  Vascular endothelial growth factor  VIC  Valvular interstitial cell  xiii  ACKNOWLEDGEMENTS I would like to say thank you to the many people who have helped me out along the way. In particular, thank you to my supervisor, Dr. Bruce McManus, for believing in me and for providing me with the opportunity to pursue a PhD under your mentorship. It has been a challenging but truly rewarding experience. To my committee members, Drs. Darryl Knight, David Granville, Chris Overall, and Chun Seow, thank you for taking the time out of your busy schedules to attend my committee meetings and for keeping my progress on track. To my fellow lab mates, Anna, Seti, David and Farshid, without your support and friendship I would never have completed this degree with my sanity in check. To my brothers and sisters, Shannon, Chris, Brady, and Jos, thank you so much for your love and support over the years, and for the many home cooked meals. To my father Mike, you are the hardest working and humblest person I have ever met, and the best role model I could have ever hoped for. And to my mother Susan, everything I have in my life I owe to you. Not a day goes by that I don’t miss you and think about you.  xiv  DEDICATION To my mother and father, Susan and Mike  xv  CHAPTER 1. INTRODUCTION  1.1  Overview of wound healing  Wound healing is a complex and dynamic process involving the interplay of many cellular and non-cellular components that ultimately restores tissue integrity after injury. The process of wound healing can be divided into four biologically defined stages that are overlapping and continuous (Figure 1, reviewed in [1,2]). The immediate response to tissue injury is generally defined by hemostasis, which is associated with platelet activation and aggregation and leads to the formation of a fibrin clot. This fibrin clot, in addition to preventing the leakage of excessive blood, also serves as a provisional matrix to which signaling mediators and cells can bind. Further, the activated platelets release a number of growth factors and chemokines that support various aspects of the repair process. An inflammatory response closely follows the immediate response, initially in the form of neutrophils and then later by the appearance of monocyte-derived macrophages. These inflammatory cells respond to the soluble mediators released at the sites of injury and themselves contribute further to the plethora of growth factors and cytokines present in the wound healing milieu. These early changes at sites of injury are supported by the sprouting of wound-edge capillaries, which leads to the formation of a microvascular network that provides nutrients and oxygen to the developing granulation tissue. The proliferative phase of wound healing is characterized by the migration and proliferation of fibroblasts into the provisional matrix, where they are responsible for replenishing the lost tissue. The synthesis of new tissue is achieved by a specialized 1  fibroblast, termed the myofibroblast, which synthesizes extracellular matrix (ECM) and acquires contractile apparatus necessary to organize this new matrix. The final phase of wound healing, generally referred to as the resolution phase, involves the continued remodeling of the provisional matrix and the death of inflammatory cells and wound myofibroblasts. During this stage, a delicate balance between collagen synthesis, bundling, and degradation is achieved, which culminates in the formation of a collagenrich scar that can restore tissue integrity to roughly 80% of its unwounded state [3].  Wound healing is clearly a critical response to injury that restores tissue integrity and homeostasis. However, dysregulated or aberrant wound healing can lead to tissue destruction by fibrosis and is associated with a number of chronic inflammatory diseases that affect the heart, lung, liver, kidney and skin [4].  1.2  Myofibroblasts  The myofibroblast is a specialized cell type that forms in granulation tissue during wound healing which has a phenotype that is characterized by increased ECM synthesis and contractile ability [5,6]. These cells are important to normal wound healing, but are also thought to be the main cell type responsible for the detrimental architectural changes seen in fibrotic conditions [7]. As such, much recent work has focused on defining the mechanisms that regulate the formation and survival of these cells. Myofibroblasts may originate from a number of sources, depending on the tissue type and the physiological or pathological environment present there [6]. There is evidence to suggest myofibroblasts  2  originate from epithelial cells, endothelial cells, smooth muscle cells, pericytes, hepatic perisinusoidal cells, mesenchymal stem cells, as well as fibrocytes [6-8], although the likely source of myofibroblasts in most settings appears to be from locally recruited fibroblasts that have undergone differentiation [6,9]. Whatever their source, myofibroblasts are differentiated from normal fibroblasts by ultrastructural changes to their cytoskeleton [5]. In addition to their increased synthetic capacity, these cells develop well defined cytoplasmic stress fibres that are characterized by the neoexpression of smooth muscle α-actin, which is the actin isoform normally expressed by vascular smooth muscle cells [8]. Incorporation of smooth muscle α-actin into their stress fibres gives myofibroblasts a more than two-fold increase in contractile ability compared with α-actin negative fibroblasts (in culture) [10]. This increased force can be transmitted to the surrounding ECM through specialized integrin containing adhesion complexes that are called fibronexus or supermature focal adhesions [11]. As such, myofibroblasts posses a significant capacity to contract and remodel their surrounding matrix.  Because of their importance to normal and aberrant wound healing, much work has been directed towards elucidating the mechanisms involved in myofibroblast differentiation. It appears at least three events are necessary for full induction of the myofibroblast phenotype: 1) the cytokine transforming growth factor-β (particularly TGF-β1) [12,13], 2) expression of the extra domain A (EDA) isoform of fibronectin (cellular fibronectin) [14], and 3) mechanical tension caused by extracellular stress [15]. In addition to these, a number of other factors have been shown to modify myofibroblast differentiation, including coagulation factors, growth factors and cytokines, membrane receptors and  3  matrix components. Clearly this is a complex process that is not entirely understood at present, which may account for part of the reason why there are no effective therapies available to treat fibrotic diseases [16].  1.3  The extracellular matrix  The ECM is probably best described as a reinforced composite of collagen and elastic fibers embedded in a viscoelastic gel constituted by proteoglycans, hyaluronan, glycoproteins, and water [17]. The main components of the matrix include fibrous structural proteins, such as collagens, laminins, fibronectin, vitronectin and elastin, specialized proteins, such as growth factors, matricellular proteins, and glyocproteins, as well as proteoglycans and glycosaminoglycans [18]. Through entanglement and crosslinking, these components interact to form an active polymer network that, depending on its composition, gives specific properties to the various tissues in the body [17]. In simple terms, the matrix is the scaffold that provides the architecture to tissues and supports its cellular constituents. However, recent studies have demonstrated that the matrix is much more than simply a supporting structure, and it is in fact a highly dynamic and biologically active substance that has a significant influence over the properties of the tissue and cells it supports [18]. Evidence suggests the matrix participates in the regulation of cell adhesion, migration, proliferation, survival, shape, and differentiation, as well as binding and modifying the activities of plasma proteins, growth factors, cytokines, and enzymes [19]. Thus, the matrix not only provides the structure to tissues, it also regulates key biochemical events involved in their physiology, and many of the  4  properties of a tissue are critically dependent on the integrity and composition of its matrix [20]. This is highlighted during the dynamic changes seen to the ECM over the course of wound healing and remodeling events.  1.4  Extracellular matrix changes during wound healing  The many cellular events that orchestrate a wound healing response are supported by a highly regulated ECM that evolves and is remodeled over the course of repair. The early matrix, termed the provisional matrix, is transient and composed of fibrin and glycoproteins such as fibronectin and vitronectin, as well as the large proteoglycan versican and the glycosaminoglycan hyaluronan [1,21]. This provisional matrix supports the migration and proliferation of mesenchymal cells, and acts as a reservoir for growth factors and cytokines [1]. Versican and hyaluronan also provide binding sites for inflammatory cells through their interaction with the membrane receptor CD44 [22,23], and may stimulate the release of inflammatory cytokines by activating toll-like receptors on macrophages [24,25]. The predominant collagen expressed in the provisional matrix is type III collagen, a genetically distinct collagen that is normally associated with embryonic development [26]. As wound healing progresses, the early matrix is degraded, type III collagen is replaced by type I collagen, and there is increased expression of the small leucine-rich proteoglycans biglycan and decorin [27]. These later changes are associated with cross-linking of type I collagen and wound contraction [28]. In uncomplicated dermal wounds, the healing and associated matrix remodeling are completed in as little as 2 weeks; however, a number of chronic inflammatory conditions  5  are characterized by the continued presence of inflammatory cells and myofibroblasts in a matrix that remains rich in versican and hyaluronan [1]. As an example of this, wound healing following stenting of the coronary arteries was shown to require at least 18 months, and the presence of a stent and underlying atherosclerosis even further delayed this process [27].  1.5  Proteoglycans  Proteoglycans are unique molecules characterized by the presence of long, unbranched polysaccharides, called glycosaminoglycans (GAGs), which are covalently linked to a core protein [29]. The GAG chains are composed of repeating disaccharide units that vary depending on the type of GAG chain(s) present (dermatan sulphate, chondroitin sulfate, or heparin sulfate) [30]. The GAG chains are modified by sulphation to yield highly negatively charged binding sites that attract many different positively charged ligands, including cytokines, growth factors, enzymes, protease inhibitors, and other matrix components [29,31]. As such, these molecules can have a significant influence over many normal and pathological processes. Proteoglycans can be classified based on their localization to those that are secreted into the matrix, those associated with the cell surface, and intracellular proteoglycans. The matrix proteoglycans can be further divided into three groups: basement membrane proteoglycans, small leucine-rich proteoglycans, and hyalactins [29].  6  1.6  Versican  1.6.1 Family and structure  Versican is a member of the hyalectin family of ECM proteoglycans that are so named because of their ability to bind hyaluronic acid through their N-terminal domains and their presence of a lectin-like C-terminal domain [32,33]. Other members of the hyalectin family include aggrecan, which is prominent in cartilage, and brevican and neurocan, which are localized to the central nervous system. Versican, on the other hand, has a wide distribution and is found mainly in soft tissues [33].  Structurally, versican is organized into two globlular domains, the G1 domain at the Nterminus, and the G3 domain at the C-terminus, that are separated by a central region which contains the attachment sites for the chondroitin sulphate GAG chains [34,35]. The G1 domain of versican contains the hyaluronan-binding region [36,37], and the Cterminal domain of versican contains a lectin-like domain adjacent to a set of two epidermal growth factor (EGF)-like sequences and a complement regulatory region [34]. Due to differential splicing of the central GAG binding region, versican can be expressed as 4 different isoforms (V0-V3) that all share identical N- and C-terminal globular domains, but differ in the length of their core protein and the number of attached GAG chains (Figure 2, reviewed in [33,38]). The central GAG binding region of versican consists of two exons, termed GAG-α and GAG-β, with the V0 isoform of versican containing both these exons, the V1 isoform containing only the GAG-β exon, V2 contains only the GAG-α exon, and V3 contains neither GAG-α nor GAG-β. 7  Consequently, the V0 isoform of versican is the largest and contains between 17-23 GAG chains, V1 is the second largest isoform and has 12-15 GAG chains, V2 is the third largest isoform and has 5-8 GAG chains, and V3 is the smallest versican isoform and does not have any GAG chains (and is not truly a proteoglycan). Thus, due to alterations in the length of its core protein and the number of attached GAG chains, versican is considered to be a highly 'versatile' proteoglycan in terms of its biological roles, which is what forms the basis of its name [33].  1.6.2 Binding interactions and function  Versican is a highly interactive proteoglycan and it appears to achieve much of its function through binding interactions with other components in the ECM (Figure 3). Through its interaction with hyaluronic acid, versican forms high molecular weight aggregates that attract water and may provide hygroscopic properties to the ECM [35]. In addition to binding hyaluronan (at its N-terminus), versican binds a number of other ECM components at its C-terminus, including fibrillin-1 [39], fibulin-1 and -2 [40,41], tenascin-R [42], type I collagen [43], and fibronectin [43]. Thus, versican is thought to play an important role in regulating the assembly and organization of the ECM. Further, versican has been shown to bind and regulate the activity of a number of chemokines and growth factors through its GAG chains [31,44]. In addition to binding other matrix proteins and soluble mediators, versican has been shown to interact with cell surface molecules including P- and L-selectin [22,31], CD44 [22], integrin β1 [45], and the epidermal growth factor receptor (EGFR) [45,46]. There is evidence to suggest that versican may stimulate intracellular signaling cascades through these interactions, most 8  notably by activating the EGFR to induce MAPK signaling through the EGF-like repeats in versican’s C-terminal globular domain [47,48]. Further, through its interaction with CD44, versican may provide binding sites for inflammatory cells within the ECM, thereby stabilizing their retention in inflammatory conditions [49]. Adding to this, versican was recently identified as an endogenous ligand of toll-like receptors, and was shown to promote the release of pro-inflammatory cytokines by activating toll-like receptor 2 on macrophages [24].  In addition to inflammation, evidence suggests versican modifies a number of other cellular processes, including cell adhesion [50,51], migration [52-54], proliferation [47,52], and apoptosis [47,55]. These functions of versican are critically involved during development and disease. A versican knockout mouse, named the ‘heart defect mouse’, is an embryonic lethal due to a failure of endocardial cells to migrate during cardiac development [56]. Versican has been shown to form a pericellular coat with hyaluronan that supports the migration and proliferation of prostate cells and vascular smooth muscle cells in culture [52-54]. Studies have demonstrated that a high level of versican is associated with the metastatic spread of prostate and breast cancers [38], and also promotes neointimal formation in atherosclerotic arteries [57,58]. Therefore, there is sufficient evidence to suggest versican influences cell phenotype during development and disease.  9  1.6.3 Regulation of versican expression  Versican expression derives largely from myofibroblasts and smooth muscle cells [5961], with limited expression also apparent in endothelial cells [62-64] and macrophages [65]. The synthesis of versican can be regulated at the level of transcription, translation, and post-translation modification through a host of soluble mediators present within the local microenvironment [66]. Studies have demonstrated that TGF-β1 and PDGF each increase versican mRNA levels, core protein synthesis and GAG chain length [60]. Other factors known to regulate versican expression include EGF, bFGF, IL-1β [35], and physical stimuli, including cell density and mechanical stress [67,68]. Our laboratory has recently demonstrated that versican is a target of canonical Wnt signaling through its transcription mediators β-catenin/T-cell factor [69]. This finding has been confirmed by other groups [70,71] and identifies versican as one of a growing list of provisional matrix components that are regulated by Wnt signaling. As Wnt signaling is upregulated during injury and repair events in many tissues and organs, there is reason to suggest Wnt family members may be important contributors to a wound healing response.  1.7  Wnt signaling  The canonical Wnt signaling pathway is best recognized for its role in development of multi-cellular organisms [72,73]. The Wnt family is comprised of 19 secreted glycoproteins that bind the Frizzled receptor and its co-receptor LRP5/6 (lipoprotein receptor-related proteins 5 or 6) to initiate an intracellular signaling cascade that controls  10  the turnover of β-catenin (Figure 4, reviewed in [74]). In the absence of Wnt ligand, βcatenin is targeted for ubiquitin-mediated degradation by the 26S proteasome. Upon ligand stimulation, the canonical Wnt signaling pathway triggers a series of phosphorylation events that lead to the accumulation of cytosolic β-catenin, which then translocates to the nucleus where it binds the T-cell factor (TCF) or lymphoid enhancer binding factor (LEF) transcription factors to initiate transcription of target genes. As such, Wnt ligands can elicit a rapid and specific response in target cells.  In addition to its role in early embryogenesis, growing evidence points to an active role for Wnt signaling in normal wound repair and in a number of human diseases. Increased Wnt signaling has been observed during cutaneous wound repair [75-77], hypertrophic scarring [78], aberrant blood vessel remodeling [79], pulmonary fibrosis [80] and aging [81], as well as being recognized as a contributor to a multitude of malignant disorders [82-84]. Despite this, very little is known about the effect of Wnt ligands on cell phenotype in the context of wound repair and remodeling events. Further, the function of many important Wnt target genes, such as versican, remains poorly understood in this context.  