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Versican in the wound healing matrix : cellular interactions and degradation by matrix metalloproteinases Pourmalek, Saloumeh 2009

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VERSICAN IN THE WOUND HEALING MATRIX: CELLULAR INTERACTIONS AND DEGRADATION BY MATRIX METALLOPROTEINASES by Saloumeh Pourmalek B.Sc., The University of British Columbia, 2003  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Dental Science)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) April 2009 © Saloumeh Pourmalek, 2009  ABSTRACT In wound healing, versican is a component of the provisional matrix laid down at the site of injury by proliferating myofibroblasts. Versican interacts with a variety of matrix molecules and is believed to interact with the cell surface. The mechanism of interaction of versican with the cell surface, however, is not well documented. Return to normal tissue structure, at late stages of wound healing, involves degradation of versican and concomitant fibroblast apoptosis. Macrophage enzymes are candidates for versican degradation; however, the mechanisms of actions of these enzymes on versican and the rates and cleavage sites are not yet known. This thesis tests several hypotheses: 1) Versican interacts with cell surface receptors of myofibroblasts and macrophages; 2) Versican influences myofibroblast cell morphology during wound contraction; and 3) Macrophage matrix metalloproteinases degrade versican during wound resolution. We first attempted to identify macrophage and fibroblast versican-binding cell surface ligands. Using biotinylated constructs of the C-terminal domain of versican as baits, we identified versican and versican fragments as the main ligands for the C-terminal construct. However, we found that most versican could be released from the cell surface by hyaluronidase treatment, and concluded that versican is held at the fibroblast cell surface mainly through its interaction with hyaluronan. Next, we examined the influence of versican and hyaluronan on the physical properties of a collagenous matrix, and the cells embedded within the matrix, using a novel 3-dimensional collagen/versican/hyaluronan matrix model. We found that fibroblast cells in matrices containing versican express smooth muscle actin and take on a contractile morphology. Finally, we hypothesized that macrophage metalloproteinases degrade versican. The macrophage matrix metalloproteinases (MMPs), MMP-2, MMP-7, and MMP-12 were chosen as candidate enzymes, which we localized to the resolving phase of wound healing in the human lung. We found that MMP-7 and MMP-12 cleave versican at multiple sites in vitro, whereas MMP-2 cleaves versican at a limited number of sites. These macrophage enzymes may be important in clearing versican in vivo. A better understanding of the mechanism of versican degradation could enable therapeutic modification of the disease process in fibrosis, cancer, and nervous system regeneration.  ii  TABLE OF CONTENTS ABSTRACT....................................................................................................... ii TABLE OF CONTENTS ................................................................................. iii LIST OF TABLES ............................................................................................ v LIST OF FIGURES ......................................................................................... vi LIST OF ABBREVIATIONS ......................................................................... vii ACKNOWLEDGEMENTS ........................................................................... viii DEDICATION.................................................................................................. ix CO-AUTHORSHIP STATEMENT.................................................................. x 1. LITERATURE REVIEW ............................................................................. 1 1.1 WOUND HEALING .....................................................................................................................1 1.1.1 Molecular Mechanisms of Wound Contraction and Remodeling.................................2 1.1.2 Pulmonary Fibrosis: An Example of Aberrant Wound Healing ...................................6 1.1.3 Study of Wound Healing In Vitro ...................................................................................8 1.2 VERSICAN ............................................................................................................................... 11 1.2.1 Lectican Family of Proteoglycans................................................................................ 14 1.2.2 Expression Profile ......................................................................................................... 17 1.2.3 Structure and Interactions of Versican with Other Matrix Components ................... 18 1.2.4 Versican Function in Pulmonary Fibrosis .................................................................. 21 1.3 METALLOPROTEINASES ......................................................................................................... 24 1.3.1 Matrix Metalloproteinases .….….………………………………………………….25 1.3.2 MMP-7 (Matrilysin)...................................................................................................... 27 1.3.2 MMP-12 (Macrophage Metalloelastase) ..................................................................... 30 1.3.3 MMP-2 (Gelatinase A) ................................................................................................. 31 1.4 REFERENCES........................................................................................................................... 34  2. VERSICAN INTERACTION WITH HUMAN LUNG FIBROBLAST AND MACROPHAGE CELL SURFACE ..................................................... 65 2.1 INTRODUCTION....................................................................................................................... 65 2.2 MATERIALS AND METHODS................................................................................................... 68 2.3 RESULTS ................................................................................................................................. 75 2.3.1 Purification and Characterization of Versican from HFL1 Fibroblast Conditioned Media ............................................................................................................................. 76 2.3.2 Purified Versican C-terminal (LC) Constructs Form Aggregates in Solution .......... 78 2.3.3 Biotinylated HisLC interacts with versican-like molecules in fibroblast cell lysate 80 2.3.4 Versican intermolecular interactions modulated by its C-terminal domain .............. 84 2.3.5 Biotinylated HisG3 interacts with proteins in fibroblast and macrophage cell membrane ...................................................................................................................... 86 2.3.6 HisG3-coated magnetic beads interact with each other and with the surface of fibroblasts ...................................................................................................................... 88 2.3.7 Hyaluronidase treatment of cell cultured fibroblasts leads to the release of versican from fibroblast cell surface .......................................................................................... 92 2.3.8 Hyaluronidase treatment of fibroblasts induces changes in cell morphology........... 95 2.4 DISCUSSION ............................................................................................................................ 97 2.5 REFERENCES......................................................................................................................... 100  iii  3. FIBROBLAST CELL MORPHOLOGY IN 3-DIMENSIONAL COLLAGEN-VERSICAN-HYALURONAN MATRIX: A MODEL TO STUDY MYOFIBROBLAST CELL BEHAVIOR .......................................105 3.1 INTRODUCTION..................................................................................................................... 105 3.2 MATERIALS AND METHODS................................................................................................. 109 3.3 RESULTS ............................................................................................................................... 114 3.3.1 Physical properties and polymerization rate of collagen I matrix is influenced by versican and hyaluronan ............................................................................................. 115 3.3.2 Fibroblast cell morphology is affected by the composition of pericellular matrix . 115 3.3.3 Fibroblast cell apoptosis at 72 hours of incubation is independent of collagen matrix composition ................................................................................................................. 119 3.4 DISCUSSION .......................................................................................................................... 121 3.5 REFERENCES......................................................................................................................... 126  4. VERSICAN DEGRADATION BY MACROPHAGE MATRIX METALLOPROTEINASES MMP-7, MMP-12, AND MMP-2 ...................133 4.1 INTRODUCTION..................................................................................................................... 133 4.2 MATERIALS AND METHODS................................................................................................. 134 4.3 RESULTS ............................................................................................................................... 140 4.3.1 Macrophages surround versican-rich lesions in BOOP ............................................ 140 4.3.2 Macrophages surrounding versican-rich lesions express high levels of MMPs ..... 140 4.3.3 Versican degradation assays by macrophage metalloproteinases............................ 142 4.3.4 MMP-7 degrades versican at multiple sites............................................................... 142 4.3.5 Recombinant human MMP-12 degrades C-terminal domain of versican .............. 144 4.3.6 MMP-2 degradation of versican is limited ................................................................ 146 4.3.7 Versican C-terminal (G3 domain) is cleaved by MMP-7......................................... 148 4.4 DISCUSSION .......................................................................................................................... 150 4.5 REFERENCES......................................................................................................................... 154  5. CONCLUSIONS AND FUTURE DIRECTIONS .....................................158 5.1 SUMMARY AND SIGNIFICANCE OF RESULTS ....................................................................... 159 5.1.1 Generation of Versican C-terminal Constructs to Study Versican Interactions with the Cell Surface........................................................................................................... 159 5.1.2 Three-Dimensional Proteoglycan-Glycosaminoglycan-Collagen Gel Matrix as a Model of Wound Healing Matrix .............................................................................. 160 5.1.3 Matrix Metalloproteinase Degradation of Versican.................................................. 161 5.2 FUTURE STUDIES .................................................................................................................. 166 5.3 REFERENCES......................................................................................................................... 167  iv  LIST OF TABLES Table 1.1 Different Factors that Influence Matrix Deposition and Myofibroblast Phenotype...........4  v  LIST OF FIGURES Figure 1.1 Contraction of Floating versus Anchored Collagen Matrices............................9 Figure 1.2 Structure of Lectican Family of Proteoglycans..................................................12 Figure 1.3 Structure of Versican Splice Variants (V0, V1, V2, V3) ..................................21 Figure 1.4 Structure of MMP-2, MMP-12, and MMP-7 .....................................................25 Figure 2.1 Structure of HisG3 and HisLC versican constructs ...........................................76 Figure 2.2 Versican Purified from HFL1 Cell Culture Form Aggregates in Solution ......77 Figure 2.3 Purification of HisLC ..........................................................................................79 Figure 2.4 Versican G3 construct (HisLC) interacts with ligands in HFL1 cell lysate....81 Figure 2.5 Versican G3 construct (HisLC) interacts with versican and other fragments containing versican C-terminal domain in the fibroblast cell lysate .........................83 Figure 2.6 Biotinylated HisLC Interacts with HisLC monomer and multimers ...............85 Figure 2.7 Versican HisG3 Interacts with HFL1 and U937 Cell Membrane Ligands .....87 Figure 2.8 HisG3 coated magnetic beads interact with HFL1 and U937 cell membrane 89 Figure 2.9 HisG3 coated magnetic beads interact with each other and HFL1 cells .........91 Figure 2.10 Versican is released from HFL1 cell surface on treatment with hyaluronidase ........................................................................................................................................93 Figure 2.11 Versican is released from HFL1 cell surface upon treatment with hyaluronidase ................................................................................................................94 Figure 2.12 Fibroblast nuclear morphology is altered upon hyaluronidase treatment ......96 Figure 3.1 Illustration of 3D Collagen Experiment ...........................................................108 Figure 3.2 HFL1 Cell Morphology in 3D Collagen Matrices Under 20X Magnification ......................................................................................................................................117 Figure 3.3 HFL1 Cell Morphology in 3D Collagen Matrices Under 63X Magnification ......................................................................................................................................118 Figure 3.4 Fibroblast cell apoptosis depends on the composition of 3D collagen matrices (40X Magnification) ...................................................................................................120 Figure 4.1 MMP expressing macrophages surround fibroproliferative lesions in human IPF................................................................................................................................141 Figure 4.2 Versican degradation pattern by MMP-7 enzyme ..........................................143 Figure 4.3 Versican degradation pattern by MMP-12 enzyme ........................................145 Figure 4.4 Versican degradation pattern by MMP-2 enzyme ..........................................147 Figure 4.5 HisG3 is degraded by rhMMP-7 enzyme........................................................149 Figure 5.1 Schematic of Wound Healing ...........................................................................165  vi  LIST OF ABBREVIATIONS α-SMA 3D AGG ARDS BOOP CCL CNS CRP CS CSPG CXCL ECM ET-1 G1 G2 G3 GAG GM-CSF HA Has Hdf HFL HisG3 HisLC Ig IPF MCP MMP NCC PBS PDGF SMC TGF-β TIMP UIP VC  α-Smooth Muscle Actin Three Dimensional Aggrecan Adult Respiratory Disease Syndrome Bronchiolitis Obliterans Organizing Pneumonia C-C Ligand Central Nervous System Complementary Repeat Protein Chondroitin Sulfate Chondroitin Sulfate Proteoglycan CXC Chemokine Ligand Extracellular Matrix endothelin-1 N-terminal Domain of Lecticans Link Binding Modules in Aggrecan C-terminal Domain of Lecticans Glycosaminoglycan granulocyte monocyte colony stimulating factor Hyaluronan Hyaluronan Synthase Heart Defect Human Fetal Lung Histidine-tagged G3 Domain Histidine-tagged LC domain Immunoglobulin Idiopathic Pulmonary Fibrosis Monocyte Chemotactic Protein Matrix Metalloproteinase Neural Crest Cell Phosphate Buffer Saline Platelet-Derived Growth Factor Smooth Muscle Cell Transforming Growth Factor-β Tissue Inhibitor of Metalloproteinase Usual Interstitial Pneumonia Versican  vii  ACKNOWLEDGEMENTS I would like to express my deep and sincere gratitude to my supervisor Clive R. Roberts, Ph.D., whose extensive knowledge in the field of science and understanding of the human condition helped me through the toughest obstacles I faced not only in completing the laboratory work for this thesis, but in my personal life as well. I am deeply grateful to my supervisory committee, Professor Edward Putnins and Doug Waterfield, Ph.D., for their continuous guidance and encouraging words. My sincere thanks are due to Professor Chris Overall and the Overall lab for their intellectual and technical support which assisted me in completing the laboratory work for this dissertation. I offer my gratitude to the official referees for their detailed review, constructive criticism and excellent advice during the preparation of this thesis. During this work I have collaborated with many colleagues for whom I have great regard, and I wish to extend my warmest thanks to all those who have helped me with my work. Also, I would not have lasted this long in academia if it were not for the lessons I learned along the way, on perseverance and rational thinking, from the most influential teachers in my life: Mrs. Mesdaghi, Mrs. Ahmadian, Dr. Maurice, Dr. Robert Maurus, Professor Gordon Slade, and Dr. Don Brunette. And above all, a big special thank you to my beautiful family, my parents Malihe Nikravan Mofrad and Esmaiel Pourmalek and my wonderful sisters Shadi and Nazanin, for their intellectual, emotional and moral support throughout my education, and for believing in me more than I did. Finally, no words can explain my feelings towards my beloved, Ciaran Aiken, who made it pleasurable to continue working on this thesis and helped me live outside of the ivory tower I felt I was trapped in. The financial support of the University of British Columbia, Faculty of Dentistry, the Canadian Institute of Health Research (CIHR), and British Columbia Lung Association is gratefully acknowledged.  viii  Dedication  To My Beloved Parents  ix  CO-AUTHORSHIP STATEMENT Early immunohistochemical studies in our laboratory, under the supervision of Clive R. Roberts, showed the accumulation of macrophages around versican-rich lesions in fibrotic lung disease through immunohistochemistry. Observation that versican degradation occurs concomitantly with fibroblast cell apoptosis lead to the hypothesis that macrophage enzymes may be involved in the degradation of versican.  I purified versican expressed by human lung fibroblast cells and performed the  degradation assays with macrophage matrix metalloproteinases (MMPs). MMP-2 and MMP-12 were kind gifts from Chris Overall laboratory.  The manuscript was written by me, and edited by my  supervisor, Clive Roberts.  Clive Roberts  Saloumeh Pourmalek  x  1. LITERATURE REVIEW 1.1 Wound Healing Wound healing is an intricate mechanism involving a myriad of factors and cell types. The wound healing process is classically divided into four phases which include homeostasis, inflammation, proliferation and remodeling (reviewed in1, 2). Homeostasis is marked by vasoconstriction of injured blood vessels and activation of intrinsic coagulation pathway that leads to formation of a blood clot and release of growth factors and cytokines by platelets.  In the inflammatory phase, vasodilation and platelet  disaggregation allow neutrophils to enter the wound site and remove bacteria and damaged tissue from the extracellular matrix. Macrophages, which are attracted to the wound site by chemoattractants,  continue the process of phagocytosis and recruit  fibroblast cells by adding to the pool of growth factors already present at the site. Once the matrix is cleared out, inflammation is turned off and the process of epithelialization and matrix formation is initiated by proliferating fibroblasts. Fibroblasts migrate into the cleared site and lay down a provisional matrix rich in proteoglycan versican, glycosaminoglycan hyaluronan, fibronectin, tenascin, and a number of other proteins. PDGF and activated TGF-β, released mainly by macrophages, are the main triggers for production of this transient matrix.  These two growth factors, along with other  granulation tissue components, signal fibroblast differentiation into contractile myofibroblast which are the main cell type responsible for wound closure. In the final remodeling phase of wound healing, fibroblasts express high levels of type I collagen, and an organized collagenous matrix replaces proteoglycan and fibronectin. As the provisional matrix is degraded by matrix metalloproteinases (MMPs), myofibroblasts go into apoptosis and the anatomy and function of the tissue is restored. Repeated insult and the ensuing inflammation result in prolonged myofibroblast proliferation and excessive granulation tissue synthesis at the site of injury. The accumulation of non-functional and excessive scar tissue, or fibrosis, is associated with many clinical problems such as keloid or hypertrophic scar formation in the skin, delayed nervous system regeneration, lung and liver dysfunction, and atherosclerosis3, 4. 1  My study centers around wound healing and fibrosis in the lungs. Replacement of normal lung architecture with collagenous matrix causes decreased lung air space volume and obstruction of gas exchange, which can result in considerable loss of lung function and ultimately respiratory failure and death. Persistence and magnitude of initial injury, multiple modes of tissue injury and slower than normal clot resolution are all possible causes that may lead to pulmonary fibrosis instead of normal wound healing. Whatever the cause, however, heavy proteoglycan deposition in association with proliferating myofibroblasts is central to the persistence of active lesions in most fibrotic lung diseases. What follows is a review of the cellular and molecular mechanisms involved in wound contraction and granulation tissue remodeling, and the pathophysiology of wound healing and pulmonary fibrosis. 1.1.1 Molecular Mechanisms of Wound Contraction and Remodeling The deposition of a transient granulation tissue and its subsequent maturation and remodeling is an important phase of wound healing. Multiple growth factors and cytokines regulate the process of new matrix synthesis and allow it to proceed in an orderly fashion5, 6. Platelet-derived growth factor (PDGF)7 and Transforming growth factor beta (TGF-β)8, 9 are the two main growth factors expressed by macrophages and fibroblasts during this phase of wound healing. In response to PDGF, fibroblasts begin synthesizing a provisional matrix rich in glycosaminoglycans, fibronectin and collagen type III10. PDGF stimulates hyaluronan synthesis11, versican synthesis12, and smooth muscle cell proliferation13. TGF-β is involved in organizing the extracellular matrix, scar remodeling and wound contracture14.  TGF-β enhances matrix deposition by  fibroblasts through increasing the expression of collagen15, 20, hyaluronan16,  17  ,  versican12, 17 and fibronectin18-20. This growth factor also prevents matrix degradation by lowering production of matrix degrading enzymes (MMPs)21 and increasing expression of tissue inhibitors of metalloproteinases (TIMPs)22. Major TGF-β subtypes involved in wound healing are TGF-β1 and TGF-β2, although there are no known major differences in terms of function23.  In response to this newly synthesized matrix, and under the  influence of TGF-β and PDGF, fibroblasts differentiate into a distinct cell type known as myofibrobalsts. 2  Differentiated contractile myofibroblasts are recognizable by their α-smooth muscle actin (α-SMA) expression in stress fibers24, and are responsible for contracting the granulation tissue and closing the wound25. Studies conducted in different tissues suggest that resident myofibroblasts, arising from a population of tissue specific fibroblasts activated in response to injury, are the primary cell type found in wound matrix26. Several mechanisms have been suggested for the differentiation of fibroblasts to myofibroblasts. Combined effects of TGF-β1 and mechanical tension, in particular, have been the focus of multiple reviews in recent years4,  26, 27  . TGF-β1 induces α-smooth  muscle actin expression in granulation tissue myofibroblasts on stiff 2-dimentional culture substrate28, 29, but not on compliant substrate void of tension30. This phenomenon is also observed in 3-dimensional collagen matrices, where TGF-β1 induces myofibroblast differentiation when gels are mechanically restrained31, but not in free-floating and relaxed gels24. On the other hand, mechanical stress alone does not seems to induce myofibroblast differentiation in the absence of active TGF-β131,  32  .  Although it is unclear how mechanical stress and TGF-β1 signaling converge to promote increased α-SMA expression and myofibroblast differentiation, one theory suggests that mechanical tension may regulate TGF-β1 activation by releasing it from its large latent complex33, 34. Latent complex provides a reservoir of latent TGF-β1 in the ECM by binding to other ECM components like fibrillin-1 and fibronectin35-37. It is noteworthy, however, that other mechanisms for activation of TGF-β1 have previously been reported. For example thrombospondin, a component of the provisional matrix, is also able to activate TGF-β138, as can integrin αvβ639 which releases TGF-β1 from its latency complex at remodeling sites. Another explanation for the cumulative effects of TGF-β1 and mechanical tension on myofibroblast differentiation and α-SMA expression may lie in the expression of multitude of other ECM proteins that are produced in response to both TGF-β1 and mechanical tension.  A comparison of all the major factors found in the wound  environment shows that the same factors that induce myofibroblast differentiation and α-SMA expression also increase versican expression and the expression of proteins that associate with versican (Table 1.1). 3  Table 1.1 Different Factors that Influence Matrix Deposition and Myofibroblast Phenotype Effectors / Stimulants  TGF-β1  Mechanical Tension  Increased Versican Expression  Stress Fiber Accumulation  • Fibroblasts cocultured with Keratinocytes40 • Kidney and lung Fibroblasts19 • Malignant Osteosarcoma Cells17  • Granulation tissue myofibroblasts & quiescent cultured Fibroblasts28 • Cultured Fibroblasts41  • Vascular Smooth Muscle Cells50 • Asthmatic and Normal Lung Fibroblasts51 • Arterial SMC12, 58 59 60  Induced α-SMA Expression  GM-CSF  Increased Expression of Other ECM Components  • Fibroblasts31 46 • Gingival Fibroblasts47 • Fetal Lung Fibroblasts48 • Fibroblasts49 • Reviewed in4  • Fibronectin & Collagen I20 • HA17  • Fibroblasts in 3D matrix52 53 • Cultured Fibroblasts54 55  • Granulation tissue myofibroblasts & quiescent cultured Fibroblasts28 • Quiescent human breast gland Fibroblasts42 • Cultured Mesengial cells43 • Human articular chondrocytes & meniscal cells44 • Dermal Fibroblasts45 • Fibroblast32 31, 46 56 • Granulation tissue Myofibroblasats30 • Liver Fibroblasts57  • Fibroblast32 56 4  • biglycan, perlecan in Vascular Smooth Muscle Cells50  • Fibroblasts, in vivo and in vitro 61  • Human articular chondrocytes & meniscal cells44  • Keloid Fibroblast62 • Wound Fibroblasts63 • Released by SMC of damaged arteries64 • activated Fibroblasts65 • Tumor cell66 • Lung Fibroblasts73 • Granulation Tissue Myofibroblst78 • Colonic myofibroblast79 • Myofibroblasts80  • Hyaluronan67 • Link Protein, HA/Vc Aggregates59 60 • Granulation Tissue  PDGF  ET-1  Fibroblast to Myofibroblast differentiation  • Glomerular Mesangial Cells72 • Lung Fibroblasts73  • CHO Cells74 • Astrocytes75  • Hepatic Stellate Cells76 • Lung Fibroblasts77 • Lung Fibroblasts73 • CHO Cells74  • Monocytes in Myocardial Infarction82  • Myofibroblasts  • Myofibroblasts83  83  68 69  • Fibronectin70 • Collagen71 • Collagen I, TGF-β, IL-6 in Glomerular Mesangial Cells 72 Lung Fibroblasts73 • HA/CD44 in VSMC81  • Asthmatic SMC via induction of TGFβ1 Receptor84 85 &TGFβ 86 • Myofibroblasts80  4  Versican is a regulatory proteoglycan and is thought to influence cell adhesion, proliferation, migration and extracellular matrix assembly87, 88.  Versican induces  neuronal differentiation and neurite outgrowth89, differentiation of preadipocytes90, and is essential for pre-cartilage aggregation and subsequent cartilage differentiation91, 92. There is a growing body of evidence that associates granulation tissue proteoglycans with myofibroblast cell differentiation and survival.  