1.8  Thesis overview  This thesis is focused around the themes of Wnt signaling and versican expression in the context of wound healing. I have first examined the effect of Wnt3a, a canonical Wnt ligand, on the phenotype of cultured fibroblasts and smooth muscle cells. I have then  11  examined some of the functional roles of the Wnt target gene versican, for which our laboratory has a long-standing interest.  The overarching hypothesis in our laboratory is that Wnt signaling, through the expression of key target genes and especially the proteoglycan versican, plays a pivotal role in regulating normal versus aberrant wound healing.  For this thesis, I hypothesized that Wnt signaling and its target versican are key regulators of mesenchymal cell phenotype.  The specific aims examined are as follows:  1) To determine the effect of Wnt3a on fibroblast cell phenotype. 2) To determine the effect of Wnt3a on smooth muscle cell phenotype. 3) To examine the expression of versican in an in vitro model of injury and repair. 4) To determine the effect of versican expression on fibroblast cell phenotype. 5) To investigate intracellular functions for versican in mesenchymal cells.  12  Figure 1. Phases of wound healing. The wound healing response can be broken into overlapping phases as depicted in this figure. See text for details. (Adapted from [85])  13  Figure 2. Structure of the versican isoforms. Alternative splicing of the central GAG attachment region gives rise to 4 versican isoforms in humans, V0, V1, V2 and V3. V0 represents the full-length protein, containing both GAG-α and GAG-β. V1 and V2 have GAG-α and GAG-β spliced out, respectively, and V3 does not have either GAG-binding regions. Differential splicing leads to versican core proteins of variable molecular weight and GAG-binding capacity, and may result in functional differences between these isoforms. (Adapted from [86])  14  Figure 3. The interaction of versican with its binding partners. Versican follows a domain template consisting of an N-terminal (G1) domain that binds hyaluronan, and a C-terminal (G3) domain that resembles the selectin family of proteins, consisting of a C-type lectin adjacent to two epidermal growth factor-like repeats and a complement regulatory region. These two globular domains are separated by an attachment region for the chondroitin sulfate side chains of versican. Through binding sites located in its core protein and GAG chains, versican binds growth factors, enzymes, lipoproteins, membrane receptors and a variety of other extracellular matrix components. (Modified from [46]) Ig, immunoglobulin-like; GAG, glycosaminoglycan; CS, chondroitin sulfate; EGF, epidermal growth factor; CRD, carbohydrate recognition domain; CBP, complement binding protein.  15  Figure 4. Overview of canonical Wnt signaling. In the absence of Wnt ligand (left panel), cytosolic β-catenin is phosphorylated by GSK3β and targeted for degradation by the ubiquitin pathway. Upon Wnt ligand binding (right panel), a series of phosphorylation events is initiated which culminates in the phosphorylation and inactivation of GSK-3β (deactivation of the destruction complex). This in turns leads to the accumulation of cytosolic β-catenin, which translocates to the nucleus where it binds the TCF family of transcription factors to initiate transcription of target genes. (Modified from [85]) LRP, lipoprotein receptor-related protein; APC, adenomatous polyposis coli; GSK, glycogen synthase kinase; TCF, T-cell factor.  16  CHAPTER 2. WNT3A INDUCES MYOFIBROBLAST DIFFERENTIATION IN CULTURED FIBROBLASTS  2.1  Introduction  Wound healing is a complex and dynamic process involving the interplay of many cellular and non-cellular components, typically culminating in the replacement of injured tissue with a fibrotic scar [2]. A number of soluble mediators released at the site of injury act as molecular cues that guide cellular responses during repair [87]. Evidence suggests the Wnt family of secreted glycoproteins and their associated signaling pathways, linked to development, are recapitulated during wound repair and regeneration events [88,89]. However, the role of Wnt signaling in such settings remains unclear.  In this aim, I examined the effect of Wnt3a, a canonical Wnt ligand, on fibroblast morphology and function. My data suggests Wnt3a stimulates a tentacular, spindle-like morphology in murine fibroblasts characterized by the increased expression of smooth muscle α-actin-positive stress fibres. These changes appear to be mediated, at least in part, by Wnt3a upregulating the expression of TGF-β signaling through SMAD2 in a βcatenin-dependent manner. Collectively, this data suggests Wnt3a promotes the formation of a myofibroblast-like phenotype in cultured fibroblasts. As myofibroblasts are critical regulators of a wound healing response, these findings suggest a central role for Wnt signaling in normal and aberrant injury and repair events.  17  2.2  Materials and methods  2.2.1 Cell culture  Mouse embryonic fibroblasts (Clontech, product number 630914) were cultured in DMEM containing 10% FBS and 100 U/mL penicillin/streptomycin. Cells were maintained in a humidified incubator at 37°C with 5% CO2 and used for experiments between passages 6-12. Recombinant murine Wnt3a (Peprotech, product number 315-20) was added to cells at a concentration of 250 ng/mL for 72 hours prior to performing functional studies or collecting cells for analysis. Morphological changes were photographed with a Nikon 50i series upright microscope equipped with a digital camera. Confirmation of Wnt pathway activation was performed using a TOPFlash reporter assay, as well as qPCR gene expression of axin2, as our lab has previously described [69], using predesigned primers to axin2 and β-actin (Applied Biosystems, assay numbers Mm00443610_m1 and Mm00607939_s1, respectively). In certain experiments, a TGF-β neutralizing antibody (Abcam, product number ab64715) was added during Wnt3a incubation at a concentration of 1 µg/mL. All experiments were performed in triplicate and repeated a minimum of 3 independent times.  2.2.2 Cell proliferation and migration assays  Cell proliferation was measured by MTS assay (Promega) 72 hours after Wnt3a treatment. Migration was measured using the in vitro scratch wound assay, as previously described [90]. Briefly, cells were treated for 72 hours with Wnt3a and confluent 18  monolayers were scratched using a dental device to create a cell-free area where migration could be measured.  2.2.3 Collagen gel contraction assay  Twelve-well culture dishes were coated with 1% bovine serum albumin (BSA) and incubated for 1 hour at 37°C to create a non-stick surface that prevents gels from attaching to the dishes. Prior to performing contraction assays, fibroblasts were treated for 72 hours with Wnt3a. Cells were then trypsinized, counted and seeded into a 0.5 mg/mL type I collagen solution (BD Biosciences, product number 354236) in growth media at a concentration of 1X105 cells/mL. The collagen/cell suspension was vortexed, and 1 mL per well was added to the BSA-coated dishes and the solution was allowed to polymerize for 45 minutes at 37°C. Fresh growth media was added to the solidified collagen gels and plates were returned to the incubator. Collagen gel contraction was monitored over a period of 24 hours and the surface area of contracted gels was measured using Image-Pro Plus software (Media Cybernetics, Bethesda, USA).  2.2.4 Western blotting  Cell lysates were collected in lysis buffer (10mM HEPES (pH 7.4), 50 mM Na4P2O7, 50 mM NaF, 50 mM NaCl, 5 mM EDTA, 5 mM EGTA, 2 mM Na3VO4, and 1 mM phenylmethylsulfonyl fluoride, with 0.1% Triton X-100 and 10 µg/mL leupeptin) followed by centrifugation at high speed (14000 X g at 4ºC for 10 minutes) to recover  19  proteins. The protein concentration of samples was measured by a Bradford protein assay. Equal amounts of protein from each sample were separated with sodium dodecylsufate-polyacrylamide gel electrophoresis (SDS-PAGE; 10% polyacrylamide) and transferred to a nitrocellulose membrane. Membranes were blocked for 1 hour in 5% milk/TBST and incubated overnight at 4°C with primary antibody in 2.5% milk/TBST. Following 3 washes in TBST, secondary antibody (Santa Cruz biotechnology) at a concentration of 1:2000 in 2.5% milk/TBST was added for 1 hour at room temperature. Antibody binding was visualized with the enhanced chemiluminescence detection system (Thermo Fischer Scientific). Images were captured with a Chemigenius2 system (Syngene, Frederick, USA) and band intensities were calculated with GeneTools software (Syngene).  Antibodies used were as follows: β-catenin (BD Biosciences, product number 610154), TGF-β (BD Biosciences, product number 555053), pSMAD2 (Cell Signaling, product number 3108), SMAD2/3 (Cell Signaling, product number 3102), smooth muscle α-actin (Santa Cruz Biotechnology, product number sc-32251).  2.2.5 Immunohistochemistry  Cells were fixed for 20 minutes in 3.7% formaldehyde, permeabilized with 0.1% triton X-100 for 20 minutes, blocked for 30 minutes with 1% BSA in PBS and incubated overnight at 4°C with the indicated primary antibody at a concentration of 1:100 in 1% BSA. Following primary antibody, cells were washed with PBS and incubated with anti-  20  mouse Alexa-fluor488 conjugated secondary antibody (Invitrogen) at a concentration of 1:200 in 1% BSA for 1 hour at room temperature in the dark. To visualize f-actin, permeabilized cells were stained for 20 minutes with phalloidin conjugated to Alexafluor594 (Invitrogen). Cells were coverslipped with VectaShield mounting medium containing DAPI (Vector Laboratories) and images were captured using a Leica AOBS SP2 confocal microscope as our lab has previously described [91,92].  2.2.6 siRNA transfections  Cells were seeded into 24-well culture dishes and siRNA transfection was performed using Oligofectamine (Invitrogen) as per the manufacturer’s instructions. Control siRNA or mouse β-catenin siRNA (Santa Cruz Biotechnology, product numbers 37007 and 29210, respectively) were added to the cells at a concentration of 60 pmol/well for 24 hours prior to treating with Wnt3a. Cell lysates were harvested for Western blotting after 72 hours of Wnt3a treatment.  2.2.7 Statistical analysis  Results are represented as the mean ± standard deviation. Significant differences in treatment groups were determined using the unpaired Student’s t-test. For all analyses, p<0.05 was considered statistically significant.  21  2.3  Results  2.3.1 Wnt3a induces canonical Wnt signaling in mouse fibroblasts  Mouse fibroblasts were treated for 24 hours with 250 ng/mL Wnt3a (or the vehicle control) to determine if recombinant Wnt3a induces nuclear accumulation of β-catenin. Immunohistochemistry demonstrated a strong nuclear signal for β-catenin in Wnt3atreated fibroblasts while control cells remained negative for nuclear β-catenin (Figure 5A), suggesting Wnt3a activates canonical Wnt signaling. To confirm activation of canonical Wnt signaling by Wnt3a treatment, cells were transfected with a TOPFlash reporter construct prior to treatment, and a luciferase assay demonstrated that Wnt3a activated the TOPFlash reporter 5.3 ± 1.6 fold after a 24 hour treatment (Figure 5B, p<0.05). Further, mRNA expression of axin2, an early immediate target of canonical Wnt signaling, was measured after 24 hours of Wnt3a treatment. Wnt3a induced a 255 ± 71 fold increase in axin2 mRNA expression compared with vehicle treated cells (Figure 5C, p<0.05).  2.3.2 Wnt3a alters the morphology of mouse fibroblasts  Wnt3a induced a marked change in fibroblast morphology after 72 hours of treatment. The Wnt-treated cells appeared spindle-shaped and organized into parallel sheets as visualized by light microscopy (Figure 6A). Consistent with this, confocal microscopy showed Wnt3a-treated cells appeared larger and had altered cytoskeletons characterized 22  by dramatically increased stress fibre formation (Figure 6B). The increased formation of stress fibres in Wnt3a-treated fibroblasts is best visualized in low density cultures of the cells (Figure 6C).  2.3.3 Wnt3a inhibited fibroblast proliferation, but increased cell migration and contraction of collagen gels  Cell proliferation was observed to be significantly decreased after 72 hours of Wnt3a treatment (Figure 7A, proliferation rate: 77.4 ± 4.5% of vehicle-treated cells, p<0.05). In contrast, Wnt3a significantly increased cell migration as measured by in vitro scratch wound assay (Figure 7B, 78.1 ± 2.1% vs 61.9 ± 3.8%, p<0.05). Fibroblast contraction, as measured by a fibroblast-populated collagen lattice contraction model, was also found to be significantly increased following the 72 hour Wnt3a treatment (Figure 7C, 16.1 ± 0.6% vs 29.4 ± 1.3% of initial gel area, p<0.05).  2.3.4 Wnt3a increases TGF-β expression, SMAD2 phosphorylation and smooth muscle α-actin expression  Morphologically and functionally, this data suggests Wnt3a stimulates a myofibroblastlike phenotype in cultured fibroblasts. I next examined whether Wnt3a alters the TGF-β signaling axis in these cells. Western blot demonstrated expression of TGF-β to be upregulated after 72 hours Wnt3a treatment, and densitometry showed this change to be significant (Figure 8A). Consistent with this, SMAD2 phosphorylation, a downstream signaling target of TGF-β, was shown to be significantly increased following Wnt3a  23  treatment (Figure 8B). Smooth muscle α-actin, the most commonly used marker of myofibroblast differentiation, was also found to be significantly upregulated by Wnt3a (Figure 8C). Immunohistochemistry and confocal microscopy confirmed this, and showed the spindle-shaped fibroblasts displayed clearly visible smooth muscle α-actinpositive stress fibres following Wnt3a treatment (Figure 8D).  2.3.5 Wnt3a-induced smooth muscle α-actin expression is dependent on βcatenin To determine if the altered cell phenotype induced by Wnt3a was dependent on β-catenin, siRNA was used to knock down β-catenin expression prior to treating cells with Wnt3a. Western blot showed siRNA significantly decreased β-catenin expression in both vehicleand Wnt3a-treated cells (Figure 9A). Knockdown of β-catenin resulted in a 48.5 ± 8.4% decrease in Wnt3a-induced axin2 mRNA expression (p<0.05, data not shown), suggesting β-catenin siRNA significantly inhibited signaling through the canonical Wnt pathway. In the absence of Wnt3a, no change in SMAD2 phosphorylation was observed in cells transfected with scrambled or β-catenin siRNA. Upon stimulation with Wnt3a, however, β-catenin siRNA significantly inhibited the Wnt3a-induced SMAD2 phosphorylation (Figure 9B). The decreased SMAD2 phosphorylation was associated with significantly decreased smooth muscle α-actin expression in Wnt3a-treated cells that had been transfected with β-catenin siRNA (Figure 9C). No change in smooth muscle αactin expression was observed in vehicle-treated cells. Immunohistochemistry and confocal microscopy confirmed that β-catenin knockdown inhibited the Wnt3a-induced smooth muscle α-actin expression in mouse fibroblasts (Figure 9D). 24  2.3.6 Wnt3a-induced change in cell phenotype is dependent on TGF-β expression To better characterize the role of TGF-β signaling in Wnt3a-treated fibroblasts, a time course experiment was performed over 72 hours. Representative Western blots are shown in Figure 10A and the relative densitometry values are plotted over time in Figure 10B. Wnt3a treatment led to a rapid induction of TGF-β expression, which was highest between 12 and 24 hours after treatment. Phosphorylation of SMAD2 appeared highest between 24 and 48 hours, which was followed by the strongest expression of smooth muscle α-actin after 72 hours of treatment, indicating a sequential activation of this pathway following Wnt3a treatment. To determine if the Wnt3a-induced SMAD2 phosphorylation and smooth muscle α-actin expression were dependent on TGF-β expression, a neutralizing antibody to TGF-β was added during Wnt3a treatment. TGF-β neutralization significantly inhibited both the Wnt3a-induced phosphorylation of SMAD2 (Figure 10C) and smooth muscle α-actin expression (Figure 10D). No change in SMAD2 phosphorylation or smooth muscle α-actin expression was seen in the vehicle-treated cells.  2.4  Discussion  In this study, I have shown that Wnt3a alters the phenotype of mouse fibroblasts. Structurally, Wnt3a induced a spindle-shaped morphology characterized by increased expression and incorporation of smooth muscle α-actin into stress fibres. Functionally, 25  Wnt3a inhibited fibroblast proliferation, but increased cell migration and contraction. These changes were mediated, at least in part, by Wnt3a-induced TGF-β expression and signaling through SMAD2 in a β-catenin-dependent mechanism. Collectively, this data suggests Wnt3a stimulates the formation of a myofibroblast-like phenotype in cultured fibroblasts.  My data is consistent with recent studies that are suggestive of an interaction between Wnt/β-catenin and TGF-β/SMAD signaling in controlling gene transcription and cell phenotype [77,93,94]. A recent report has demonstrated that Wnt3a controls transcriptional regulation of SM22α in mesenchymal cells via convergence with TGFβ/SMAD signaling at a novel regulatory element in the SM22α promoter [94]. SM22α is a calponin-like protein that exhibits a similar expression pattern as smooth muscle α-actin [95], both of which are smooth muscle cell contractile proteins commonly viewed as markers of an activated myofibroblast phenotype [96]. In a separate study that documented the gene expression profile induced by Wnt3a in fibroblasts, TGF-β was identified as one of the genes upregulated more than two fold after a 6 hour treatment [77,97]. Consistent with this, Wnt3a was found to stimulate TGF-β and collagen I mRNA expression in cultures of fetal and post-natal fibroblasts [77]. My data add to this story by showing Wnt3a stimulates TGF-β protein expression and activation of its downstream signaling, culminating in increased smooth muscle α-actin expression. In another study, Laeremans et al showed overexpression of the Frizzled 1 receptor in combination with Wnt3a treatment stimulated the expression of myofibroblast markers in cardiac fibroblasts, changes that occurred in a β-catenin-independent pathway [98]. Interestingly,  26  Wnt3a treatment alone was actually found to decrease the expression of smooth muscle α-actin in their study. The cause of such discrepancy is not clear at present. However, when taken together, these studies support a role for Wnt3a in modifying cell phenotype, with my data strongly suggesting Wnt3a promotes a myofibroblast-like phenotype in cultured fibroblasts.  Growing evidence points to an active role for Wnt signaling in normal wound repair and in a number of human diseases. Increased canonical Wnt signaling has been observed during cutaneous wound repair [75-77], but is also well recognized as a contributor to a multitude of malignant disorders [82-84], as well as hypertrophic scarring [78], aberrant blood vessel remodeling [79], pulmonary fibrosis [80] and aging [81], among others. After tissue injury, fibroblasts differentiate into contractile and secretory myofibroblasts that participate in the synthesis and remodeling of granulation tissue during repair [99]. However, these myofibroblasts can severely impair organ function when contraction and ECM secretion become excessive [8]. Moreover, myofibroblasts present in the stroma reaction of epithelial tumors may promote the progression of cancer invasion [8,100]. TGF-β is a known and potent inducer of myofibroblast differentiation [12,101], however the regulation of TGF-β expression remains relatively understudied. The finding that Wnt3a upregulates TGF-β expression and stimulates smooth muscle α-actin expression provides a link between Wnt signaling and myofibroblasts in wound repair and disease. If Wnt3a also upregulates TGF-β expression and myofibroblast differentiation in vivo, the Wnt signaling pathway may be shown as a critical regulator of the wound healing  27  response. More work will be needed to determine how manipulating the Wnt pathway alters injury and repair events in vivo.  Wnt proteins are believed to signal through three distinct pathways, of which the canonical Wnt/β-catenin cascade is the best understood. The other pathways include the noncanonical planar cell polarity pathway and the Wnt/Ca2+ pathway [73]. By using siRNA, I was able to demonstrate that knocking down β-catenin expression reversed the Wnt3a-induced smooth muscle α-actin expression, suggesting these changes were mediated by canonical Wnt signaling through β-catenin. In addition, I demonstrated that Wnt3a also upregulated TGF-β expression. Previous reports have identified TGF-β as one of the genes whose mRNA expression is rapidly induced by Wnt3a treatment [97]. Thus, it may be reasonable to speculate that TGF-β is a target of canonical Wnt signaling. To my knowledge, however, there are no published reports that have examined whether TGF-β gene transcription is regulated by Wnt signaling or whether its promoter contains functionally important TCF/LEF binding sites. It will be important to determine whether TGF-β is one of a growing list of direct targets genes for Wnt signaling, as this information might provide new therapeutic targets for controlling TGF-β levels in disease settings.  In summary, this study presents data on a novel role for Wnt3a in stimulating myofibroblast differentiation in cultured fibroblasts. The data suggests Wnt3a treatment promotes a contractile and migratory fibroblast phenotype that is characterized by increased expression of smooth muscle α-actin. These changes appear to be mediated by  28  increased expression of TGF-β and its signaling through SMAD2 in a β-catenindependent manner. As myofibroblasts play a central role in normal and aberrant injury and repair events, this data suggests Wnt3a may be critically involved in a wound healing response.  29  Figure 5. Wnt3a induces canonical Wnt signaling in mouse fibroblasts. A, Confocal images of fibroblasts treated for 24 hours with vehicle (top panels) or 250 ng/mL Wnt3a (bottom panels) and immunonstained for β-catenin (green) and nuclei (blue). Wnt3a treatment induced clear nuclear accumulation of β-catenin in murine fibroblasts (arrows). B, TOPFlash reporter assay demonstrated Wnt3a significantly increased luciferase activity 5.3 ± 1.6 fold after a 24 hour treatment (p<0.05). C, Wnt3a treatment induced a 255 ± 71 fold increase in the mRNA expression of axin2, a target of canonical Wnt signaling (p<0.05). (Scale bar = 23.00 µm in A, * denotes p<0.05)  30  Figure 6. Wnt3a induces a spindle-like morphology with increased stress fibre formation after 72 hours of treatment. A, Light microscope images of mouse fibroblasts that had been treated for 72 hours with vehicle (left panel) or 250 ng/mL Wnt3a (right panel). Wnt3a treatment induced a spindle-like morphology in fibroblasts. B, Confocal images of vehicle-treated (left panel) 31  or Wnt3a-treated (right panel) fibroblasts immunostained for f-actin (red) and nuclei (blue) showing the increased formation and parallel organization of stress fibres following 72 hours Wnt3a treatment. C, Low density culture of vehicle-treated (left panel) or Wnt3a-treated (right panel) fibroblasts highlights the increased formation of stress fibres seen after Wnt3a treatment. (Scale bars = 47.00 µm in B, 23.00 µm in C)  32  Figure 7. Wnt3a inhibits cell proliferation, but increases cell migration and contraction after 72 hour treatment. A, Cell proliferation was measured after 72 hours of treatment with Wnt3a or vehicle. Wnt-treated cells grew at 77.4 ± 4.5% the rate of vehicle treated cells (p<0.05). B, Cells were treated for 72 hours with Wnt3a or vehicle, and then a scratch wound assay was performed to measure cell migration. Wnt-treated cells closed the scratch wound at a significantly faster rate than vehicle-treated cells, as measured 48 hours after injury (78.1 ± 2.1% vs 61.9 ± 3.8%, p<0.05). C, Cells were treated for 72 hours with Wnt3a or vehicle and then a fibroblast-populated collagen lattice contraction assay was performed. Images of contracted gels taken at 24 hours are shown along with the quantified surface areas of contracted gels. Wnt3a treatment significantly increased the fibroblast-mediated contraction of collagen gels (16.1 ± 0.6% vs 29.4 ± 1.3% of initial surface area, p<0.05). (* denotes p<0.05)  33  34  Figure 8. Wnt3a increases TGF-β expression, SMAD2 phosphorylation and smooth muscle α-actin expression. A, Representative Western blot of TGF-β expression in vehicle-treated and Wnt3atreated fibroblasts after 72 hours. Densitometry showed TGF-β expression to be significantly increased after Wnt3a treatment (p<0.05). B, Western blot of SMAD2 phosphorylation after 72 hours of vehicle or Wnt3a treatment. Densitometry showed Wnt3a significantly increased SMAD2 phosphorylation at 72 hours (p<0.05). C, Western blot of smooth muscle α-actin expression in vehicle-treated or Wnt3a-treated cells. Wnt3a-treatment significantly increased the expression of smooth muscle α-actin expression in mouse fibroblasts, as measured by densitometry (p<0.05). D, Confocal images of fibroblasts immunostained for smooth muscle α-actin (green) and nuclei (blue). Wnt3a-treated fibroblasts had clearly visible smooth muscle α-actin positive stress fibres while the vehicle-treated cells did not display prominent expression of smooth muscle αactin in their stress fibres. (Scale bar = 47.00 µm in D, * denotes p<0.05)  35  Figure 9. Wnt3a-induced change in cell phenotype is dependent on β-catenin. A, Western blot demonstrated β-catenin siRNA significantly decreased β-catenin expression in vehicle- and Wnt-treated fibroblasts when compared to a scrambled siRNA (p<0.05). B, Western blot showed knockdown of β-catenin expression significantly inhibited the Wnt3a-induced SMAD2 phosporylation (p<0.05). No difference in SMAD2 phosporylation was detected in vehicle treated cells (p=0.25). C, Western blot of smooth 36  muscle α-actin expression demonstrated that β-catenin siRNA significantly decreased smooth muscle α-actin expression in Wnt3a-treated fibroblasts (p<0.05). No significant difference was seen in the vehicle-treated cells (p=0.27). D, Immunohistochemistry showed Wnt3a promoted smooth muscle α-actin stress fibre formation in control siRNA transfected cells (green, arrows), but β-catenin siRNA completely inhibited the Wnt3ainduced smooth muscle α-actin expression. Cell nuclei are stained blue with DAPI. (Scale bar = 23.00 µm in D, * denotes p<0.05)  37  Figure 10. Wnt3a-induced change in cell phenotype is dependent on TGF-β expression. A, Representative Western blots of vehicle- and Wnt3a-treated fibroblasts showing TGFβ expression, SMAD2 phosphorylation, and smooth muscle α-actin expression at 12, 24, 48, and 72 hours of treatment. B, Graphical representation of the densitometry results for the blots in A shows, in a sequential manner, that TGF-β expression peaks between 12 and 24 hours, followed by SMAD2 phosphorylation peaking between 24 and 48 hours, which is then followed by smooth muscle α-actin expression peaking after 72 hours of treatment. C, Western blot of SMAD2 phosphorylation in fibroblasts treated with or without Wnt3a and a TGF-β neutralizing antibody. Densitometry demonstrated the TGFβ neutralizing antibody significantly inhibited Wnt3a-induced SMAD2 phosphorylation (p<0.05). No change was seen in the vehicle-treated cells (p=0.74). D, Western blot of smooth muscle α-actin expression in fibroblasts treated with or without Wnt3a and the TGF-β neutralizing antibody. Densitometry confirmed TGF-β neutralization significantly inhibited the Wnt3a-induced smooth muscle α-actin expression (p<0.05). No change was seen in vehicle-treated cells (p=0.71). (* denotes p<0.05) 38  CHAPTER 3. WNT3A INDUCES A CONTRACTILE AND SECRETORY PHENOTYPE IN CULTURED VASCULAR SMOOTH MUSCLE CELLS  3.1  Introduction  Numerous studies have demonstrated that after arterial injury, vessel walls follow a response-to-injury pattern of wound healing leading to stenosis secondary to the neointimal accumulation of smooth muscle cells and ECM [27,102-104]. A number of soluble mediators released at the site of vascular injury act as molecular cues that guide smooth muscle cell responses during repair [87,105]. Evidence suggests a role for the Wnt family of secreted glycoproteins and their associated signaling pathways in regulating many of the processes involved in vascular wound repair and remodeling events [79,106-112]. However, the precise mechanisms and outcomes of Wnt signaling in such settings remain unclear. Moreover, the effect of specific Wnt family members on vascular cell phenotype remains undefined.  In the current study, I examined the effect of Wnt3a, a canonical Wnt ligand, on vascular smooth muscle cell morphology and function. My data suggests Wnt3a stimulates a contractile and secretory phenotype in smooth muscle cells, characterized by decreased proliferation but increased expression of the contractile proteins, calponin and smooth muscle α-actin, as well as the ECM genes, collagen I and III. The altered cell phenotype caused by Wnt3a was associated with increased expression of the gap junction protein connexin 43 and increased cell-cell communication. Use of the Wnt antagonist, dickkopf39  related protein 1 (DKK1), completely reversed the Wnt3a-mediated change in cell phenotype, suggesting these changes were dependent on the canonical Wnt signaling pathway. Collectively, this data suggests Wnt signaling modifies vascular smooth muscle cell phenotype and, as such, may have important implications for our understanding of vascular development and disease.  3.2  Materials and methods  3.2.1 Cell culture  Mouse vascular smooth muscle cells (ATCC, product number CRL-2797) were cultured in DMEM containing 10% FBS and 100 U/mL penicillin/streptomycin. Cells were maintained in a humidified incubator at 37°C with 5% CO2 and used for experiments between passages 6-12. Recombinant murine Wnt3a (Peprotech, product number 315-20) was added to the cells (at 50% confluency) at a concentration of 250 ng/mL for 72 hours prior to performing functional studies or collecting cells for analysis. To test that Wnt3a activated canonical Wnt signaling in the smooth muscle cells, experiments were performed where Wnt3a was only added for 24 hours prior to measuring nuclear βcatenin or target gene expression. In certain experiments, TGF-β (Peprotech, product number 100-21) was added alone or in combination with Wnt3a at a concentration of 5 ng/mL. In other experiments, recombinant murine DKK1 (R&D systems, product number 5897-DK) was added during Wnt3a incubation at a concentration of 250 ng/mL. In these experiments, cells were pretreated for 1 hour with DKK1 prior to adding Wnt3a. All  40  experiments were performed in triplicate (unless otherwise stated) and repeated a minimum of 3 independent times.  3.2.2 qPCR analysis  RNA extraction, cDNA synthesis and qPCR analysis were performed as our lab has previously described [69]. Predesigned primers to axin2, collagen I, collagen III, and βactin were purchased from Applied Biosystems (assay numbers Mm00443610_m1, Mm00483888_m1, Mm01254476_m1, and Mm00607939_s1, respectively).  3.2.3 Cell proliferation  Cell proliferation was measured by MTS assay (Promega) 72 hours after Wnt3a treatment as our lab has previously described [113].  3.2.4 Western blotting  Cell lysates were collected in lysis buffer (10mM HEPES (pH 7.4), 50 mM Na4P2O7, 50 mM NaF, 50 mM NaCl, 5 mM EDTA, 5 mM EGTA, 2 mM Na3VO4, and 1 mM phenylmethylsulfonyl fluoride, with 0.1% Triton X-100 and 10 µg/mL leupeptin) followed by centrifugation at high speed (14000 X g at 4ºC for 10 minutes) to recover proteins. The protein concentration of samples was measured by a Bradford protein assay. Equal amounts of protein from each sample were separated with SDS-PAGE (10%  41  polyacrylamide) and transferred to a nitrocellulose membrane. Membranes were blocked for 1 hour in 5% milk/TBST and incubated overnight at 4°C with primary antibody in 2.5% milk/TBST. Following 3 washes in TBST, secondary antibody (Santa Cruz biotechnology) at a concentration of 1:2000 in 2.5% milk/TBST was added for 1 hour at room  temperature.  Antibody  binding  was  visualized  with  the  enhanced  chemiluminescence detection system (Thermo Fischer Scientific). Images were captured with a Chemigenius2 system (Syngene, Frederick, USA) and band intensities were calculated with GeneTools software (Syngene).  Antibodies used were as follows: calponin (Abcam, product number ab46794), connexin 43 (Cell Signaling, product number 3512) smooth muscle α-actin (Santa Cruz Biotechnology, product number sc-32251), β-actin (Abcam, product number ab6276), MMP2 (Abcam, product number 3158), MMP9 (Abcam, product number ab38898), TIMP2 (Cell Signaling Technology, product number 5738).  3.2.5 Collagen gel contraction assay  Twelve-well culture dishes were coated with 1% BSA and incubated for 1 hour at 37°C to create a non-stick surface that prevents gels from attaching to the dishes. Prior to performing contraction assays, smooth muscle cells were treated for 72 hours with Wnt3a. Cells were then trypsinized, counted and seeded into a 0.5 mg/mL type I collagen solution (BD Biosciences, product number 354236) in growth media at a concentration of 1X105 cells/mL. The collagen/cell suspension was vortexed, and 1 mL per well was  42  added to the BSA-coated dishes and the solution was allowed to polymerize for 45 minutes at 37°C. Fresh growth media was added to the solidified collagen gels and plates were returned to the incubator. Collagen gel contraction was monitored over a period of 48 hours and the surface area of contracted gels was measured using Image-Pro Plus software (Media Cybernetics, Bethesda, USA).  3.2.6 Immunohistochemistry  Cells were fixed for 20 minutes in 3.7% formaldehyde, permeabilized with 0.1% triton X-100 for 20 minutes, blocked for 30 minutes with 1% BSA in PBS and incubated overnight at 4°C with the indicated primary antibody at a concentration of 1:100 in 1% BSA. Following primary antibody, cells were washed with PBS and incubated with antimouse Alexa-fluor conjugated secondary antibody (Invitrogen) at a concentration of 1:200 in 1% BSA for 1 hour at room temperature in the dark. To visualize f-actin, permeabilized cells were stained for 20 minutes with phalloidin conjugated to Alexafluor594 (Invitrogen). Cells were coverslipped with VectaShield mounting medium containing DAPI (Vector Laboratories) and images were captured using a Leica AOBS SP2 confocal microscope as our lab has previously described [91,92].  Antibodies used for staining were as follows: β-catenin (BD Biosciences, product number 610154) and connexin 43 (Cell Signaling, product number 3512)  43  3.2.7 Scrape loading dye transfer assay  Intercellular communication was measured by using the scrape loading dye transfer method, as previously described [114]. Briefly, cells were cultured on glass coverslips in 6-well dishes until confluent in the presence or absence of Wnt3a. Cells were rinsed twice with PBS, and 0.05% Lucifer Yellow was then added to the dishes. Scrapes were made through the confluent cultures with a sterile scalpel, and the cells were incubated in the dye mix for precisely 1 minute, rinsed quickly three times with PBS, and fixed with formaldehyde. Cells were coverslipped and visualized by confocal microscopy as described above.  3.2.8 Statistical analysis  Results are represented as the mean ± standard deviation. Significant differences in treatment groups were determined using the unpaired Student’s t-test. For all analyses, p<0.05 was considered statistically significant.  3.3  Results  3.3.1 Wnt3a activates canonical Wnt signaling in mouse vascular smooth muscle cells.  Smooth muscle cells were treated for 24 hours with 250 ng/mL of Wnt3a (or the vehicle control) and immunohistochemistry for β-catenin was performed. A clear nuclear 44  accumulation of β-catenin was observed in the Wnt3a-treated cells (Figure 11A), suggesting Wnt3a activates canonical Wnt signaling. The vehicle-treated cells remained negative for nuclear β-catenin. To confirm activation of canonical Wnt signaling by Wnt3a, mRNA expression of axin2, an early immediate target of canonical Wnt signaling, was measured after 24 hours of Wnt3a treatment. Wnt3a induced a 71.2 ± 9.6 fold increase in axin2 mRNA expression compared to vehicle-treated control cells (Figure 11B, p<0.05).  3.3.2 Wnt3a alters the phenotype of vascular smooth muscle cells  Wnt3a induced a marked change in smooth muscle cell phenotype following 72 hours of treatment. The Wnt3a-treated cells appeared larger, flatter, and had increased formation of stress fibres than the vehicle-treated control cells when visualized by actin staining and confocal microscopy (Figure 12A). This change in morphology was associated with significantly decreased proliferation after 72 hours incubation with Wnt3a (Figure 12B, relative rate = 0.79 ± 0.13 of control cells, p<0.05).  I next examined this phenotypic change in more detail. The Wnt3a-treated cells demonstrated increased expression of the smooth muscle contractile proteins, calponin and smooth muscle α-actin (Figure 13A). Consistent with this, the Wnt3a-treated cells contracted a collagen lattice significantly faster than control cells, following a 72 hour treatment (Figure 13B, 24.5 ± 0.8% vs 36.7 ± 0.9% of initial gel area, p<0.05). To determine if there was a dose response to Wnt3a, I tested the expression of contractile  45  proteins following treatment over a range of Wnt3a concentrations (0-300 ng/mL). Wnt3a induced a dramatic increase in the calponin and smooth muscle α-actin expression at all concentrations tested (Figure 13C). As growing evidence suggests cross-talk between Wnt signaling and TGF-β signaling [94,115], I next examined the effect of a combined treatment of Wnt3a and TGF-β on the expression of these proteins. The data suggest that, while Wnt3a and TGF-β both increased the expression of calponin and smooth muscle αactin individually, the combination of these growth factors increased the expression of these proteins even further and in a dramatic fashion (Figure 13D).  