The structural, mechanical, and  biochemical properties of versican-rich matrix and its influence on myofibroblast cell phenotype are further investigated in this thesis; and the results have lead me to develop a new hypothesis for the role of proteoglycan versican in wound healing. My hypothesis implicates versican as more than just a structural support in the temporary scaffold of granulation tissue. A detailed description of versican and its binding partners, and an examination of their role in wound healing is given in the next section of this chapter. In the later stages of wound remodeling, differentiated myofibrobalsts lay down a collagenous matrix that replaces the provisional proteoglycan-rich matrix and shapes the fibrous tissue generally observed in fibrosis93-95. Once the process of wound contraction by differentiated myofibroblasts is completed, α-SMA expressing myofibroblasts disappear from the scar25 through the process of apoptosis96.  Although it is well  established that regression of granulation tissue occurs by apoptosis42, 97, 98, factors and mechanisms that lead to myofibroblast apoptosis are not well understood.  One  hypothesis considers release of mechanical tension, cytoskeletal disruptions, and growth factor withdrawal as key stimulants of fibroblast apoptosis in an in vitro model of granulation tissue99. As granulation tissue proteoglycans, such as versican can greatly influence myofibroblast cell phenotype, and as myofibroblast apoptosis occurs concomitantly with granulation tissue degradation in vivo; the possible role of proteoglycan degradation in myofibroblast apoptosis is worthy of further investigation. Matrix metalloproteinases (MMPs), which are involved in all aspects of wound healing, are prime candidates for reshaping the matrix and influencing cell morphology. MMPs regulate inflammation through processing of chemokines and cytokines, mediating cell-cell and cell-matrix interactions during re-epithelialization, and remodeling the scar extracellular matrix either directly by proteolytic degradation of  5  proteins or indirectly by influencing cell behavior (reviewed in100, 101). In the granulation tissue, macrophages and fibroblasts release MMPs that can activate TGF-β, which in turn stimulates further fibroblast proliferation and matrix deposition. TGF-β causes the release of tissue inhibitors of metalloproteinases (TIMPs), and downregulates expression of MMPs known to degrade components of the provisional matrix22 (reviewed in1, 5). Although the large MMP family consists of 24 secreted and membrane-bound enzymes that are known to act on a variety of bioactive molecules and other substrates in the extracellular matrix, substrate specificity and compartmentalization allow them to function as regulators of extracellular matrix remodeling and cell morphology in many aspects of normal physiology and pathology102. For example, MMP-2 and MMP-9 of the gelatinase family act on cleaved collagen better than other MMPs103, MMP-7 (Matrilysin) is a more potent proteoglycanase than MMP-3 or MMP-9104, and MMP-12 (Macrophage Metalloelastase) is the most elastolytic enzyme of the MMP family105 capable of efficiently degrading fibronectin and chondroitin sulfate chains of proteoglycans106. Also, cells do not release these proteases indiscriminately. Anchoring of MMPs to the cell membrane targets their catalytic activity to specific substrates within the pericellular space. For example, MMP-2 binds to αvβ3 integrin107, MMP-9 interacts with CD44108, and MMP-7 binds to cell surface proteoglycans109. The structure and function of MMPs and their role in wound healing is more extensively described in a following section of this chapter. 1.1.2 Pulmonary Fibrosis: An Example of Aberrant Wound Healing Extracellular matrix in the lungs is composed of basal lamina, underlying the epithelium, and the interstitial matrix. The epithelium of the lung acts as a barrier that allows regulated passage of inflammatory cells into the airways. The basal lamina underlies the epithelium, and provides attachment for epithelial cells through anchorage molecules such as laminin, fibronectin, and type IV collagen110,  111  . The basal lamina is also  connected, through its components, to the deeper connective tissues such as elastic fibers112. Some constituents of the extracellular matrix, such as collagens I, II, III and IV, elastic fibers, and proteoglycans maintain tissue structure, while others such as laminin  6  and fibronectin are involved in cell attachment and signaling113, 114. The interstitium of the lung contains many cell types including fibroblasts, myofibroblasts, smooth muscle cells, macrophages, and some undifferentiated fibroblast-like cells. Significance of intact basal lamina for tissue repair was shown by a study which demonstrated that regeneration and function can be restored if basal lamina remains intact115. In the lungs, for example, major damage to basal lamina results in altered architecture and the degree of remodeling depends on severity of original injury116. Injury to the epithelium causes the release of interstitial content through the basement membrane into the alveolar space of the lung117. Inflammation is marked by an increase in the number of a variety of immune cells including alveolar macrophages, neutrophils, eosinophils, T-cells, B-cells, basophils and mast cells. Constituents of normal ECM, both molecular and cellular, cause the recruitment of many of these immune cells through the formation and release of cytokines and chemokines118.  The immune cells that are  recruited to the site of injury in turn enhance the inflammatory process by releasing more chemokines7, 8. There are many parallels between wound healing and pulmonary fibrosis119. Similar to wound healing, PDGF7, 120, 121 and TGF-β8, 9 are the two main growth factors that are localized to centers of fibroblast proliferation in fibrotic lung.  PDGF stimulates  hyaluronan synthesis11, smooth muscle cell proliferation13, and versican synthesis12. TGF-β promotes growth and differentiation of connective tissue cells.  Fibroblasts  differentiate into contractile, smooth muscle actin-rich myofibroblasts as their expression of alpha actin protein is induced by TGF-β29.  TGF-β also induces expression of  hyaluronan16, 17, versican12, 17, and protease inhibitors that prevent matrix degradation122. Altered cellular and molecular ECM composition leads to the creation of a ‘provisional’ matrix where mesenchymal cell proliferation occurs. Overall, there is an increase in the amount  of  fibronectin123  and  glycosaminoglycan hyaluronan93, collagen type I, III, and VI  124-128  94  proteoglycans  versican,  biglycan,  and  the  in the active lesions, and an eventual increase in  in the fibrotic lung. Thus, pulmonary fibrosis is a disease  process driven by chronic inflammation and matrix degradation, cellular migration and  7  proliferation, that ultimately leads to the accumulation of collagenous matrix in the interstitium and alveolar spaces of the lung129, 130. Regardless of the cause of pulmonary fibrosis, degradation of granulation tissue components along with concomitant myofibroblast apoptosis leaves a collagenous-rich matrix that causes thickening of the alveoli walls and filling up the alveolar spaces119. The expression of several matrix metalloproteinases (MMPs) also increases in pulmonary fibrotic diseases as a result of injury. Production of MMPs may contribute to the removal of versican-rich provisional matrix, a process that must occur in the evolution of the lesions to collagen-rich and proteoglycan-poor fibrotic tissue93, 94. Persistence of granulation tissue, rich in proteoglycans and contractile myofibroblasts, and excessive deposition of collagenous matrix is a hallmark of fibrosis in different tissues in the body. The similarity of the process in different tissues suggests universal functions for these proteoglycans in the provisional matrix. Our study centers around pulmonary fibrosis and the role of proteoglycan versican, its effects on myofibroblast cell morphology and its subsequent degradation, in the aberrant process leading to fibrosis. 1.1.3 Study of Wound Healing In Vitro Three dimensional (3D) collagen or fibrin matrices, containing cultured fibroblasts, have become popular as in vitro models of wound contraction132. Early experiments with collagen133 and fibrin134 matrices, floating in culture media, showed that fibroblast cells cultured in 3D matrices phenotypically resemble cells found in the wound matrix more closely than cells grown in a planer culture plate. Three models of collagen matrix contraction are currently in use, and they are floating matrix, anchored matrix, and initially anchored and subsequently released “stress-relaxation” matrix (Figure 1.1). Although contraction occurs in all three types of matrices135 as a consequence of motile activity of migrating cells136, the fibroblast phenotype that develops as a result of this contraction differs depending on the matrix mechanics.  8  Relaxed Matrix Floating  Anchored  StressRelease  Contracted Matrix Figure 1.1 Contraction of Floating versus Anchored Collagen Matrices Contraction of floating collagen matrices results in a tissue resembling the dermis, in which fibroblasts develop long processes and a cytoskeletal meshwork132 138-140  their ability to proliferate  .  137  and loose  Decreased collagen biosynthesis and increased  collagenase production is another hallmark of cells in floating matrices14,  141-143  .  Anchored collagen gels, on the other hand, develop into a stressed tissue resembling granulation tissue, with a tensional force comparable to that of contracting skin wound144-146. In these stiff matrices, fibroblast cell orient along lines of tension147 137, and develop prominent stress fibers resembling myofibroblasts31, 148, 149. Cells in anchored matrices continue to proliferate138,  139  and synthesize collagen. Ensuing fibroblast cell  contraction resembles that of a smooth muscle cell requiring intact stress fibers, regulated by serum factors148, retraction of cell pseudopodia and collapse of actin filament bundles148,  149  .  In the third model, the reaction of cells to stress release or matrix  relaxation may represent an in vitro model of the transition from granulation to scar tissue that happens in the later stages of wound healing and fibrosis135. Cell proliferation and collagen synthesis decline rapidly as cells switch from an activated to resting phenotype149, 150. In summary, studies of the 3D models suggest that mechanical tension in anchored matrices leads to intracellular tension and formation of stress fibers as the fibroblast cells differentiate first into proto-myofibroblasts with organized stress fibers and then into  9  myofibroblasts with α-SMA decorated stress fibers24. Increased expression of other matrix components, such as fibronectin and collagen I, and inhibition of MMPs also accompany myofibroblast contractile phenotype in response to outside stress. Once the gels are contracted or released from their support (free-floating gels), cells go into a quiescent state135,  151  and the process of apoptosis begins152.  It is important to  acknowledge, however, the dependence of contractile cell phenotype on extracellular factors in these matrices.  Collagen matrix contraction requires serum153,  154  , whose  activity can be replaced or enhanced by purified growth factors. TGF-β, for example, stimulates contraction of both floating and anchored collagen matrices14, 155, 156. PDGF also stimulates matrix contraction157,  158  by a mechanism independent of TGF-β159.  Mechanical tension combined with extracellular factors join forces to transform quiescent fibroblasts into contractile myofibroblasts. How the downstream signaling mechanisms of growth factors and mechanical tension converge to stimulate this transformation is not known yet.  In this thesis, we will focus on one such possible converging factor  downstream of both growth factor and mechanical tension stimulation, namely versican.  10  1.2 Versican Versican, a large proteoglycan with an estimated molecular mass of more than 1000 kDa, is a member of the lectican, also known as hyalectan, family of proteoglycans which includes aggrecan (abundant in cartilage), brevican and neurocan (nervous system proteoglycans; reviewed in  87, 160  ). These proteoglycans share structural similarities at  both the genomic and protein level. Their tri-domain structure consists of globular N- and C-termini, which is homologous among the members of this family, and a central glycosaminoglycan (GAG) binding region (Figure 1.2). The name hyalectan is attributed to the interactions of their N-terminal domain with hyaluronan and the interactions of C-terminal lectin-like domain with a variety of other ECM molecules that helps to stabilize the matrix through formation of supra-molecular aggregates. Although lecticans share a very similar N-terminal hyaluronan binding domain and a common C-terminal domain, differences in the number and type of GAG chains bound to the core protein adds diversity to the structure and function of proteoglycans. GAGs are composed of repeating disaccharide units161, and variations in sulfation patterns affects the charge density on GAG chains, and thus the shape and properties of the proteoglycan.  11  GAG-α  GAG-β  N-Terminal  C-Terminal  Versican  Aggrecan  N-Terminal  C-Terminal  N-Terminal  C-Terminal  Neurocan  N-Terminal  C-Terminal  Brevican  Immunoglobulin type repeat 2 Link protein type modules 2 EGF-like modules C-type Lectin like module Complement regulatory protein Keratan sulfate Chondroitin sulfate Glycosaminoglycan bidning  region  Figure 1.2 Structure of Lectican Family of Proteoglycans The N-terminal domain (G1 domain) of lecticans consists of an immunoglobulin (Ig)-like loop and two copies of a hyaluronan-binding motif, or link modules (also called the proteoglycan tandem repeats).  Only aggrecan has an additional globular domain  (G2 domain) near the N-terminal, which consists of two more link modules. In cartilage,  12  aggrecan forms a ternary complex with hyaluronan and the link protein. The link protein plays a crucial role in stabilizing the complex by binding to both aggrecan and hyaluronan162. HA binding properties are shown to be present in all lecticans including versican163. Similar to aggrecan, interactions between the versican G1 domain, link protein and HA have been reported164. The C-terminal globular domain (G3 domain) consists of one or two epidermal growth factor-like (EGF) repeats, a C-type lectin domain and a complementary repeat protein (CRP)-like domain. The C-terminal domain of lecticans interact with a variety of ligands both in vivo and in vitro. Binding partners for the G3 domain of versican will be discussed in more detail in another section of this chapter. Although their N- and C-terminal domains are highly conserved, the central domain of lecticans is quite diverse in terms of size and sequence. Unlike the N-terminal and C-terminal domains of lecticans, the central domain lacks any cysteine residues and probably has a highly extended three dimensional structure due to the fact that most of the GAG attachment sites are present in the central domain. The numbers of potential GAG attachment sites are quite variable among lecticans, with about 120 chondroitin sulfate attachment sites for aggrecan and 24 chondroitin sulfate chains for versican. In addition, aggrecan carries keratan sulfate chains as well as the mentioned chondroitin sulfate chains165, 166. The two smallest members of lectican family, namely neurocan and brevican, contain 3-7 and 1-3 chondroitin sulfate chains respectively. Because of the negatively charged sulfate or carboxyl groups, chondroitin sulfate chains can attract various positively charged molecules such as certain growth factors, cytokines, and chemokines167-169. This interaction in the extracellular matrix or on the cell surface is important in the formation of immobilized gradients of these factors, their protection from proteolytic cleavage, and their presentation to specific cell-surface receptors 170-172. Another property of negatively charged GAGs, observed most clearly with aggrecan in cartilage, is their interaction with water molecules and large hydrodynamic volume when subjected to compressive forces.  13  1.2.1 Lectican Family of Proteoglycans The expression of lecticans varies widely with respect to time and space, in development and disease. The role of lecticans as growth inhibitory molecules in the central nervous system (CNS) has been well documented. Neurocan, one of the brain specific lecticans, interacts with the neural cell adhesion molecules in vitro173, and inhibits neuronal adhesion and neurite outgrowth following injury to the CNS. It has also been suggested that neurocan, and other chondroitin sulfate proteoglycans, may act as a barrier to neurite extension in developing retina174. The effects of neurocan, however, depends on the nature of the neuron and the precise molecular context in which it is embedded. Brevican, another small neural-specific lectican of the brain extracellular matrix, is expressed by neuronal and glial cells in the terminally differentiated CNS175. Unlike neurocan, brevican is strongly up-regulated during postnatal development and its level peaks particularly after terminal differentiation of the brain176. As a part-time proteoglycan, brevican has bifunctional effects on neurite outgrowth. Brevican can be found both in the matrix in secreted form and on the cell membrane in glycosyl-phosphatidylinositol (GPI) anchored form.  The reason for brevican’s  bifunctionality has been linked to both its chondroitin sulfate (CS) chains and core protein features. For example, brevican is able to inhibit cerebellar neurite outgrowth on laminin through its CS chains and promote outgrowth of hippocampal neurons via its protein core177  178  . Thus, presence of GAGs have a great influence in determining the  function of this lectican. On the other hand, the growth promoting effect of brevican appears to come from its lectin domain178, suggesting that other lecticans are capable of producing the same effects through their lectin domains. Aggrecan, discovered as a major structural component of cartilage, is present mostly as a CS proteoglycan in the CNS and seems to strongly inhibit neurite extension on a variety of substrata, in vitro179. The effects of aggrecan on neurite outgrowth seems to be influenced by both the composition of the surrounding matrix and the ability of neurons to modulate cellular receptors in response to aggrecan180.  The anti-adhesive and  anti-migratory effect of chondroitin sulfate proteoglycan (CSPG) aggrecan, from juvenile  14  or adult cartilage, was demonstrated by addition of this molecule to the ECM in vitro181, 182. It was also shown that the inhibitory effect of PGs were sensitive to chondroitinase ABC digestion, but not to hyaluronidase, suggesting the involvement of CSPG183. Versican is unique in that it can be expressed as one of 4 distinct structural variants which differ in their potential number of glycosaminoglycan attachment sites184, 185. The structural and functional diversity of versican is increased by this variation in glycosaminoglycan content, and this topic is further investigated in this thesis, under section 1.2.4. Another interesting observation is that although versican and aggrecan appear to share a close N- and C-terminal structural homology, they are different in that aggrecan contains far more glycosaminoglycan attachment sites and a greater variety of GAGs than versican. Not surprisingly, their expression pattern and proposed functions suggest opposing roles for these two lecticans in a number of different processes such as neural crest cell (NCC) migration, chondrogenesis, and development of the heart. Functional differences between versican and aggrecan have been extensively studied in the context of NCC migration180,  186, 187  . For example, in the larvae of white mutant  axolotl unlike the normal dark ones, migration of trunk pigment cells is restricted188, 189. Developmental studies revealed a decrease in the quantity of versican mRNA in the mutant embryo compared to the wild type190. Also. the synthesis of versican during NCC migration was shown to be critical in the wild type axolotl embryo and antibodies against versican stained the subepidermal matrix of dark axolotl embryos. A study of NCC migration alongside embedded micromembranes which were coated with either versican or aggrecan discovered that in contrast to versican-coated micromembranes, those covered with aggrecan completely inhibit the movement of NCCs191. Permissiveness of versican to NCC movement has also been illustrated in a collagen type I-versican substrata, a movement which is not permitted through a three-dimensional collagen type I-aggrecan substrata in vitro191. Difference in the inhibitory effects of aggrecan and versican may be as a result of the different CS chains bound to each molecule and the presence of KS chains in aggrecan but not versican. Aggrecan seems to bind the cell  15  surface through HA and inhibit NCC movement through its KS chains, possibly by influencing integrin function182. A number of other experiments have centered around the role of proteoglycans in the development of the heart.  In 1998, Mjaatvedt mapped the recessive lethal heart  defect (hdf) gene in mouse to versican192.  The disrupted gene was shown  to be  important in the formation of right ventricle and the endocardial cushions. Immunohistochemistry studies with an antibody against a GAG epitope on versican confirmed the absence of versican in the homozygous mutant embryos when compared to the normal wild-type embryos.  In situ hybridization and immunohistochemistry on  sectioned mouse embryos show that the mRNA and protein for versican are expressed in a dynamic pattern during development of the heart193. From these studies, it is possible to infer that versican is involved in specification of the ventricular chambers, in growth and fusion of the atrial and ventricular septa, and in the transformation from epithelium to mesenchyme that characterizes development of the endocardial cushions.  In 1999,  another group of researchers mapped versican to the myocardium and the myocardial basement membrane194. Finally, in developing cartilage, versican is transiently expressed at a high level in the mesenchymal  condensation  area  and  rapidly  disappears  during  cartilage  development 195 196. Recent immunohistochemical studies on developing limb bud cartilage revealed that an area positive for versican is gradually replaced by an area positive for aggrecan. These reciprocal patterns of versican and aggrecan expression suggest that versican serves as a temporary framework in developing cartilage matrix. Although the aggrecan aggregate is the major component of cartilage ECM and versican has not been detected by immunohistochemical studies197, constitutive low level transcription of the versican gene is reported in cartilage198 and chondrocytes198,  199  .  In addition, extracts of human adult articular cartilage contain versican200, suggesting its distinct role in that tissue.  An investigation on versican expression, localization, and  aggregate formation in cartilage showed that versican is mainly localized in the interterritorial zone of the articular surface, whereas aggrecan is rather diffused, especially with dense staining in the territorial zone of pre-hypertrophic chondrocytes201.  16  Although transcription of the versican gene dramatically decreases after birth, versican remains in the articular cartilage in the form of the proteoglycan aggregate. Based on these observations, it has been suggested that the versican aggregate is present in the articular surface and may provide ECM properties distinct from deeper zones where aggrecan aggregates are abundant201. Above are three examples illustrating opposing functions for versican and aggrecan in different tissues during development. We also present data showing different effects of these two proteoglycans in the context of myofibroblast cell morphology in a 3D model of wound contraction, and we will look back at these functional difference in more detail in chapter 3. 1.2.2 Expression Profile Although versican is mainly expressed during embryonic development in various tissues197 which includes the nervous system, it is also expressed in some adult tissues, such as the heart, blood vessels, and brain. Versican is constitutively expressed and serves as a structural macromolecule of the ECM197. Smooth muscle cells of blood vessels, epithelial cells of skin, and the cells of central and peripheral nervous system are a few examples of cell types that express versican physiologically197.  The role of  versican in cell adhesion202, migration203, proliferation, and differentiation204 has also been studied in development and disease. Versican is involved in guiding the migration of embryonic cells involved in the formation of the heart192, 193, 205 and outlining the path for neural crest cell migration191 in development. Versican expression is also high in the developing mesenchyme during limb development and is subsequently down-regulated during mesenchymal condensation as aggrecan replaces versican in the pre-chondrogenic core206. In disease, versican is a key factor in inflammation through interactions with adhesion molecules on the surfaces of inflammatory leukocytes207,  208  and interactions  with chemokines that are involved in recruiting inflammatory cell207. Increased versican expression is often observed in association with proliferating cells within remodeling tissue in lung and cardiovascular diseases and in cancer. For example, versican is heavily deposited in the lung interstitium during the development of many forms of lung diseases, including asthma, adult respiratory disease syndrome (ARDS), and idiopathic pulmonary fibrosis (IPF)209 119. Versican is involved in tumor growth in tissues such as breast210-213,  17  brain214, ovary215, gastrointestinal tract216, prostate217, 218, and melanoma219, Sarcoma210, and mesothelioma220. Versican can also inhibit nervous system regeneration and axonal growth following an injury to the central nervous system221-223,176, 224. 1.2.3 Structure and Interactions of Versican with Other Matrix Components The N-terminal (G1) globular domain consists of Ig-like loop and two link module, and has HA binding properties. The N-terminal of versican is thought to be involved in maintaining the integrity of the ECM by interacting with hyaluronan (HA)163.  Its  interactions with the link protein has also been studied164. Hyaluronan plays an important role in homeostasis and its expression is increased in epidermal injury225, 226, along with its cell surface receptor CD44227. Formation of versican-hyaluronan complex at the cell surface is essential for proliferation and migration of smooth muscle cells in vitro 59, 60. Versican-hyaluronan pericellular coat also promotes cancer cell motility228. Hyaluronan is required for matrix expansion and initiation of cell migration in the developing heart229, 230. The heart defects in the hyaluronan synthase-2 (has-2), which is the most widely expressed hyaluronan synthase during mid-gestation in the mouse230, are very similar to that of the hdf mutant (versican deficient) mouse231. This is a good indication that both versican and HA are necessary in the migration of cardiac NCC. The C-terminal (G3) globular domain consists of one or two EGF repeats, a C-type lectin domain and complement regulatory protein (CRP)-like domain. In recent years, a direct role for versican in cell proliferation has been suggested based on the interaction of its C-terminal EGF modules with the cell surface EGF-receptors232. The C-terminal domain also binds a variety of ligands in ECM which contribute significantly to the functions of versican. One important family of ligands is the tenascin family which due to their complex multi-domain structure can possess both growth promoting and inhibitory activities on the same molecule233, and are reported to have anti-adhesive properties234. A study of tenascin knockout mouse identified tenascin as a significant player in corneal wound healing under mechanical stress235. The C-lectin domain of versican interacts with tenascin-R through its fibronectin type III (FnIII) repeat 3-5 domain in a calcium dependant manner, in vivo236 and in vitro237. Different tenascin domains interact with a  18  wide range of cellular receptors, including integrins, cell adhesion molecules and members of the syndecan and glypican proteoglycan families233. Tenascin-C, a major versican binding partner during tissue repair238, is a transient glycoprotein that promotes fibroblast migration and differentiation in injured tissue239 and at the tumor invasion front of cancers240.  