Concomitant with the increased contractile protein expression, ECM synthesis was also increased in Wnt3a-treated fibroblasts, as measured by collagen I and III mRNA expression. The Wnt3a-treated cells had 1.20 ± 0.04 fold increase in collagen I mRNA expression and a 1.50 ± 0.04 fold increase in collagen III mRNA expression, when compared to the vehicle-treated control cells (Figures 14A and B, respectively, p<0.05). These cells also displayed increased expression of MMP2 and MMP9, but decreased TIMP2 levels (Figure 14C), suggesting Wnt3a induced a shift in balance in these matrix proteases and one of their inhibitors.  3.3.3 Wnt3a increases connexin 43 expression and intercellular communication in smooth muscle cells  I next examined the expression of connexin 43, a gap junction protein and the principal connexin expressed in arterial smooth muscle cells [116-118]. The Wnt3a-treated smooth muscle cells displayed increased connexin 43 expression, as measured by Western blot 46  (Figure 15A). Densitometry showed Wnt3a increased connexin 43 expression 1.71 ± 0.09 fold when compared to control cells (Figure 15B, p<0.05). Immunohistochemistry confirmed the increased connexin 43 expression seen in the Wnt3a-treated cells, and showed the connexin 43 to localize at cell-cell junctions between neighboring cells (Figure 15C). To determine if the increased connexin 43 expression in Wnt3a-treated cells correlated with increased intercellular communication, a scrape-loading dye transfer assay was performed. Representative images of dye transfer are shown in Figure 16A. Quantification showed the relative distance of dye transfer to be significantly increased in Wnt3a-treated cells (Figure 16B, relative distance = 0.86 ± 0.09 in Wnt3a cells versus 0.16 ± 0.03 in control cells, p<0.05, N=9 for each treatment group).  3.3.4 Wnt3a-induced change in smooth muscle cell phenotype is dependent on canonical Wnt signaling  To determine if the altered cell phenotype induced by Wnt3a is dependent on canonical Wnt signaling, DKK1 was added during Wnt3a treatment. Incubation with DKK1 eliminated the Wnt3a-induced expression of axin2 mRNA at 24 hours, confirming it effectively antagonized canonical Wnt signaling (Figure 17A, fold change in axin2 mRNA expression, compared to vehicle-treated control cells, was 1.07 ± 0.97 for DKK1 alone, 421.21 ± 136.39 for Wnt3a alone, and 10.18 ± 11.69 for the combined Wnt3a and DKK1 treatment). Following a 72 hour incubation with Wnt3a, DKK1 completely inhibited the Wnt3a-induced expression of calponin and smooth muscle α-actin, as demonstrated by Western blot (Figure 17B). Further, the increased expression of connexin 43 that was seen in Wnt3a-treated cells was completely eliminated in the 47  presence of DKK1, as demonstrated by immunohistochemistry and confocal microscopy (Figure 17C), indicating the Wnt3a-induced change in cell phenotype was dependent on canonical Wnt signaling.  3.4  Discussion  In this study, I have shown that Wnt3a alters the phenotype of vascular smooth muscle cells. Structurally, Wnt3a induced a larger morphology in smooth muscle cells that was characterized by increased stress fibre formation. These cells displayed upregulated expression of the smooth muscle contractile proteins, calponin and smooth muscle αactin, along with increased expression of the ECM genes collagen I and III, and an altered balance in the expression of matrix proteases and their inhibitors. Functionally, the Wnt3a-treated cells proliferated slower than control cells but contracted a collagen lattice faster. Collectively, this data suggests Wnt3a induced a contractile and synthetic phenotype in vascular smooth muscle cells. This change in cell phenotype induced by Wnt3a was associated with an increase in expression of the gap junction protein connexin 43 and increased cell-cell communication. These changes appeared to occur via canonical Wnt signaling, as DKK1 blocked the Wnt3a-mediated change in cell phenotype. Altogether, this data suggests Wnt3a alters the phenotype of vascular smooth muscle cells. As Wnt signaling is upregulated after vascular injury [79,106], this data highlights a potential role for Wnt3a in controlling smooth muscle cell phenotype during vascular wound repair and remodeling processes.  48  The current study is consistent with previous reports that have suggested a role for Wnt signaling in modifying cell phenotype and fate [77,113,119]. My data suggests Wnt3a induced a smooth muscle cell phenotype with increased expression of contractile proteins as well as increased expression of ECM genes. Thus, it appears Wnt3a simultaneously induced a contractile and synthetic phenotype in smooth muscle cells. Normally, vascular smooth muscle cells form the medial layer of blood vessels where they are found in a differentiated contractile phenotype [120]. However, numerous vascular diseases are characterized by the accumulation of smooth muscle cells in the intimal layer of blood vessels where they are surrounded by newly synthesized ECM [27,57,104]. These cells are generally thought of as dedifferentiated and synthetic, in that they display a loss of contractile differentiation, but an increased capacity to synthesize matrix proteins [120]. While traditionally it has been thought that smooth muscle cells existed in either a synthetic or contractile state, it is now being recognized that there is considerable heterogeneity in smooth muscle cell populations and in some instances, contractile differentiation markers may be expressed simultaneously with matrix synthesis [121123]. My data is in support of this idea that contractile differentiation and increased matrix synthesis are not mutually exclusive in vascular smooth muscle cells. In fact, these cells appear to resemble a myofibroblast-like cell type in nature, which is a specialized wound repair fibroblast characterized by increased contractile ability and matrix synthesis [8]. My previous work has suggested that Wnt3a induces myofibroblast differentiation in murine fibroblasts [113], and it is tempting to speculate that a similar process may be occurring in vascular smooth muscle cells. More work will be needed to determine  49  whether this relationship between Wnt signaling and smooth muscle cell phenotype exists in vivo, and how such a cell phenotype modifies vascular development and disease.  Gap junctions are clusters of transmembrane channels which directly link adjacent cells and allow for the passage of ions and small signaling molecules [124]. Thus, gap junctions allow cells to coordinate their functions. These channels are comprised of subunit proteins encoded by the multigene connexin family, of which connexin 43 is the predominant connexin expressed by vascular smooth muscle cells [118]. It has previously been reported that connexin 43 expression is upregulated in smooth muscle cells in response to injury in rodent models and in early human atherosclerosis, situations in which elevated Wnt signaling has been reported [79,106] and in which increased ECM synthesis is well documented [27,125]. Reducing connexin 43 expression has been shown to inhibit lesion development in rodent models of atherosclerosis and acute vascular injury [126-128], suggesting potential pro-atherogenic functions for gap junction communication. My results suggest Wnt3a increases the expression of connexin 43 in vascular smooth muscle cells, and this increase in connexin 43 expression is associated with increased intercellular communication and matrix synthesis. A recent report has demonstrated that TGF-β also induced a contractile and synthetic phenotype in vascular smooth muscle cells which correlated with increased connexin 43 expression and intercellular communication [123]. When taken together, these studies imply a role for connexin 43 in the Wnt3a- and TGF-β-mediated smooth muscle cell differentiation and matrix synthesis. In support of this idea, recent studies have highlighted a potential role for connexin 43 hemichannels in regulating vascular smooth muscle cell phenotype in  50  response to balloon injury and other mitogenic stimuli [128,129]. Adding to this, my data suggests that the presence of both Wnt3a and TGF-β may have an additive effect on cell phenotype, as I observed their combined effects to be much greater than the effect of either protein individually.  Wnt proteins are believed to signal through three distinct pathways, of which the classical or canonical Wnt/β-catenin cascade is the best understood. The other pathways include the noncanonical planar cell polarity pathway and the Wnt/Ca2+ pathway [73]. By using DKK1, I was able to demonstrate that blocking canonical Wnt signaling reversed the Wnt3a-induced change in cell phenotype, suggesting these changes were mediated through the classical Wnt signaling pathway. There is sufficient evidence to suggest a role for canonical Wnt signaling in the development of atheromatous vascular disease [112]. A link between β-catenin stabilization and smooth muscle cell proliferation has been demonstrated both in vitro and after balloon catheter injury [79,109,130]. Oxidized LDL has also been shown to stimulate smooth muscle cell migration in a mechanism that was dependent on β-catenin signaling [131]. A number of confirmed Wnt target genes, including versican [69], fibronectin [132] and matrix metalloproteases [133], are upregulated after vascular injury and appear to promote inflammation and atheroma formation [24,65,134]. My data add to this story by suggesting canonical Wnt signaling promotes a contractile and secretory phenotype in vascular smooth muscle cells. I also observed an alteration to the balance of protease expression, as both MMP2 and MMP9 expression were increased while the protease inhibitor TIMP2 was decreased, signifying Wnt3a may stimulate an active remodeling process in smooth muscle cells. While a link  51  between Wnt signaling and vascular calcification has been established [135], to my knowledge, there are no functional studies that have assessed the impact of inhibiting Wnt signaling in experimental models of vascular wound repair and disease. Clearly this is an area that should be explored in the future.  In summary, I provide data on a novel role for Wnt3a in altering the phenotype of cultured vascular smooth muscle cells. The data suggests Wnt3a promotes a contractile and secretory phenotype in smooth muscle cells that is associated with the increased expression of connexin 43. These changes appear to be mediated by canonical Wnt signaling. As smooth muscle cells are critically involved in normal and aberrant vascular injury and repair events, this data suggests a potential role for Wnt3a in vascular wound repair and remodeling events.  52  Figure 11. Wnt3a activates canonical Wnt signaling in vascular smooth muscle cells. A, Smooth muscle cells immunostained for cell nuclei (blue) and β-catenin (green) showing nuclear accumulation of β-catenin in the Wnt3a-treated cells (arrows). B, Wnt3a-treated cells had a 71.2 ± 9.6 fold increase in axin2 mRNA expression (p<0.05), an immediate target gene of canonical Wnt signaling. (Scale bar = 23.00 µm in A, * denotes p<0.05)  53  Figure 12. Wnt3a alters the phenotype of vascular smooth muscle cells. A, Confocal images of smooth muscle cells stained for cell nuclei (blue) and f-actin (red) showing the altered morphology of cells following Wnt3a treatment. The Wnt3a-treated cells appeared larger and acquired a flattened morphology characterized by increased stress fibre formation. B, Cell proliferation was found to be significantly decreased in Wnt3a-treated cells (relative rate = 0.79 ± 0.13 of control cells, p<0.05). (Scale bar = 46.00 µm, * denotes p<0.05)  54  Figure 13. Wnt3a increases contractile properties of vascular smooth muscle cells. A, Representative Western blot images showing the increased expression of calponin and smooth muscle α-actin in Wnt3a-treated cells. B, Wnt3a increased cell-mediated contraction of a collagen lattice, after a 72 hour treatment (24.5 ± 0.8% vs 36.7 ± 0.9% of initial gel area, p<0.05). C, Dose response shows Wnt3a increased calponin and smooth muscle α-actin at all concentrations tested. D, Combined treatment of Wnt3a and TGF-β increased calponin and smooth muscle α-actin expression more than either treatment alone. (* denotes p<0.05)  55  Figure 14. Wnt3a inceases collagen synthesis and protease expression in smooth muscle cells. A, Wnt3a significantly increased the expression of collagen I mRNA (1.20 ± 0.04 fold increase, p<0.05), as well as (B) collagen III mRNA (1.50 ± 0.04 fold increase, p<0.05). C, Representative Western blot images showing the increased expression of MMP2 and MMP9, but decreased expression of TIMP2, in Wnt3a-treated cells. (* denotes p<0.05)  56  Figure 15. Wnt3a increases connexin 43 expression in smooth muscle cells. A, Representative Western blot showing the increased connexin 43 expression in Wnt3atreated smooth muscle cells. B, Densitometry showed the change in connexin 43 expression to be significant (1.71 ± 0.09 fold increase, p<0.05). C, Confocal images of cells immunostained for cell nuclei (blue) and connexin 43 (red) showing the increased expression and localization of connexin 43 at cell-cell junctions (arrows). (Scale bar = 23.00 µm in C, * denotes p<0.05) 57  Figure 16. Wnt3a increases gap junction communication in vascular smooth muscle cells. A, Representative images showing the increased transfer of lucifer yellow in Wnt3atreated cells, as measured by scrape-loading dye transfer. B, Quantification showed the relative distance of dye transfer to be significantly increased in Wnt3a-treated cells (relative distance = 0.86 ± 0.09 in Wnt3a cells versus 0.16 ± 0.03 in control cells, p<0.05). (*denotes p<0.05)  58  Figure 17. Wnt3a-mediated change in smooth muscle cell phenotype and connexin 43 expression is dependent on canonical Wnt signaling. A, Treatment of smooth muscle cells with DKK1 significantly decreased the Wnt3ainduced axin2 mRNA expression (fold change in mRNA expression, compared to vehicle-treated cells, was 1.07 ± 0.97 for DKK1, 421.21 ± 136.39 for Wnt3a, and 10.18 ± 11.69 for the combined Wnt3a and DKK1 treatment). B, Representative Western blots show DKK1 decreased the Wnt3a-induced calponin and smooth muscle α-actin expression. C, Confocal images of smooth muscle cells stained for cell nuclei (blue) and connexin 43 (red) shows the increased expression of connexin 43 in Wnt3a-treated cells 59  (arrows) is eliminated in the presence of DKK1. (Scale bar = 23.00 µm in C, * denotes p<0.05)  60  CHAPTER 4. VERSICAN AND ITS MEMBRANE RECEPTOR CD44 ARE EXPRESSED FOLLOWING INJURY TO VALVE MYOFIBROBLASTS  4.1  Introduction  Valvular heart disease is a major contributor to the overall burden of cardiovascular disease [136]. Pathological analysis of most diseased heart valves suggests a response to injury process is involved in the development of valvular heart disease, generally characterized by an accumulation of valve interstitial cells (VIC) and ECM, along with various intensities of inflammation, neovascularization and fibrosis, ultimately leading to valve sclerosis, possible calcification and dysfunction [137,138]. Being the predominant cell type found in the interstitium of heart valves, VICs are likely critical regulators of this repair process. In response to valve injury, VICs become activated and consistently display features of myofibroblasts, including increased contraction, stress fibre formation, and smooth muscle α-actin expression [139,140]. As myofibroblasts regulate wound repair in many organs [8], a better understanding of how VICs respond to injury will help us to gain insight into the pathobiology of valvular heart disease.  The ECM is now widely recognized as more than simply a support scaffold, and is emerging as one of the key regulators of cell phenotypes. This function is evidenced by the significant remodeling of tissues during injury, inflammation, and repair processes [1,18]. The proteoglycan versican is one of the key ECM components upregulated after injury in a number of tissue and cell types [27,104,141-144], such as in diseased 61  myxomatous heart valves [145,146]. Versican is a large multidomain chondroitin sulphate proteoglycan that binds hyaluronic acid at its N-terminus [36], and binds a variety of other matrix proteins at its C-terminus, including fibrillin-1 [39], fibulin-1 and -2 [40,41], tenascin-R [42], type I collagen [43], and fibronectin [43]. These terminal domains are separated by a central GAG attachment region consisting of two exons named GAG-α and GAG-β. As a result of differential splicing to these central domains, the versican gene codes for four variants (namely V0-V3) that differ in their length and number of attached GAG side chains [147,148]. Because versican connects hyaluronan at the cell surface to other matrix proteins at its C-terminus, it is believed that versican plays a role in the assembly and organization of the ECM. In addition, this versatile proteoglycan modifies a number of cell processes that are thought to contribute to repair, including cell adhesion [50,51], migration [52-54], proliferation [47,52], and apoptosis [47,55]. Therefore, it is likely that versican is a key regulator of cell phenotype during normal or aberrant wound healing.  Despite the apparent importance of versican to injury and repair processes, the role of this versatile proteoglycan in heart valve repair has not been studied. In this study, I sought to determine whether versican is expressed by cultured VICs after injury. I used a scratch assay to mechanically injure confluent monolayers of human VICs and then followed repair over a period of 48 hours. The results suggest that human VICs synthesize and secrete versican into the ECM following injury. These cells also upregulate the expression of CD44, a membrane receptor involved in cell adhesion and migration that binds versican and hyaluronan [22,23]. When a neutralizing antibody was used to block  62  CD44, wound repair in our model was significantly impaired. Further, blocking CD44 disrupted the organization of versican in the pericellular matrix and inhibited stress fibre formation in VICs. Collectively, this data suggests versican expression and organization are important to VIC injury and repair events, and that CD44 may serve as a link between the actin cytoskeleton and the provisional wound repair ECM.  4.2  Materials and methods  4.2.1 Cell culture  This study was approved by the UBC/Providence Health Care Research Ethics Board and conforms with the principles outlined in the Declaration of Helsinki for use of human tissues or subjects. Human aortic valve leaflets were collected by the Cardiovascular Registry, St. Paul’s Hospital, Vancouver, Canada, from the explanted heart at the time of cardiac transplantation from a 47 year old male patient with no previous history of heart valve disease. Valvular interstitial cells were isolated from non-calcified valve leaflets by enzymatic digestion and phenotyped by immunofluorescence using a panel of antibodies against cytoskeletal components. Valve interstitial cells were cultured in MCDB 131 (GIBCO) containing 15% FBS and 100U/mL penicillin/streptomycin. Cells were maintained in a humidified incubator at 37°C with 5% CO2. Cells were used for experiments between passages 2-4. Subconfluent cultures that had been grown for 3-4 days and confluent cultures grown for 11 days were fixed in their non-wounded states with 3.7% formaldehyde for 20 minutes prior to immunohistochemistry. The in vitro  63  wound model is described below. For antibody treatments, cells were trypsinized and incubated for 45 minutes at 4°C with 20µg/mL of the CD44 blocking antibody (BD Pharmingen, clone IM7, product number: 553130) and then seeded onto glass coverslips and cultured for 24 hours prior to fixation and immunohistochemistry. All experiments were repeated a minimum of 3 independent times.  4.2.2 In vitro scratch injury model  The in vitro scratch injury model was performed according to the protocol described previously [90]. Briefly, VICs were seeded on glass coverslips that had been placed in 6well culture dishes, and grown to confluence (approximately 11 days). A sterile P200 pipette tip was used to make a linear wound across the confluent monolayers. Cells were subsequently fixed at the indicated timepoints with 3.7% formaldehyde.  4.2.3 Immunohistochemistry  Fixed cells were permeabilized with 0.1% triton X-100 for 20 minutes, blocked for 30 minutes with 1% BSA in PBS, and incubated overnight at 4°C with the versican (US Biologicals, product number: L1350) or CD44 (Santa Cruz Biotechnology, product number: sc-9960) primary antibodies at a concentration of 1:100 in 1% BSA. Following primary antibodies, cells were incubated with anti-mouse Alexa-fluor488 conjugated secondary antibody (Invitrogen) at a concentration of 1:200 in 1% BSA for 1 hour at room temperature in the dark. Cells were then stained for 20 minutes with phalloidin  64  conjugated to Alexa-fluor594 (Invitrogen) to visualize f-actin and coverslipped with VectaShield mounting medium containing DAPI (Vector Labs). Cells were imaged using a Leica AOBS SP2 confocal microscope as our lab has previously described [91,92,149].  4.2.4 Collagen gel contraction assay  Twelve-well culture dishes were coated with 1% BSA and incubated for 1 hour at 37°C to create a non-stick surface that prevents gels from attaching to the dishes. Cells were trypsinized and incubated at 4ºC in the presence or absence of CD44 blocking antibody (20µg/mL) and then seeded into a 0.5 mg/mL type I collagen solution (BD Biosciences, product number: 354236) in growth media at a concentration of 2.5x104 cells/mL. The collagen/cell suspension was then vortexed, and 1 mL per well was added to the BSAcoated dishes and the solution was allowed to polymerize for 45 minutes at 37°C. Fresh growth media was then added to the solidified collagen gels and plates were returned to the incubator. Collagen gel contraction was monitored over a period of 24 hours and the surface area of contracted gels was measured using Image-Pro Plus software (Media Cybernetics, Bethesda, USA). The collagen gels were then fixed and immunostained as described above.  65  4.2.5 Statistics  Results are represented as the mean ± standard deviation. Significant differences in treatment groups were determined using the unpaired Student’s t-test. For all analyses, p<0.05 was considered statistically significant.  4.3  Results  4.3.1 Phenotypic analysis of valvular interstitial cells  A panel of antibodies to cytoskeletal elements was used to phenotype the VICs used in this study by immunohistochemistry. The valve cells demonstrated positive staining for smooth muscle α-actin, vimentin and desmin, but were negative for smooth muscle myosin heavy chain (Figure 18), suggesting these cells display a myofibroblast-like phenotype in culture.  4.3.2 Versican immunostaining in valvular interstitial cells  Immunohistochemistry demonstrated that cultured VICs produce an abundance of versican. In subconfluent cultures of VICs, versican was clearly observed intracellularly in the perinuclear region, as well as along the cell membrane and was deposited in the ECM (Figure 19A, B). In contrast, confluent monolayers of VICs displayed little detectable intracellular versican. In these cultures, versican was localized to the 66  extracellular space between cells where it was organized into fibrils that aligned in parallel to the f-actin stress fibres (Figure 19C, D). This staining pattern suggests that versican is involved in VIC proliferation and migration and is downregulated in quiescent (confluent) cells. A negative control experiment, in which no primary antibody was added, demonstrated the specificity of the versican antibody (data not shown).  Wounding of the confluent monolayers led to the migration of elongated VICs moving outward from the wound edge as has previously been described [137,150]. There was a loss of versican reactivity at the wound edge 4 hours after injury, but migrating VICs clearly showed cytoplasmic staining for versican in the perinuclear region (Figure 20A). Twenty-four hours after wounding, the migrating VICs showed increased versican expression along the cell membrane and in the ECM (Figure 20B). The newly synthesized versican was deposited at the trailing edge of migrating cells, suggesting the cells leave behind a trail of versican as they move into the wound. Forty-eight hours after injury, as cells begin to close the wound, versican was predominantly detected in the ECM where it was organized into small thin fibrils (Figure 20C). These versican fibrils were not as dense as those seen in confluent cultures of VICs, nor did they display the same degree of organization.  4.3.3 CD44 immunostaining in valvular interstitial cells  I next studied the expression pattern of CD44, a membrane receptor that binds versican and is involved in cell migration [22,151]. In subconfluent cultures of VICs, CD44 was  67  localized exclusively to the cell membrane, and expression was often stronger on one side of the cell as compared to the other in a polarized fashion (Figure 21A, B). Interestingly, little immunostaining for CD44 was observed in confluent cultures of VICs (Figure 21C, D), suggesting this protein, like versican, is involved in the proliferation and migration of VICs, but is downregulated in quiescent cells.  Early after injury (at 4 hours) the cells had started migrating into the wound, yet the CD44 expression remained scarcely detectable, similar to what was seen in the confluent monolayers (Figure 22A, B). Twenty-four hours after wounding, however, CD44 was detected intracellularly and along the cell membrane in migrating cells (Figure 22C, D). Forty-eight hours after wounding, the CD44 immunoreactivity was increased and was clearly seen along the cell membrane, predominantly localized to the trailing edge of migrating cells (Figure 22E, F).  4.3.4 Blocking CD44 disrupts pericellular matrix organization and stress fibre formation in valvular interstitial cells  To examine the relationship between versican and CD44 in VICs, I used a blocking antibody to CD44 and observed any changes in cell phenotype. In the absence of CD44 blocking antibody, immunohistochemistry demonstrated that VICs formed a highly organized versican pericellular matrix (Figure 23A) and were characterized by prominent intracellular stress fibres (Figure 23B). In contrast, in the presence of CD44 blocking antibody, the pericellular versican organization was clearly disrupted and versican tended to clump as deposits under the cells (Figure 23C). Likewise, the actin cytoskeleton 68  appeared disordered with little stress fibre formation following treatment with CD44 blocking antibody (Figure 23D), highlighting a potential role for CD44 as a link between the actin cytoskeleton and versican in the ECM.  4.3.5 Blocking CD44 inhibits valvular interstitial cell-mediated contraction of type I collagen gels  To investigate the functional effect of blocking CD44, I first performed a scratch wound assay in the presence or absence of CD44 neutralizing antibody. Interestingly, I did not detect any difference in the wound closure rate seen in cells treated with CD44 antibody when compared to control cells (p>0.05, data not shown). As CD44 blocking disrupted stress fibre formation in VICs, I next performed a collagen gel contraction assay, a commonly employed in vitro model which can be used to measure the contractile properties of a population of cells. Valve interstitial cells were seeded into type I collagen gels in the presence or absence of CD44 blocking antibody, and gel contraction was followed over a period of 24 hours by measuring the surface area of contracted gels. Representative images and the quantified surface area of contracted gels are shown in Figure 24. Blocking CD44 significantly inhibited VIC-mediated contraction of the type I collagen gels (35.7 ± 0.7% vs 23.3 ± 1.4% of initial gel area, p<0.01).  Immunohistochemistry on VICs cultured in collagen gels showed that in the absence of CD44 blocking antibody, VICs appeared elongated, interconnected and had clearly visible stress fibres (Figure 25A-D). These cells also displayed strong immunoreactivity for versican in the pericellular environment and in the ECM. In contrast, blocking CD44 69  was associated with a decrease in extracellular versican as detected by immunostaining, as well as a decrease in stress fibre formation (Figure 25E-H). This was apparent in high power magnification images (Figure 25I, J), where versican appeared as clumps that deposited at the cell membrane in the CD44 antibody treated cells, with very little versican being secreted into the ECM.  4.4  Discussion  In this study, I have made the first efforts to characterize the interactions between VICs and versican during injury and repair processes. By studying cells in three conditions, namely subconfluent, confluent, and wounded cultures, I was able to gain insight into the relationship between versican and VICs in quiescent and activated states. In subconfluent cultures of actively proliferating VICs, versican was observed intracellularly, along the cell membrane, and was deposited into the ECM at the trailing edge of migrating cells. Once cells reached confluence, versican was no longer detected intracellularly but rather had been organized into fibrils present at the cell membrane that filled the space between adjacent cells. This staining pattern suggests versican is involved in VIC proliferation and migration, and, as cells reach confluence and become quiescent, they stop producing de novo versican. In wounded cultures, versican was first detected intracellularly during the initiation of repair as cells undergo the transition from a quiescent to an activated state. As time progressed, the expression of versican increased and was deposited into the ECM at the trailing edge of migrating cells. Collectively, this data suggests versican is  70  expressed and secreted by activated VICs where it forms part of the provisional wound repair ECM.  These findings are consistent with reports in other cell and tissue types that suggest versican promotes cell migration and proliferation. Studies have demonstrated that a high level of versican is associated with the metastatic spread of prostate and breast cancers [38], and also promotes neointimal formation in atherosclerotic arteries [57,58]. Versican has been shown to form a pericellular coat with hyaluronan that supports the migration and proliferation of prostate cells and vascular smooth muscle cells [52-54]. The importance of this pericellular matrix to cell movement may be best exemplified during early heart development, when disruption of the versican gene is associated with a failure of endocardial cushion cells to migrate, and with a failure of the heart to develop [152]. At present, the mechanism by which versican alters cell behaviour remains poorly understood, however, the structure of this proteoglycan lends itself to a number of binding interactions that may be involved. Versican has a high affinity for water [35], and it has been suggested that expression of versican following injury promotes the formation of a hydrated ECM that better supports migration and proliferation. Versican has also been shown to bind and sequester potent mitogenic growth factors and chemokines, thereby potentially regulating their activity [44]. Further, it has been reported that versican itself directly stimulates growth factor receptors to activate MAPK signaling, likely through two EGF repeats in its C-terminal globular domain [153]. The finding that versican is secreted at the trailing edge of migrating cells suggests it may stimulate these growth factor receptors in a polarized fashion, possibly imparting a degree of  71  directionality to migrating cells. Further experiments will be needed to explore this possibility.  Very little is known about the relationship between VICs and the ECM during injury and repair processes. A recent report suggested that VICs secrete fibronectin and form fibrillar adhesions in response to injury [150]. Interestingly, it was reported in the study that VICs secrete fibronectin at the leading edge of migrating cells, whereas my data suggests versican is deposited at the trailing edge in migrating cells. Versican and fibronectin have been reported to interact with each other in fibroblasts and endothelial cells [43,154]. In endothelial cells, versican was shown to upregulate the expression of fibronectin and these two molecules formed a complex with VEGF that enhanced angiogenesis-associated activities [154]. As both versican and fibronectin appear to form part of the provisional wound repair ECM in VICs, a better understanding of how these molecules interact with each other to regulate valve cell phenotype and wound repair should be an important focus of future studies.  CD44 is a cell-surface glycoprotein involved in cell adhesion and migration which has been shown to bind versican [22], and is perhaps best known as a receptor for hyaluronic acid [23]. Immunostaining showed that VICs express CD44 at the cell membrane in both sub-confluent cultures and following wounding, when the cells are in an activated state. In contrast, I did not detect any CD44 expression in confluent cultures of quiescent VICs. The expression pattern for CD44 mirrored that of versican in the wounded cultures, where CD44 was upregulated after injury and localized to the trailing edge of migrating  72  cells. Interestingly, I did not observe any difference in the rate of wound closure in VICs treated with a CD44 blocking antibody. There are conflicting reports in the literature about the role of CD44 in cell migration, and a recent report showed that CD44 knockout fibroblasts actually had a higher rate of migration, but the migration was directionless and disorganized in these cells and resulted in less efficient wound closure [155]. The polarized expression pattern seen for CD44 in VICs supports the idea that CD44 may influence the direction of migration. It seems plausible to suggest that CD44 expression results in a slower rate of migration, but promotes a more coordinated and efficient wound closure response.  The similar localization of CD44 and versican observed in activated VICs suggests CD44 may be involved in organizing the versican-rich provisional matrix secreted by these cells. Consistent with this idea, cellular expression of CD44 has been shown to be required for the formation of a hyaluronan/versican pericellular sheath during migration of prostate cancer cells [54]. The cytoplasmic tail of CD44 is anchored to cytoskeletal elements via ankyrin [156], and therefore may provide a link between the actin cytoskeleton and versican in the pericellular matrix, and could have important implications to VIC function. Indeed, the current study has demonstrated that blocking CD44 inhibited the organization of versican in the pericellular matrix of VICs and was associated with a decreased ability of VICs to form stress fibres. Functionally, the change in cell structure induced by blocking CD44 was associated with a decreased contractile ability of VICs when seeded into type I collagen gels. These results support findings from studies in chronic inflammatory diseases where CD44 has been suggested to play a role  73  in tissue remodeling and fibrosis [155], and suggest valve myofibroblasts require CD44 to remodel extracellular matrices.  In summary, I have shown that activated VICs secrete versican into the ECM following wounding where it forms part of the provisional wound repair ECM. The immunohistochemistry results also suggest valve cells upregulate the expression of CD44 following injury, a membrane receptor that binds versican. The similar localization of versican and CD44 at the trailing edge of migrating valve cells suggests this receptor is involved in organizing the provisional versican-rich ECM. Blocking antibodies to CD44 confirmed this idea, highlighting CD44 as an important link between the actin cytoskeleton and the ECM. In functional studies, these changes were associated with decreased contraction of collagen gels by VICs treated with CD44 blocking antibody. As VICs are the predominant cell type found in heart valves, a better understanding of how VICs respond to injury will help us to gain insight into valvular wound repair and the pathological complications that arise from abnormalities in this process.  