Full length tenascin-C also promotes fibroblast migration within  fibrin-fibronectin 3D matrices241, and tenascin and fibronectin promote corneal fibroblast migration and adhesion242.  Yet another C-terminal binding partner for versican,  fibronectin, plays an important role in cellular proliferation and migration243 modulates cell-matrix interactions during tissue repair  247  .  244-246  and  ED-A fibronectin splice  variant, a crucial factor in myofibroblast differentiation by TGF-β1, is a major component of myofibroblast matrix49. ED-A fibronectin is expressed by fibroblastic cells in culture248 and vascular SMC in vivo and in vitro249. Fibulin is another ligand for versican’s C-lectin domain237, a protein whose expression is associated with that of versican in the developing heart.  Fibulin is also implicated in wound healing and  development250. The C-lectin domain of Versican also interacts with fibrillin-1251. The interaction with fibrillin maintains the structural integrity of the matrix and links extracellular microfibrils to other connective tissue networks251. The central GAG-binding domain of versican is decorated with chondroitin sulfate glycosaminoglycans (GAGs).  The structural and functional diversity of versican is  increased by variations in GAG sulfation patterns and the type of GAG chains bound to the core protein. Although there is only a single versican gene, alternative splicing of its mRNA produces 4 distinct versican isoforms that differ in their potential number of GAG chains184, 185 (Figure 1.3). All isoforms have homologous N-terminal (HA binding) and C-terminal (lectin-like) domains. The central domain of versican V0 contains both the GAG-α and GAG-β domains. V1 isoform has the GAG-β domain, V2 has the GAG-α domain, and V3 is void of any GAG attachment domains, and only consists of the N-terminal and C-terminal globular domains. The GAGs, being composed of repeating disaccharide units161, contribute to the negative charge and many other properties of versican.  The binding of versican with leukocyte adhesion molecules L-selectin,  P-selectin, and CD44 is mediated by the interaction of CS chains of versican with the  19  carbohydrate-binding domain of these molecules167. Both CD44 and L-selectin have been implicated in leukocyte trafficking252, 253. The ability of versican to bind a large panel of chemokines and the biological consequences of such binding has also been examined207. It has been suggested that versican can bind specific chemokines through its CS chains and this interaction down-regulates the chemokines function207. Recently, in lights of results that V1 and V2 isoforms of versican have opposite effects on cell proliferation,  glycosaminoglycan  domain  GAG-β  has  been  implicated  in  versican-enhanced cell proliferation and versican-induced reduction of cell apoptosis254.  20  GAG-α  V0  GAG-β  N-Terminal  C-Terminal  GAG-β  V1  C-Terminal  N-Terminal  GAG-α  V2  N-Terminal  C-Terminal  Immunoglobulin type repeat 2 Link protein type modules 2 EGF-like modules  V3  N-Terminal C-Terminal  C-type Lectin like module Complement regulatory protein Chondroitin sulfate chains Glycosaminoglycan binding region - α Glycosaminoglycan binding region - β  Figure 1.3 Structure of Versican Splice Variants (V0, V1, V2, V3) 1.2.4 Versican Function in Pulmonary Fibrosis Many functions of versican have already been discussed in the context of its tissue specific expression pattern in development or disease, the interactions with its binding partners, and in case of gene deletions that influenced versican’s expression and cell function. It is becoming more apparent that versican affects cell and tissue function in a number of different ways. In addition to serving as a structural molecule affecting mechanics of tissue, versican can interact with a number of different ECM ligands and  21  possibly with the cell surface and thus regulate processes such as cell adhesion, proliferation, and migration. Versican’s ability to bind hyaluronan and form highly hydrated, supra-molecular aggregates  255  , can contribute to swelling pressure and thus influence the mechanical  properties of matrices such as blood vessel walls or remodeling tissue which are under pressure. This function of versican resembles the role of its family member, aggrecan, in resisting compressive forces put on cartilage. In the lungs, degradation of human airway smooth muscle-associated matrix is associated with decreased passive tension and alterations in smooth muscle contractility256. The large size (>1000 kDa) and hydration capability of versican, may also sterically hinder the interaction of integrins (large family of cell adhesion molecules) with their cell surface receptors202,  257  .  Along with its  anti-adhesive binding partners, hyaluronan and tenascin-C, versican is expressed in the active firboproliferative lesions of the remodeling lung and may additionally function as a scaffold on which growth factors and chemokines concentrate. Versican association with proliferating cells has been observed in a number of tissues and cell types, including atherosclerotic lesions258, stromal reaction to tumors211, proliferating vascular smooth muscle cells259, contractile ∝-SMA positive cells, and elastic fibers in the airway of normal lung. The spatial and temporal association of large splice-variants of versican, namely V0 and V1, with proliferating and contracting myofibroblasts (as defined by ∝-SMA and collagen type-1 expression) has also been observed in all fibrotic lung diseases119. Versican can be found in association with fibroblasts that migrate into airspaces, the hyperplastic epithelium, and in the alveolar wall thickening in the very early stage of idiopathic pulmonary fibrosis. Research shows that versican may play a direct and an indirect role in cell proliferation. Indirectly, interactions of G1 domain of versican with HA results in the formation of a pericellular matrix that is required for the proliferation of arterial smooth muscle cells259. Dissolution of the pericellular matrix by treatment of the cells with HA oligosaccharides inhibits SMC proliferation259. CD44, the main cell receptor for hyaluronan, also interacts  22  with versican through its chondroitin-sulfate chains167, and versican interaction may complement or modulate CD44 mediated adhesion and migration. Evidence for a direct role of versican in cellular proliferation is more controversial, and relies upon vector-driven overexpression of versican constructs or deletion of segments within such constructs (Comprehensively examined in Chapter 2 of this thesis). For example, an engineered chimeric molecule named “mini-versican” has been shown to modestly stimulate NIH 3T3 cell proliferation through its EGF-like modules in the G3 domain232. Deletion of the G3 domain or the EGF-like repeats eliminated the effect of overexpression or addition of versican products on cell proliferation. This group has also reported that addition of antisense against EGF receptor could block the effect of added versican, although versican has not been shown to bind or activate the EGF receptor. In contrast, studies of rat SMC that were retrovirally transduced to express versican V3, which lacks the GAG binding domains, showed decreased cell proliferation and migration and increased cell adhesion260. It has been suggested that the effects of V3 on SMC indicate that over-expression of versican G3 domain constructs do not universally promote cell proliferation. Effects of V3 on cell behavior may be as a result of competition for binding to cell surface-associated endogenous versican (V0/V1) ligands, such as HA87. This suggestion is supported by the observation that the formation of the HA-versican V0 and V1 pericellular matrix is inhibited in cells that express versican V3. This implies that the anti-adhesive chondroitin sulfate GAG chains of V0 and V1 versican isoforms may be most influential in regulating cellular phenotype through binding growth factors, inhibiting cell-cell cell-matrix interactions, or influencing the mechanical properties of the matrix. The precise roles of versican in wound healing and fibrosis in the lung remain open to further investigation, and I hope my present research sheds some light on the cellular biology and biochemistry of versican in association with myofibroblast cell type.  23  1.3 Metalloproteinases There are eight clans and forty families in the Metalloproteinase class based on the three dimensional protein folding and evolutionary relations275. These endopeptidases depend on a zinc or calcium ion in their active site, and include the metzincins (serralysins, astacins, adamalysins) and matrixins (matrix metalloproteinases or MMPs) family. Considering the increasing interest on the function of adamalysins (ADAM – a disintegrin and metalloproteinase domain) and matrixins in recent years, my review will focus on these two families of enzymes. There are more than 20 matrix metalloproteinases known to this day, all of which depend on zinc at their active site for catalytic function274. The common names for MMPs are based on their substrate specificity, and thus traditionally, MMPs are classified as “collagenases”, “gelatinases”, “stromelysin”, “membrane-type MMPs”, and “other MMPs”. All of the members of this family contain a consensus motif of three histidines (HExxHxxGxxH) that bind zinc at the catalytic site and a conserved “Met-turn” motif below the active site zinc276. Most MMPs contain five basic domains: a pre-domain or signal sequence to direct secretion from the cell, a latency or pro-domain, a zinc-binding catalytic domain, and a hinge region followed by a hemopexin domain with sequence homology to both hemopexin, a plasma heme-binding protein, and vitronectin, a cell adhesive protein. The propeptide region contains the ‘cysteine switch’, within the sequence PRCG(V/N)PD, in which the cysteine residue binds the catalytic Zn2+ ion in the proenzyme. Latency is maintained through coordination of the active site zinc with the thiol of this conserved cysteine in the pro-domain277. ADAMs contain a disintegrin or integrin-binding domain, and a metalloproteinase domain that is similar to the conserved MMP zinc-binding catalytic domain396. The primary function of these transmembrane proteases is to cleave extracellular domains of many membrane proteins from the cell surface396,  397  . ADAMTS (a disintegrin and  metalloproteinase with thrombospondin motifs) is a recently described family of proteinases closely related to the ADAM family, except that the ADAMTSs are secreted enzymes capable of binding to the ECM398, 399. Several members of the ADAMTS have  24  been shown to cleave versican N-terminal domain, leading to the generation of the well-known glial hyaluronate-binding protein (GHAP) among other fragments. GHAP was first identified as a 60kDa hyaluronate-binding fragment in the brain white matter, with protein sequence identity to the hyaluronate-binding region of versican400, 401. In this context, it was suggested to be a naturally occurring versican degradation product, possibly generated by the action of MMP-1, MMP-2, and/or MMP-3402 on the V1 versican variant.  With the knowledge that V2 is the major versican variant of the  brain403, it was shown that native GHAP is actually generated from versican V2 core by digestion with ADAMTS-4 and that the cleavage site is at Glu405-Gln406 in the GAG-alpha domain of versican V2404.  This discovery followed similar results of  versican V0 and V1 degradation by ADAMTS-1 and -4 in aorta405, and others have shown versican N-terminal cleavage by ADAMTS-9406 and ADAMTS-1 in atherosclerotic lesions407. 1.3.1 Matrix Metalloproteinases There are some differences within the family between individual MMPs. The structures of three members of this family, that will be discussed extensively in this thesis, namely MMP-2 MMP-7 and MMP-12 are illustrated in the following figure (Figure 1.4). Gelatinase-A (MMP-2)  Signal peptide Propeptide Catalytic domain  Metalloelastase (MMP-12)  Fibronectin type II modules Hemopexin domain  Matrilysin (MMP-7)  Zn2+ binding domain Linker peptide  Figure 1.4 Structure of MMP-2, MMP-12, and MMP-7 There are notable differences in the structure of these three MMPs. MMP-7 is one of the few MMPs that is synthesized without a hemopexin-like C-terminal domain, and thus it is comparably small in size. Although the hemopexin-C domain is void of any catalytic activity, it plays an important role in substrate recognition, conferring specificity, and in  25  binding to the tissue inhibitors of metalloproteinases (TIMPs)278. MMP-2, a member of the gelatinase subgroup, contains three additional fibronectin-type II modules which are hydrophobic in nature and are thought to be involved in substrate binding279, 280. The most important difference is, however, among the catalytic domains of MMPs and the size of the S1’ specificity pocket, which will be discussed in more detail for each of MMP-7, MMP-12 and MMP-2 in the sections that follow. Proteolysis of the extracellular matrix and cell surface molecules is a critical requirement in processes such as morphogenesis, regeneration, tumorigenesis, and wound healing. The family of matrix metalloproteinases (MMPs) plays a major role in remodeling of ECM in many physiological processes261-263 such as embryonic development264,  265  ,  skeletal growth266 and ovulation267. In disease, the imbalance of these proteases and their corresponding inhibitors, tissue inhibitor of metalloproteinases (TIMPS), could result in the break down of ECM and development of pathological diseases such as cancer invasion and metastasis268, arthritis269, and cardiovascular diseases270. MMPs promote tumor progression and metastasis in invasive cancers by degrading ECM comprised of many proteins such as laminin-5, proteoglycans, entactin, osteonectin, and collagen IV. In addition to their role in matrix degradation, recent research has shown that MMPs are capable of performing highly specific and limited cleavages of a number of bioactive molecules to modulate many aspects of cell behavior (reviewed in271, 100, 272) such as cell proliferation, differentiation, migration, apoptosis, angiogenesis, bone morphogenesis and the immune response272, 273. The proteolytic activity of MMPs is strictly regulated through a number of mechanisms. Their expression is controlled at the level of transcription, post-transcriptional modification, and protein secretion281 by several growth factors and cytokines. Among other growth factors, PDGF is a strong inducer of MMP expression in many cell types, while TGF-β can both induce and suppress MMP expression282.  The extracellular  matrix can also regulate MMP gene expression through binding cell receptors and activating intracellular signaling pathways. For example, collagen type I stimulates the expression of MMP-1283, while fragments of laminin-5284 and fibronectin285 upregulate MMP-1 and MMP-13 respectively.  Tenascin-fibronectin complexes also stimulate  26  secretion of MMPs by fibroblasts131. Proenzyme activation and release from latency, and the action of natural tissue inhibitors of MMPs (TIMPs) provide other means of regulating MMPs286-288. Another important regulatory mechanism is how these enzymes are anchored outside the cell. Anchoring MMPs to the cell surface or extracellular matrix could not only provide a reservoir by preventing them from rapidly diffusing away, but also enable the cell to keep them under close regulatory control109. In vivo study of wound healing in different tissues have implicated MMPs and their natural tissue inhibitors (TIMPs) in normal wound repair (Reviewed in 100). Multiple cell types including fibroblasts, keratinocytes, and macrophages release MMPs at the wound site and contribute to ECM remodeling289. The expression of most MMPs and TIMPs are perturbed among cells involved in skin repair290-293. MMPs are also induced in a number diseases that leads to pulmonary fibrosis.  Levels of MMP-8294, MMP-2 and  MMP-9295-297 are all elevated in human idiopathic pulmonary fibrosis. Cancer drug, bleomycin, can induce MMP-2, MMP-9, and MMP-12123,  298  in the lungs. Epithelial  derived MMP-7 is also highly upregulated in human idiopathic pulmonary fibrosis299. Recently, it was shown that the levels of MMP-2, MMP-7, MMP-9, MMP-12 and MMP-13 increase in a pulmonary fibrosis model caused by asbestos injury, and it was suggested that MMP inhibition protects against asbestos-induced fibrosis300. We have also studied MMP expression in lung fibrosis, and based on the results of our research and findings of others will focus here on three of these enzymes, namely MMP-7, MMP-12 and MMP-2. 1.3.2 MMP-7 (Matrilysin) MMP-7, also known as matrilysin, is the smallest of the MMPs which was first identified and purified from involuting rat uterus301, 302. The molecular weights of the latent and active form of this enzyme are 28 kDa and 19 kDa, respectively303. MMP-7 has a simple structure consisting of a propeptide and a catalytic domain, and lacks a hemopexin domain which is rather a rarity among members of the MMP family. Since MMP-7 has a limited sized substrate pocket304, the enzyme prefers residues with aliphatic or aromatic side chain305. This enzyme is responsible for the degradation of an array of molecules  27  (complete list306), including components of basement membrane such as collagen IV, entactin307, fibronectin and laminin, and components of extracellular matrix such as aggrecan308, versican104, proteoglycan link protein309, fibulin-2310, and tenascin-C. In fact, MMP-7 is a more potent proteoglycanase than other MMPs, including Stromelysin-1309 307 or MMP-2104. It can also activate proMMP-2311-313 and is involved in the auto-cleavage of proMMP-7314, 315. In adult human lung, MMP-7 is an epithelial cell product that tends to be released lumenally316, 317 in peribronchial and conducting airways. MMP-7 is also produced by blood monocytes318 and by tissue macrophages in atherosclerotic lesions104. Besides tissue-specific expression of MMP-7, specific cell surface interactions such as binding of MMP-7 to cell-surface proteoglycans109 localizes MMP-7 even further. Such anchoring of proMMP-7 to either cell surface or nearby basement membrane would retain the enzyme near the cell for proteolytic activation. This regulated compartmentalization may allow the proteinase to serve multiple functions by acting on spatially distinct substrates. Although there are no known functions for MMP-7 during embryonic development319, 320, its expression in healthy adult tissue signifies its role in tissue homeostasis and innate immunity.  All tissues with constitutive expression of MMP-7 are open to the  environment and vulnerable to bacterial exposure. For example, expression of MMP-7 by exocrine and mucosal epithelial cells lining peribronchial glands and conducting airways316 is upregulated in response to bacterial exposure in the adult lung317. MMP-7 can also activate the pro-form of α-defensins321, a class of secreted antimicrobial peptides expressed by specialized epithelial cells in small intestine321 which can kill bacteria by membrane disruption322. The full capacity of MMP-7 in wound healing is perhaps best observed in knock-out mutant mice phenotype. The mutant mouse phenotype is not lethal, though it does present innate immunity defects in response to bacteria321, inability to repair mucosal epithelial wounds due to decreased re-epithelialization after lung injury316, and reduced tumorigenesis323. Let us look at each of these phenotypes in more detail. In MMP-7  28  knockout mice, absence of the active form of α-defensins otherwise processed by MMP-7, results in impaired bacteriocidal activity321. Also in the absence of MMP-7, neutrophils migration past the epithelial barrier is attenuated due to a lack of the neutrophil attractant chemokine (Keratinocyte-derived chemokine or CXCL1) in the fluid of the alveolar lumens324. Another role of MMP-7 is in re-epithelialization in the injured lungs316, 325 and other mucosal epithelia in stomach and intestinal ulcers, injured epithelial cells in the kidney, and basal epithelial cells during corneal wound healing328-330. The process of re-epithelialization of tracheal wounds is almost completely abrogated due to impaired cleavage of E-cadherin and thus epithelial migration in mice lacking MMP-7316, 325. On the other hand, over-expression of constitutively active MMP-7 in lung epithelial cells in vitro leads to E-cadherin shedding and increased epithelial cell migration325. Although expression of MMP-7 in adult human tissue is clearly regulated by bacterial exposure, the production of this enzyme is also induced in response to injury. Impaired airway epithelial cell differentiation in a human tracheal xenograft model has confirmed a role for this MMP during later stages of wound healing, when epithelial cell differentiation occurs333. Expression of MMP-7 is also seen in migrating airway and alveolar epithelial cells at sites of overt damage in several lung diseases such as emphysema, lungs with diffuse alveolar damage, idiopathic pneumonia syndrome, and cystic  fibrosis316.  MMP-7  is  upregulated  during  the  fibrotic  phase  in  bleomycin-induced299, 326, silica-induced327, and asbestos-induced models of pulmonary fibrosis300. It has been suggested that, at this capacity, MMP-7 may contribute to further inflammation by regulating chemokine activity and establishing a chemotactic gradient, thus inducing influx of neutrophils into the lung and airspaces324.  This is mediated by  the cleavage of syndecan-1, which releases CXCL1 bound to the heparan sulfate glycosaminoglycan chains on syndecan-1, thus establishing a chemotactic gradient for neutrophils316, 324. The mechanism of how matrilysin facilities repair is not known. In the absence of bacteria, wound-induced matrilysin is released basally towards the underlying matrix, suggesting that matrilysin may act on components of extracellular matrix. Furthermore,  29  matrilysin may not be the only MMP involved in repair of airway epithelial wounds331, 332. MMP-2 is also expressed by injured epithelial cells in distal airways, and deficiency of this MMP leads to excessive bronchiolization334. In addition, the activity of MMP-2 is required for the migration of isolated airway epithelial cells over a matrix substratum335. Several MMPs may thus act concurrently on different substrates to facilitate repair. 1.3.3 MMP-12 (Macrophage Metalloelastase) MMP-12, also known as macrophage metalloelastase, was first identified as an elastolytic metalloproteinase secreted by inflammatory macrophages 30 years ago336,  337  .  As a  macrophage metalloeslastase, its expression is appropriately restricted to macrophages338. MMP-12 has a general structure and is composed of a propeptide which maintains enzyme latency, a catalytic zinc binding domain, the linker region, and a hemopexin-like module which is involved in substrate specificity. The latent enzyme (54 kDa) is self-activating and produces the 45 kDa and 22 kDa active forms of the enzyme after autolytic processing339,  340  .  The major substrate for MMP-12 is elastin, a highly  proteinase-resistant molecule abundant in the lung and arterial wall341 that normally lasts a life time342.  MMP-12 is also capable of degrading a broad spectrum of other  extracellular matrix components, including type IV collagen, fibronectin, laminin, vitronectin, proteoglycans, chondroitin sulfate, and myelin basic protein338 (reviewed in306). In addition to degrading its extracellular substrates, another important function of active MMP-12 in vivo is its ability to activate other MMPs such as MMP-2 and MMP-3, through which MMP-12 may exaggerates the cascade of proteolytic processes343 or play a role in innate immunity344. Abnormal regulation of MMP-12 expression has been implicated in abdominal aortic aneurysm345, atherosclerosis346, and emphysema347. Studies in MMP-12 mutant mice show key roles for this enzyme in tissue invasion and extracellular degradation338.  Although not lethal, MMP-12 deficiency results in  diminished recovery from spinal cord injury348, increased angiogenesis due to decreased angiostatin349, delayed wound repair in cut ligament due to an inability to recruit macrophages to the injured site350, reduced elastolytic activity by macrophages341,  351  ,  30  reduced ability of macrophages to migrate through the matrix341, protection from smoke induced emphysema347. expression after lung injury in bleomycin  , and reduced  Other studies have also implicated  MMP-12 in the development of emphysema347, 123, 298  351  352  , and have reported increased  and asbestos treated fibrotic lung300.  Collagen replaces the elastin content of the lung parenchyma in emphysematous patients353.  Several other MMPs are also produced in human emphysema354,  355  .  For example, the expression of MT1-MMP, involved in activation of MMP-2, and the expressin of MMP-2 markedly increase in pneumocytes, fibroblasts and alveolar macrophages of emphysematous lung355. 1.3.4 MMP-2 (Gelatinase A) The nascent form of MMP-2, also known as gelatinase A, contains an N-terminal signal sequence (pre-domain) followed by a pro-domain that maintains enzyme-latency, and a catalytic domain that contains the conserved zinc-binding region. A hemopexin and vitronectin-like domain is connected to the catalytic domain by a hinge or linker region. The hemopexin domain is involved in TIMP binding356 and membrane activation in the case of MMP-2. Though unlike other MMPs, the hemopexin C domain of MMP-2 is not involved in substrate binding358, 359. Instead, the three fibronectin type II modules within the catalytic domain which resemble the collagen-binding type II repeats of fibronectin bind and cleave denatured collagen360. MMP-2 proteolytically digests gelatin103 (denatured collagen) better than other MMPs, and is capable of degrading collagen type IV, V, VII, IX and X, and several chemokines including CCL7 and CXCL12357. The regulation of MMP-2 activity occurs at many levels. Pro-MMP-2 activation is seen by complex signaling induced by ECM proteins like osteopontin, various cytokines, and other factors. Specific cell–MMP interactions, such as the binding of MMP-2 to the integrin αvβ3107, have also been reported in recent years. Another intricate regulatory mechanism is the interaction of latent MMP-2 with tissue inhibitor of metalloproteinases (TIMP)-2 and MT1-MMP on the cell surface, and formation of a trimeric complex, essential for activation of this gelatinase361, 362. Physiologically, MMP-2 plays a role in normal tissue remodeling events such as 31  embryonic development, angiogenesis363,  364  , ovulation365, mammary gland involution  and wound healing. MMP-2 knockout mice, although not lethal, exhibit reduced body size366, reduced neovascularization364, decreased primary ductal invasion in the mammary gland367, reduced lung saccular development368, and reduced angiogenesis and tumor growth369. The role of MMP-2 in allergen induced inflammation is well documented. MMP-2 establishes chemokine gradients and recruits neutrophils and eosinophils, as observed with the immune cells recruited from the lung parenchyma into the airway370, 371. On the other hand, MMP-2 can also participate in a regulatory loop that dampens allergic inflammation (Reviewed in101). MMP-2 protects against inflammation of the brain and spinal cord372, and in asthma models, eosinophils from MMP2-deficient mice fail to migrate into the airways and accumulate in the interstitium370,  371  .  The interstitial  eosinophil accumulation have been explained by the disruption of transepithelial chemokine gradients.  