74  Figure 18. Cultured valvular interstitial cells display a myofibroblast-like phenotype. Confocal images of VICs immunostained for (A) smooth muscle α-actin, (B) desmin, (C) vimentin and (D) smooth muscle myosin heavy chain. These cells had positive immunostaining for smooth muscle α-actin, desmin and vimentin (green) but were negative for smooth muscle myosin heavy chain. (Scale bar = 47.62 µm)  75  Figure 19. Versican expression in cardiac valvular interstitial cells. Confocal images of subconfluent VICs immunostained for (A) versican and (B) Alexafluor594-labeled phalloidin (to identify f-actin stress fibres). Versican was detected in the cytoplasm (arrow), along the cell membrane and in cell protrusions (arrowhead), and was deposited in the ECM (empty arrow). C, In confluent cultures, versican was present in the extracellular space between cells with no apparent intracellular staining, and was organized into fibrils (arrows) that aligned in parallel with the f-actin stress fibres (D). (Scale bar = 47.62 µm)  76  Figure 20. Versican expression during injury and repair in cardiac valvular interstitial cells. Confocal images of cells at the wound edge immunostained to detect versican at 4 hours (A), 24 hours (B), and 48 hours (C) after wounding. At 4 hours (A), cells had started migrating into the wound (w) and synthesized de novo versican that was present in the cytoplasm in the perinuclear region (arrows). Twenty-four hours after wounding (B), versican was apparent along the cell membrane of migrating cells and was beginning to be deposited into the ECM (arrows). Forty-eight hours after wounding (C), versican appeared predominantly in the ECM where it was organized into fibrils (arrows). Collectively, these results suggest versican is synthesized and secreted into the ECM following injury to VICs. (Scale bar = 37.50 µm)  77  Figure 21. Expression of CD44 in cardiac valvular interstitial cells. Confocal images of subconfluent VICs immunostained for (A) CD44 and (B) f-actin showing immunostaining for CD44 predominantly along the cell membrane (arrows) in non-confluent cultures. C, In confluent cultures of valve cells, expression of CD44 was minimal with no clearly observable pattern. Actin staining is shown in (D). (Scale bar = 37.50 µm)  78  Figure 22. Expression of CD44 during injury and repair in cardiac valvular interstitial cells. Confocal images of cells at the wound edge immunostained to detect CD44 (A, C, E) and f-actin (B, D, F) at 4 hours (A, B), 24 hours (C, D), and 48 hours (E, F) after wounding. At 4 hours (A, B), cells had started migrating into the wound (w) and demonstrated very little immunostaining for CD44. Twenty-four hours after wounding (C, D), cells upregulated the expression of CD44, which localized to the cell membrane in migrating cells (arrows). Forty-eight hours after wounding (E, F), migrating cells demonstrated the highest expression of CD44 (arrows), localized predominantly along the cell membrane in trailing cell processes. (Scale bar = 75.00 µm)  79  Figure 23. Blocking CD44 inhibits pericellular matrix organization and stress fibre formation in valvular interstitial cells. Confocal images of cells incubated in the absence (A, B) or presence (C, D) of a CD44 blocking antibody and immunostained for versican (A, C) and f-actin (B, D). In the absence of a CD44 blocking antibody, the cells displayed a versican pericellular matrix that was highly organized and deposited into the ECM as fibrils (A, arrows). These cells clearly demonstrated the formation of well-organized f-actin stress fibres (B, arrows). In the presence of a CD44 blocking antibody, there was a lack of pericellular matrix organization and the versican tended to clump as deposits under the cell (C, arrows). These cells displayed a poorly organized actin cytoskeleton with little stress fibre formation (D, arrows). (Scale bar = 23.81 µm)  80  Figure 24. Blocking CD44 inhibits valvular interstitial cell-mediated contraction of type I collagen gels. Valve interstitial cells were seeded into type I collagen gels in the presence or absence of CD44 blocking antibody and gel contraction was followed for a period of 24 hours. Representative images of contracted gels showing VIC-mediated collagen contraction is inhibited in the presence of CD44 blocking antibody. Quantification of the surface areas of contracted gels confirmed collagen gel contraction was significantly inhibited by blocking CD44 (35.7 ± 0.7% vs 23.3 ± 1.4% of initial gel area, p<0.01).  81  Figure 25. Blocking CD44 inhibits stress fibre formation and versican deposition into the ECM in valvular interstitial cells cultured in collagen gels. Immunohistochemistry was performed on VICs cultured in collagen gels in the absence (A-D) or presence (E-H) of CD44 blocking antibody. Cells were stained for nuclei (A, E), f-actin (B, F), and versican (C, G). Overlay images (D, H) show the cells to be elongated and interconnected in the absence of CD44 blocking antibody, with clearly visible f-actin stress fibres and versican deposition around the cell membrane and in the ECM (D, arrows). In contrast, cells treated with CD44 blocking antibody lacked clear stress fibres, did not appear interconnected, and had less versican present in the ECM (H, arrows). (I, J) High power confocal images highlight the absence of extracellular versican in cells treated with CD44 blocking antibody as compared to control cells. In the CD44 antibody treated cells, versican tended to clump around the cell membrane (J, arrows) instead of being secreted into the ECM as seen in control cells (I, arrows). The antibody treated cells also lacked clear stress fibres as compared to control cells. (Scale bars = 47.62 µm in A, 23.81 µm in I)  82  CHAPTER 5. VERSICAN EXPRESSION INDUCES A MYOFIBROBLAST-LIKE PHENOTYPE IN CULTURED FIBROBLASTS  5.1  Introduction  Versican is a large, multidomain, chondroitin sulphate proteoglycan that is found in the ECM of many tissues in the body. Versican is important developmentally, as a knockout of this gene results in embryonic lethality due to a failure of the heart and blood vessels to develop [56]. Versican is also upregulated after injury where it forms part of the provisional wound repair matrix [27,104,141-144], and accumulates in a number of chronic inflammatory disorders including atherosclerosis [57,58], pulmonary fibrosis [157] and cancers [158,159]. Despite the importance of versican to development and disease, the function of this proteoglycan and its role in wound repair remain poorly understood.  In this study, I examined the effect of a versican-rich matrix on cell phenotype by cloning the V1 isoform of human versican and setting up an expression model in mouse fibroblasts. My data suggests versican increases the expression of N-cadherin, integrin β1, and smooth muscle α-actin, and promotes fibroblast contraction of a collagen lattice. These changes appear to be mediated, at least in part, by increased activation of TGF-β signaling in the versican-transfected cells, as measured by the phosphorylation and nuclear localization of SMAD2. Collectively, this data suggests versican expression alters  83  the phenotype of fibroblasts and, as such, may play a role in regulating fibroblastmediated injury and repair events.  5.2  Materials and methods  5.2.1 Cell culture  Mouse embryonic fibroblasts (Clontech, product number: 630914) were cultured in DMEM containing 10% FBS and 100 U/mL penicillin/streptomycin. Cells were maintained in a humidified incubator at 37°C with 5% CO2 and used for experiments between passages 6-12. All experiments were repeated a minimum of 3 independent times.  5.2.2 Creation of the versican V1 cell line  The full length human versican cDNA for the V1 isoform (accession number: X15998.1) was synthesized by Epoch Biosciences (Houston, USA) with unique restriction sites for MLUI and NOTI created at the N- and C-termini, respectively. This cDNA was then subcloned into the pTRE2hyg-6xHN vector (Clontech, product number: 631053) using the abovementioned restriction sites to generate the versican expression construct. Subcloning was confirmed by digesting this construct with EcoRV, and the full length versican V1 cDNA was sequenced for accuracy. The versican construct (or the empty vector control) was then stably transfected using Fugene 6 (Roche) according to the 84  manufacturer’s instructions, and clones were selected by adding 500 µg/mL hygromycin to the growth media. Western blot with a monoclonal antibody against the V0/V1 isoforms of human versican (US Biological, product number: L1350) was used to confirm gene expression, and clones with high expression of versican were selected for experimental purposes. Stable cell lines were maintained in growth media supplemented with 100 µg/mL G418 and 100 µg/mL hygromycin, which was removed from the growth media for experiments.  5.2.3 Western blotting  Cell lysates were collected in lysis buffer (10mM HEPES (pH 7.4), 50 mM Na4P2O7, 50 mM NaF, 50 mM NaCl, 5 mM EDTA, 5 mM EGTA, 2 mM Na3VO4, and 1 mM phenylmethylsulfonyl fluoride, with 0.1% Triton X-100 and 10 µg/mL leupeptin) followed by centrifugation at high speed (14000 X g at 4ºC for 10 minutes) to recover proteins. The protein concentration of samples was measured by a Bradford protein assay. Equal amounts of protein from each sample were separated with SDS-PAGE (10% polyacrylamide) and transferred to a nitrocellulose membrane. Membranes were blocked for 1 hour in 5% milk/TBST and incubated overnight at 4°C with primary antibody in 2.5% milk/TBST. Following 3 washes in TBST, secondary antibody (Santa Cruz biotechnology) at a concentration of 1:2000 in 2.5% milk/TBST was added for 1 hour at room  temperature.  Antibody  binding  was  visualized  with  the  enhanced  chemiluminescence detection system (Thermo Fischer Scientific). Images were captured with a Chemigenius2 system (Syngene, Frederick, USA) and band intensities were  85  calculated with GeneTools software (Syngene). Antibodies used were as follows: Ncadherin (Abcam, product number 18203), integrin β1 (Cell Signaling Technologies, product number 4706), pFAK397 (BD Biosciences, product number 611723), smooth muscle α-actin (Santa Cruz Biotech, product number sc-32251), and pSMAD2 (Cell Signaling Technologies, product number 3108).  For detecting versican by Western blot, the procedure was modified slightly by using non-reducing sample buffer and a 5% polyacrylamide separating gel, followed by incubating the membrane with anti-versican (US Biological, product number: L1350) at a concentration of 1:1000 as described above.  5.2.4 Immunohistochemistry and confocal microscopy  Cells were fixed for 20 minutes in 3.7% formaldehyde, washed with PBS, permeabilized with 0.1% triton X-100 for 20 minutes, blocked for 30 minutes with 1% BSA in PBS and incubated overnight at 4°C with the indicated primary antibody at a concentration of 1:100 in 1% BSA. Following incubation with primary antibody, cells were washed with PBS and incubated with anti-mouse Alexa-fluor488 or –fluor594 conjugated secondary antibody (Invitrogen) at a concentration of 1:200 in 1% BSA for 1 hour at room temperature in the dark. In certain experiments, cells washed and then stained for 20 minutes with Alexa-fluor594 phalloidin (Invitrogen) to visualize f-actin. Cells were washed a final time in PBS and coverslipped with VectaSheild mounting medium  86  containing DAPI (Vector Labs). Images were captured using a Leica AOBS SP2 confocal microscope as our lab has previously described [91,92].  5.2.5 Cell proliferation, migration and adhesion assays  Cell proliferation was measured by MTS assay (Promega) according to the manufacturer’s instructions. Migration was measured using the in vitro scratch wound assay, as previously described [90]. Briefly, cells were grown to confluence and monolayers were scratched using a dental device to create a cell-free area where migration could be measured. Adhesion was measured by trypsinizing and seeding cells into culture dishes at a concentration of 2X105 cells/mL. Cells were allowed to adhere for 30 minutes prior to taking photographs. For migration and adhesion images, cells were photographed with a Nikon 50i series upright microscope equipped with a digital camera.  5.2.6 Collagen gel contraction assay  Twelve-well culture dishes were coated with 1% BSA and incubated for 1 hour at 37°C to create a non-stick surface that prevents gels from attaching to the dishes. Cells were trypsinized, counted and seeded into a 0.5 mg/mL type I collagen solution (BD Biosciences, product number: 354236) in growth media at a concentration of 1X105 cells/mL. The collagen/cell suspension was then vortexed, and 1 mL per well was added to the BSA-coated dishes and the solution was allowed to polymerize for 45 minutes at 37°C. Fresh growth media was then added to the solidified collagen gels and plates were  87  returned to the incubator. Collagen gel contraction was monitored over a period of 7 days and the surface area of contracted gels was measured using Image-Pro Plus software (Media Cybernetics, Bethesda, USA). The collagen gels were fixed and immunostained as described above.  5.2.7 qPCR analysis  RNA extraction, cDNA synthesis and qPCR analysis were performed as our lab has previously described [69]. Predesigned primers to smooth muscle α-actin and β-actin were purchased from Applied Biosystems (assay numbers and Mm00725412_s1 and Mm00607939_s1, respectively).  5.2.8 Statistical analysis  Results are represented as the mean ± standard deviation. Significant differences in treatment groups were determined using the unpaired Student’s t-test. For all analyses, p<0.05 was considered statistically significant.  88  5.3  Results  5.3.1 Expression and secretion of human versican in mouse fibroblasts  Fibroblasts that had been stably transfected with the human versican V1 construct (or empty vector control) were seeded into 6-well dishes and grown for 48 hours prior to harvesting cell lysates and conditioned media for Western blot. Recombinant versican was detected in the cell lysate and conditioned media of versican-transfected cells, suggesting the recombinant protein is both synthesized and secreted (Figure 26A). As versican achieves much of its function through the GAG chains attached to the central domains of its core protein [46], I next determined if the recombinant versican was synthesized with GAG chains by treating cell lysates with chondroitinase ABC prior to Western blotting. In the absence of chondroitinase treatment, versican appeared as a large smear, representing molecules of different molecular weights with GAG chains attached. After chondroitinase treatment, versican appeared as a tight and compact band running at the size of the smallest versican molecules from the smear, confirming the presence of GAG chains on the recombinant protein (Figure 26B). Immunohistochemistry showed versican is deposited into the ECM as cells move across the plate (Figure 26C). Collectively, this data suggests the recombinant versican is synthesized, secreted, and deposited into the ECM in the versican-transfected fibroblasts.  89  5.3.2 Versican alters the phenotype of cultured fibroblasts  A recent report has suggested that versican induced the formation of epithelial-like islands in cultured fibroblasts that were characterized by decreased N-cadherin expression. Interestingly, I did not detect a noticeable difference in the morphology of versican-transfected cells at either sub-confluent or confluent cell densities (Figure 27A). Further, I actually observed N-cadherin expression to be increased in the versican expressing  fibroblasts,  as  shown  by  Western  blot  (Figure  27B)  and  immunohistochemistry (Figure 27C).  Although no major change in morphology was observed, the versican-expressing fibroblasts were found to function much differently than control cells. The versican fibroblasts proliferated significantly faster than control cells (Figure 28A, relative rate = 1.44 ± 0.07 of control cells, p<0.05). In contrast, the versican fibroblasts migrated slower than control cells (Figure 28B). These cells also showed increased adhesion to culture dishes (Figure 28C). Concomitant with increased cell adhesion, the versican fibroblasts displayed increased expression of integrin β1 as well as phosphorylation of focal adhesion kinase (FAK) (Figure 28D).  5.3.3 Versican induces a myofibroblast-like phenotype in cultured fibroblasts I next investigated whether versican altered smooth muscle α-actin expression in cultured fibroblasts. Versican significantly increased smooth muscle actin mRNA expression  90  (Figure 29A, 1.38 ± 0.15 fold increase, p<0.05) and protein expression, as shown by Western blot (Figure 29B, 1.47 ± 0.10 fold increase, p<0.05) and immunohistochemistry (Figure 29C). In addition to the increased smooth muscle α-actin expression, the versican transfected fibroblasts showed a 2.93 ± 0.22 fold increase in collagen III mRNA levels, compared to control cells (Figure 29D, p<0.05).  To investigate whether the increased contractile protein expression seen in veriscan expressing fibroblasts translated functionally into increased contractile properties, I employed a collagen gel contraction assay. Versican-transfected fibroblasts (or empty vector control cells) were seeded into 0.5 mg/mL type I collagen gels, and contraction was monitored over the period of 7 days. Representative images and the quantified surface area of contracted gels are shown in Figure 30A. Versican expression significantly increased fibroblast-mediated contraction of the type I collagen gels as compared to control cells (14.9 ± 0.7% vs 24.8 ± 1.8% of initial gel area, p<0.05). Immunohistochemistry was performed on versican and control fibroblasts cultured in the collagen gels and a clear change in cell phenotype was observed in the versican expressing cells. These cells exhibited an elongated morphology, were interconnected and had clearly visible f-actin stress fibres, while the control cells did not display prominent cell protrusions or stress fibres (Figure 30B). Versican was found to form a pericellular coat around the cell membrane in the versican-transfected cells that was clearly visible in Z-stack confocal images as a ring around cell protrusions (Figure 30C, suggesting it may be well localized to influence the bioavailability or activity of growth factors or cytokines present in the pericellular environment.  91  5.3.4 Versican mediates increased TGF-β signaling in cultured fibroblasts A recent report has suggested versican localizes TGF-β and increases its signaling in chondrocytes during joint formation [160]. Therefore, I hypothesized that versican may also alter the TGF-β signaling axis in cultured fibroblasts. Immunohistochemistry demonstrated increased expression and incorporation of smooth muscle α-actin in versican-transfected fibroblasts that were cultured in type I collagen gels (Figure 31A). These cells also displayed nuclear accumulation of phosphorylated SMAD2, a direct target of TGF-β signaling, when cultured in collagen gels (Figure 31B). To confirm activation of TGF-β signaling in versican expressing fibroblasts, Western blotting was performed on cultured fibroblasts and representative blots are shown in Figure 31C. The versican expressing fibroblasts displayed increased SMAD2 phosphorylation as compared to control cells. The increased nuclear accumulation of phosphorylated SMAD2 was again observed in 2D cultures of versican-transfected fibroblasts, confirming activation of this signaling pathway (Figure 31D).  