MMP-2 also processes neutrophil- and macrophage-specific  chemokines that are found in the bronchioalveolar fluid of asthmatic mice373. In addition, truncation of macrophage-derived chemokine, CCL7, by MMP-2 results in the formation of peptides that can bind to the CC chemokine receptor and function as antagonists374, 375. In other experiments, a screen for MMP-2 substrates lead to the discovery of monocyte chemoattractant protein-3 (MCP-3), which when cleaved acts as a general chemokine antagonist and dampens inflammation376. Elevated expression of MMP-2 is usually seen in invasive and highly tumorigenic cancers such as colorectal tumors377, gastric carcinoma378, pancreatic carcinoma379,  380  ,  breast cancer381 and breast cancer metastasis to lungs382, oral cancer383, melanoma384-386, malignant gliomas387,  388  , Chondrosarcoma389, and osteosarcoma390. MMPs promote  tumor progression and metastasis in invasive cancers by degradation of the ECM, which consists of two main components: basement membranes and interstitial connective tissue. MMP-2 efficiently degrades collagen IV and laminin-5 which are components of the basement membrane, thereby assisting the metastatic cancerous cells to pass through it. MMP-2, also promotes angiogenesis369, a critical process required for tumor cell survival and restoration of healing wound.  32  MMP-2 is also considered as a key enzyme in tissue remodeling during inflammation and wound healing391,  392  .  MMP-2 deficient mice have reduced inflammation in their  airspaces compared to wild-type mice in an asthma model of lung disease, indicating a role for this MMP in pulmonary inflammation  371  . In addition, MMP-2 is expressed by  fibroblasts and has been hypothesized to play a role in the migration of fibroblasts in the lung (reviewed in393) which may contribute to fibrosis. MMP-2 can also have negative effects on cell proliferation (reviewed in100). For example, MMP-2 produced by cartilage from the trachea and bronchus decreases respiratory epithelial cell proliferation in vitro394 and addition of a broad spectrum MMP inhibitor rescues epithelial cell proliferation394. It has also been suggested that interaction between keratinocytes and fibroblasts, which occurs during wound healing, can regulate MMP-2 expression by these cells392. Although both keratinocytes and fibroblasts express MMP-2 in culture392, a co-culture of both keratinocytes and fibroblasts leads to increased MMP-2 expression. Due to prolonged and sustained MMP-2 expression during dermal wound repair, a role for MMP-2 in angiogenesis and matrix remodeling has also been suggested292, 300, 395. In an asbestos model of lung inflammation and fibrosis, it was shown that MMP-2 strongly associates with time points revealing development of fibrosis300, and fibroblast production of MMP-2 assists in fibroblast migration and perhaps fibrosis300. The importance of MMP-2 in cell proliferation and fibrosis suggests that roles for MMP-2, outside of inflammation, should not be overlooked.  33  1.4 References 1.  Diegelmann RF, Evans MC: Wound healing: an overview of acute, fibrotic and delayed healing, Front Biosci 2004, 9:283-289  2.  Broughton G, 2nd, Janis JE, Attinger CE: Wound healing: an overview, Plast Reconstr Surg 2006, 117:1e-S-32e-S  3.  Gabbiani G: The myofibroblast in wound healing and fibrocontractive diseases, J Pathol 2003, 200:500-503  4.  Hinz B: Formation and function of the myofibroblast during tissue repair, J Invest Dermatol 2007, 127:526-537  5.  Broughton G, 2nd, Janis JE, Attinger CE: The basic science of wound healing, Plast Reconstr Surg 2006, 117:12S-34S  6.  Agren MS, Werthen M: The extracellular matrix in wound healing: a closer look at therapeutics for chronic wounds, Int J Low Extrem Wounds 2007, 6:82-97  7.  Martinet Y, Rom WN, Grotendorst GR, Martin GR, Crystal RG: Exaggerated spontaneous release of platelet-derived growth factor by alveolar macrophages from patients with idiopathic pulmonary fibrosis, N Engl J Med 1987, 317:202-209  8.  Khalil N, O'Connor RN, Unruh HW, Warren PW, Flanders KC, Kemp A, Bereznay OH, Greenberg AH: Increased production and immunohistochemical localization of transforming growth factor-beta in idiopathic pulmonary fibrosis, Am J Respir Cell Mol Biol 1991, 5:155-162  9.  Broekelmann TJ, Limper AH, Colby TV, McDonald JA: Transforming growth factor beta 1 is present at sites of extracellular matrix gene expression in human pulmonary fibrosis, Proc Natl Acad Sci U S A 1991, 88:6642-6646  10.  Pierce GF, Vande Berg J, Rudolph R, Tarpley J, Mustoe TA: Platelet-derived growth factor-BB and transforming growth factor beta 1 selectively modulate glycosaminoglycans, collagen, and myofibroblasts in excisional wounds, Am J Pathol 1991, 138:629-646  11.  Papakonstantinou E, Karakiulakis G, Roth M, Block LH: Platelet-derived growth factor stimulates the secretion of hyaluronic acid by proliferating human vascular smooth muscle cells, Proc Natl Acad Sci U S A 1995, 92:9881-9885  12.  Schonherr E, Jarvelainen HT, Sandell LJ, Wight TN: Effects of platelet-derived growth factor and transforming growth factor-beta 1 on the synthesis of a large versican-like chondroitin sulfate proteoglycan by arterial smooth muscle cells, J Biol Chem 1991, 266:17640-17647  13.  Ross R, Raines EW, Bowen-Pope DF: The biology of platelet-derived growth factor, Cell 1986, 46:155-169  14.  Fukamizu H, Grinnell F: Spatial organization of extracellular matrix and fibroblast activity: effects of serum, transforming growth factor beta, and fibronectin, Exp Cell Res 1990, 190:276-282 34  15.  Sporn MB, Roberts AB, Roche NS, Kagechika H, Shudo K: Mechanism of action of retinoids, J Am Acad Dermatol 1986, 15:756-764  16.  Westergren-Thorsson G, Sarnstrand B, Fransson LA, Malmstrom A: TGF-beta enhances the production of hyaluronan in human lung but not in skin fibroblasts, Exp Cell Res 1990, 186:192-195  17.  Nikitovic D, Zafiropoulos A, Katonis P, Tsatsakis A, Theocharis AD, Karamanos NK, Tzanakakis GN: Transforming growth factor-beta as a key molecule triggering the expression of versican isoforms v0 and v1, hyaluronan synthase-2 and synthesis of hyaluronan in malignant osteosarcoma cells, IUBMB Life 2006, 58:47-53  18.  Roberts AB, McCune BK, Sporn MB: TGF-beta: regulation of extracellular matrix, Kidney Int 1992, 41:557-559  19.  Bassols A, Massague J: Transforming growth factor beta regulates the expression and structure of extracellular matrix chondroitin/dermatan sulfate proteoglycans, J Biol Chem 1988, 263:3039-3045  20.  Ignotz RA, Endo T, Massague J: Regulation of fibronectin and type I collagen mRNA levels by transforming growth factor-beta, J Biol Chem 1987, 262:64436446  21.  Goldman R: Growth factors and chronic wound healing: past, present, and future, Adv Skin Wound Care 2004, 17:24-35  22.  Hall MC, Young DA, Waters JG, Rowan AD, Chantry A, Edwards DR, Clark IM: The comparative role of activator protein 1 and Smad factors in the regulation of Timp-1 and MMP-1 gene expression by transforming growth factor-beta 1, J Biol Chem 2003, 278:10304-10313  23.  Massague J: The TGF-beta family of growth and differentiation factors, Cell 1987, 49:437-438  24.  Tomasek JJ, Gabbiani G, Hinz B, Chaponnier C, Brown RA: Myofibroblasts and mechano-regulation of connective tissue remodelling, Nat Rev Mol Cell Biol 2002, 3:349-363  25.  Darby I, Skalli O, Gabbiani G: Alpha-smooth muscle actin is transiently expressed by myofibroblasts during experimental wound healing, Lab Invest 1990, 63:21-29  26.  Kisseleva T, Brenner DA: Mechanisms of fibrogenesis, Exp Biol Med (Maywood) 2008, 233:109-122  27.  Rhee S, Grinnell F: Fibroblast mechanics in 3D collagen matrices, Adv Drug Deliv Rev 2007, 59:1299-1305  28.  Desmouliere A, Geinoz A, Gabbiani F, Gabbiani G: Transforming growth factorbeta 1 induces alpha-smooth muscle actin expression in granulation tissue myofibroblasts and in quiescent and growing cultured fibroblasts, J Cell Biol 1993, 122:103-111  29.  Hautmann MB, Madsen CS, Owens GK: A transforming growth factor beta (TGFbeta) control element drives TGFbeta-induced stimulation of smooth muscle  35  alpha-actin gene expression in concert with two CArG elements, J Biol Chem 1997, 272:10948-10956 30.  Goffin JM, Pittet P, Csucs G, Lussi JW, Meister JJ, Hinz B: Focal adhesion size controls tension-dependent recruitment of alpha-smooth muscle actin to stress fibers, J Cell Biol 2006, 172:259-268  31.  Arora PD, Narani N, McCulloch CA: The compliance of collagen gels regulates transforming growth factor-beta induction of alpha-smooth muscle actin in fibroblasts, Am J Pathol 1999, 154:871-882  32.  Hinz B, Mastrangelo D, Iselin CE, Chaponnier C, Gabbiani G: Mechanical tension controls granulation tissue contractile activity and myofibroblast differentiation, Am J Pathol 2001, 159:1009-1020  33.  Wipff PJ, Rifkin DB, Meister JJ, Hinz B: Myofibroblast contraction activates latent TGF-beta1 from the extracellular matrix, J Cell Biol 2007, 179:1311-1323  34.  Wipff PJ, Hinz B: Integrins and the activation of latent transforming growth factor beta1 - An intimate relationship, Eur J Cell Biol 2008,  35.  Annes JP, Munger JS, Rifkin DB: Making sense of latent TGFbeta activation, J Cell Sci 2003, 116:217-224  36.  Hyytiainen M, Penttinen C, Keski-Oja J: Latent TGF-beta binding proteins: extracellular matrix association and roles in TGF-beta activation, Crit Rev Clin Lab Sci 2004, 41:233-264  37.  Koli K, Hyytiainen M, Ryynanen MJ, Keski-Oja J: Sequential deposition of latent TGF-beta binding proteins (LTBPs) during formation of the extracellular matrix in human lung fibroblasts, Exp Cell Res 2005, 310:370-382  38.  Schultz-Cherry S, Chen H, Mosher DF, Misenheimer TM, Krutzsch HC, Roberts DD, Murphy-Ullrich JE: Regulation of transforming growth factor-beta activation by discrete sequences of thrombospondin 1, J Biol Chem 1995, 270:7304-7310  39.  Annes, JP, Chen, Y, Munger, JS, Rifkin, DB: Integrin alphaVbeta6-mediated activation of latent TGF-beta requires the latent TGF-beta binding protein-1, J Cell Biol 2004, 165: 723-734  40.  Werner S, Krieg T, Smola H: Keratinocyte-fibroblast interactions in wound healing, J Invest Dermatol 2007, 127:998-1008  41.  Malmstrom J, Lindberg H, Lindberg C, Bratt C, Wieslander E, Delander EL, Sarnstrand B, Burns JS, Mose-Larsen P, Fey S, Marko-Varga G: Transforming growth factor-beta 1 specifically induce proteins involved in the myofibroblast contractile apparatus, Mol Cell Proteomics 2004, 3:466-477  42.  Ronnov-Jessen L, Petersen OW: Induction of alpha-smooth muscle actin by transforming growth factor-beta 1 in quiescent human breast gland fibroblasts. Implications for myofibroblast generation in breast neoplasia, Lab Invest 1993, 68:696-707  36  43.  Stephenson LA, Haney LB, Hussaini IM, Karns LR, Glass WF, 2nd: Regulation of smooth muscle alpha-actin expression and hypertrophy in cultured mesangial cells, Kidney Int 1998, 54:1175-1187  44.  Zaleskas JM, Kinner B, Freyman TM, Yannas IV, Gibson LJ, Spector M: Growth factor regulation of smooth muscle actin expression and contraction of human articular chondrocytes and meniscal cells in a collagen-GAG matrix, Exp Cell Res 2001, 270:21-31  45.  Goldberg MT, Han YP, Yan C, Shaw MC, Garner WL: TNF-alpha suppresses alpha-smooth muscle actin expression in human dermal fibroblasts: an implication for abnormal wound healing, J Invest Dermatol 2007, 127:2645-2655  46.  Narani N, Arora PD, Lew A, Luo L, Glogauer M, Ganss B, McCulloch CA: Transforming growth factor-beta induction of alpha-smooth muscle actin is dependent on the deformability of the collagen matrix, Curr Top Pathol 1999, 93:47-60  47.  Sobral LM, Montan PF, Martelli-Junior H, Graner E, Coletta RD: Opposite effects of TGF-beta1 and IFN-gamma on transdifferentiation of myofibroblast in human gingival cell cultures, J Clin Periodontol 2007, 34:397-406  48.  Gu L, Zhu YJ, Guo ZJ, Xu XX, Xu WB: Effect of IFN-gamma and dexamethasone on TGF-beta1-induced human fetal lung fibroblast-myofibroblast differentiation, Acta Pharmacol Sin 2004, 25:1479-1488  49.  Serini G, Bochaton-Piallat ML, Ropraz P, Geinoz A, Borsi L, Zardi L, Gabbiani G: The fibronectin domain ED-A is crucial for myofibroblastic phenotype induction by transforming growth factor-beta1, J Cell Biol 1998, 142:873-881  50.  Lee RT, Yamamoto C, Feng Y, Potter-Perigo S, Briggs WH, Landschulz KT, Turi TG, Thompson JF, Libby P, Wight TN: Mechanical strain induces specific changes in the synthesis and organization of proteoglycans by vascular smooth muscle cells, J Biol Chem 2001, 276:13847-13851  51.  Le Bellego F, Plante S, Chakir J, Hamid Q, Ludwig MS: Differences in MAP kinase phosphorylation in response to mechanical strain in asthmatic fibroblasts, Respir Res 2006, 7:68  52.  Tamariz E, Grinnell F: Modulation of fibroblast morphology and adhesion during collagen matrix remodeling, Mol Biol Cell 2002, 13:3915-3929  53.  Marenzana M, Wilson-Jones N, Mudera V, Brown RA: The origins and regulation of tissue tension: identification of collagen tension-fixation process in vitro, Exp Cell Res 2006, 312:423-433  54.  Discher DE, Janmey P, Wang YL: Tissue cells feel and respond to the stiffness of their substrate, Science 2005, 310:1139-1143  55.  Yeung T, Georges PC, Flanagan LA, Marg B, Ortiz M, Funaki M, Zahir N, Ming W, Weaver V, Janmey PA: Effects of substrate stiffness on cell morphology, cytoskeletal structure, and adhesion, Cell Motil Cytoskeleton 2005, 60:24-34  37  56.  Hinz B, Gabbiani G: Cell-matrix and cell-cell contacts of myofibroblasts: role in connective tissue remodeling, Thromb Haemost 2003, 90:993-1002  57.  Wells PB, Thomsen S, Jones MA, Baek S, Humphrey JD: Histological evidence for the role of mechanical stress in modulating thermal denaturation of collagen, Biomech Model Mechanobiol 2005, 4:201-210  58.  Schonherr E, Kinsella MG, Wight TN: Genistein selectively inhibits plateletderived growth factor-stimulated versican biosynthesis in monkey arterial smooth muscle cells, Arch Biochem Biophys 1997, 339:353-361  59.  Evanko SP, Johnson PY, Braun KR, Underhill CB, Dudhia J, Wight TN: Plateletderived growth factor stimulates the formation of versican-hyaluronan aggregates and pericellular matrix expansion in arterial smooth muscle cells, Arch Biochem Biophys 2001, 394:29-38  60.  Ogawa H, Oohashi T, Sata M, Bekku Y, Hirohata S, Nakamura K, Yonezawa T, Kusachi S, Shiratori Y, Ninomiya Y: Lp3/Hapln3, a novel link protein that colocalizes with versican and is coordinately up-regulated by platelet-derived growth factor in arterial smooth muscle cells, Matrix Biol 2004, 23:287-298  61.  Desmouliere A, Rubbia-Brandt L, Grau G, Gabbiani G: Heparin induces alphasmooth muscle actin expression in cultured fibroblasts and in granulation tissue myofibroblasts, Lab Invest 1992, 67:716-726  62.  Haisa M, Okochi H, Grotendorst GR: Elevated levels of PDGF alpha receptors in keloid fibroblasts contribute to an enhanced response to PDGF, J Invest Dermatol 1994, 103:560-563  63.  Pierce GF, Tarpley JE, Tseng J, Bready J, Chang D, Kenney WC, Rudolph R, Robson MC, Vande Berg J, Reid P, et al.: Detection of platelet-derived growth factor (PDGF)-AA in actively healing human wounds treated with recombinant PDGF-BB and absence of PDGF in chronic nonhealing wounds, J Clin Invest 1995, 96:1336-1350  64.  Walker LN, Bowen-Pope DF, Ross R, Reidy MA: Production of platelet-derived growth factor-like molecules by cultured arterial smooth muscle cells accompanies proliferation after arterial injury, Proc Natl Acad Sci U S A 1986, 83:7311-7315  65.  Paulsson Y, Bywater M, Heldin CH, Westermark B: Effects of epidermal growth factor and platelet-derived growth factor on c-fos and c-myc mRNA levels in normal human fibroblasts, Exp Cell Res 1987, 171:186-194  66.  Mukaratirwa S, Koninkx JF, Gruys E, Nederbragt H: Mutual paracrine effects of colorectal tumour cells and stromal cells: modulation of tumour and stromal cell differentiation and extracellular matrix component production in culture, Int J Exp Pathol 2005, 86:219-229  67.  Heldin P, Laurent TC, Heldin CH: Effect of growth factors on hyaluronan synthesis in cultured human fibroblasts, Biochem J 1989, 258:919-922  38  68.  Grotendorst GR, Martin GR, Pencev D, Sodek J, Harvey AK: Stimulation of granulation tissue formation by platelet-derived growth factor in normal and diabetic rats, J Clin Invest 1985, 76:2323-2329  69.  Sprugel KH, McPherson JM, Clowes AW, Ross R: Effects of growth factors in vivo. I. Cell ingrowth into porous subcutaneous chambers, Am J Pathol 1987, 129:601-613  70.  Blatti SP, Foster DN, Ranganathan G, Moses HL, Getz MJ: Induction of fibronectin gene transcription and mRNA is a primary response to growth-factor stimulation of AKR-2B cells, Proc Natl Acad Sci U S A 1988, 85:1119-1123  71.  Canalis E: Effect of platelet-derived growth factor on DNA and protein synthesis in cultured rat calvaria, Metabolism 1981, 30:970-975  72.  Mishra R, Leahy P, Simonson MS: Gene expression profile of endothelin-1induced growth in glomerular mesangial cells, Am J Physiol Cell Physiol 2003, 285:C1109-1115  73.  Shi-Wen X, Chen Y, Denton CP, Eastwood M, Renzoni EA, Bou-Gharios G, Pearson JD, Dashwood M, du Bois RM, Black CM, Leask A, Abraham DJ: Endothelin-1 promotes myofibroblast induction through the ETA receptor via a rac/phosphoinositide 3-kinase/Akt-dependent pathway and is essential for the enhanced contractile phenotype of fibrotic fibroblasts, Mol Biol Cell 2004, 15:2707-2719  74.  Kawanabe Y, Okamoto Y, Nozaki K, Hashimoto N, Miwa S, Masaki T: Molecular mechanism for endothelin-1-induced stress-fiber formation: analysis of G proteins using a mutant endothelin(A) receptor, Mol Pharmacol 2002, 61:277-284  75.  Koyama Y, Yoshioka Y, Matsuda T, Baba A: Focal adhesion kinase is required for endothelin-induced cell cycle progression of cultured astrocytes, Glia 2003, 43:185189  76.  Klonowski-Stumpe H, Reinehr R, Fischer R, Warskulat U, Luthen R, Haussinger D: Production and effects of endothelin-1 in rat pancreatic stellate cells, Pancreas 2003, 27:67-74  77.  Tokuriki S, Ohshima Y, Yamada A, Ohta N, Tsukahara H, Mayumi M: Leukotriene D(4) enhances the function of endothelin-1-primed fibroblasts, Clin Immunol 2007, 125:88-94  78.  Tomasek JJ, Vaughan MB, Kropp BP, Gabbiani G, Martin MD, Haaksma CJ, Hinz B: Contraction of myofibroblasts in granulation tissue is dependent on Rho/Rho kinase/myosin light chain phosphatase activity, Wound Repair Regen 2006, 14:313-320  79.  Kernochan LE, Tran BN, Tangkijvanich P, Melton AC, Tam SP, Yee HF, Jr.: Endothelin-1 stimulates human colonic myofibroblast contraction and migration, Gut 2002, 50:65-70  39  80.  Shephard P, Hinz B, Smola-Hess S, Meister JJ, Krieg T, Smola H: Dissecting the roles of endothelin, TGF-beta and GM-CSF on myofibroblast differentiation by keratinocytes, Thromb Haemost 2004, 92:262-274  81.  Tanaka Y, Sung KC, Tsutsumi A, Ohba S, Ueda K, Morrison WA: Tissue engineering skin flaps: which vascular carrier, arteriovenous shunt loop or arteriovenous bundle, has more potential for angiogenesis and tissue generation? Plast Reconstr Surg 2003, 112:1636-1644  82.  Toeda K, Nakamura K, Hirohata S, Hatipoglu OF, Demircan K, Yamawaki H, Ogawa H, Kusachi S, Shiratori Y, Ninomiya Y: Versican is induced in infiltrating monocytes in myocardial infarction, Mol Cell Biochem 2005, 280:47-56  83.  Vyalov S, Desmouliere A, Gabbiani G: GM-CSF-induced granulation tissue formation: relationships between macrophage and myofibroblast accumulation, Virchows Arch B Cell Pathol Incl Mol Pathol 1993, 63:231-239  84.  Chen G, Grotendorst G, Eichholtz T, Khalil N: GM-CSF increases airway smooth muscle cell connective tissue expression by inducing TGF-beta receptors, Am J Physiol Lung Cell Mol Physiol 2003, 284:L548-556  85.  Rubbia-Brandt L, Sappino AP, Gabbiani G: Locally applied GM-CSF induces the accumulation of alpha-smooth muscle actin containing myofibroblasts, Virchows Arch B Cell Pathol Incl Mol Pathol 1991, 60:73-82  86.  Xing Z, Tremblay GM, Sime PJ, Gauldie J: Overexpression of granulocytemacrophage colony-stimulating factor induces pulmonary granulation tissue formation and fibrosis by induction of transforming growth factor-beta 1 and myofibroblast accumulation, Am J Pathol 1997, 150:59-66  87.  Kinsella MG, Bressler SL, Wight TN: The regulated synthesis of versican, decorin, and biglycan: extracellular matrix proteoglycans that influence cellular phenotype, Crit Rev Eukaryot Gene Expr 2004, 14:203-234  88.  Wight TN: Versican: a versatile extracellular matrix proteoglycan in cell biology, Curr Opin Cell Biol 2002, 14:617-623  89.  Wu Y, Sheng W, Chen L, Dong H, Lee V, Lu F, Wong CS, Lu WY, Yang BB: Versican V1 isoform induces neuronal differentiation and promotes neurite outgrowth, Mol Biol Cell 2004, 15:2093-2104  90.  Zizola CF, Julianelli V, Bertolesi G, Yanagishita M, Calvo JC: Role of versican and hyaluronan in the differentiation of 3T3-L1 cells into preadipocytes and mature adipocytes, Matrix Biol 2007, 26:419-430  91.  Williams DR, Jr., Presar AR, Richmond AT, Mjaatvedt CH, Hoffman S, Capehart AA: Limb chondrogenesis is compromised in the versican deficient hdf mouse, Biochem Biophys Res Commun 2005, 334:960-966  92.  Kamiya N, Watanabe H, Habuchi H, Takagi H, Shinomura T, Shimizu K, Kimata K: Versican/PG-M regulates chondrogenesis as an extracellular matrix molecule crucial for mesenchymal condensation, J Biol Chem 2006, 281:2390-2400  40  93.  Bensadoun ES, Burke AK, Hogg JC, Roberts CR: Proteoglycan deposition in pulmonary fibrosis, Am J Respir Crit Care Med 1996, 154:1819-1828  94.  Bensadoun ES, Burke AK, Hogg JC, Roberts CR: Proteoglycans in granulomatous lung diseases, Eur Respir J 1997, 10:2731-2737  95.  Roberts CR, Burke AK: Remodelling of the extracellular matrix in asthma: proteoglycan synthesis and degradation, Can Respir J 1998, 5:48-50  96.  Desmouliere A, Redard M, Darby I, Gabbiani G: Apoptosis mediates the decrease in cellularity during the transition between granulation tissue and scar, Am J Pathol 1995, 146:56-66  97.  Skalli O, and Gabbiani, G.: The biology of the myofibroblast in relationship to wound contraction and fibrocontractive disease. Edited by Henson RAFCaPM. New York, Plenum, 1988, p. pp. 373-402  98.  Rudolph R, Berg, J. V., and Ehrlich, H. P.: Wound contraction and scar contracture. Edited by I. K. Cohen RFD, and W. J. Lindbald. Philadelphia, Saunders, 1992, p. pp. 96-114  99.  Grinnell F, Zhu M, Carlson MA, Abrams JM: Release of mechanical tension triggers apoptosis of human fibroblasts in a model of regressing granulation tissue, Exp Cell Res 1999, 248:608-619  100. Gill SE, Parks WC: Metalloproteinases and their inhibitors: Regulators of wound healing, Int J Biochem Cell Biol 2007, 40:1334-1347 101. Page-McCaw A, Ewald AJ, Werb Z: Matrix metalloproteinases and the regulation of tissue remodelling, Nat Rev Mol Cell Biol 2007, 8:221-233 102. Parks WC, Shapiro SD: Matrix metalloproteinases in lung biology, Respir Res 2001, 2:10-19 103. Mackay AR, Hartzler JL, Pelina MD, Thorgeirsson UP: Studies on the ability of 65-kDa and 92-kDa tumor cell gelatinases to degrade type IV collagen, J Biol Chem 1990, 265:21929-21934 104. Halpert I, Sires UI, Roby JD, Potter-Perigo S, Wight TN, Shapiro SD, Welgus HG, Wickline SA, Parks WC: Matrilysin is expressed by lipid-laden macrophages at sites of potential rupture in atherosclerotic lesions and localizes to areas of versican deposition, a proteoglycan substrate for the enzyme, Proc Natl Acad Sci U S A 1996, 93:9748-9753 105. Shapiro SD: Elastolytic metalloproteinases produced by human mononuclear phagocytes. Potential roles in destructive lung disease, Am J Respir Crit Care Med 1994, 150:S160-164 106. Gronski TJ, Jr., Martin RL, Kobayashi DK, Walsh BC, Holman MC, Huber M, Van Wart HE, Shapiro SD: Hydrolysis of a broad spectrum of extracellular matrix proteins by human macrophage elastase, J Biol Chem 1997, 272:12189-12194 107. Brooks PC, Stromblad S, Sanders LC, von Schalscha TL, Aimes RT, StetlerStevenson WG, Quigley JP, Cheresh DA: Localization of matrix metalloproteinase  41  MMP-2 to the surface of invasive cells by interaction with integrin alpha v beta 3, Cell 1996, 85:683-693 108. Yu Q, Stamenkovic I: Cell surface-localized matrix metalloproteinase-9 proteolytically activates TGF-beta and promotes tumor invasion and angiogenesis, Genes Dev 2000, 14:163-176 109. Yu WH, Woessner JF, Jr.: Heparan sulfate proteoglycans as extracellular docking molecules for matrilysin (matrix metalloproteinase 7), J Biol Chem 2000, 275:4183-4191 110. Kawanami O, Ferrans VJ, Crystal RG: Anchoring fibrils in the normal canine respiratory system, Am Rev Respir Dis 1979, 120:595-611 111. Olsen BRaN, Y.: Basement membrane collagens (type IV). Edited by Vale TKaR. Oxford, Oxford University Press, 1993, p. pp. 32-48 112. Wasano K, Yamamoto T: Microthread-like filaments connecting the epithelial basal lamina with underlying fibrillar components of the connective tissue in the rat trachea. A real anchoring device? Cell Tissue Res 1985, 239:485-495 113. Yurchenco PD, Ruben GC: Basement membrane structure in situ: evidence for lateral associations in the type IV collagen network, J Cell Biol 1987, 105:25592568 114. van der Rest M, Garrone R: Collagen family of proteins, Faseb J 1991, 5:28142823 115. Vracko R: Basal lamina scaffold-anatomy and significance for maintenance of orderly tissue structure, Am J Pathol 1974, 77:314-346 116. Vracko R: Significance of basal lamina for regeneration of injured lung, Virchows Arch A Pathol Pathol Anat 1972, 355:264-274 117. Fukuda Y, Ferrans VJ, Schoenberger CI, Rennard SI, Crystal RG: Patterns of pulmonary structural remodeling after experimental paraquat toxicity. The morphogenesis of intraalveolar fibrosis, Am J Pathol 1985, 118:452-475 118. Wolff G, Worgall S, van Rooijen N, Song WR, Harvey BG, Crystal RG: Enhancement of in vivo adenovirus-mediated gene transfer and expression by prior depletion of tissue macrophages in the target organ, J Virol 1997, 71:624-629 119. Roberts CR: Versican in the Cell Biology of Pulmonary Fibrosis. Edited by Hari G. Garg PJR, and Charles A. Hales. New York, Marcel Dekker, Inc., 2002, p. pp. 191212 120. Limper AH, Broekelmann TJ, Colby TV, Malizia G, McDonald JA: Analysis of local mRNA expression for extracellular matrix proteins and growth factors using in situ hybridization in fibroproliferative lung disorders, Chest 1991, 99:55S-56S 121. Antoniades HN, Bravo MA, Avila RE, Galanopoulos T, Neville-Golden J, Maxwell M, Selman M: Platelet-derived growth factor in idiopathic pulmonary fibrosis, J Clin Invest 1990, 86:1055-1064  42  122. Yue J, Mulder KM: Transforming growth factor-beta signal transduction in epithelial cells, Pharmacol Ther 2001, 91:1-34 123. Swiderski RE, Dencoff JE, Floerchinger CS, Shapiro SD, Hunninghake GW: Differential expression of extracellular matrix remodeling genes in a murine model of bleomycin-induced pulmonary fibrosis, Am J Pathol 1998, 152:821-828 124. Raghu G, Striker LJ, Hudson LD, Striker GE: Extracellular matrix in normal and fibrotic human lungs, Am Rev Respir Dis 1985, 131:281-289 125. Saldiva PH, Delmonte VC, de Carvalho CR, Kairalla RA, Auler Junior JO: Histochemical evaluation of lung collagen content in acute and chronic interstitial diseases, Chest 1989, 95:953-957 126. Specks U, Nerlich A, Colby TV, Wiest I, Timpl R: Increased expression of type VI collagen in lung fibrosis, Am J Respir Crit Care Med 1995, 151:1956-1964 127. Kuhn C, McDonald JA: The roles of the myofibroblast in idiopathic pulmonary fibrosis. Ultrastructural and immunohistochemical features of sites of active extracellular matrix synthesis, Am J Pathol 1991, 138:1257-1265 128. Zhang K, Rekhter MD, Gordon D, Phan SH: Myofibroblasts and their role in lung collagen gene expression during pulmonary fibrosis. A combined immunohistochemical and in situ hybridization study, Am J Pathol 1994, 145:114125 129. Basset F, Ferrans VJ, Soler P, Takemura T, Fukuda Y, Crystal RG: Intraluminal fibrosis in interstitial lung disorders, Am J Pathol 1986, 122:443-461 130. Brass DM, Hoyle GW, Poovey HG, Liu JY, Brody AR: Reduced tumor necrosis factor-alpha and transforming growth factor-beta1 expression in the lungs of inbred mice that fail to develop fibroproliferative lesions consequent to asbestos exposure, Am J Pathol 1999, 154:853-862 131. Tremble P, Chiquet-Ehrismann R, Werb Z: The extracellular matrix ligands fibronectin and tenascin collaborate in regulating collagenase gene expression in fibroblasts, Mol Biol Cell 1994, 5:439-453 132. Bell E, Ivarsson B, Merrill C: Production of a tissue-like structure by contraction of collagen lattices by human fibroblasts of different proliferative potential in vitro, Proc Natl Acad Sci U S A 1979, 76:1274-1278 133. Elsdale T, Bard J: Collagen substrata for studies on cell behavior, J Cell Biol 1972, 54:626-637 134. Niewiarowski S, Goldstein S: Interaction of cultured human fibroblasts with fibrin: modification by drugs and aging in vitro, J Lab Clin Med 1973, 82:605-610 135. Grinnell F: Fibroblasts, myofibroblasts, and wound contraction, J Cell Biol 1994, 124:401-404 136. Harris AK, Stopak D, Wild P: Fibroblast traction as a mechanism for collagen morphogenesis, Nature 1981, 290:249-251  43  137. Bellows CG, Melcher AH, Aubin JE: Contraction and organization of collagen gels by cells cultured from periodontal ligament, gingiva and bone suggest functional differences between cell types, J Cell Sci 1981, 50:299-314 138. Nishiyama T, Tsunenaga M, Nakayama Y, Adachi E, Hayashi T: Growth rate of human fibroblasts is repressed by the culture within reconstituted collagen matrix but not by the culture on the matrix, Matrix 1989, 9:193-199 139. Nakagawa S, Pawelek P, Grinnell F: Long-term culture of fibroblasts in contracted collagen gels: effects on cell growth and biosynthetic activity, J Invest Dermatol 1989, 93:792-798 140. Kono T, Tanii T, Furukawa M, Mizuno N, Kitajima J, Ishii M, Hamada T, Yoshizato K: Cell cycle analysis of human dermal fibroblasts cultured on or in hydrated type I collagen lattices, Arch Dermatol Res 1990, 282:258-262 141. Nusgens B, Merrill C, Lapiere C, Bell E: Collagen biosynthesis by cells in a tissue equivalent matrix in vitro, Coll Relat Res 1984, 4:351-363 142. Unemori EN, Werb Z: Reorganization of polymerized actin: a possible trigger for induction of procollagenase in fibroblasts cultured in and on collagen gels, J Cell Biol 1986, 103:1021-1031 143. Paye M, Nusgens BV, Lapiere CM: Modulation of cellular biosynthetic activity in the retracting collagen lattice, Eur J Cell Biol 1987, 45:44-50 144. Kasugai S, Suzuki S, Shibata S, Yasui S, Amano H, Ogura H: Measurements of the isometric contractile forces generated by dog periodontal ligament fibroblasts in vitro, Arch Oral Biol 1990, 35:597-601 145. Delvoye P, Wiliquet P, Leveque JL, Nusgens BV, Lapiere CM: Measurement of mechanical forces generated by skin fibroblasts embedded in a three-dimensional collagen gel, J Invest Dermatol 1991, 97:898-902 146. Kolodney MS, Wysolmerski RB: Isometric contraction by fibroblasts and endothelial cells in tissue culture: a quantitative study, J Cell Biol 1992, 117:73-82 147. Stopak D, Harris AK: Connective tissue morphogenesis by fibroblast traction. I. Tissue culture observations, Dev Biol 1982, 90:383-398 148. Tomasek JJ, Haaksma CJ, Eddy RJ, Vaughan MB: Fibroblast contraction occurs on release of tension in attached collagen lattices: dependency on an organized actin cytoskeleton and serum, Anat Rec 1992, 232:359-368 149. Mochitate K, Pawelek P, Grinnell F: Stress relaxation of contracted collagen gels: disruption of actin filament bundles, release of cell surface fibronectin, and downregulation of DNA and protein synthesis, Exp Cell Res 1991, 193:198-207 150. Iwig M, Glaesser D, Bethge M: Cell shape-mediated growth control of lens epithelial cells grown in culture, Exp Cell Res 1981, 131:47-55 151. Fringer J, Grinnell F: Fibroblast quiescence in floating or released collagen matrices: contribution of the ERK signaling pathway and actin cytoskeletal organization, J Biol Chem 2001, 276:31047-31052  44  152. Fluck J, Querfeld C, Cremer A, Niland S, Krieg T, Sollberg S: Normal human primary fibroblasts undergo apoptosis in three-dimensional contractile collagen gels, J Invest Dermatol 1998, 110:153-157 153. Steinberg BM, Smith K, Colozzo M, Pollack R: Establishment and transformation diminish the ability of fibroblasts to contract a native collagen gel, J Cell Biol 1980, 87:304-308 154. Guidry C, Grinnell F: Studies on the mechanism of hydrated collagen gel reorganization by human skin fibroblasts, J Cell Sci 1985, 79:67-81 155. Montesano R, Orci L: Transforming growth factor beta stimulates collagen-matrix contraction by fibroblasts: implications for wound healing, Proc Natl Acad Sci U S A 1988, 85:4894-4897 156. Finesmith TH, Broadley KN, Davidson JM: Fibroblasts from wounds of different stages of repair vary in their ability to contract a collagen gel in response to growth factors, J Cell Physiol 1990, 144:99-107 157. Clark RA, Folkvord JM, Hart CE, Murray MJ, McPherson JM: Platelet isoforms of platelet-derived growth factor stimulate fibroblasts to contract collagen matrices, J Clin Invest 1989, 84:1036-1040 158. Gullberg D, Tingstrom A, Thuresson AC, Olsson L, Terracio L, Borg TK, Rubin K: Beta 1 integrin-mediated collagen gel contraction is stimulated by PDGF, Exp Cell Res 1990, 186:264-272 159. Tingstrom A, Reuterdahl C, Lindahl P, Heldin CH, Rubin K: Expression of platelet-derived growth factor-beta receptors on human fibroblasts. Regulation by recombinant platelet-derived growth factor-BB, IL-1, and tumor necrosis factoralpha, J Immunol 1992, 148:546-554 160. Yamaguchi Y: Lecticans: organizers of the brain extracellular matrix, Cell Mol Life Sci 2000, 57:276-289 161. Ruoslahti E: Structure and biology of proteoglycans, Annu Rev Cell Biol 1988, 4:229-255 162. Nieduszynski IA, Sheehan JK, Phelps CF, Hardingham TE, Muir H: Equilibriumbinding studies of pig laryngeal cartilage proteoglycans with hyaluronate oligosaccharide fractions, Biochem J 1980, 185:107-114 163. LeBaron RG, Zimmermann DR, Ruoslahti E: Hyaluronate binding properties of versican, J Biol Chem 1992, 267:10003-10010 164. Matsumoto K, Shionyu M, Go M, Shimizu K, Shinomura T, Kimata K, Watanabe H: Distinct interaction of versican/PG-M with hyaluronan and link protein, J Biol Chem 2003, 278:41205-41212 165. Heinegard D: Polydispersity of cartilage proteoglycans. Structural variations with size and buoyant density of the molecules, J Biol Chem 1977, 252:1980-1989 166. Heinegard D, Axelsson I: Distribution of keratan sulfate in cartilage proteoglycans, J Biol Chem 1977, 252:1971-1979  45  167. Kawashima H, Hirose M, Hirose J, Nagakubo D, Plaas AH, Miyasaka M: Binding of a large chondroitin sulfate/dermatan sulfate proteoglycan, versican, to L-selectin, P-selectin, and CD44, J Biol Chem 2000, 275:35448-35456 168. Kjellen L, Lindahl U: Proteoglycans: structures and interactions, Annu Rev Biochem 1991, 60:443-475 169. Schlessinger J, Lax I, Lemmon M: Regulation of growth factor activation by proteoglycans: what is the role of the low affinity receptors? Cell 1995, 83:357-360 170. Ruoslahti E, Yamaguchi Y: Proteoglycans as modulators of growth factor activities, Cell 1991, 64:867-869 171. Tanaka Y, Adams DH, Shaw S: Proteoglycans on endothelial cells present adhesion-inducing cytokines to leukocytes, Immunol Today 1993, 14:111-115 172. Tanaka Y, Adams DH, Shaw S: Regulation of leukocyte recruitment by proadhesive cytokines immobilized on endothelial proteoglycan, Curr Top Microbiol Immunol 1993, 184:99-106 173. Retzler C, Gohring W, Rauch U: Analysis of neurocan structures interacting with the neural cell adhesion molecule N-CAM, J Biol Chem 1996, 271:27304-27310 174. Li H, Leung TC, Hoffman S, Balsamo J, Lilien J: Coordinate regulation of cadherin and integrin function by the chondroitin sulfate proteoglycan neurocan, J Cell Biol 2000, 149:1275-1288 175. Seidenbecher CI, Smalla KH, Fischer N, Gundelfinger ED, Kreutz MR: Brevican isoforms associate with neural membranes, J Neurochem 2002, 83:738-746 176. Milev P, Maurel P, Chiba A, Mevissen M, Popp S, Yamaguchi Y, Margolis RK, Margolis RU: Differential regulation of expression of hyaluronan-binding proteoglycans in developing brain: aggrecan, versican, neurocan, and brevican, Biochem Biophys Res Commun 1998, 247:207-212 177. Miura R, Aspberg A, Ethell IM, Hagihara K, Schnaar RL, Ruoslahti E, Yamaguchi Y: The proteoglycan lectin domain binds sulfated cell surface glycolipids and promotes cell adhesion, J Biol Chem 1999, 274:11431-11438 178. Miura R, Ethell IM, Yamaguchi Y: Carbohydrate-protein interactions between HNK-1-reactive sulfoglucuronyl glycolipids and the proteoglycan lectin domain mediate neuronal cell adhesion and neurite outgrowth, J Neurochem 2001, 76:413424 179. Snow DM, Letourneau PC: Neurite outgrowth on a step gradient of chondroitin sulfate proteoglycan (CS-PG), J Neurobiol 1992, 23:322-336 180. Condic ML, Lemons ML: Extracellular matrix in spinal cord regeneration: getting beyond attraction and inhibition, Neuroreport 2002, 13:A37-48 181. Newgreen DF: Adhesion to extracellular materials by neural crest cells at the stage of initial migration, Cell Tissue Res 1982, 227:297-317  46  182. Perris R, Perissinotto D, Pettway Z, Bronner-Fraser M, Morgelin M, Kimata K: Inhibitory effects of PG-H/aggrecan and PG-M/versican on avian neural crest cell migration, Faseb J 1996, 10:293-301 183. Newgreen DF, Scheel M, Kastner V: Morphogenesis of sclerotome and neural crest in avian embryos. In vivo and in vitro studies on the role of notochordal extracellular material, Cell Tissue Res 1986, 244:299-313 184. Ito K, Shinomura T, Zako M, Ujita M, Kimata K: Multiple forms of mouse PG-M, a large chondroitin sulfate proteoglycan generated by alternative splicing, J Biol Chem 1995, 270:958-965 185. Zako M, Shinomura T, Ujita M, Ito K, Kimata K: Expression of PG-M(V3), an alternatively spliced form of PG-M without a chondroitin sulfate attachment in region in mouse and human tissues, J Biol Chem 1995, 270:3914-3918 186. Ruoslahti E: Brain extracellular matrix, Glycobiology 1996, 6:489-492 187. Iozzo RV: Matrix proteoglycans: from molecular design to cellular function, Annu Rev Biochem 1998, 67:609-652 188. Keller RE, Lofberg J, Spieth J: Neural crest cell behavior in white and dark embryos of Ambystoma mexicanum: epidermal inhibition of pigment cell migration in the white axolotl, Dev Biol 1982, 89:179-195 189. Keller RE, Spieth J: Neural crest cell behavior in white and dark larvae of Ambystoma mexicanum: time-lapse cinemicrographic analysis of pigment cell movement in vivo and in culture, J Exp Zool 1984, 229:109-126 190. Stigson M, Lofberg J, Kjellen L: PG-M/versican-like proteoglycans are components of large disulfide-stabilized complexes in the axolotl embryo, J Biol Chem 1997, 272:3246-3253 191. Perissinotto D, Iacopetti P, Bellina I, Doliana R, Colombatti A, Pettway Z, Bronner-Fraser M, Shinomura T, Kimata K, Morgelin M, Lofberg J, Perris R: Avian neural crest cell migration is diversely regulated by the two major hyaluronan-binding proteoglycans PG-M/versican and aggrecan, Development 2000, 127:2823-2842 192. Mjaatvedt CH, Yamamura H, Capehart AA, Turner D, Markwald RR: The Cspg2 gene, disrupted in the hdf mutant, is required for right cardiac chamber and endocardial cushion formation, Dev Biol 1998, 202:56-66 193. Henderson DJ, Copp AJ: Versican expression is associated with chamber specification, septation, and valvulogenesis in the developing mouse heart, Circ Res 1998, 83:523-532 194. Zanin MK, Bundy J, Ernst H, Wessels A, Conway SJ, Hoffman S: Distinct spatial and temporal distributions of aggrecan and versican in the embryonic chick heart, Anat Rec 1999, 256:366-380 195. Kimata K, Oike Y, Tani K, Shinomura T, Yamagata M, Uritani M, Suzuki S: A large chondroitin sulfate proteoglycan (PG-M) synthesized before chondrogenesis in the limb bud of chick embryo, J Biol Chem 1986, 261:13517-13525 47  196. Shibata S, Fukada K, Imai H, Abe T, Yamashita Y: In situ hybridization and immunohistochemistry of versican, aggrecan and link protein, and histochemistry of hyaluronan in the developing mouse limb bud cartilage, J Anat 2003, 203:425432 197. Bode-Lesniewska B, Dours-Zimmermann MT, Odermatt BF, Briner J, Heitz PU, Zimmermann DR: Distribution of the large aggregating proteoglycan versican in adult human tissues, J Histochem Cytochem 1996, 44:303-312 198. Grover J, Roughley PJ: Versican gene expression in human articular cartilage and comparison of mRNA splicing variation with aggrecan, Biochem J 1993, 291 (Pt 2):361-367 199. Kolettas E, Buluwela L, Bayliss MT, Muir HI: Expression of cartilage-specific molecules is retained on long-term culture of human articular chondrocytes, J Cell Sci 1995, 108 (Pt 5):1991-1999 200. Sztrolovics R, Grover J, Cs-Szabo G, Shi SL, Zhang Y, Mort JS, Roughley PJ: The characterization of versican and its message in human articular cartilage and intervertebral disc, J Orthop Res 2002, 20:257-266 201. Matsumoto K, Kamiya N, Suwan K, Atsumi F, Shimizu K, Shinomura T, Yamada Y, Kimata K, Watanabe H: Versican/PG-M aggregates in cartilage: identification and characterization, J Biol Chem 2006, 202. Yamagata M, Saga S, Kato M, Bernfield M, Kimata K: Selective distributions of proteoglycans and their ligands in pericellular matrix of cultured fibroblasts. Implications for their roles in cell-substratum adhesion, J Cell Sci 1993, 106 (Pt 1):55-65 203. Landolt RM, Vaughan L, Winterhalter KH, Zimmermann DR: Versican is selectively expressed in embryonic tissues that act as barriers to neural crest cell migration and axon outgrowth, Development 1995, 121:2303-2312 204. Kishimoto J, Ehama R, Wu L, Jiang S, Jiang N, Burgeson RE: Selective activation of the versican promoter by epithelial- mesenchymal interactions during hair follicle development, Proc Natl Acad Sci U S A 1999, 96:7336-7341 205. Yamamura H, Zhang M, Markwald RR, Mjaatvedt CH: A heart segmental defect in the anterior-posterior axis of a transgenic mutant mouse, Dev Biol 1997, 186:58-72 206. Shinomura T, Nishida Y, Ito K, Kimata K: cDNA cloning of PG-M, a large chondroitin sulfate proteoglycan expressed during chondrogenesis in chick limb buds. Alternative spliced multiforms of PG-M and their relationships to versican, J Biol Chem 1993, 268:14461-14469 207. Hirose J, Kawashima H, Yoshie O, Tashiro K, Miyasaka M: Versican interacts with chemokines and modulates cellular responses, J Biol Chem 2001, 276:5228-5234 208. Kawashima H, Watanabe N, Hirose M, Sun X, Atarashi K, Kimura T, Shikata K, Matsuda M, Ogawa D, Heljasvaara R, Rehn M, Pihlajaniemi T, Miyasaka M: Collagen XVIII, a basement membrane heparan sulfate proteoglycan, interacts with  48  L-selectin and monocyte chemoattractant protein-1, J Biol Chem 2003, 278:1306913076 209. Juul SE, Kinsella MG, Jackson JC, Truog WE, Standaert TA, Hodson WA: Changes in hyaluronan deposition during early respiratory distress syndrome in premature monkeys, Pediatr Res 1994, 35:238-243 210. Isogai Z, Shinomura T, Yamakawa N, Takeuchi J, Tsuji T, Heinegard D, Kimata K: 2B1 antigen characteristically expressed on extracellular matrices of human malignant tumors is a large chondroitin sulfate proteoglycan, PG-M/versican, Cancer Res 1996, 56:3902-3908 211. Nara Y, Kato Y, Torii Y, Tsuji Y, Nakagaki S, Goto S, Isobe H, Nakashima N, Takeuchi J: Immunohistochemical localization of extracellular matrix components in human breast tumours with special reference to PG-M/versican, Histochem J 1997, 29:21-30 212. Brown LF, Guidi AJ, Schnitt SJ, Van De Water L, Iruela-Arispe ML, Yeo TK, Tognazzi K, Dvorak HF: Vascular stroma formation in carcinoma in situ, invasive carcinoma, and metastatic carcinoma of the breast, Clin Cancer Res 1999, 5:10411056 213. Ricciardelli C, Brooks JH, Suwiwat S, Sakko AJ, Mayne K, Raymond WA, Seshadri R, LeBaron RG, Horsfall DJ: Regulation of stromal versican expression by breast cancer cells and importance to relapse-free survival in patients with nodenegative primary breast cancer, Clin Cancer Res 2002, 8:1054-1060 214. Paulus W, Baur I, Dours-Zimmermann MT, Zimmermann DR: Differential expression of versican isoforms in brain tumors, J Neuropathol Exp Neurol 1996, 55:528-533 215. Voutilainen K, Anttila M, Sillanpaa S, Tammi R, Tammi M, Saarikoski S, Kosma VM: Versican in epithelial ovarian cancer: relation to hyaluronan, clinicopathologic factors and prognosis, Int J Cancer 2003, 107:359-364 216. Theocharis AD, Vynios DH, Papageorgakopoulou N, Skandalis SS, Theocharis DA: Altered content composition and structure of glycosaminoglycans and proteoglycans in gastric carcinoma, Int J Biochem Cell Biol 2003, 35:376-390 217. Sakko AJ, Ricciardelli C, Mayne K, Tilley WD, Lebaron RG, Horsfall DJ: Versican accumulation in human prostatic fibroblast cultures is enhanced by prostate cancer cell-derived transforming growth factor beta1, Cancer Res 2001, 61:926-930 218. Ricciardelli C, Mayne K, Sykes PJ, Raymond WA, McCaul K, Marshall VR, Horsfall DJ: Elevated levels of versican but not decorin predict disease progression in early-stage prostate cancer, Clin Cancer Res 1998, 4:963-971 219. Touab M, Villena J, Barranco C, Arumi-Uria M, Bassols A: Versican is differentially expressed in human melanoma and may play a role in tumor development, Am J Pathol 2002, 160:549-557  49  220. Gulyas M, Hjerpe A: Proteoglycans and WT1 as markers for distinguishing adenocarcinoma, epithelioid mesothelioma, and benign mesothelium, J Pathol 2003, 199:479-487 221. Fidler PS, Schuette K, Asher RA, Dobbertin A, Thornton SR, Calle-Patino Y, Muir E, Levine JM, Geller HM, Rogers JH, Faissner A, Fawcett JW: Comparing astrocytic cell lines that are inhibitory or permissive for axon growth: the major axon-inhibitory proteoglycan is NG2, J Neurosci 1999, 19:8778-8788 222. Niederost BP, Zimmermann DR, Schwab ME, Bandtlow CE: Bovine CNS myelin contains neurite growth-inhibitory activity associated with chondroitin sulfate proteoglycans, J Neurosci 1999, 19:8979-8989 223. Schmalfeldt M, Bandtlow CE, Dours-Zimmermann MT, Winterhalter KH, Zimmermann DR: Brain derived versican V2 is a potent inhibitor of axonal growth, J Cell Sci 2000, 113 (Pt 5):807-816 224. Asher RA, Morgenstern DA, Shearer MC, Adcock KH, Pesheva P, Fawcett JW: Versican is upregulated in CNS injury and is a product of oligodendrocyte lineage cells, J Neurosci 2002, 22:2225-2236 225. Tammi R, Pasonen-Seppanen S, Kolehmainen E, Tammi M: Hyaluronan synthase induction and hyaluronan accumulation in mouse epidermis following skin injury, J Invest Dermatol 2005, 124:898-905 226. Jiang D, Liang J, Noble PW: Hyaluronan in tissue injury and repair, Annu Rev Cell Dev Biol 2007, 23:435-461 227. Oksala O, Salo T, Tammi R, Hakkinen L, Jalkanen M, Inki P, Larjava H: Expression of proteoglycans and hyaluronan during wound healing, J Histochem Cytochem 1995, 43:125-135 228. Ricciardelli C, Russell DL, Ween MP, Mayne K, Suwiwat S, Byers S, Marshall VR, Tilley WD, Horsfall DJ: Formation of hyaluronan- and versican-rich pericellular matrix by prostate cancer cells promotes cell motility, J Biol Chem 2007, 282:10814-10825 229. Camenisch TD, Schroeder JA, Bradley J, Klewer SE, McDonald JA: Heart-valve mesenchyme formation is dependent on hyaluronan-augmented activation of ErbB2-ErbB3 receptors, Nat Med 2002, 8:850-855 230. Camenisch TD, Spicer AP, Brehm-Gibson T, Biesterfeldt J, Augustine ML, Calabro A, Jr., Kubalak S, Klewer SE, McDonald JA: Disruption of hyaluronan synthase-2 abrogates normal cardiac morphogenesis and hyaluronan-mediated transformation of epithelium to mesenchyme, J Clin Invest 2000, 106:349-360 231. McDonald JA, Camenisch TD: Hyaluronan: genetic insights into the complex biology of a simple polysaccharide, Glycoconj J 2002, 19:331-339 232. Zhang Y, Cao L, Yang BL, Yang BB: The G3 domain of versican enhances cell proliferation via epidermial growth factor-like motifs, J Biol Chem 1998, 273:21342-21351  50  233. Jones PL, Jones FS: Tenascin-C in development and disease: gene regulation and cell function, Matrix Biol 2000, 19:581-596 234. Kuhn C, Mason RJ: Immunolocalization of SPARC, tenascin, and thrombospondin in pulmonary fibrosis, Am J Pathol 1995, 147:1759-1769 235. Matsuda A, Yoshiki A, Tagawa Y, Matsuda H, Kusakabe M: Corneal wound healing in tenascin knockout mouse, Invest Ophthalmol Vis Sci 1999, 40:10711080 236. Aspberg A, Binkert C, Ruoslahti E: The versican C-type lectin domain recognizes the adhesion protein tenascin-R, Proc Natl Acad Sci U S A 1995, 92:10590-10594 237. Olin AI, Morgelin M, Sasaki T, Timpl R, Heinegard D, Aspberg A: The proteoglycans aggrecan and Versican form networks with fibulin-2 through their lectin domain binding, J Biol Chem 2001, 276:1253-1261 238. Chiquet-Ehrismann R, Chiquet M: Tenascins: regulation and putative functions during pathological stress, J Pathol 2003, 200:488-499 239. Tamaoki M, Imanaka-Yoshida K, Yokoyama K, Nishioka T, Inada H, Hiroe M, Sakakura T, Yoshida T: Tenascin-C regulates recruitment of myofibroblasts during tissue repair after myocardial injury, Am J Pathol 2005, 167:71-80 240. De Wever O, Nguyen QD, Van Hoorde L, Bracke M, Bruyneel E, Gespach C, Mareel M: Tenascin-C and SF/HGF produced by myofibroblasts in vitro provide convergent pro-invasive signals to human colon cancer cells through RhoA and Rac, Faseb J 2004, 18:1016-1018 241. Trebaul A, Chan EK, Midwood KS: Regulation of fibroblast migration by tenascinC, Biochem Soc Trans 2007, 35:695-697 242. Schmidinger G, Hanselmayer G, Pieh S, Lackner B, Kaminski S, Ruhswurm I, Skorpik C: Effect of tenascin and fibronectin on the migration of human corneal fibroblasts, J Cataract Refract Surg 2003, 29:354-360 243. Briggs SL: The role of fibronectin in fibroblast migration during tissue repair, J Wound Care 2005, 14:284-287 244. Greiling D, Clark RA: Fibronectin provides a conduit for fibroblast transmigration from collagenous stroma into fibrin clot provisional matrix, J Cell Sci 1997, 110 (Pt 7):861-870 245. Clark RA, An JQ, Greiling D, Khan A, Schwarzbauer JE: Fibroblast migration on fibronectin requires three distinct functional domains, J Invest Dermatol 2003, 121:695-705 246. Sottile J, Hocking DC: Fibronectin polymerization regulates the composition and stability of extracellular matrix fibrils and cell-matrix adhesions, Mol Biol Cell 2002, 13:3546-3559 247. Midwood KS, Mao Y, Hsia HC, Valenick LV, Schwarzbauer JE: Modulation of cell-fibronectin matrix interactions during tissue repair, J Investig Dermatol Symp Proc 2006, 11:73-78  51  248. Dugina V, Fontao L, Chaponnier C, Vasiliev J, Gabbiani G: Focal adhesion features during myofibroblastic differentiation are controlled by intracellular and extracellular factors, J Cell Sci 2001, 114:3285-3296 249. Glukhova MA, Frid MG, Shekhonin BV, Vasilevskaya TD, Grunwald J, Saginati M, Koteliansky VE: Expression of extra domain A fibronectin sequence in vascular smooth muscle cells is phenotype dependent, J Cell Biol 1989, 109:357-366 250. Lee MJ, Roy NK, Mogford JE, Schiemann WP, Mustoe TA: Fibulin-5 promotes wound healing in vivo, J Am Coll Surg 2004, 199:403-410 251. Isogai Z, Aspberg A, Keene DR, Ono RN, Reinhardt DP, Sakai LY: Versican interacts with fibrillin-1 and links extracellular microfibrils to other connective tissue networks, J Biol Chem 2002, 277:4565-4572 252. Arbones ML, Ord DC, Ley K, Ratech H, Maynard-Curry C, Otten G, Capon DJ, Tedder TF: Lymphocyte homing and leukocyte rolling and migration are impaired in L-selectin-deficient mice, Immunity 1994, 1:247-260 253. Naor D, Sionov RV, Ish-Shalom D: CD44: structure, function, and association with the malignant process, Adv Cancer Res 1997, 71:241-319 254. Sheng W, Wang G, Wang Y, Liang J, Wen J, Zheng PS, Wu Y, Lee V, Slingerland J, Dumont D, Yang BB: The roles of versican V1 and V2 isoforms in cell proliferation and apoptosis, Mol Biol Cell 2005, 16:1330-1340 255. Roughley PJ, Lee ER: Cartilage proteoglycans: structure and potential functions, Microsc Res Tech 1994, 28:385-397 256. Bramley AM, Roberts CR, Schellenberg RR: Collagenase increases shortening of human bronchial smooth muscle in vitro, Am J Respir Crit Care Med 1995, 152:1513-1517 257. Yamagata M, Kimata K: Repression of a malignant cell-substratum adhesion phenotype by inhibiting the production of the anti-adhesive proteoglycan, PGM/versican, J Cell Sci 1994, 107 (Pt 9):2581-2590 258. Fraser R. G. PJA, Pare P. D., Fraser R. S., and Genereux G. P.: Tuberculosis. Edited by Saunders WB. 1991, p. 259. Evanko SP, Angello JC, Wight TN: Formation of hyaluronan- and versican-rich pericellular matrix is required for proliferation and migration of vascular smooth muscle cells, Arterioscler Thromb Vasc Biol 1999, 19:1004-1013 260. Lemire JM, Merrilees MJ, Braun KR, Wight TN: Overexpression of the V3 variant of versican alters arterial smooth muscle cell adhesion, migration, and proliferation in vitro, J Cell Physiol 2002, 190:38-45 261. Woessner JF, Jr.: Matrix metalloproteinases and their inhibitors in connective tissue remodeling, Faseb J 1991, 5:2145-2154 262. Birkedal-Hansen H: Role of cytokines and inflammatory mediators in tissue destruction, J Periodontal Res 1993, 28:500-510  52  263. Birkedal-Hansen H, Moore WG, Bodden MK, Windsor LJ, Birkedal-Hansen B, DeCarlo A, Engler JA: Matrix metalloproteinases: a review, Crit Rev Oral Biol Med 1993, 4:197-250 264. Brenner CA, Adler RR, Rappolee DA, Pedersen RA, Werb Z: Genes for extracellular-matrix-degrading metalloproteinases and their inhibitor, TIMP, are expressed during early mammalian development, Genes Dev 1989, 3:848-859 265. Nomura S, Hogan BL, Wills AJ, Heath JK, Edwards DR: Developmental expression of tissue inhibitor of metalloproteinase (TIMP) RNA, Development 1989, 105:575-583 266. Sellers A, Reynolds JJ, Meikle MC: Neutral metallo-proteinases of rabbit bone. Separation in latent forms of distinct enzymes that when activated degrade collagen, gelatin and proteoglycans, Biochem J 1978, 171:493-496 267. Brannstrom M, Woessner JF, Jr., Koos RD, Sear CH, LeMaire WJ: Inhibitors of mammalian tissue collagenase and metalloproteinases suppress ovulation in the perfused rat ovary, Endocrinology 1988, 122:1715-1721 268. Chambers AF, Matrisian LM: Changing views of the role of matrix metalloproteinases in metastasis, J Natl Cancer Inst 1997, 89:1260-1270 269. Cawston T: Matrix metalloproteinases and TIMPs: properties and implications for the rheumatic diseases, Mol Med Today 1998, 4:130-137 270. Gunja-Smith Z, Morales AR, Romanelli R, Woessner JF, Jr.: Remodeling of human myocardial collagen in idiopathic dilated cardiomyopathy. Role of metalloproteinases and pyridinoline cross-links, Am J Pathol 1996, 148:1639-1648 271. McCawley LJ, Matrisian LM: Matrix metalloproteinases: they're not just for matrix anymore! Curr Opin Cell Biol 2001, 13:534-540 272. Sternlicht MD, Werb Z: How matrix metalloproteinases regulate cell behavior, Annu Rev Cell Dev Biol 2001, 17:463-516 273. Egeblad M, Werb Z: New functions for the matrix metalloproteinases in cancer progression, Nat Rev Cancer 2002, 2:161-174 274. Nagase H, Woessner JF, Jr.: Matrix metalloproteinases, J Biol Chem 1999, 274:21491-21494 275. Rawlings ND, Barrett AJ: Evolutionary families of metallopeptidases, Methods Enzymol 1995, 248:183-228 276. Stocker W, Bode W: Structural features of a superfamily of zinc-endopeptidases: the metzincins, Curr Opin Struct Biol 1995, 5:383-390 277. Van Wart HE, Birkedal-Hansen H: The cysteine switch: a principle of regulation of metalloproteinase activity with potential applicability to the entire matrix metalloproteinase gene family, Proc Natl Acad Sci U S A 1990, 87:5578-5582 278. Murphy G, Knauper V: Relating matrix metalloproteinase structure to function: why the "hemopexin" domain? Matrix Biol 1997, 15:511-518  53  279. Briknarova K, Grishaev A, Banyai L, Tordai H, Patthy L, Llinas M: The second type II module from human matrix metalloproteinase 2: structure, function and dynamics, Structure 1999, 7:1235-1245 280. Gehrmann M, Briknarova K, Banyai L, Patthy L, Llinas M: The col-1 module of human matrix metalloproteinase-2 (MMP-2): structural/functional relatedness between gelatin-binding fibronectin type II modules and lysine-binding kringle domains, Biol Chem 2002, 383:137-148 281. Opdenakker G, Van Damme J: Cytokine-regulated proteases in autoimmune diseases, Immunol Today 1994, 15:103-107 282. Uria JA, Jimenez MG, Balbin M, Freije JM, Lopez-Otin C: Differential effects of transforming growth factor-beta on the expression of collagenase-1 and collagenase-3 in human fibroblasts, J Biol Chem 1998, 273:9769-9777 283. Vogel W, Gish GD, Alves F, Pawson T: The discoidin domain receptor tyrosine kinases are activated by collagen, Mol Cell 1997, 1:13-23 284. Utani A, Momota Y, Endo H, Kasuya Y, Beck K, Suzuki N, Nomizu M, Shinkai H: Laminin alpha 3 LG4 module induces matrix metalloproteinase-1 through mitogenactivated protein kinase signaling, J Biol Chem 2003, 278:34483-34490 285. Lehti K, Valtanen H, Wickstrom SA, Lohi J, Keski-Oja J: Regulation of membrane-type-1 matrix metalloproteinase activity by its cytoplasmic domain, J Biol Chem 2000, 275:15006-15013 286. Fahling M, Steege A, Perlewitz A, Nafz B, Mrowka R, Persson PB, Thiele BJ: Role of nucleolin in posttranscriptional control of MMP-9 expression, Biochim Biophys Acta 2005, 1731:32-40 287. Han YP, Tuan TL, Hughes M, Wu H, Garner WL: Transforming growth factor-beta - and tumor necrosis factor-alpha -mediated induction and proteolytic activation of MMP-9 in human skin, J Biol Chem 2001, 276:22341-22350 288. Maskos K, Bode W: Structural basis of matrix metalloproteinases and tissue inhibitors of metalloproteinases, Mol Biotechnol 2003, 25:241-266 289. Clark R: The Molecular and Cellular Biology of Wound Repair. Edited by New York, Plenum Press, 1996, p 290. Madlener M, Parks WC, Werner S: Matrix metalloproteinases (MMPs) and their physiological inhibitors (TIMPs) are differentially expressed during excisional skin wound repair, Exp Cell Res 1998, 242:201-210 291. Parks WC: Matrix metalloproteinases in repair, Wound Repair Regen 1999, 7:423432 292. Soo C, Shaw WW, Zhang X, Longaker MT, Howard EW, Ting K: Differential expression of matrix metalloproteinases and their tissue-derived inhibitors in cutaneous wound repair, Plast Reconstr Surg 2000, 105:638-647  54  293. Klein SA, Anderson GL, Kennedy AB, Bond SJ: The effects of a broad-spectrum matrix metalloproteinase inhibitor on characteristics of wound healing, J Invest Surg 2002, 15:199-207 294. Henry MT, McMahon K, Mackarel AJ, Prikk K, Sorsa T, Maisi P, Sepper R, Fitzgerald MX, O'Connor CM: Matrix metalloproteinases and tissue inhibitor of metalloproteinase-1 in sarcoidosis and IPF, Eur Respir J 2002, 20:1220-1227 295. Suga M, Iyonaga K, Okamoto T, Gushima Y, Miyakawa H, Akaike T, Ando M: Characteristic elevation of matrix metalloproteinase activity in idiopathic interstitial pneumonias, Am J Respir Crit Care Med 2000, 162:1949-1956 296. Fukuda Y, Ishizaki M, Kudoh S, Kitaichi M, Yamanaka N: Localization of matrix metalloproteinases-1, -2, and -9 and tissue inhibitor of metalloproteinase-2 in interstitial lung diseases, Lab Invest 1998, 78:687-698 297. Hayashi T, Stetler-Stevenson WG, Fleming MV, Fishback N, Koss MN, Liotta LA, Ferrans VJ, Travis WD: Immunohistochemical study of metalloproteinases and their tissue inhibitors in the lungs of patients with diffuse alveolar damage and idiopathic pulmonary fibrosis, Am J Pathol 1996, 149:1241-1256 298. Swaisgood CM, French EL, Noga C, Simon RH, Ploplis VA: The development of bleomycin-induced pulmonary fibrosis in mice deficient for components of the fibrinolytic system, Am J Pathol 2000, 157:177-187 299. Zuo F, Kaminski N, Eugui E, Allard J, Yakhini Z, Ben-Dor A, Lollini L, Morris D, Kim Y, DeLustro B, Sheppard D, Pardo A, Selman M, Heller RA: Gene expression analysis reveals matrilysin as a key regulator of pulmonary fibrosis in mice and humans, Proc Natl Acad Sci U S A 2002, 99:6292-6297 300. Tan RJ, Fattman CL, Niehouse LM, Tobolewski JM, Hanford LE, Li Q, Monzon FA, Parks WC, Oury TD: Matrix metalloproteinases promote inflammation and fibrosis in asbestos-induced lung injury in mice, Am J Respir Cell Mol Biol 2006, 35:289-297 301. Sellers A, Woessner JF, Jr.: The extraction of a neutral metalloproteinase from the involuting rat uterus, and its action on cartilage proteoglycan, Biochem J 1980, 189:521-531 302. Woessner JF, Jr., Taplin CJ: Purification and properties of a small latent matrix metalloproteinase of the rat uterus, J Biol Chem 1988, 263:16918-16925 303. Woessner JF, Jr.: Matrilysin, Methods Enzymol 1995, 248:485-495 304. Browner MF, Smith WW, Castelhano AL: Matrilysin-inhibitor complexes: common themes among metalloproteases, Biochemistry 1995, 34:6602-6610 305. Netzel-Arnett S, Sang QX, Moore WG, Navre M, Birkedal-Hansen H, Van Wart HE: Comparative sequence specificities of human 72- and 92-kDa gelatinases (type IV collagenases) and PUMP (matrilysin), Biochemistry 1993, 32:6427-6432 306. Lynch CC, Matrisian LM: Matrix metalloproteinases in tumor-host cell communication, Differentiation 2002, 70:561-573  55  307. Sires UI, Griffin GL, Broekelmann TJ, Mecham RP, Murphy G, Chung AE, Welgus HG, Senior RM: Degradation of entactin by matrix metalloproteinases. Susceptibility to matrilysin and identification of cleavage sites, J Biol Chem 1993, 268:2069-2074 308. Fosang AJ, Neame PJ, Last K, Hardingham TE, Murphy G, Hamilton JA: The interglobular domain of cartilage aggrecan is cleaved by PUMP, gelatinases, and cathepsin B, J Biol Chem 1992, 267:19470-19474 309. Nguyen Q, Murphy G, Hughes CE, Mort JS, Roughley PJ: Matrix metalloproteinases cleave at two distinct sites on human cartilage link protein, Biochem J 1993, 295 (Pt 2):595-598 310. Sasaki T, Mann K, Murphy G, Chu ML, Timpl R: Different susceptibilities of fibulin-1 and fibulin-2 to cleavage by matrix metalloproteinases and other tissue proteases, Eur J Biochem 1996, 240:427-434 311. Crabbe T, O'Connell JP, Smith BJ, Docherty AJ: Reciprocated matrix metalloproteinase activation: a process performed by interstitial collagenase and progelatinase A, Biochemistry 1994, 33:14419-14425 312. Crabbe T, Smith B, O'Connell J, Docherty A: Human progelatinase A can be activated by matrilysin, FEBS Lett 1994, 345:14-16 313. von Bredow DC, Cress AE, Howard EW, Bowden GT, Nagle RB: Activation of gelatinase-tissue-inhibitors-of-metalloproteinase complexes by matrilysin, Biochem J 1998, 331 (Pt 3):965-972 314. Crabbe T, Willenbrock F, Eaton D, Hynds P, Carne AF, Murphy G, Docherty AJ: Biochemical characterization of matrilysin. Activation conforms to the stepwise mechanisms proposed for other matrix metalloproteinases, Biochemistry 1992, 31:8500-8507 315. Itoh M, Masuda K, Ito Y, Akizawa T, Yoshioka M, Imai K, Okada Y, Sato H, Seiki M: Purification and refolding of recombinant human proMMP-7 (pro-matrilysin) expressed in Escherichia coli and its characterization, J Biochem 1996, 119:667673 316. Dunsmore SE, Saarialho-Kere UK, Roby JD, Wilson CL, Matrisian LM, Welgus HG, Parks WC: Matrilysin expression and function in airway epithelium, J Clin Invest 1998, 102:1321-1331 317. Lopez-Boado YS, Wilson CL, Hooper LV, Gordon JI, Hultgren SJ, Parks WC: Bacterial exposure induces and activates matrilysin in mucosal epithelial cells, J Cell Biol 2000, 148:1305-1315 318. Busiek DF, Baragi V, Nehring LC, Parks WC, Welgus HG: Matrilysin expression by human mononuclear phagocytes and its regulation by cytokines and hormones, J Immunol 1995, 154:6484-6491 319. Karelina TV, Goldberg GI, Eisen AZ: Matrilysin (PUMP) correlates with dermal invasion during appendageal development and cutaneous neoplasia, J Invest Dermatol 1994, 103:482-487  56  320. Wilson CL, Heppner KJ, Rudolph LA, Matrisian LM: The metalloproteinase matrilysin is preferentially expressed by epithelial cells in a tissue-restricted pattern in the mouse, Mol Biol Cell 1995, 6:851-869 321. Wilson CL, Ouellette AJ, Satchell DP, Ayabe T, Lopez-Boado YS, Stratman JL, Hultgren SJ, Matrisian LM, Parks WC: Regulation of intestinal alpha-defensin activation by the metalloproteinase matrilysin in innate host defense, Science 1999, 286:113-117 322. D'Armiento J, Dalal SS, Okada Y, Berg RA, Chada K: Collagenase expression in the lungs of transgenic mice causes pulmonary emphysema, Cell 1992, 71:955-961 323. Wilson CL, Heppner KJ, Labosky PA, Hogan BL, Matrisian LM: Intestinal tumorigenesis is suppressed in mice lacking the metalloproteinase matrilysin, Proc Natl Acad Sci U S A 1997, 94:1402-1407 324. Li Q, Park PW, Wilson CL, Parks WC: Matrilysin shedding of syndecan-1 regulates chemokine mobilization and transepithelial efflux of neutrophils in acute lung injury, Cell 2002, 111:635-646 325. McGuire JK, Li Q, Parks WC: Matrilysin (matrix metalloproteinase-7) mediates Ecadherin ectodomain shedding in injured lung epithelium, Am J Pathol 2003, 162:1831-1843 326. Yaguchi T, Fukuda Y, Ishizaki M, Yamanaka N: Immunohistochemical and gelatin zymography studies for matrix metalloproteinases in bleomycin-induced pulmonary fibrosis, Pathol Int 1998, 48:954-963 327. Perez-Ramos J, de Lourdes Segura-Valdez M, Vanda B, Selman M, Pardo A: Matrix metalloproteinases 2, 9, and 13, and tissue inhibitors of metalloproteinases 1 and 2 in experimental lung silicosis, Am J Respir Crit Care Med 1999, 160:12741282 328. Lu PC, Ye H, Maeda M, Azar DT: Immunolocalization and gene expression of matrilysin during corneal wound healing, Invest Ophthalmol Vis Sci 1999, 40:2027 329. Salmela MT, Pender SL, Karjalainen-Lindsberg ML, Puolakkainen P, Macdonald TT, Saarialho-Kere U: Collagenase-1 (MMP-1), matrilysin-1 (MMP-7), and stromelysin-2 (MMP-10) are expressed by migrating enterocytes during intestinal wound healing, Scand J Gastroenterol 2004, 39:1095-1104 330. Surendran K, Simon TC, Liapis H, McGuire JK: Matrilysin (MMP-7) expression in renal tubular damage: association with Wnt4, Kidney Int 2004, 65:2212-2222 331. Pilcher BK, Dumin JA, Sudbeck BD, Krane SM, Welgus HG, Parks WC: The activity of collagenase-1 is required for keratinocyte migration on a type I collagen matrix, J Cell Biol 1997, 137:1445-1457 332. Lochter A, Galosy S, Muschler J, Freedman N, Werb Z, Bissell MJ: Matrix metalloproteinase stromelysin-1 triggers a cascade of molecular alterations that leads to stable epithelial-to-mesenchymal conversion and a premalignant phenotype in mammary epithelial cells, J Cell Biol 1997, 139:1861-1872  57  333. Coraux C, Martinella-Catusse C, Nawrocki-Raby B, Hajj R, Burlet H, Escotte S, Laplace V, Birembaut P, Puchelle E: Differential expression of matrix metalloproteinases and interleukin-8 during regeneration of human airway epithelium in vivo, J Pathol 2005, 206:160-169 334. Betsuyaku T, Fukuda Y, Parks WC, Shipley JM, Senior RM: Gelatinase B is required for alveolar bronchiolization after intratracheal bleomycin, Am J Pathol 2000, 157:525-535 335. Legrand C, Gilles C, Zahm JM, Polette M, Buisson AC, Kaplan H, Birembaut P, Tournier JM: Airway epithelial cell migration dynamics. MMP-9 role in cellextracellular matrix remodeling, J Cell Biol 1999, 146:517-529 336. Werb Z, Gordon S: Elastase secretion by stimulated macrophages. Characterization and regulation, J Exp Med 1975, 142:361-377 337. Banda MJ, Werb Z: Mouse macrophage elastase. Purification and characterization as a metalloproteinase, Biochem J 1981, 193:589-605 338. Overall CM: Molecular determinants of metalloproteinase substrate specificity: matrix metalloproteinase substrate binding domains, modules, and exosites., Mol Biotechnol 2002, 22:51-86 339. Shapiro SD, Griffin GL, Gilbert DJ, Jenkins NA, Copeland NG, Welgus HG, Senior RM, Ley TJ: Molecular cloning, chromosomal localization, and bacterial expression of a murine macrophage metalloelastase, J Biol Chem 1992, 267:46644671 340. Shapiro SD, Kobayashi DK, Ley TJ: Cloning and characterization of a unique elastolytic metalloproteinase produced by human alveolar macrophages, J Biol Chem 1993, 268:23824-23829 341. Maskos K: Crystal structures of MMPs in complex with physiological and pharmacological inhibitors, Biochimie 2005, 87:249-263 342. Shapiro SD, Endicott SK, Province MA, Pierce JA, Campbell EJ: Marked longevity of human lung parenchymal elastic fibers deduced from prevalence of D-aspartate and nuclear weapons-related radiocarbon, J Clin Invest 1991, 87:1828-1834 343. Ducharme A, Frantz S, Aikawa M, Rabkin E, Lindsey M, Rohde LE, Schoen FJ, Kelly RA, Werb Z, Libby P, Lee RT: Targeted deletion of matrix metalloproteinase-9 attenuates left ventricular enlargement and collagen accumulation after experimental myocardial infarction, J Clin Invest 2000, 106:5562 344. Senft AP, Korfhagen TR, Whitsett JA, Shapiro SD, LeVine AM: Surfactant protein-D regulates soluble CD14 through matrix metalloproteinase-12, J Immunol 2005, 174:4953-4959 345. Curci JA, Liao S, Huffman MD, Shapiro SD, Thompson RW: Expression and localization of macrophage elastase (matrix metalloproteinase-12) in abdominal aortic aneurysms, J Clin Invest 1998, 102:1900-1910  58  346. Matsumoto S, Kobayashi T, Katoh M, Saito S, Ikeda Y, Kobori M, Masuho Y, Watanabe T: Expression and localization of matrix metalloproteinase-12 in the aorta of cholesterol-fed rabbits: relationship to lesion development, Am J Pathol 1998, 153:109-119 347. Hautamaki RD, Kobayashi DK, Senior RM, Shapiro SD: Requirement for macrophage elastase for cigarette smoke-induced emphysema in mice, Science 1997, 277:2002-2004 348. Wells JE, Rice TK, Nuttall RK, Edwards DR, Zekki H, Rivest S, Yong VW: An adverse role for matrix metalloproteinase 12 after spinal cord injury in mice, J Neurosci 2003, 23:10107-10115 349. Houghton AM, Grisolano JL, Baumann ML, Kobayashi DK, Hautamaki RD, Nehring LC, Cornelius LA, Shapiro SD: Macrophage elastase (matrix metalloproteinase-12) suppresses growth of lung metastases, Cancer Res 2006, 66:6149-6155 350. Wright RW, Allen T, El-Zawawy HB, Brodt MD, Silva MJ, Gill CS, Sandell LJ: Medial collateral ligament healing in macrophage metalloelastase (MMP-12)deficient mice, J Orthop Res 2006, 24:2106-2113 351. Shipley JM, Wesselschmidt RL, Kobayashi DK, Ley TJ, Shapiro SD: Metalloelastase is required for macrophage-mediated proteolysis and matrix invasion in mice, Proc Natl Acad Sci U S A 1996, 93:3942-3946 352. Morris DG, Huang X, Kaminski N, Wang Y, Shapiro SD, Dolganov G, Glick A, Sheppard D: Loss of integrin alpha(v)beta6-mediated TGF-beta activation causes Mmp12-dependent emphysema, Nature 2003, 422:169-173 353. Mercer RR, Crapo JD: Spatial distribution of collagen and elastin fibers in the lungs, J Appl Physiol 1990, 69:756-765 354. Finlay GA, O'Driscoll LR, Russell KJ, D'Arcy EM, Masterson JB, FitzGerald MX, O'Connor CM: Matrix metalloproteinase expression and production by alveolar macrophages in emphysema, Am J Respir Crit Care Med 1997, 156:240-247 355. Ohnishi K, Takagi M, Kurokawa Y, Satomi S, Konttinen YT: Matrix metalloproteinase-mediated extracellular matrix protein degradation in human pulmonary emphysema, Lab Invest 1998, 78:1077-1087 356. Bigg HF, Shi YE, Liu YE, Steffensen B, Overall CM: Specific, high affinity binding of tissue inhibitor of metalloproteinases-4 (TIMP-4) to the COOH-terminal hemopexin-like domain of human gelatinase A. TIMP-4 binds progelatinase A and the COOH-terminal domain in a similar manner to TIMP-2, J Biol Chem 1997, 272:15496-15500 357. Overall CM, McQuibban GA, Clark-Lewis I: Discovery of chemokine substrates for matrix metalloproteinases by exosite scanning: a new tool for degradomics, Biol Chem 2002, 383:1059-1066  59  358. Allan JA, Docherty AJ, Barker PJ, Huskisson NS, Reynolds JJ, Murphy G: Binding of gelatinases A and B to type-I collagen and other matrix components, Biochem J 1995, 309 (Pt 1):299-306 359. Wallon UM, Overall CM: The hemopexin-like domain (C domain) of human gelatinase A (matrix metalloproteinase-2) requires Ca2+ for fibronectin and heparin binding. Binding properties of recombinant gelatinase A C domain to extracellular matrix and basement membrane components, J Biol Chem 1997, 272:7473-7481 360. Steffensen B, Wallon UM, Overall CM: Extracellular matrix binding properties of recombinant fibronectin type II-like modules of human 72-kDa gelatinase/type IV collagenase. High affinity binding to native type I collagen but not native type IV collagen, J Biol Chem 1995, 270:11555-11566 361. Wang Z, Juttermann R, Soloway PD: TIMP-2 is required for efficient activation of proMMP-2 in vivo, J Biol Chem 2000, 275:26411-26415 362. Caterina JJ, Yamada S, Caterina NC, Longenecker G, Holmback K, Shi J, Yermovsky AE, Engler JA, Birkedal-Hansen H: Inactivating mutation of the mouse tissue inhibitor of metalloproteinases-2(Timp-2) gene alters proMMP-2 activation, J Biol Chem 2000, 275:26416-26422 363. Brooks PC, Silletti S, von Schalscha TL, Friedlander M, Cheresh DA: Disruption of angiogenesis by PEX, a noncatalytic metalloproteinase fragment with integrin binding activity, Cell 1998, 92:391-400 364. Kato T, Kure T, Chang JH, Gabison EE, Itoh T, Itohara S, Azar DT: Diminished corneal angiogenesis in gelatinase A-deficient mice, FEBS Lett 2001, 508:187-190 365. Irwin JC, Kirk D, Gwatkin RB, Navre M, Cannon P, Giudice LC: Human endometrial matrix metalloproteinase-2, a putative menstrual proteinase. Hormonal regulation in cultured stromal cells and messenger RNA expression during the menstrual cycle, J Clin Invest 1996, 97:438-447 366. Itoh T, Ikeda T, Gomi H, Nakao S, Suzuki T, Itohara S: Unaltered secretion of beta-amyloid precursor protein in gelatinase A (matrix metalloproteinase 2)deficient mice, J Biol Chem 1997, 272:22389-22392 367. Wiseman BS, Sternlicht MD, Lund LR, Alexander CM, Mott J, Bissell MJ, Soloway P, Itohara S, Werb Z: Site-specific inductive and inhibitory activities of MMP-2 and MMP-3 orchestrate mammary gland branching morphogenesis, J Cell Biol 2003, 162:1123-1133 368. Kheradmand F, Rishi K, Werb Z: Signaling through the EGF receptor controls lung morphogenesis in part by regulating MT1-MMP-mediated activation of gelatinase A/MMP-2, J Cell Sci 2002, 115:839-848 369. Itoh T, Tanioka M, Yoshida H, Yoshioka T, Nishimoto H, Itohara S: Reduced angiogenesis and tumor progression in gelatinase A-deficient mice, Cancer Res 1998, 58:1048-1051 370. Corry DB, Kiss A, Song LZ, Song L, Xu J, Lee SH, Werb Z, Kheradmand F: Overlapping and independent contributions of MMP2 and MMP9 to lung allergic  60  inflammatory cell egression through decreased CC chemokines, Faseb J 2004, 18:995-997 371. Corry DB, Rishi K, Kanellis J, Kiss A, Song Lz LZ, Xu J, Feng L, Werb Z, Kheradmand F: Decreased allergic lung inflammatory cell egression and increased susceptibility to asphyxiation in MMP2-deficiency, Nat Immunol 2002, 3:347-353 372. Esparza J, Kruse M, Lee J, Michaud M, Madri JA: MMP-2 null mice exhibit an early onset and severe experimental autoimmune encephalomyelitis due to an increase in MMP-9 expression and activity, Faseb J 2004, 18:1682-1691 373. Greenlee KJ, Corry DB, Engler DA, Matsunami RK, Tessier P, Cook RG, Werb Z, Kheradmand F: Proteomic identification of in vivo substrates for matrix metalloproteinases 2 and 9 reveals a mechanism for resolution of inflammation, J Immunol 2006, 177:7312-7321 374. McQuibban GA, Butler GS, Gong JH, Bendall L, Power C, Clark-Lewis I, Overall CM: Matrix metalloproteinase activity inactivates the CXC chemokine stromal cellderived factor-1, J Biol Chem 2001, 276:43503-43508 375. McQuibban GA, Gong JH, Wong JP, Wallace JL, Clark-Lewis I, Overall CM: Matrix metalloproteinase processing of monocyte chemoattractant proteins generates CC chemokine receptor antagonists with anti-inflammatory properties in vivo, Blood 2002, 100:1160-1167 376. McQuibban GA, Gong JH, Tam EM, McCulloch CA, Clark-Lewis I, Overall CM: Inflammation dampened by gelatinase A cleavage of monocyte chemoattractant protein-3, Science 2000, 289:1202-1206 377. Zeng ZS, Cohen AM, Guillem JG: Loss of basement membrane type IV collagen is associated with increased expression of metalloproteinases 2 and 9 (MMP-2 and MMP-9) during human colorectal tumorigenesis, Carcinogenesis 1999, 20:749-755 378. Sier CF, Kubben FJ, Ganesh S, Heerding MM, Griffioen G, Hanemaaijer R, van Krieken JH, Lamers CB, Verspaget HW: Tissue levels of matrix metalloproteinases MMP-2 and MMP-9 are related to the overall survival of patients with gastric carcinoma, Br J Cancer 1996, 74:413-417 379. Musso O, Theret N, Campion JP, Turlin B, Milani S, Grappone C, Clement B: In situ detection of matrix metalloproteinase-2 (MMP2) and the metalloproteinase inhibitor TIMP2 transcripts in human primary hepatocellular carcinoma and in liver metastasis, J Hepatol 1997, 26:593-605 380. Theret N, Musso O, Campion JP, Turlin B, Loreal O, L'Helgoualc'h A, Clement B: Overexpression of matrix metalloproteinase-2 and tissue inhibitor of matrix metalloproteinase-2 in liver from patients with gastrointestinal adenocarcinoma and no detectable metastasis, Int J Cancer 1997, 74:426-432 381. Tetu B, Brisson J, Lapointe H, Bernard P: Prognostic significance of stromelysin 3, gelatinase A, and urokinase expression in breast cancer, Hum Pathol 1998, 29:979985  61  382. Minn AJ, Gupta GP, Siegel PM, Bos PD, Shu W, Giri DD, Viale A, Olshen AB, Gerald WL, Massague J: Genes that mediate breast cancer metastasis to lung, Nature 2005, 436:518-524 383. Ikebe T, Shinohara M, Takeuchi H, Beppu M, Kurahara S, Nakamura S, Shirasuna K: Gelatinolytic activity of matrix metalloproteinase in tumor tissues correlates with the invasiveness of oral cancer, Clin Exp Metastasis 1999, 17:315-323 384. Hofmann UB, Westphal JR, Waas ET, Zendman AJ, Cornelissen IM, Ruiter DJ, van Muijen GN: Matrix metalloproteinases in human melanoma cell lines and xenografts: increased expression of activated matrix metalloproteinase-2 (MMP-2) correlates with melanoma progression, Br J Cancer 1999, 81:774-782 385. Kurschat P, Zigrino P, Nischt R, Breitkopf K, Steurer P, Klein CE, Krieg T, Mauch C: Tissue inhibitor of matrix metalloproteinase-2 regulates matrix metalloproteinase-2 activation by modulation of membrane-type 1 matrix metalloproteinase activity in high and low invasive melanoma cell lines, J Biol Chem 1999, 274:21056-21062 386. Vaisanen A, Tuominen H, Kallioinen M, Turpeenniemi-Hujanen T: Matrix metalloproteinase-2 (72 kD type IV collagenase) expression occurs in the early stage of human melanocytic tumour progression and may have prognostic value, J Pathol 1996, 180:283-289 387. Forsyth PA, Wong H, Laing TD, Rewcastle NB, Morris DG, Muzik H, Leco KJ, Johnston RN, Brasher PM, Sutherland G, Edwards DR: Gelatinase-A (MMP-2), gelatinase-B (MMP-9) and membrane type matrix metalloproteinase-1 (MT1MMP) are involved in different aspects of the pathophysiology of malignant gliomas, Br J Cancer 1999, 79:1828-1835 388. Friedberg MH, Glantz MJ, Klempner MS, Cole BF, Perides G: Specific matrix metalloproteinase profiles in the cerebrospinal fluid correlated with the presence of malignant astrocytomas, brain metastases, and carcinomatous meningitis, Cancer 1998, 82:923-930 389. Sakamoto A, Oda Y, Iwamoto Y, Tsuneyoshi M: Expression of membrane type 1 matrix metalloproteinase, matrix metalloproteinase 2 and tissue inhibitor of metalloproteinase 2 in human cartilaginous tumors with special emphasis on mesenchymal and dedifferentiated chondrosarcoma, J Cancer Res Clin Oncol 1999, 125:541-548 390. Bjornland K, Winberg JO, Odegaard OT, Hovig E, Loennechen T, Aasen AO, Fodstad O, Maelandsmo GM: S100A4 involvement in metastasis: deregulation of matrix metalloproteinases and tissue inhibitors of matrix metalloproteinases in osteosarcoma cells transfected with an anti-S100A4 ribozyme, Cancer Res 1999, 59:4702-4708 391. Parks WCaM, R. P.: Matrix Metalloproteinases. Edited by San Diego, Academic Press, 1998, p 392. Sawicki G, Marcoux Y, Sarkhosh K, Tredget EE, Ghahary A: Interaction of keratinocytes and fibroblasts modulates the expression of matrix  62  metalloproteinases-2 and -9 and their inhibitors, Mol Cell Biochem 2005, 269:209216 393. Corbel M, Belleguic C, Boichot E, Lagente V: Involvement of gelatinases (MMP-2 and MMP-9) in the development of airway inflammation and pulmonary fibrosis, Cell Biol Toxicol 2002, 18:51-61 394. Sigurdson L, Sen T, Hall L, 3rd, Rubenfeld A, Hard R, Gardella J, Bright F, Hicks WL, Jr.: Possible impedance of luminal reepithelialization by tracheal cartilage metalloproteinases, Arch Otolaryngol Head Neck Surg 2003, 129:197-200 395. Ravanti L, Kahari VM: Matrix metalloproteinases in wound repair (review), Int J Mol Med 2000, 6:391-407 396. Kheradmand F, Werb Z: Shedding light on sheddases: role in growth and development, BioEssays 2002, 24:8-12 397. Moss ML, Bartsch JW: Therapeutic benefits from targeting of ADAM family members, Biochemistry 2004, 43(23):7227-7235 398. Tang BL: ADAMTS: a novel family of extracellular matrix proteases, Int J Biochem Cell Biol 2001, 33:33-44 399. Kuno K, Matsushima K: ADAMTS-1 protein anchors at the extracellular matrix through the thrombospondin type I motifs and its spacing region, J Biol Chem 1998, 273:13912-7 400. Zimmermann DR, Ruoslahti E: Multiple domains of the large fibroblast proteoglycan, versican, EMBO J 1989, 8:2975-2981 401. Perides G, Rahemtulla F, Lane WS, Asher RA, Bignami A: Isolation of a large aggregating proteoglycan from human brain, J Biol Chem 1992, 267:23883-7 402. Perides G, Asher RA, Lark MW, Lane WS, Robinson RA, Bignami A: Glial hyaluronate-binding protein: a product of metalloproteinase digestion of versican?, Biochem J 1995, 312:377-384 403. Schmalfeldt M, Dours-Zimmermann MT, Winterhalter KH, Zimmermann DR: Versican V2 is a major extracellular matrix component of the mature bovine brain, J Biol Chem 1998, 273:15758-64 404. Westling J, Gottschall PE, Thompson VP, Cockburn A, Perides G, Zimmermann DR, Sandy JD: ADAMTS4 (aggrecanase-1) cleaves human brain versican V2 at Glu405-Gln406 to generate glial hyaluronate binding protein, Biochem J 2004, 377:787-795 405. Sandy JD, Westling J, Kenagy RD, Iruela-Arispe ML, Verscharen C, RodrigueMazaneque JC, Zimmerman D, Lemire JM, Fischer JW, Wight TN, Clowes AW: Versican V1 proteolysis in human aorta in vivo occurs at the Glu441-Ala442 bond, a site which is cleaved by recombinant ADAMTS-1 and ADAMTS-4, J Biol Chem 2001, 276:13372-13378 406. Somerville R, Longpre JM, Engle M, Jungers KA, Ross M, Evanko S, Wight TN, Leduc R, Apte SS: Characterization of ADAMTS-9 and ADAMTS-20 as a distinct  63  ADAMTS subfamily related to Caenorhabditis elegans GON-1, J Biol Chem 2003, 278:9503-9513 407. Jonsson-Rylander AC, Nilsson T, Fritsche-Danielson R, Hammarstrom A, Behrendt M, Andersson JO, Lindgren K, Andersson AK, Wallbrandt P, Rosengren B, Brodin P, Thelin A, Westin A, Hurt-Camejo E, Lee-Sogaard CH: Role of ADAMTS-1 in Atherosclerosis: Remodeling of Carotid Artery, Immunohistochemistry, and Proteolysis of Versican, Arterio Thrombo Vasc Biol 2005, 25:180-185  64  2. VERSICAN INTERACTION WITH HUMAN FIBROBLAST AND MACROPHAGE CELL SURFACE1  LUNG  2.1 Introduction Increased versican expression is often observed in association with proliferating cells within remodeling tissue in many diseases including pulmonary fibrosis, cardiovascular diseases, and cancer. In many forms of pulmonary fibrosis, such as organizing diffuse alveolar damage and idiopathic pulmonary fibrosis, the early accumulation of versican1, 2 and its binding partner hyaluronan3 occurs in association with proliferating myofibroblasts prior to deposition of a collagenous matrix4, 5. Versican accumulates in atherosclerotic lesions (reviewed in6), and is found in association with proliferating vascular and arterial smooth muscle cells7. Versican is also associated with tumor growth in tissues such as breast8-11, brain12, ovary13, gastrointestinal tract14, prostate15,  16  ,  melanoma17, and mesothelioma18. Versican, a large proteoglycan with an estimated molecular mass of more than 1000 kDa, is a member of the hyalectan (hyaluronan and lectican binding) family of proteoglycans which also includes aggrecan, abundant in cartilage, and brevican and neurocan, the two nervous system proteoglycans (reviewed in19-21). Although there is a single versican gene, alternative splicing of its mRNA produces 4 distinct versican isoforms that carry different number of glycosaminoglycan (GAG) chains22, 23.  All isoforms have  homologous N-terminal (HA binding) and C-terminal (lectin-like) domains. The central domain of versican V0 contains both the GAG-α and GAG-β GAG-bearing protein domains. The V1 isoform has the GAG-β domain, V2 has the GAG-α domain, and V3 is devoid of any GAG domains and only consists of the N-terminal and C-terminal globular domains.  Versican is consistently found in association with its binding partners:  hyaluronan, fibronectin, and tenascin24-27. The N-terminal domain of versican interacts with hyaluronan, and its C-terminal lectin-like domain may interact with a variety of other extracellular matrix molecules. These interactions may help to stabilize the matrix through formation of supra-molecular aggregates26-28. 1  A version of this chapter will be submitted for publication. Pourmalek, S, and Roberts, C. Versican interaction with human lung fibroblast and macrophage cell surface. 65  The N-terminal (G1) globular domain consists of an Immunoglobulin-like loop and two link module with hyaluronan binding properties.  It has been suggested that the  N-terminal domain of versican may play a role in maintaining the integrity of the extracellular matrix by interacting with both hyaluronan29 and link protein30. In epidermal injury, the expression of hyaluronan31, 32 along with its cell surface receptor CD4433 is increased. It has been shown that hyaluronan-CD44 signaling can lead to actin cytoskeleton reorganization34-36 and cell proliferation37 in tumor cells.  