5.4  Discussion  In this study, I have cloned the full length V1 isoform of human versican and expressed the recombinant protein in mouse fibroblasts. Using this system, I found the versicantransfected fibroblasts to have an altered cell phenotype when compared with the empty vector control cells. Functionally, the versican expressing fibroblasts proliferated faster  92  and displayed increased cell adhesion, but migrated slower than control cells. These changes in cell function were associated with increased N-cadherin and integrin β1 expression, along with increased FAK phosphorylation. In addition, the versican expressing fibroblasts displayed increased expression of smooth muscle α-actin, a marker of myofibroblast differentiation [8]. Consistent with this, the versican fibroblasts had increased synthetic activity, as measure by collagen III expression, as well as an increased capacity to contract a collagen lattice. These changes appear to be mediated, at least in part, by versican increasing TGF-β signaling through SMAD2. Collectively, this data suggests a role for versican in promoting myofibroblast differentiation in cultured fibroblasts.  Growing evidence suggests versican is associated with normal and aberrant injury and repair events, but the function of this proteoglycan remains poorly understood. Versican has consistently been shown to be expressed in injured and healing tissues, including after skin injury by incision [143] or burn [141], following myocardial infarction [144], in stented arteries [27,104] and in cancers [158,159], among others. Other groups have reported the accumulation of a versican- and hyaluronan-rich ECM around actively proliferating and migrating mesenchymal cells [52,54], and a knockdown of versican in smooth muscle cells inhibited these processes [53]. In these settings, versican expression appears to be regulated by growth factor activation of receptor tyrosine kinase signaling. Versican is dramatically upregulated by PDGF [161,162] and TGF-β [163,164], and our lab has previously shown a role for Wnt signaling through β-catenin in regulating versican expression [69]. My data add to this story by suggesting versican promotes the  93  formation of a contractile, myofibroblast-like phenotype in cultured fibroblasts with increased expression of smooth muscle α-actin. In addition, versican was shown to alter the proliferation, migration and adhesion of cultured fibroblasts. As these cell-mediated processes are all critically involved in a wound healing response, this data suggests versican may significantly influence the outcomes of repair. When taken together, these studies suggest locally released growth factors and cytokines that are released at sites of injury promote the formation of a versican-rich ECM that regulates the phenotype of the cells embedded within it.  In addition to the increased smooth muscle α-actin expression, my data shows versican increases the levels of integrin β1 and phosphorylated FAK in cultured fibroblasts. This finding is consistent with previous work in which a ‘mini’ versican construct or expression of its c-terminal globular domain increased integrin β1 levels in an astrocytoma cell line [45,55]. In an elegant series of papers in the setting of fibrotic lung disorders, Bensaduon et al demonstrated that collagen synthesis takes place in a versicanrich provisional matrix during early repair events, and suggested versican initiates the process of matrix remodeling following lung injury [157,165]. My data are in support of this idea, as I observed increased collagen III expression in the versican-transfected fibroblasts as well as increased contractile properties in these cells. The upregulated expression of integrin signaling seen in the versican fibroblasts may provide a mechanism to facilitate cellular interaction with the early collagen matrix. How versican increases integrin expression is not clear from the present study, although the localization of versican to the pericellular space in three-dimensional gels suggests it is well situated to  94  modify a number of biological processes that are occurring at the cell membrane and may be involved. For example, versican has been shown to stimulate MAPK signaling by directly activating the EGFR through two EGF-like repeats in its C-terminal domain [47,153]. Versican has also been shown to bind growth factors and may regulate their activity [44]. In light of this, a recent paper has shown that versican is necessary to localize TGF-β in the pericellular matrix and thereby regulates its signaling during chondrogenesis [160]. My data are consistent with this finding, as I observed increased phosphorylation and nuclear accumulation of SMAD2, a downstream signaling target of TGF-β, in the versican expressing fibroblasts. As TGF-β is the most potent known inducer of myofibroblast differentiation, it is likely that activation of its signaling is responsible for the myofibroblast-like phenotype observed in the versican-transfected fibroblasts. However, future experiments that block specific aspects of this pathway will be needed to confirm this.  Interestingly, a recent report in which versican V1 was expressed in NIH3T3 fibroblasts showed expression of versican promoted an epithelial-like phenotype in these cells [166]. In the study, the versican expressing fibroblasts were found to aggregate and form ‘epithelial islands’ that were distinctly observable by light microscopy. Further, the authors found versican induced a cadherin switch in fibroblasts that was characterized by a loss of N-cadherin expression but an increase in E-cadherin. My data would appear to contradict these findings. I did not observe the formation of epithelial-like aggregates in my model of versican overexpression, and actually observed increased N-cadherin expression when versican was overexpressed. Versican is a complex molecule which  95  forms numerous interactions with other proteins in the matrix and it is difficult to determine the cause of these discrepancies. At the time of writing this dissertation, a report was published in which the pericellular accumulation of versican in ADAMTS5 knockout fibroblasts (versican protease) was found to promote myofibroblast differentiation [167], data which are in support of my findings. More work is clearly necessary to better understand the effects of versican expression on cell behavior. Further, the development of conditional models for versican expression will allow for the study of these phenomena in vivo.  In summary, I have shown that versican promotes the formation of a myofibroblast-like cell phenotype in cultured fibroblasts. These results lend support to the idea that versican is an important component of the provisional wound healing matrix that is expressed following injury. However, if versican also promotes myofibroblast differentiation and increased contractile properties in vivo, the relative amount of versican expressed at sites of chronic inflammation could promote excessive tissue remodeling and fibrosis.  96  Figure 26. Versican expression in murine fibroblasts. A, Western blot of recombinant versican expression in the cell lysate and conditioned medium of versican-transfected fibroblasts, suggesting the recombinant protein is synthesized and secreted. B, Cell lysates were digested with chondroitinase ABC prior to Western blotting to confirm the presence of GAG chains on the recombinant versican. In the absence of chondroitinase, versican appeared as a large smear representing molecules of different molecular weights; after chondroitinase treatment, versican appeared as a compact band at the size of the smallest versican molecules from the smear, suggesting the GAG chains were present and had been removed. C, Immunohistochemistry showed recombinant versican was deposited into the ECM in versican-transfected cells (green, arrows). (Scale bar = 47.00 µm)  97  Figure 27. Versican increases N-cadherin expression. A, Light microscopy shows no major change in cell morphology in versican-transfected fibroblasts at both sub-confluent and confluent densities. B, Representative Western blot showing increased expression of N-cadherin in versican-transfected cells. C, Confocal microscopy confirmed the increased N-cadherin expression in versican-transfected cells. (Scale bar = 12.00 µm)  98  Figure 28. Versican alters the function of fibroblasts. A, Versican increased cell proliferation 1.44 ± 0.07 fold over control cells, p<0.05. B, Versican expressing fibroblasts showed decreased cell migration, as measure by in vitro scrape wound assay. C, Cell adhesion was found to be increased in versican-transfected fibroblasts, measured at 30 minutes after seeding. D, Representative Western blot showing increased integrin β1 expression, as well as phosphorylation of FAK-397, in versican-transfected fibroblasts. (* denotes p<0.05)  99  Figure 29. Versican increases smooth muscle α-actin expression. A, Versican induced a 1.38 ± 0.15 fold increase in smooth muscle α-actin mRNA expression, p<0.05. B, Versican induced a 1.47 ± 0.10 fold increase in smooth muscle αactin protein expression, p<0.05. C, Immunohistochemistry confirmed smooth muscle αactin expression was increased in the versican-transfected cells. D, Versican induced a 2.93 ± 0.22 fold increase in collagen III mRNA expression, p<0.05. (Scale bar = 47.00 µm, * denotes p<0.05)  100  Figure 30. Versican increases fibroblast-mediated contraction of a collagen lattice. A, Representative images of contracted collagen gels are shown along with the quantified surface areas of contracted gels. Versican significantly increased the fibroblast-mediated contraction of collagen gels (14.9 ± 0.7% vs 24.8 ± 1.8% of initial gel area, p<0.05). B, Confocal imaging demonstrated the versican-transfected fibroblasts to be elongated, interconnected, and to have increased stress fibre formation in collagen gels (arrows, red in overlay). Versican was found to localize to the pericellular matrix surrounding elongated cells (arrowheads, green in overlay). C, A Z-stack image reveals versican 101  (green) forms a pericellular coat around cell protrusions in versican-transfected fibroblasts, suggesting it may be well-situated to influence biological events at the cell membrane. (Scale bars = 23.00 µm in B, 12.00 µm in C, * denotes p<0.05)  102  Figure 31. Versican increases TGF-β signaling in cultured fibroblasts. A, Confocal imaging of contracted gels demonstrated the versican-transfected fibroblasts to have increased expression and incorporation of smooth muscle α-actin into their stress fibres (arrows, yellow in overlay). B, The versican-transfected fibroblasts also displayed increased staining and nuclear localization of phosphorylated SMAD2 in contracted collagen gels (arrows, red in overlay). C, Representative Western blot shows increased phosphorylation of SMAD2 in cultures of versican-transfected fibroblasts. D, Confocal microscopy confirmed the increased SMAD2 phosphorylation and nuclear accumulation in cultures of versican-transfected fibroblasts (arrows). (Scale bars = 23.00 µm in A, 12.00 µm in B, D)  103  CHAPTER 6. THE INTRACELLULAR LOCALIZATION OF VERSICAN IN MESENCHYMAL CELLS  6.1  Introduction  Versican is a large, multidomain, chrondroitin sulphate proteoglycan that is found in the ECM of many tissues in the body. Versican is critical to normal development of the heart [56], but also accumulates in a number of cardiovascular diseases including atherosclerosis [58], post-angioplasty restenosis [27], cardiac allograft vasculopathy [57], myxomatous valve disease [146], and others. Versican was originally named due to its versatility; it is expressed by almost every cell type in the body, and exists as four different isoforms in humans that differ in their length and number of attached GAG side chains [33]. These isoforms (named V0-V3) arise due to differential splicing of the two central GAG-attachment exons, such that all isoforms share identical N- and C-terminal globular domains, but have central regions of variable length and in terms of the number of attached GAGs [147,148]. Despite the prominence of versican to cardiovascular development and disease, the function of this proteoglycan remains poorly understood. It is known that versican appears to achieve much of its functionality through binding interactions with other molecules in the ECM [46]. Versican binds hyaluronic acid at its N-terminus [36], and binds a number of other ECM components at its C-terminus, including fibrillin-1 [39], fibulin-1 and -2 [40,41], tenascin-R [42], type I collagen [43], and fibronectin [43]. Thus, versican is thought to play an important role in regulating the assembly and organization of the ECM. Through its interaction with hyaluronic acid,  104  versican forms high molecular weight aggregates that attract water and may provide hygroscopic properties to the ECM [35]. Pathologically, versican is thought of as proatherogenic because of its ability to bind and retain lipoproteins within the vessel wall [168-174]. Versican has also been known to promote smooth muscle cell migration and proliferation [52] and may possess pro-inflammatory properties [24]. All of the functions attributed to versican are related to its role in the ECM. In the current work, I present data that strongly suggests versican has intracellular functions in addition to its better known roles in the ECM.  6.2  Materials and methods  6.2.1 Reagents  Rat aortic smooth muscle cells were purchased from ATCC (clone A7r5, product number CRL-1444). Mouse embryonic fibroblasts were purchased from Clontech (product number 630914). Purified 3T3 mouse embryonic fibroblast nuclear extracts were from Active Motif (product number 36001). Versican antibodies were purchased from Affinity Bioreagents (product number PA1-1748A) and US Biologicals (product number L1350), and were used to detect versican in rat/mouse or human cells, respectively. The Cu/Zn SOD antibody was from Stressgen (product number SOD-100), nucleolin was from Abcam (product number ab22758), and RHAMM was from Abcam (product number ab108339). The synthetic peptide to neutralize the PA1-1748A versican antibody was from Affinity Bioreagents (product number PEP-235). Alexa-fluor conjugated secondary  105  antibodies were from Invitrogen. VectaShield mounting medium containing DAPI was from Vector Labs. Horseradish peroxidase-conjugated secondary antibodies for Western blotting were from Santa Cruz Biotechnology. The chemiluminescence detection system was from Thermo Fischer Scientific and the Chemigenius2 system was from Syngene. Pre-designed control and versican siRNA were purchased from Santa Cruz Biotechnology (product numbers 37007 and 41903, respectively) and Oligofectamine was from Invitrogen. All other chemicals were of analytical grade.  6.2.2 Cell culture  Cells  were  cultured  in  DMEM  containing  10%  FBS  and  100  U/mL  penicillin/streptomycin. Cells were maintained in a humidified incubator at 37°C with 5% CO2 and used for experiments between passages 4-10. All experiments were repeated a minimum of 3 independent times.  For certain experiments, valvular interstitial cells were used that had been isolated from non-calcified aortic valve leaflets (collected by the Cardiovascular Registry, St. Paul’s Hospital, Vancouver, Canada) by enzymatic digestion, as our lab has previously described [175-177]. Valve interstitial cells were cultured in MCDB 131 containing 15% FBS and maintained in a humidified incubator as described above. The use of these cells for experimental purposes was approved by the UBC/Providence Health Care Research Ethics Board.  106  6.2.3 Immunohistochemistry  Cells were seeded onto glass coverslips that had been placed in 6-well culture dishes, and were allowed to adhere overnight prior to fixation with 3.7% formaldehyde. After fixation, cells were washed 3X with PBS and then permeabilized with 0.1% triton X-100 for 20 minutes, blocked for 30 minutes with 1% BSA in PBS, and incubated overnight at 4°C with the indicated primary antibodies at a concentration of 1:100 in 1% BSA. Cells were then washed 3X in PBS and incubated with Alexa-fluor conjugated secondary antibodies at a concentration of 1:200 in 1% BSA for 1 hour at room temperature in the dark. Cells were washed again with PBS and coverslipped using VectaShield mounting medium containing DAPI. Images were captured using a Leica AOBS SP2 confocal microscope as our lab has previously described [91,92,149].  6.2.4 Western blotting  Cell lysates were collected in lysis buffer (10mM HEPES (pH 7.4), 50 mM Na4P2O7, 50 mM NaF, 50 mM NaCl, 5 mM EDTA, 5 mM EGTA, 2 mM Na3VO4, and 1 mM phenylmethylsulfonyl fluoride, with 0.1% Triton X-100 and 10 µg/mL leupeptin) followed by centrifugation at high speed (14000 X g at 4ºC for 10 minutes) to recover proteins. Proteins from each sample were separated with SDS-PAGE (10% polyacrylamide) and transferred to a nitrocellulose membrane. Membranes were blocked for 1 hour in 5% milk/TBST and incubated overnight in primary antibody at a concentration of 1:1000 in 2.5% milk/TBST at 4°C. Following 3 washes in TBST, secondary antibody at a concentration of 1:2000 in 2.5% milk/TBST was added for 1 107  hour at room temperature. Antibody binding was visualized with the enhanced chemiluminescence detection system. Images were captured with a Chemigenius2 system.  For detection of versican by Western blotting, the procedure was modified slightly by using non-reducing sample buffer and a 5% polyacrylamide separating gel, followed by incubation of the membrane with anti-versican at a concentration of 1:1000 as described above.  6.2.5 siRNA transfections  Cells were seeded into 24-well culture dishes and siRNA transfection was performed using Oligofectamine as per the manufacturer’s instructions. Control siRNA or versican siRNA were added to the cells for 72 hours prior to harvesting lysates for Western blot or performing experiments.  6.3  Results  6.3.1 Intracellular localization of versican  I have consistently observed an intracellular staining pattern for versican in cultured smooth muscle cells; thus, I sought to investigate this localization of versican in more detail. I first stained rat smooth muscle cells with the PA1-1748A versican antibody and 108  used confocal microscopy to create 3-dimensional images. Versican was detected throughout the cytosol and a strong signal for versican was present in cell nuclei (Figure 32A, arrow).  To determine the specificity of the PA1-1748A versican antibody, a synthetic peptide corresponding to residues 436-441 of versican, which reacts with the versican antibody, was added during immunostaining according to the manufacturer’s instructions. In the absence of neutralizing peptide, versican was detected in the nucleus and cytosol in a similar staining pattern to that observed in Figure 32A (Figure 32B, arrow). Addition of the synthetic peptide during incubation of the primary antibody competitively inhibited the versican antibody-protein binding interaction, which completely eliminated versican immunoreactivity and confirmed the versican antibody was specifically reactive with versican in my experiments (Figure 32C).  