Versican  colocalization with hyaluronan and CD44 in pericellular matrix of cultured fibroblasts38, in lesions of atherosclerosis and restenosis6, 1, 2, 40  diseases  39  , and in a variety of fibrotic lung  suggests a possible role for this proteoglycan in the signaling process that  leads to migration and proliferation of these different cell types. In addition, versican synthesis is upregulated by proliferating smooth muscle cells in vitro41, and it has been suggested that the formation of versican-hyaluronan complex at the cell surface may facilitate the migration and proliferation of smooth muscle cells42, 43. The C-terminal (G3) globular domain of versican consists of one or two EGF repeats, a C-type lectin domain and complement regulatory protein (CRP)-like domain. The C-terminal domain binds to a variety of ligands in ECM which may contribute significantly to the functions of versican. A recent study has shown that complexes of G3, fibronectin and vascular endothelial growth factor enhance proliferation and angiogenesis of astrocytoma cells44. Moreover, it has been shown that versican G3 motif is involved in the formation of intermolecular disulfide bonds that stabilize the matrix, and disruption of these interactions can affect cell adhesion and cell-matrix stability45. Studies using a chimeric construct of versican have indicated a direct role for versican in cell proliferation based on the interaction of EGF-like module in the C-terminal domain of versican with the EGF receptors of fibroblasts46. However, versican has not been shown to bind or activate the EGF receptors, and other studies using versican V3 isoform seem to suggest otherwise. The central glycosaminoglycan-containing domain of versican is decorated with covalently-bound chondroitin sulfate glycosaminoglycan chains.  The structural and  functional diversity of versican is increased by variations in the number of  66  glycosaminoglycan chains bound to the core protein, as observed with four versican isoforms that exist due to alternate splicing of their glycosaminoglycan binding region. Versican’s chondroitin sulfate chains can interact with and localize a variety of growth factors47,  48  behavior21, 49.  and cytokines in the ECM, which can indirectly regulate cellular Studies of self-assembling50 hyaluronan molecules have shown that  hyaluronan, in association with aggregating chondroitin sulfate proteoglycans, increases the viscosity of the matrix51, 52 and adds swelling pressure through the negatively-charged chondroitin sulfate chains53, 54.  Chondroitin sulfate proteoglycan aggregation can  potentially stiffen the hyaluronan network55 and influence cell behavior. The receptors for three of the four major components of provisional matrix, all of which interact with versican, have been identified. To name a few, α9β1 integrin interacts with tenascin56, α(2-5)β1 integrins are receptors for fibronectin57, and CD44 for hyaluronan33. However, there is no evidence for direct interaction between versican and a cell surface ligand. In this study, we tested the hypothesis that versican interacts with the cell surface of fibroblasts and macrophages through its C-terminal domain, considering the Nterminal of versican is generally found in association with hyaluronan and link protein in vivo. Our aim was to identify possible versican cell surface receptors using modified G3 constructs as ligands. We employed the following techniques in achieving the results presented in this study: i.  Biotinylated G3 constructs were used as ligands in “far western blotting” experiments (refer to sections 2.3.3 – 2.3.5).  ii.  G3 bound to magnetic beads was used as a tool to pull down interacting proteins in the cell membrane fraction of fibroblast and macrophage cells (2.3.6).  iii.  G3-bound magnetic beads were incubated with fibroblast cells to determine any interactions with the cell surface (2.3.6).  We were unable to identify a cell surface receptor that directly interacts with C-terminal domain of versican.  However, we have shown that: G3 domain of versican  homodimerizes and forms aggregates; G3 interacts with versican and that these interactions are dependant on disulfide bond formation; and G3-coated beads bind to the  67  surface of fibroblast cells. We have also identified hyaluronan, bound to fibroblast cell surface, as the main ligand for versican and have shown that versican-hyaluronan interaction at the cell surface is important for the survival of fetal lung fibroblasts in vitro. In conclusion, we were unable to detect a versican-specific receptor against a background of these interactions.  2.2 Materials and Methods Expression and Purification of Versican – Human fetal lung (HFL1) fibroblast cells American Type Culture Collection (Manassas, VA) were cultured in 75-cm2 flasks (Sarstedt; Quebec, Canada) in Dulbecco’s Modified Eagle Medium (DMEM; Life Technologies, Inc., Gaithersburg, MD) supplemented with 10% (v/v) fetal bovine serum (FBS; HyClone, Logan, UT) to 80% confluence. Cells from two confluent 75-cm2 flasks were trypsinized and transferred to a 850-cm2 tissue culture roller bottle (Becton Dickinson) with 200ml of DMEM and incubated at 37˚C in a BELCO Biotechnology Roll-in incubator. Serum free conditioned medium from fibroblast cultures (CM) was collected and centrifuged at 1500 X g for 15 minutes to remove cellular debris. Then, Urea and salt concentrations in HFL1 conditioned media were adjusted to 7M Urea and 0.4M NaCl and loaded onto Q-Sepharose Fast Flow ion exchange resin (Amersham Biosciences, Piscataway, NJ) at approximately 1 litre CM per 5 mls resin. The column was washed with a 10 fold bed volume of 7M Urea, 0.4M NaCl, 0.1M NaOAc, pH 6.0 before elution with 7M Urea, 1.5M NaCl, 0.1M NaOAc, pH 6.0. Peak fractions were pooled and dialyzed against PBS (140mM NaCl, 2.7mM KCl, 10mM Na2HPO4, 1.8mM NaH2PO4, pH7.4) exhaustively, then flash frozen with liquid nitrogen and stored at -70˚C. Fractions were monitored for versican content by alcian blue (Sigma, St. Louis, MO) staining of SDS-PAGE gels58 and by Western blotting.  Purified versican  concentration was estimated using the dimethylmethylene blue (DMMB) assay (Serva, Heidelberg)59 to quantify sulfated glycosaminoglycan using known concentrations of chondroitin sulfate C as standards (Seikagaku). The concentration of versican was estimated based on an average of 1.5 mg total proteoglycan per 1 mg sulfated glycosaminoglycan detected with a resultant concentration of 1.12 mg/ml or approximately 1.12 µM versican.  68  Generation of Recombinant Constructs (HisL, HisLC, HisG3) – Before ligating the His-tagged C-terminal construct cDNA to the expression vector pGYMXC, the PCR product was amplified using the pPCR-Script strategy. Briefly, cDNA (generated from an HFL-1 mRNA library and amplified from PCR) was purified using a QIAEX II Gel Extraction Kit (Qiagen, Mississauga, Ontario). The DNA fragment was ligated into the pPCR-Script Cam SK(+) plasmid (Stratagene, CA, USA). The ligation mixture contained 1µl of 10X reaction buffer, 1µl of Srf I restriction enzyme (5U), 1µl of T4DNA ligase (4U), 0.5µl of 10mM rATP, 3µl of the HisLC insert (190ng), and 1µl of the cloning vector (10ng) for a 100:1 molar ratio of insert to cloning vector. The mixture was diluted to 10 µl with ddH2O, gently mixed, and incubated at room temperature for 1 hr before heating at 65˚C for 10min. Two µl of the ligation mixture was used for heat shock transformation of supercompetent E. coli strain DH5α. The bacteria was transferred to 50µl of 2X YT media (1.0% w/v yeast extract, 1.6%w/v tryptone, 0.5% w/v NaCl, pH 7.5) and agitated at 275rpm for 30min at 37˚C before plating on LB agar plates containing 30µg/ml chloramphenicol. Plates were incubated for 16hrs at 37˚C. Colonies were selected and grown in 5ml of terrific broth media (1.2% w/v tryptone, 2.4% w/v yeast extract, 0.231% w/v KH2PO4, 1.254% w/v K2HPO4, 0.4% glycerol) with 30µg/ml chloramphenicol at 37˚C for 16hrs at 275rpm. The plasmid was isolated using the QIAprep Spin Miniprep Kit (Qiagen, Mississauga, Ontario). The insert was removed from the CAM SK(+) plasmid by Hind III and NheI restriction enzyme digestion and purified by QIAEX II Gel Extraction Kit (Qiagen, Mississauga, Ontario). The identity of the new HisLC pGYMX construct was confirmed by Hind III and Nhe I restriction enzyme digestion and by DNA sequencing performed by Nucleic Acids-Protein Service (NAPS) at the University of British Columbia. Transformation, Expression and Purification of Engineered Versican C-terminal Domain Constructs (HisL, HisLC, and HisG3)– The pGYMX HisG3 expression vector was transformed into E.coli BL21(DE3) competent cells at a ratio of 1µl of cDNA into 50µl BL21 Gold E.coli cells. Briefly, the mixture was iced for about an hour before a 90 seconds heat shock at 42˚C. The mixture was iced for 2 minutes, and incubated at 37˚C for 30 min in shaker with 50µl of 2XYT media. The cells were then plated on Luria–  69  Bertani (LB) agar plates containing 100µg/ml ampicillin. 5ml of superbroth (0.8% w/v yeast extract, 1.0%w/v tryptone, 0.5% w/v NaCl, 0.1% glycerol, pH 7.5) and incubated in 37˚C shaker for 24 hours. Next, another plate of agar was inoculated with a single colony and incubated at 37˚C for 16 hrs at 275rpm. Aliquots of this log phase seed culture were used to innoculate 3.5L of superbroth in a1:1000 (v/v) ratio with 100µg/ml ampicillin and incubated at 37˚C for 24hrs at 275rpm. Collected cells were washed with 500ml of NET buffer (100mM NaCl, 1mM EDTA, 20mM Tris-HCl, pH 8.0) then lysed in 250ml of lysis buffer (50mM NaCl, 1mM EDTA, 20mM Na2HPO4, 1mg/ml lysozyme, 1mM PMSF, pH 8.0) for 2hrs at 37˚C and 275rpm and sonicated with 5 sec bursts. The inclusion bodies were washed with 500ml of NET buffer then dissolved in a solubilization buffer (8M Urea, 10mM Tris-HCl, 100mM Na2HPO4, pH 8.0) for 16hrs at 4˚C. Dissolved inclusion bodies were centrifuged at 20000rpm for 1hr and purified using a 30ml Ni2+- charged chelating sepharose column (Amersham Pharmacia) equilibrated in column buffer (8M Urea, 0.5M NaCl, 20mM Na2HPO4, pH 7.4). The column was washed in succession with 10-fold bed volume of column buffer, column buffer with 1M NaCl, column buffer with 1M NaCl, pH 6.0, and again with column buffer. Proteins with non-specific interactions to the Ni-chelate column were pre-eluted by a 10-fold bed volume of column buffer with 200mM imidazole. HisLC fusion protein was then eluted by a 200mM to 1M imidazole gradient over a 10-fold bed. Fractions were analyzed by SDS-PAGE and stored at –20˚C. Refolding of HisG3 Recombinant Protein – Peak fractions obtained from the imidazole gradient were pooled and diluted 20-fold before dialysis in equal volume of refolding buffer (18.2mM Na2CO3, 24mM NaHCO3, 125mM NaCl, 1:10 ration of 3mM Cysteine/Cystine, pH 10.0) with aeration at room temperature. Refolding buffer was changed every 2 hrs for 8 hrs before exhaustive dialysis with refolding buffer minus the redox pair of Cysteine/Cystine for complete removal of urea.  Because the pooled  fractions were diluted 20 fold before refolding, a 10ml Ni-chelate column equilibrated in 18.2mM Na2CO3, 24mM NaHCO3, 125mM NaCl, pH 10.0 was used to concentrate the diluted pool. HisLC was eluted with 18.2mM Na2CO3, 24mM NaHCO3, 125mM NaCl, 500mM imidazole, pH 10.0. Peak fractions were pooled and dialyzed against excess  70  volume of 18.2mM Na2CO3, 24mM NaHCO3, 125mM NaCl, pH 10.0. The protein pool was aliquoted into 1ml fractions, flash frozen with liquid nitrogen, and stored at –70˚C. HisLC Antibody Preparation – New Zealand white rabbits (3months old, 3kg each) were injected with 1ml of HisLC (0.263 mg/ml, expressed and purified in our laboratory) mixed with Freund’s Incomplete Adjuvant. In total, 64µg of HisLC was injected into each rabbit. 10ml of pre-bleed was collected from each rabbit before HisLC injection. A booster shot of the same amount was repeated at 3 weeks, and rabbits were fully bled at 1 month after initial injection. Blood was incubated at 37°C for 1 hour to promote clotting. Blood clot was removed and the rest of the mixture was centrifuged at 3000rpm for 6 minutes. The supernatant was removed and incubated at 4°C for 3 hours. Serial dilutions of supernatant (1:4 – 1:4096) were prepared in an ELISA plate that had been coated with 200µg/well HisLC and were blocked with 2.5% BSA at 4°C overnight. Each well was washed with TBS plus 0.05% Tween-20 and incubated in 1:2000 GARAP for 1 hour at room temperature. The plate was washed again and developed with 100µl/well p-nitrophenyl phosphate (pnPP) substrate, in Tris buffer, to estimate the concentration of antibody in serum. Electrophoretic Techniques – Samples in nonreducing sample buffer (125 mM Tris-HCL, pH 6.8, 2.0% SDS, 2.0 M urea, 0.05% bromophenol blue) were separated on discontinuous SDS-PAGE gels with 4% (stacking) and 12.5% (separating) acrylamide. Stacking and separating gels were kept during staining and Western blotting to monitor high molecular weight versican aggregates within the stacking gel. Relative molecular mass (measured in kDa) was estimated based on mobility of molecular weight markers: HiMark Prestained (Invitrogen), MagicMark XP (Invitrogen) and Kaleidoscope Prestained (Bio-Rad, Hercules, CA). Western blotting was performed using the XCell II blot module (Invitrogen) to PVDF membrane (Millipore). Blocking was performed with a solution of 2.5% (w/v) bovine serum albumin, 20 mM Tris, 5 mM EDTA, 0.9%NaCl, and 0.3% (v/v) Tween 20. Anti-versican 2B1(Seikagaku Corporation, Tokyo, Japan), 1:500 dilution, was used for detection of versican at an epitope near the C-terminal domain; anti-G3 antibody (anti-HisLC “LC2”), 1:10,000 dilution, for detection of the C-terminal domain of versican; and biotinylated HisLC and biotinylated HisG3 (prepared 71  in our laboratories), 1:20 dilution. Antibodies were diluted in a solution of 2% (w/v) bovine serum albumin, 20 mM Tris, pH 7.5, 0.9 % NaCl and 0.05% (v/v) Tween 20. Highly cross adsorbed goat anti-mouse horseradish peroxidase-conjugate (Bio-Rad); highly cross adsorbed goat anti-rabbit horseradish peroxidase-conjugate (Bio-Rad) and Streptavidin Alkaline Phosphate HRP were diluted to 1:5000. Visualization of the peroxidase was performed with Enhanced Chemiluminescence Plus Western blotting reagents (Amersham Biosciences) and exposed to X-ray film (Kodak, New Haven, CT) or captured using the ChemiGenius-2 bio-imaging system and Gene Snap software (Perkin Elmer, Woodbridge, ON) Release of Versican from Fibroblast Cell Surface Using Hyaluronidase– Human fetal lung fibroblast cell line (HFL1) was obtained from American Type Culture Collection (Manassas, VA) and grown in Dulbecco’s modified Eagle medium (DMEM) supplemented with 20 mM HEPES and 10% (v/v) Cosmic Calf Serum (Hyclone, Logan, UT) in cell culture flasks. Once cells were at 80% confluence, they were trypsinized and transferred into 4well chamber slides. Cells were allowed to spread on the slides in serum enriched media at 37ºC until 80% confluent. Cells were subsequently treated with hyaluronidse (Type X, from Leech, 2000 units/ml, Sigma Chemical Co. Louis, MO, USA) in phosphate buffer saline, at different concentrations (0, 3.3µg/ml, 33.3µg/ml, and 333.3µg/ml) and for different lengths of time (30 minutes, 1hour, 4hours, and 8hours). At the end of each time point, the media containing hyaluronidase enzyme and its cleavage products was collected for further analysis, and the cellular behavior was analyzed using fluorescent staining and microscopy. Products of hyaluronidase treatment in the media were analyzed with silver stained SDS-PAGE and western blotting. Cytochemistry – At the end of each time point, cells were fixed in 4% paraformaldehyde in PBS for 15 minutes in 37°C incubator.  Fixed cells were  permeabilized with 0.1% Triton X-100 and PBS for 1 minute and washed with PBS before being blocked by freshly prepared 1% bovine serum albumin (BSA) solution in PBS for 5 minutes. Versican was detected with LeBaron rabbit polyclonal antibody, a kind gift from Dr. Richard LeBaron60 and Dr. Erkki Ruoslahti (La Jolla Cancer Research Foundation, CA, USA), 1:250 dilution; and 1:250 Alexafluor-488 anti-rabbit antibody  72  (Molecular Probes, Eugene, Oregon, USA). After being washed with PBS, cells were incubated in Hoechst stain (33342, Invitrogen) at 1:2000 dilution in 1% BSA/PBS, for 10 minutes. Cells were then washed with PBS and stored with ProLong Gold antifade reagent (Invitrogen, Molecular Probes) on a covered slide. Fluorescent Light Microscopy – Fixed and stained cells were viewed using a Leica CTR fluorescent light microscope on monochrome setting. Images were captured at 40X magnification, in layers of 0.75µm thickness using the multi-channel z-stack capture option using Q-Imagine Retiga Exi mounted camera and OpenLab software (4.0.2). Emission wavelengths were set to best view each stained organelle: actin filaments were viewed at 520nm61; nuclei at 456nm (DAPI), and mitochondrion at 594nm (Texas Red). Once images were captured, each image was deconvoluted using the Nearest Neighbor DCI function to remove unfocussed light.  All layered images taken of the same  coordinates but at different points along the z-axis (height) were merged to create one 3dimentional image of cells spread in the collagen matrix. The appropriate color of the stain was then applied to the image, and images captured under different emission wavelength of different organelles were merged into one composite.  The scale bar was  set at 95µm. Biotinylation of Versican C-terminal Constructs – Versican C-terminal constructs were biotinylated using EZ-Link NHS-PEO Solid Phase Biotinylation Kit (Pierce, 21440, Rockford, IL) following the manufacturer’s instructions.  In short, HisG3 protein  constructs were bound to a charged Nickel column. The biotin solution at an appropriate concentration for the amount of protein being biotinylated was applied to column and allowed to completely immerse the capped gel bed. The biotin solution was incubated with the gel bed for 30 minutes at room temperature and then removed by allowing it to flow through the column. The biotinylated protein was eluted from the column by 0.2M Imidazole in PBS, and stored at 4°C. Production of Magnetic Beads – Protein constructs were dialyzed (Slide-A-Lyzer dialysis cassette, Pierce, Canada) in a buffer free of any amine or carboxyl groups, with an appropriate pH (4 – 6) for covalent attachment of proteins to magnetic beads. BioMag  73  Plus (BP618) with carboxyl-terminated surface (Bangs Laboratories, Inc., 6445) was used, and manufacture’s instructions were followed, in the production of HisG3-bound magnetic beads. In short, the beads were washed and activated with EDC [1-ethyl-3(3dimethylaminopropyl)-carbodiimide] and coupled with the protein solution of interest (HisLC, HisG3, or with Glycine for production of control beads). After 24 hours of incubation, the unbound protein solution (supernatant) was removed and saved for binding efficiency calculations, and the reaction was stopped by adding the quenching solution (1.0M Glycine , pH 8.0). The protein-bound beads (with a particle concentration of 5mg/ml) were washed and stored in a favorable buffer (HEPES, pH 7.0) at 4°C. This procedure yielded protein-bound magnetic beads with a 90.9% efficiency. The binding efficiency of proteins to the beads were calculated by measuring the absorption (A280) of protein solution before and after binding to the beads, using the following formula: (Absbefore binding x dilution) – (Absafter binding x dilution) / (Absbefore binding x dilution) % Examination of G3-Coated Magnetic Bead Binding to Fibroblast Cell Surface – For visualization of HisG3-bound magnetic beads interaction with HFL1 fibroblast cell surface using phase microscopy, fibroblast cells were plated on 12 well chamber slides. Cells were grown to 50% confluence, and then, HisG3-bound magnetic beads or Glycinebound control beads (50 µg/ml; diluted in HEPES buffer) were added to the cells. After 20 minutes of incubation at 37°C, cells were fixed with 40% formaldehyde solution for 20 minutes and then observed under a differential interference contrast (DIC) microscope at different magnifications (10X, 20X, 40X).  In the case of detecting cell membrane  proteins which may interact with HisG3-bound magnetic beads, the solution, containing either HFL1 fibroblast or U937 macrophage cell membrane protein fraction, was incubated with HisG3-bound magnetic beads overnight at 4°C.  The beads were  magnetically separated from the solution of unbound proteins and the supernatant was saved for electrophoretic analysis. The HisG3 binding proteins were eluted with DTT containing sample buffer and analyzed with silver stain after SDS-PAGE. Preparation of cell membrane fraction from cell lysate – Protein fraction from HFL1 fibroblast and U937 macrophage cell membranes were separated from the cell lysate using nitrogen-disruption bomb (Parr Instrument Company, 4635, Moline, IL, 74  USA) following manufacturer’s instructions. In summary, cells at a concentration of 1x106 cells/ml were centrifuged and washed in an appropriate buffer. The cells in a falcon tube were assembled in the nitrogen flask, and the flask was pressurized with nitrogen gas from a nitrogen tank to 500psi. The cells were incubated under pressure for 30 minutes before the pressure was released slowly and the lysed cells were collected in a second tube.  The cell lysate, in appropriate buffer with proteinase inhibitors, was  centrifuged at 2700 rpm for 10 minutes to separate the pellet of heavier cell solids from the solubilized membrane proteins in the supernatant.  The supernatant was  then  centrifuged at 18,500 rpm for 1 hour to separate the membrane protein pellet from the solution. The membrane protein pellet was homogenized in buffer at 11,000 rpm for 5 seconds and stored at 4°C.  2.3 Results Histidine-tagged C-terminal constructs of versican, alternatively called the HisG3 and HisLC domain (figure 2.1), were expressed in E.coli and purified in our laboratory. These C-terminal constructs were used to investigate specific interactions between versican and the cell surface through versican’s C-terminal domain.  The results  presented here are representative of at least three different trials for each given experiment.  75  His-G3 (His-EELC) His-LC 6 Histidine residues (His tag) 2 EGF-like modules C-type Lectin module Complement regulatory protein  Figure 2.1 Structure of HisG3 and HisLC versican constructs 2.3.1 Purification and Characterization of Versican from HFL1 Fibroblast Conditioned Media Versican was purified from the serum-free conditioned media of Human fetal lung (HFL1) fibroblast cells. Serum free media from 80% confluent fibroblast cultures were collected and centrifuged at 1500g for 15 minutes to remove cellular debris. Then, Urea and salt concentrations in HFL1 conditioned media were adjusted to 7M Urea and 0.4M NaCl and loaded onto Q-Sepharose Fast Flow ion exchange resin at approximately 1 litre culture media per 5 mls resin (Figure 2.2, FT). The column was washed with a 10 fold bed volume of 7M Urea, 0.4M NaCl, 0.1M NaOAc, pH 6.0 (Figure 2.2, W) before elution with 7M Urea, 1.5M NaCl, 0.1M NaOAc, pH 6.0 (Figure 2.2, Eluted Vc). Fractions were monitored for versican content by silver staining and alcian blue staining of SDS-PAGE gels (Figure 2.2).  The identity of versican as the predominant  proteoglycan was established by western blotting and mass spectrometry (data not shown). Purified versican concentration was estimated using the dimethylmethylene blue (DMMB) assay to quantify sulfated glycosaminoglycan using known concentrations of chondroitin sulfate C as standards. The concentration of versican was estimated based on an average of 1.5 mg total proteoglycan per 1 mg sulfated glycosaminoglycan detected with a resultant concentration of 1.12 mg/ml or approximately 1.12 µM versican. The pattern left by high-molecular weight versican and versican aggregates on the polyacrylamide gel is signatory, and is observed in all data presented in this thesis. Versican leaves rippling marks on the larger pored stacking gel, and hardly penetrates the smaller pored separating gel.  76  2.3.2 Purified Versican C-terminal (LC) Constructs Form Aggregates in Solution The HisLC expression vector was transformed into E.coli competent cells, and cells were grown in LB media. Cells were lysed and the inclusion bodies were separated through centrifugation, washed, then dissolved in a solubilization buffer (Figure 2.3, L). Dissolved inclusion bodies were centrifuged and purified using a Ni2+ charged chelating sepharose column equilibrated in column buffer. The column was washed in succession with 10-fold bed volume of column buffer, column buffer with 1M NaCl, column buffer with 1M NaCl, pH 6.0, and again with column buffer (Figure 2.3, FT). Proteins with non-specific interactions to the Ni-chelate column were pre-eluted by a 10-fold bed volume of column buffer with 200mM imidazole (Figure 2.3, W). HisLC fusion protein was then eluted by a 200mM to 1M imidazole gradient over a 10-fold bed (Figure 2.3, Eluted HisLC). Peak fractions obtained from the imidazole gradient were pooled and diluted 20-fold before dialysis in equal volume of refolding buffer with 1:10 ratio of 3mM Cysteine/Cystine and aeration at room temperature. Refolding buffer was changed frequently before exhaustive dialysis with refolding buffer minus the redox pair of Cysteine/Cystine for complete removal of urea.  Because the pooled fractions were  diluted 20 fold before refolding, a 10ml Ni-chelate column was used to concentrate the diluted pool. HisLC was eluted, and peak fractions were pooled and dialyzed against excess volume of Voller’s buffer, based on a test of at least 48 buffers and conditions. The full length C-terminal domain (HisG3) of versican was purified following the same procedures (by Haidi Kai, data not shown). HisLC formed higher molecular aggregates in solution which made the refolding process particularly difficult. HisG3 was refolded and verified by binding to fluorescein-heparin, by fluorescence anisotropy spectroscopy, and showed much greater degree of self-association than HisLC under the same conditions. However, the majority of molecular species were found at the appropriate molecular weight for a monomer, as observed in the following SDS gel for HisLC.  78  2.3.3 Biotinylated HisLC interacts with versican-like molecules in fibroblast cell lysate In order to determine any possible interactions between the lectin domain of versican and fibroblast cell surface, biotinylated HisLC construct was used as ligand in far western blotting with HFL1 cell lysate. To this end, HisLC construct was biotin-labeled using the EZ-Link NHS-PEO Solid phase biotinylation kits (Figure 2.4, ½ diluted in lane 5; and neat concentration in lane 6). Cell lysate of fibroblasts, grown in culture plates, contains high-molecular weight proteins that interact with biotinylated HisLC under non-reducing conditions (Figure 2.4, -DTT, lane 1, stacking gel). Biotinylated HisLC also interacts with a number of other molecules in fibroblast cell lysate, with varying molecular weights ranging from 30 kDa – 100 kDa. Three strongest protein bands that interact with biotinylated HisLC are of the same molecular weight as HisLC (29 kDa) and HisLC aggregates (Biotinylated HisLC +DTT, arrows).  Note that there is no non-specific  interaction between the secondary Avidin-HRP antibody and any of the proteins in the cell lysate (Figure 2.4, control), and that Avidin-HRP interacts only with the biotin group on biotinylated HisLC (Figure 2.4, lanes 5 and 6).  80  The pattern of these high molecular weight species, which interact with HisLC in the cell lysate (Figure 2.4), is similar to what is generally observed with purified versican on western blot (Figure 2.2). As such, we examined the possibility that these high molecular weight species may in fact be versican. We employed a number of different methods, and assessed the protein content of fibroblast cell lysate (Figure 2.5, CL) with the versican-specific 2B1 antibody, HisLC antibody, and alcian blue stain which stains for glycosaminoglycan chains, in parallel to a biotinylated HisLC far-western blot. The high molecular weight molecule detected in biotinylated HisLC blot corresponded to the pattern of molecules detected by versican 2B1 antibody and by alcian blue stain. This observation supports an interaction between the biotinylated HisLC construct and versican species in the cell lysate. This experiment also confirmed that lower molecular weight fragments in fibroblast cell lysate, as detected by biotinylated HisLC, contain the C-terminal domain of  versican as they are also recognized by HisLC antibody  (Figure 2.5, arrows between 41.3-89 kDa).  