6.3.2 Versican localizes to the nucleolus in non-dividing vascular smooth muscle cells  High magnification confocal images of rat cell nuclei that were immunostained for versican highlighted the presence of versican in the nucleus, where it aggregated in areas that did not stain with DAPI, hinting of a nucleolar localization of versican (Figure 33AC). Western blotting for versican was performed on purified nuclear extracts and whole cell lysates from mouse embryonic fibroblasts to confirm the nuclear localization of versican (Figure 33D). There was a strong signal for the V1 isoform of versican in the nuclear extracts, but interestingly, the V0 isoform was not detected. In the whole cell 109  lysates, both the V0 and V1 isoforms of versican were detected, as would be expected. Cu/Zn SOD was used as a purity control for the nuclear extracts. To confirm a nucleolar localization of versican, multi-fluorescent immunostaining for both versican and nucleolin was performed. High magnification confocal images of the cell nuclei showed strong colocalization of versican and nucleolin in overlay images (Figure 34). In addition to the nucleolar staining pattern of versican, I observed a clear association of versican staining with distinct clefts and furrows in cell nuclei (data not shown).  6.3.3 Association of intracellular versican with mitosis  In human valvular interstitial cells that were undergoing mitosis, I observed a strong signal for versican in and around the chromosomes during late prophase and early prometaphase, when the nuclear membrane breaks down and individual chromosomes become visible (Figure 35). As cells proceeded through metaphase and anaphase, versican surrounded the chromosomes at the metaphase plate and then filled the space between the separating chromosomes at anaphase. Strong staining persisted through telophase, where versican appeared to aggregate in a polarized fashion at opposing ends of dividing chromosomes (Figure 35, arrows).  6.3.4 Versican localizes to polar ends of the mitotic spindle and is required for normal spindle organization  To investigate the relationship between intracellular versican and mitosis in further detail, multi-fluorescent immunostaining for versican and RHAMM was performed in valvular 110  interstitial cells. Versican was observed to localize at polar ends of the mitotic spindle during metaphase where it colocalized with RHAMM (Figure 36). I hypothesized that versican may play a role in the organization of the mitotic spindle. To test this, siRNA was used to knock down versican expression, and mitotic spindle organization was visualized by immunostaining for RHAMM (Figure 37). When versican was knocked down by siRNA, the mitotic spindles in these cells appeared disorganized and were characterized by the formation of multiple spindle poles during metaphase (Figure 37C).  6.4  Discussion  Versican is a highly interactive chondroitin sulfate proteoglycan that is found in the ECM of many tissues and is a major component of developing and mature atherosclerotic lesions and is present in other cardiovascular diseases [57,58]. Versican is implicated in several atherogenic events including smooth muscle cell growth and migration [52], retention of lipoproteins [35], stimulation of inflammation [24] and promotion of thrombogenesis [178]. In this study, I provide clear evidence of versican’s presence inside vascular cells, and thus suggest intracellular functions for this proteoglycan in addition to its better known roles in the ECM. My data indicates that versican localizes to both the nucleus and cytosol of proliferating vascular smooth muscle cells. In the nucleus, I demonstrated by Western blot that it is specifically the V1 isoform of versican that is present, and not the V0 isoform. I also demonstrated the localization of versican in nucleoli, and observed its presence in nuclear clefts and furrows. In dividing cells, I observed a strong nuclear signal for versican in and around the chromosomes where it  111  colocalized with RHAMM in a polarized fashion at opposite ends of the mitotic spindle. Knockdown of versican expression disrupted the organization of the mitotic spindle and led to the formation of multipolar spindles in dividing cells. Collectively, these observations open a new avenue for studies of versican, implying even more versatile roles for this proteoglycan than previously described.  There are only a few reports in the literature that have hinted towards an intracellular localization of versican in different mesenchymal cells types [179,180], and none have attempted to characterize the nature and extent of intracellular versican. It is likely that the presence of intracellular versican in vascular cells has been overlooked in the past because of the overwhelming presence of protein in the ECM, particularly in injured tissues. Given the importance of versican to the normal architecture of the ECM, it is plausible that it also serves a vital role inside cells. Indeed, the current study suggests a function for versican in organizing the mitotic spindle of dividing cells. Versican expression has consistently been associated with increased cell proliferation [47,52,154], and versican knockout cells proliferate significantly slower than wild-type cells [53]. The localization of versican in the nucleus of proliferating cells, particularly in and around the visible chromosomes of dividing cells, provides a possible new mechanism whereby versican may be acting directly on nuclear elements to promote cell division. More work will be needed to explore this possibility and to better define the functional role of intracellular versican. One of the major challenges moving forward will be to differentiate between intracellular and extracellular functions of this proteoglycan. To this note, one of the limitations of the current study is that I cannot rule out the possibility  112  that the mitotic spindle changes seen in the versican knockdown cells were in fact secondary to altered versican in the extracellular environment.  Growing evidence points to the presence of other ECM components intracellularly in many cell types [181-183]. Specifically, hyaluronan has been reported in the nucleus and cytoplasm of vascular smooth muscle cells, where it appears to have important regulatory roles in such processes as cell cycle regulation, mitosis, cell motility, and RNA splicing [184,185]. This is of particular interest because versican and hyaluronan form large aggregates with each other in the ECM where they are thought to regulate cell adhesive properties during proliferation and migration [35,52]. In fact, versican is a member of the hyalectin family of proteoglycans which are recognized and named because of their ability to bind hyaluronan [33]. Intracellular hyaluronan was reported to localize to the same areas that I have now shown versican to be, namely the nucleoli, cleavage furrow and mitotic spindle of dividing cells [184,185]. With numerous reports supporting an interaction between these two macromolecules in the ECM, along with their similar distribution pattern inside vascular smooth muscle cells, it seems probable that versican and hyaluronan also interact inside of cells. Recently it was reported that versican is necessary to properly organize hyaluronan in the ECM [186]. It would be interesting to determine whether versican also plays a role in organizing hyaluornan intracellularly. Further work will be needed to explore whether there is an interaction between these two molecules there.  113  In addition to hyaluronan, other ECM proteins are being recognized to have intracellular functions. For example, an intracellular form of OPN that is associated with the CD44 complex has been identified in migrating fibroblast and metastatic cells [182]. MMP-2 and TIMP-1 have been localized to the nucleus where they appear to regulate the activity of PARP by proteolytic cleavage [183]. The presence of these proteins, along with hyaluronan and now versican, support the idea that an intracellular and/or nuclear matrix exists. There is controversy over the presence of such an entity, in part because its composition and structural components have not been identified [187]. However, data suggests nuclear function depends not only on the passive diffusion of molecules, but also on a dynamic scaffold of structural elements that establishes functional domains in the nucleus [188]. Thus, it is thought that the contents of the nuclear matrix functions in a similar manner to the ECM, in that it imparts structure and organization to the nucleus, as well as acting as an anchor for molecules [189]. Morphological changes observed in tumour cells include alterations to the nuclear structure, such as a change in its size and shape, number and size of nucleoli, and chromatin texture [190], adding further support to this idea. Whether versican forms part of this nuclear matrix is not clear from the current studies, but if such an entity exists, it is tempting to speculate that versican may play a role in its structural organization.  In summary, the current study suggests versican is one of a growing list of secreted proteins that are also found intracellularly and in the nucleus. The presence of intracellular versican appears to be a general phenomenon in mesenchymal cells, as I observed it in fibroblasts and valvular interstitial cells, in addition to smooth muscle cells.  114  As versican is an important proteoglycan in vascular development and disease, a better understanding of its function, and differentiation between its intracellular and extracellular roles, will help us to gain insight into the pathobiology of many vascular diseases.  115  Figure 32. Versican expression in vascular smooth muscle cells. A, Thee-dimensional reconstruction from confocal image stacks of a rat aortic smooth muscle cell immunostained to detect versican (red) and the nucleus (blue) showing versican immunoreactivity throughout the cytosol and in the nucleus (arrow). B, C, Neutralization of the versican antibody with a synthetic peptide confirmed its specificity for versican. In the absence of the versican synthetic peptide, versican immunoreactivity was strongly detected in smooth muscle cells (B). In the presence of the versican synthetic peptide, immunostaining for versican was eliminated (C). (Scale bar = 23.00 µm in b)  116  Figure 33. Versican localizes to the nucleus in vascular smooth muscle cells. (A-C) High power magnification images of a smooth muscle cell immunostained for the nucleus (A, blue) and versican (B, red). Overlay image shows localization of versican in nucleoli (C, arrows). D, Western blot of purified nuclear extracts confirmed the presence of the V1 isoform of versican in the nucleus, but the V0 isoform was not detected. Cu/Zn SOD was used as a control to demonstrate the purity of the nuclear extracts. (Scale bar = 5.80 µm in a)  117  Figure 34. Colocalization of versican and nucleolin confirms nucleolar localization of versican in smooth muscle cells. Immunofluorescent staining of a smooth muscle cell for the nucleus (A, blue), versican (B, red) and nucleolin (C, green) shows strong colocalization of versican and nucleolin in the nucleus (D, yellow, arrows), confirming a nucleolar localization of versican in smooth muscle cells. (Scale bar = 5.80 µm)  118  119  Figure 35. Intracellular versican is associated with mitosis. Images of nuclear versican in valve interstitial cells captured during various stages of mitosis. Versican (red) was observed to fill the nucleus and surround the chromosomes at early stages of mitosis and at the metaphase plate. As cells proceeded through anaphase, versican staining was present between the chromosomes as they move to opposite poles of the cell. Strong staining persisted through telophase, where versican specifically localized to opposing poles of the dividing chromosomes (arrows). (Scale bar = 5.80 µm)  120  Figure 36. Versican localizes to polar ends of the mitotic spindle. Immunofluorescent staining of a cell nucleus (A, blue), versican (B, red) and RHAMM (C, green) shows localization of versican at polar ends of the mitotic spindle during metaphase (D, arrows). (Scale bar = 5.80 µm)  121  Figure 37. Versican knockdown alters mitotic spindle organization. A, Western blot of versican expression in scramble and versican siRNA-treated cells, showing decreased versican expression in the knockdown cells. B, C, Mitotic spindle morphology was visualized by staining for RHAMM (green) in scramble siRNA (B) and versican knockdown cells (C). Versican depletion resulted in disorganized and multipolar spindle formation (arrows in C). (Scale bar = 5.80 µm in B)  122  CHAPTER 7. CONCLUSIONS AND FUTURE DIRECTIONS In this dissertation, I have examined the effect of a canonical Wnt ligand, Wnt3a, and a target of Wnt signaling, versican, in modifying mesenchymal cell phenotype. In chapter 2, I have shown that Wnt3a induces a myofibroblast-like phenotype in cultured fibroblasts, characterized by the increased expression and incorporation of smooth muscle α-actin into stress fibres. These changes were mediated, at least in part, by Wnt3a upregulating the expression of the cytokine TGF-β and its associated signaling through SMAD2 in a mechanism that was dependent on the canonical Wnt mediator β-catenin. In chapter 3, I demonstrated that Wnt3a alters the phenotype of cultured vascular smooth muscle cells and induces a contractile and secretory phenotype in these cells that is associated with increased protease expression and intercellular communication. These changes were also mediated through a mechanism that was dependent on the classical Wnt signaling pathway, as the antagonist DKK1 inhibited these changes. Collectively, these data suggest Wnt3a promotes the formation of a mesenchymal cell phenotype with increased contractile ability, synthetic capacity, protease expression, and cell-cell communication, all cell functions that are critically involved in a wound healing response [1,2,28]. As growing evidence demonstrates canonical Wnt signaling is activated following injury to a number of tissues [75-84], I propose that Wnt ligands such as Wnt3a play an important role in regulating cell phenotype during repair processes.  In the second part of this dissertation, I explored the functional roles of the proteoglycan versican, for which our lab and others have demonstrated is a target of canonical Wnt  123  signaling [69-71], and which has been shown to accumulate in many chronic inflammatory conditions [38,57,58,157-159]. In chapter 4, I showed versican is expressed and secreted as ECM following injury to cultures of valve myofibroblasts. This study also highlighted a role for the membrane receptor CD44 in regulating the organization of versican in the ECM, and suggests CD44 acts as an integral protein that provides a link between the actin cytoskeleton and the provisional ECM. In chapter 5, I cloned the full length V1 isoform of human versican and demonstrated that expression of this recombinant versican itself induced a myofibroblast-like phenotype in cultured fibroblasts. A recent report has shown that versican is required to localize TGF-β to the pericellular space and thereby regulates its activity [160]. My data are in support of this idea, as I found TGF-β signaling to be increased in the versican-transfected fibroblasts. Lastly, in chapter 6, I explored the intracellular localization of versican in vascular cells, and provide data to suggest versican has intracellular functions in addition to its better known roles in the ECM. In particular, it was observed that versican localized to the nucleus during cell division where it was found in close association with the mitotic spindle. Collectively, these data suggest versican is expressed following injury where it forms part of the provisional wound repair matrix that regulates cell phenotype during repair.  Based on the main findings in this dissertation, when taken in the context of previous literature, I propose a model whereby increased Wnt signaling in response to injury upregulates the expression of the cytokine TGF-β as well as the proteoglycan versican. In  124  this model, I suggest versican is necessary to localize TGF-β to the pericellular space where it can then stimulate its pro-fibrotic effects (Figure 38).  To test this model, a key future experiment will be to determine if Wnt3a is able to promote myofibroblast differentiation in the absence of versican expression (i.e. is versican expression required for myofibroblast differentiation?). Further, it will be interesting to determine how TGF-β is regulated in the pericellular matrix. For example, is this localization necessary to promote its interaction with the TGF-β receptors, or does this localization facilitate its interaction with other matrix components such as proteases, which can cleave and thus activate the latent peptide [191].  Another interesting area that is worth exploring in future studies is the potential cross-talk between the Wnt and TGF-β signaling pathways. In the current study, I observed the combined effects of Wnt3a and TGF-β to be complimentary and more dramatic than the individual effects of either of these mediators alone. Other groups have also demonstrated the potential for cross-talk between these two signaling pathways [115,192-196], with one study showing these two pathways culminate at novel regulatory elements to synergistically coregulate the transcription of profibrotic gene targets [94]. It will be important to determine whether Wnt is one of the key drivers of fibrosis, or whether it merely acts to support the profibrotic effects of TGF-β.  A major limitation to the current work is that the experiments have been performed in vitro on cultured cells. Thus, an important focus of future directions should determine  125  whether these findings can be translated into the in vivo setting. In this regard, there are a number of animal models and compounds available to activate or inhibit various aspects of the Wnt signaling pathway, in vivo (reviewed in [88,193,197,198]). Unfortunately, for versican, the knockout mouse is an embryonic lethal [56], and this has stalled in vivo studies into the functional roles of this proteoglycan. The development of a conditional knockout mouse for versican has recently been established [160], and this model should fuel future work around this area. Interestingly, there appears to be differences between humans and mice in terms of the expression pattern and tissue localization of the different proteoglycans. For example, mice express very little versican in their blood vessels, whereas versican is the predominant ECM proteoglycan found in the blood vessels of humans [199]. In light of this, it will be important to also develop a conditional ‘knockin’ approach for versican expression which may better model certain human conditions, and this remains a long term goal of our laboratory.  In summary, through the work carried out in this dissertation, I have provided novel data to suggest canonical Wnt signaling and the proteoglycan versican play key roles in regulating mesenchymal cell phenotype. These factors both promoted the formation of a myofibroblast-like phenotype in cultured fibroblasts, through mechanisms that revolved around activation of the TGF-β signaling axis. 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