82  2.3.4 Versican intermolecular interactions modulated by its C-terminal domain In the previous section, we showed that biotinylated HisLC interacted with fragments in the HFL1 fibroblast cell lysate that were the same size as the LC monomer and aggregates (Figure 2.4), and with fragments of versican that contained the C-terminal domain of versican (Figure 2.5). Based on these observations, intermolecular interactions of versican through its C-terminal domain were investigated further. Purified C-terminal construct of versican was loaded onto a polyacrylamide gel and transferred to a PVDF membrane after electrophoresis. The biotinylated versican construct was used as a ligand in far-western blotting experiments to examine intermolecular interactions. Our data showed that HisLC interacts with biotinylated HisLC construct, and confirmed our previous observation that HisLC monomer forms higher molecular weight aggregates (Figure 2.6), illustrating a specific intermolecular interaction between the G3 domains of versican molecules.  84  In summary, the HisLC domain seemed to interact with itself and the full G3 domain of versican under non-reducing conditions, suggesting that versican may self-associate through disulfide bond formation between the LC domains at its C-terminus. 2.3.5 Biotinylated HisG3 interacts with proteins in fibroblast and macrophage cell membrane To gain a better understanding of the interactions of C-terminal domain of versican with the cell surface of HFL1 fibroblasts and U937 macrophages, cell membrane proteins from both cell types were isolated from the cell lysate using the “Nitrogen Bomb” method. Biotinylated HisG3 (full C-terminal construct of versican) was used as a probe in far-western blotting experiment (Figure 2.7, Western Blot), under reducing (+DTT) and non-reducing (-DTT) conditions, to analyze the size of the cell membrane proteins that interact with it. Far fewer proteins in HFL1 fibroblast cell membrane interact with biotinylated HisG3 in comparison to the many protein bands that are detected in U937 macrophage cell membrane. There were a very large number of proteins in the cell membrane fraction that bound labeled HisG3, and we were unable to unravel the identity of HisG3 interacting proteins.  86  2.3.6 HisG3-coated magnetic beads interact with each other and with the surface of fibroblasts In order to identify cell surface molecules that interact with versican C-terminal domain, we used HisG3-coated magnetic beads.  HisG3 versican constructs were covalently  bound to carboxyl-terminated magnetic beads with about 90% efficiency. HisG3-bound magnetic beads were then used as bait to pull down any cell surface ligands that may exist in the pool of cell membrane proteins isolated from human lung fibroblasts and macrophages (Figure 2.8). The HisG3 coated magnetic bead chromatography led to the visualization of a number of proteins from the HFL1 fibroblasts and U937 macrophages cell membrane fraction. Our hypothesis was that any major G3-interacting protein would be pulled down by G3-coated beads and not by control (glycine-coated) beads. From the silver stained gels alone, no major cell surface ligands for G3 domain of versican could be detected in either cell type. The protein band pattern of the eluate from glycine-coated magnetic beads and that of HisG3-bound magnetic beads were visually identical. One caveat of using 1-dimensional gel electrophoresis to separate a complex mixture of proteins is that a single band on the gel may represent several different proteins indistinguishable by this method. However, within the limits of this technique, no major targets for protein sequencing were visualized.  88  In order to determine if HisG3-bound magnetic beads could bind to cell surface of fibroblasts, we performed cell culture experiments through which the binding patterns of HisG3-bound, and control beads, could be observed with high resolution differential interference contrast (DIC) microscopy (Figure 2.9). Images taken of the fibroblast cell interaction with the magnetic beads showed that HisG3-coated beads interact with each other, and with the fibroblast cell surface. Control glycine-bound beads, on the contrary, did not aggregate and seemed to uniformly cover the glass slide on and around the cells. Cell microscopy of magnetic beads coated with HisG3 protein construct confirmed our previous results (Figure 2.6) that HisG3 self-associates and interacts with the cell surface of fibroblast cells.  It is important to note that aggregate formation from beads  individually coated with G3 shows that HisG3-HisG3 interactions form from HisG3 monomers, rather than as an artifact of aberrant folding of the HisG3 construct.  90  2.3.7 Hyaluronidase treatment of cell cultured fibroblasts leads to the release of versican from fibroblast cell surface HFL1 fibroblast cells, plated to 75% confluence in culture flask, were treated with hyaluronidase to determine whether degradation of hyaluronan would release cell surface versican. The intensity of fluorescent immunostain to versican, in association with HFL1 fibroblast cell surface, decreases with increase in the concentration of hyaluronidase (33.3 – 333.3 µg/ml) and with increasing time of incubation of cells with hyaluronidase (4 – 8 hours). Versican was released from the fibroblast pericellular matrix into the extracellular media, as observed by fluorescence microscopy using both the LeBaron antibody (Figure 2.10) and by western blotting with the 2B1 antibody (Figure 2.11). It is important to note that no fluorescent staining was observed for versican in the control groups, which were treated only with the secondary fluorescent antibody. Both LeBaron and 2B1 antibodies have been characterized in western blots as specific for versican, both in our and other laboratories60. The release of versican was confirmed with western blot analysis using 2B1 antibody (Figure 2.11, arrow head). The silver stain of the proteins released into media also shows an increase in the concentration of a number of proteins including a species that corresponds to high molecular weight versican (Figure 2.11, arrow head). These immunofluorescence studies suggest that most versican is releasable from the cell surface by hyaluronidase treatment within 8 hours. These results strongly support the hypothesis that versican is held at fibroblast cell surface mainly through the interaction of its N-terminus with hyaluronan.  92  2.3.8 Hyaluronidase treatment of fibroblasts induces changes in cell morphology Fibroblast treatment with hyaluronidase lead to another observation, that as versican-hyaluronan complex is released from fibroblast cell surface, the morphology of nucleus is changed from a healthy dividing state into one marked by chromatin clumping and nucleus swelling, perhaps indicative of necrosis (Figure 2.12). This is particularly visible in the cells treated with higher concentration of hyaluronidase (333.3 µl/ml) and with higher incubation periods (8 hours). Versican association with the cell surface is essential for normal fibroblast cell morphology, and release of versican from the cell surface can force fibroblasts to conform to the lines of stress laid out by collagen fibers.  95  2.4 Discussion Increased versican expression is often observed in association with proliferating cells within remodeling tissue in lung and cardiovascular diseases and in cancer. In this study, we tested the hypothesis that versican interacts with the cell surface of fibroblasts and macrophages through its C-terminal domain. Our results have failed to identify any ligands on the macrophage cell surface, and have lead us to believe that versican is held near the fibroblast cell surface mainly through the interaction of its N-terminus with hyaluronan. These results are in concert with other research that suggests interactions of G1 domain of versican with HA results in the formation of a pericellular matrix that is required for the proliferation of arterial smooth muscle cells7. We have shown that hyaluronidase treatment of cultured fibroblast cells leads to shedding of versican from the cell surface and alteration of fibroblast phenotype, in vitro. Similarly, other studies have shown that dissolution of the pericellular matrix by treatment of the cells with HA oligosaccharides inhibits SMC proliferation7. CD44, the main cell receptor for hyaluronan, may also interact with versican through its chondroitin-sulfate chains48. The binding of versican with CD44 may be mediated by the interaction of CS chains of versican with the carbohydrate-binding domain of these molecules48, and it has been suggested that versican interaction may complement or modulate CD44 mediated adhesion and migration. Nonetheless, hyaluronidase treatment seems to release most versican from the cell surface. Our data are consistent with a model where versican can self-associate through G3 domain and can form large aggregates45. Our G3 constructs could also interact with versican through interactions with the C-terminal domain. It has been hypothesized that versican G3 motif is involved in the formation of intermolecular disulfide bonds that stabilize the matrix, and disruption of these interactions can affect cell adhesion and cell-matrix stability45. Our search for cell surface receptors that would bind versican  97  C-terminal domain, using G3 constructs, did not lead to identification of possible novel ligands. Evidence in the literature for a direct interaction of versican with the cell surface relies upon vector-driven over-expression of versican constructs or deletion of segments within such constructs. For example, an engineered chimeric molecule named “mini-versican” has been shown to modestly stimulate NIH 3T3 cell proliferation, and it has been suggested that the EGF-like modules in the G3 domain are responsible for the observed enhanced cellular proliferation46. Deletion of the G3 domain or the EGF-like repeats, they reported, eliminated the effect of over-expression or addition of versican products on cell proliferation. It is important to note, however, that the G3 construct used to show enhanced cellular proliferation contained an MRGS-His tag, while the G3 mutant which did not result in cell proliferation had the MRGS-His tag removed along with the EGF sequences. MRGS sequence is similar to the RGD (Arg-Gly-Asp) tri-peptide which is frequently found in proteins that interact with integrin cell adhesion receptors (Reviewed in63, 64). Possible interaction of MRGS with cell surface integrin receptors could explain the observed difference in cell behavior in the presence of G3 and G3-mutant constructs. This group has also reported that addition of anti-sense against EGF receptor could block the effect of added versican, although versican has not been shown to bind or activate the EGF receptor. In contrast, studies of rat SMC that were retrovirally transduced to express versican V3, which lacks the GAG binding domains, showed decreased cell proliferation and migration and increased cell adhesion65. It has been suggested that the effects of V3 on SMC indicate that over-expression of versican G3 domain constructs do not universally promote cell proliferation. Effects of V3 on cell behavior may be as a result of competition for binding to cell surface-associated endogenous versican (V0/V1) ligands, such as HA66. This suggestion is supported by the observation that the formation of the HA-versican V0 and V1 pericellular matrix is inhibited in cells that express versican V366. Taken together, the data in the literature suggest that versican has important functions at the cell surface that influences cell behavior, but more studies are needed to elucidate the mechanism of its action on the cells. Our data are consistent with versican-versican and versican-hyaluronan interactions at the cell surface found in other studies. Against a 98  background of versican-versican and versican-hyaluronan interactions, with these cell types, we were unable to find evidence for other major receptor-ligand interactions. The deposition of a transient granulation tissue and its subsequent maturation and remodeling is the most clinically significant phase of wound healing. Myofibroblasts proliferate in a matrix rich in fibronectin67, tenascin, proteoglycans versican, biglycan, and the glycosaminoglycan hyaluronan1, 2. Versican is believed to play a significant role in this matrix through interactions with the cell surface and with its binding partners, and thus has been the focus of much research in many different tissues. Our findings support previous research, and suggest that versican is held at fibroblast cell surface predominantly through its interactions with hyaluronan, and that formation of this pericellular matrix is essential for the maintenance of  fibroblast cell phenotype.  Homodimerization of G3 through formation of disulfide bonds may also be significant, as it may contribute to cell adhesion and cell-matrix stability. In addition, lack of any direct interactions between fibroblast cell surface proteins and versican C-terminal constructs lead us to believe that versican may exert its effects on the cells either as a structural molecule in the matrix through interacting with its binding partners, or through biochemical properties of its glycosaminoglycan chains. In this context, it is interesting that versican exists in four isoforms that differ principally in the number of covalently bound GAG chains. This reflects different biological functions for these splice variants, possibly through influencing charge densities in the extracellular matrix. The biological significance of GAG density and the resulting charge density that may influence versican interaction with various elements in the wound healing matrix is unknown, but worthy of further investigation.  99  2.5 References 1.  Bensadoun ES, Burke AK, Hogg JC, Roberts CR: Proteoglycan deposition in pulmonary fibrosis, Am J Respir Crit Care Med 1996, 154:1819-1828  2.  Bensadoun ES, Burke AK, Hogg JC, Roberts CR: Proteoglycans in granulomatous lung diseases, Eur Respir J 1997, 10:2731-2737  3.  Juul SE, Kinsella MG, Jackson JC, Truog WE, Standaert TA, Hodson WA: Changes in hyaluronan deposition during early respiratory distress syndrome in premature monkeys, Pediatr Res 1994, 35:238-243  4.  Roberts CR: Versican in the Cell Biology of Pulmonary Fibrosis. Edited by Hari G. Garg PJR, and Charles A. Hales. New York, Marcel Dekker, Inc., 2002, p. pp. 191-212  5.  Roberts CR, Burke AK: Remodelling of the extracellular matrix in asthma: proteoglycan synthesis and degradation, Can Respir J 1998, 5:48-50  6.  Wight TN, Merrilees MJ: Proteoglycans in atherosclerosis and restenosis: key roles for versican, Circ Res 2004, 94:1158-1167  7.  Evanko SP, Angello JC, Wight TN: Formation of hyaluronan- and versican-rich pericellular matrix is required for proliferation and migration of vascular smooth muscle cells, Arterioscler Thromb Vasc Biol 1999, 19:1004-1013  8.  Isogai Z, Shinomura T, Yamakawa N, Takeuchi J, Tsuji T, Heinegard D, Kimata K: 2B1 antigen characteristically expressed on extracellular matrices of human malignant tumors is a large chondroitin sulfate proteoglycan, PG-M/versican, Cancer Res 1996, 56:3902-3908  9.  Nara Y, Kato Y, Torii Y, Tsuji Y, Nakagaki S, Goto S, Isobe H, Nakashima N, Takeuchi J: Immunohistochemical localization of extracellular matrix components in human breast tumours with special reference to PG-M/versican, Histochem J 1997, 29:21-30  10.  Brown LF, Guidi AJ, Schnitt SJ, Van De Water L, Iruela-Arispe ML, Yeo TK, Tognazzi K, Dvorak HF: Vascular stroma formation in carcinoma in situ, invasive carcinoma, and metastatic carcinoma of the breast, Clin Cancer Res 1999, 5:10411056  11.  Ricciardelli C, Brooks JH, Suwiwat S, Sakko AJ, Mayne K, Raymond WA, Seshadri R, LeBaron RG, Horsfall DJ: Regulation of stromal versican expression by breast cancer cells and importance to relapse-free survival in patients with node-negative primary breast cancer, Clin Cancer Res 2002, 8:1054-1060  12.  Paulus W, Baur I, Dours-Zimmermann MT, Zimmermann DR: Differential expression of versican isoforms in brain tumors, J Neuropathol Exp Neurol 1996, 55:528-533  13.  Voutilainen K, Anttila M, Sillanpaa S, Tammi R, Tammi M, Saarikoski S, Kosma VM: Versican in epithelial ovarian cancer: relation to hyaluronan, clinicopathologic factors and prognosis, Int J Cancer 2003, 107:359-364 100  14.  Theocharis AD, Vynios DH, Papageorgakopoulou N, Skandalis SS, Theocharis DA: Altered content composition and structure of glycosaminoglycans and proteoglycans in gastric carcinoma, Int J Biochem Cell Biol 2003, 35:376-390  15.  Sakko AJ, Ricciardelli C, Mayne K, Tilley WD, Lebaron RG, Horsfall DJ: Versican accumulation in human prostatic fibroblast cultures is enhanced by prostate cancer cell-derived transforming growth factor beta1, Cancer Res 2001, 61:926-930  16.  Ricciardelli C, Mayne K, Sykes PJ, Raymond WA, McCaul K, Marshall VR, Horsfall DJ: Elevated levels of versican but not decorin predict disease progression in early-stage prostate cancer, Clin Cancer Res 1998, 4:963-971  17.  Touab M, Villena J, Barranco C, Arumi-Uria M, Bassols A: Versican is differentially expressed in human melanoma and may play a role in tumor development, Am J Pathol 2002, 160:549-557  18.  Gulyas M, Hjerpe A: Proteoglycans and WT1 as markers for distinguishing adenocarcinoma, epithelioid mesothelioma, and benign mesothelium, J Pathol 2003, 199:479-487  19.  Iozzo RV: Matrix proteoglycans: from molecular design to cellular function, Annu Rev Biochem 1998, 67:609-652  20.  Yamaguchi Y: Lecticans: organizers of the brain extracellular matrix, Cell Mol Life Sci 2000, 57:276-289  21.  Kinsella MG, Bressler SL, Wight TN: The regulated synthesis of versican, decorin, and biglycan: extracellular matrix proteoglycans that influence cellular phenotype, Crit Rev Eukaryot Gene Expr 2004, 14:203-234  22.  Ito K, Shinomura T, Zako M, Ujita M, Kimata K: Multiple forms of mouse PGM, a large chondroitin sulfate proteoglycan generated by alternative splicing, J Biol Chem 1995, 270:958-965  23.  Zako M, Shinomura T, Ujita M, Ito K, Kimata K: Expression of PG-M(V3), an alternatively spliced form of PG-M without a chondroitin sulfate attachment in region in mouse and human tissues, J Biol Chem 1995, 270:3914-3918  24.  Yamagata M, Yamada KM, Yoneda M, Suzuki S, Kimata K: Chondroitin sulfate proteoglycan (PG-M-like proteoglycan) is involved in the binding of hyaluronic acid to cellular fibronectin, J Biol Chem 1986, 261:13526-13535  25.  Aspberg A, Binkert C, Ruoslahti E: The versican C-type lectin domain recognizes the adhesion protein tenascin-R, Proc Natl Acad Sci U S A 1995, 92:10590-10594  26.  Kohda D, Morton CJ, Parkar AA, Hatanaka H, Inagaki FM, Campbell ID, Day AJ: Solution structure of the link module: a hyaluronan-binding domain involved in extracellular matrix stability and cell migration, Cell 1996, 86:767-775  27.  Wu YJ, La Pierre DP, Wu J, Yee AJ, Yang BB: The interaction of versican with its binding partners, Cell Res 2005, 15:483-494  101  28.  Aspberg A, Miura R, Bourdoulous S, Shimonaka M, Heinegard D, Schachner M, Ruoslahti E, Yamaguchi Y: The C-type lectin domains of lecticans, a family of aggregating chondroitin sulfate proteoglycans, bind tenascin-R by protein-protein interactions independent of carbohydrate moiety, Proc Natl Acad Sci U S A 1997, 94:10116-10121  29.  LeBaron RG, Zimmermann DR, Ruoslahti E: Hyaluronate binding properties of versican, J Biol Chem 1992, 267:10003-10010  30.  Matsumoto K, Shionyu M, Go M, Shimizu K, Shinomura T, Kimata K, Watanabe H: Distinct interaction of versican/PG-M with hyaluronan and link protein, J Biol Chem 2003, 278:41205-41212  31.  Tammi R, Pasonen-Seppanen S, Kolehmainen E, Tammi M: Hyaluronan synthase induction and hyaluronan accumulation in mouse epidermis following skin injury, J Invest Dermatol 2005, 124:898-905  32.  Jiang D, Liang J, Noble PW: Hyaluronan in tissue injury and repair, Annu Rev Cell Dev Biol 2007, 23:435-461  33.  Underhill C: CD44: the hyaluronan receptor, J Cell Sci 1992, 103 (Pt 2):293-298  34.  Bourguignon LY, Zhu H, Shao L, Zhu D, Chen YW: Rho-kinase (ROK) promotes CD44v(3,8-10)-ankyrin interaction and tumor cell migration in metastatic breast cancer cells, Cell Motil Cytoskeleton 1999, 43:269-287  35.  Zhu D, Bourguignon LY: Interaction between CD44 and the repeat domain of ankyrin promotes hyaluronic acid-mediated ovarian tumor cell migration, J Cell Physiol 2000, 183:182-195  36.  Oliferenko S, Kaverina I, Small JV, Huber LA: Hyaluronic acid (HA) binding to CD44 activates Rac1 and induces lamellipodia outgrowth, J Cell Biol 2000, 148:1159-1164  37.  Bourguignon LY, Zhu H, Chu A, Iida N, Zhang L, Hung MC: Interaction between the adhesion receptor, CD44, and the oncogene product, p185HER2, promotes human ovarian tumor cell activation, J Biol Chem 1997, 272:27913-27918  38.  Yamagata M, Saga S, Kato M, Bernfield M, Kimata K: Selective distributions of proteoglycans and their ligands in pericellular matrix of cultured fibroblasts. Implications for their roles in cell-substratum adhesion, J Cell Sci 1993, 106 (Pt 1):55-65  39.  Wight TN, Lara S, Riessen R, Le Baron R, Isner J: Selective deposits of versican in the extracellular matrix of restenotic lesions from human peripheral arteries, Am J Pathol 1997, 151:963-973  40.  Roberts CR: Versican in the Cell Biology of Pulmonary Fibrosis. Edited by Garg HG, Roughley, P. J., and Hales, C. A. New York, NY, Marcel Dekker, 2003, p  41.  Schonherr E, Jarvelainen HT, Sandell LJ, Wight TN: Effects of platelet-derived growth factor and transforming growth factor-beta 1 on the synthesis of a large versican-like chondroitin sulfate proteoglycan by arterial smooth muscle cells, J Biol Chem 1991, 266:17640-17647 102  42.  Evanko SP, Johnson PY, Braun KR, Underhill CB, Dudhia J, Wight TN: Plateletderived growth factor stimulates the formation of versican-hyaluronan aggregates and pericellular matrix expansion in arterial smooth muscle cells, Arch Biochem Biophys 2001, 394:29-38  43.  Ogawa H, Oohashi T, Sata M, Bekku Y, Hirohata S, Nakamura K, Yonezawa T, Kusachi S, Shiratori Y, Ninomiya Y: Lp3/Hapln3, a novel link protein that colocalizes with versican and is coordinately up-regulated by platelet-derived growth factor in arterial smooth muscle cells, Matrix Biol 2004, 23:287-298  44.  Zheng PS, Vais D, Lapierre D, Liang YY, Lee V, Yang BL, Yang BB: PGM/versican binds to P-selectin glycoprotein ligand-1 and mediates leukocyte aggregation, J Cell Sci 2004, 117:5887-5895  45.  Chen L, Yang BL, Wu Y, Yee A, Yang BB: G3 domains of aggrecan and PGM/versican form intermolecular disulfide bonds that stabilize cell-matrix interaction, Biochemistry 2003, 42:8332-8341  46.  Zhang Y, Cao L, Yang BL, Yang BB: The G3 domain of versican enhances cell proliferation via epidermial growth factor-like motifs, J Biol Chem 1998, 273:21342-21351  47.  Hirose J, Kawashima H, Yoshie O, Tashiro K, Miyasaka M: Versican interacts with chemokines and modulates cellular responses, J Biol Chem 2001, 276:52285234  48.  Kawashima H, Hirose M, Hirose J, Nagakubo D, Plaas AH, Miyasaka M: Binding of a large chondroitin sulfate/dermatan sulfate proteoglycan, versican, to Lselectin, P-selectin, and CD44, J Biol Chem 2000, 275:35448-35456  49.  Ruoslahti E, Yamaguchi Y: Proteoglycans as modulators of growth factor activities, Cell 1991, 64:867-869  50.  Scott JE, Cummings C, Brass A, Chen Y: Secondary and tertiary structures of hyaluronan in aqueous solution, investigated by rotary shadowing-electron microscopy and computer simulation. Hyaluronan is a very efficient networkforming polymer, Biochem J 1991, 274 (Pt 3):699-705  51.  Mow VC, Mak AF, Lai WM, Rosenberg LC, Tang LH: Viscoelastic properties of proteoglycan subunits and aggregates in varying solution concentrations, J Biomech 1984, 17:325-338  52.  Soby L, Jamieson AM, Blackwell J, Choi HU, Rosenberg LC: Viscoelastic and rheological properties of concentrated solutions of proteoglycan subunit and proteoglycan aggregate, Biopolymers 1990, 29:1587-1592  53.  Knudson W, Bartnik E, Knudson CB: Assembly of pericellular matrices by COS7 cells transfected with CD44 lymphocyte-homing receptor genes, Proc Natl Acad Sci U S A 1993, 90:4003-4007  54.  Lee GM, Johnstone B, Jacobson K, Caterson B: The dynamic structure of the pericellular matrix on living cells, J Cell Biol 1993, 123:1899-1907  103  55.  Morgelin M, Paulsson M, Heinegard D, Aebi U, Engel J: Evidence of a defined spatial arrangement of hyaluronate in the central filament of cartilage proteoglycan aggregates, Biochem J 1995, 307 (Pt 2):595-601  56.  Yokosaki Y, Palmer EL, Prieto AL, Crossin KL, Bourdon MA, Pytela R, Sheppard D: The integrin alpha 9 beta 1 mediates cell attachment to a non-RGD site in the third fibronectin type III repeat of tenascin, J Biol Chem 1994, 269:26691-26696  57.  Midwood KS, Mao Y, Hsia HC, Valenick LV, Schwarzbauer JE: Modulation of cell-fibronectin matrix interactions during tissue repair, J Investig Dermatol Symp Proc 2006, 11:73-78  58.  Krueger RC, Jr., Schwartz NB: An improved method of sequential alcian blue and ammoniacal silver staining of chondroitin sulfate proteoglycan in polyacrylamide gels, Anal Biochem 1987, 167:295-300  59.  Farndale RW, Buttle DJ, Barrett AJ: Improved quantitation and discrimination of sulphated glycosaminoglycans by use of dimethylmethylene blue, Biochim Biophys Acta 1986, 883:173-177  60.  du Cros DL, LeBaron RG, Couchman JR: Association of versican with dermal matrices and its potential role in hair follicle development and cycling, J Invest Dermatol 1995, 105:426-431  61.  Fitch MT, Silver J: CNS injury, glial scars, and inflammation: Inhibitory extracellular matrices and regeneration failure, Exp Neurol 2007,  62.  Kawashima H, Watanabe N, Hirose M, Sun X, Atarashi K, Kimura T, Shikata K, Matsuda M, Ogawa D, Heljasvaara R, Rehn M, Pihlajaniemi T, Miyasaka M: Collagen XVIII, a basement membrane heparan sulfate proteoglycan, interacts with L-selectin and monocyte chemoattractant protein-1, J Biol Chem 2003, 278:13069-13076  63.  Ruoslahti E: RGD and other recognition sequences for integrins, Annu Rev Cell Dev Biol 1996, 12:697-715  64.  Ruoslahti E, Pierschbacher MD: New perspectives in cell adhesion: RGD and integrins, Science 1987, 238:491-497  65.  Lemire JM, Merrilees MJ, Braun KR, Wight TN: Overexpression of the V3 variant of versican alters arterial smooth muscle cell adhesion, migration, and proliferation in vitro, J Cell Physiol 2002, 190:38-45  66.  Lemire JM, Braun KR, Maurel P, Kaplan ED, Schwartz SM, Wight TN: Versican/PG-M isoforms in vascular smooth muscle cells, Arterioscler Thromb Vasc Biol 1999, 19:1630-1639  67.  Swiderski RE, Dencoff JE, Floerchinger CS, Shapiro SD, Hunninghake GW: Differential expression of extracellular matrix remodeling genes in a murine model of bleomycin-induced pulmonary fibrosis, Am J Pathol 1998, 152:821-828  104  3. FIBROBLAST CELL MORPHOLOGY IN 3-DIMENSIONAL COLLAGEN-VERSICAN-HYALURONAN MATRIX: A MODEL TO STUDY MYOFIBROBLAST CELL BEHAVIOR2 3.1 Introduction The wound healing process is classically divided into four phases which include homeostasis, inflammation, proliferation and remodeling (reviewed in1 2). Once inflammation is turned off, the process of epithelialization and matrix formation is initiated by proliferating fibroblasts. Fibroblasts migrate into the former clot and lay down a provisional matrix rich in proteoglycan versican, glycosaminoglycan hyaluronan, fibronectin, tenascin, and a number of other proteins. PDGF and activated TGF-β, along with other granulation tissue components, signal differentiation of fibroblasts into contractile myofibroblasts which are the main cell type responsible for wound closure. In the final remodeling phase of wound healing, fibroblasts express high levels of type-I collagen, and an organized collagenous matrix replaces proteoglycan and fibronectin. As the provisional matrix is degraded, myofibroblasts go into apoptosis and the anatomy and function of the tissue is restored. However, repeated insult and the ensuing inflammation result in prolonged myofibroblast proliferation and excessive deposition of granulation tissue at the site of injury. The accumulation of non-functional and excessive scar tissue, or fibrosis, is associated with many clinical problems such as keloid or hypertrophic scar formation in the skin, delayed nervous system regeneration, lung and liver dysfunction, and atherosclerosis3, 4. The tensile forces that develop within the wound matrix during repair are normally relieved through a combination of biosynthetic activity and wound contraction. Fibroblasts in the granulation tissue are responsible for both biosynthesis of the new connective tissue matrix, and contraction of the matrix4-6 by differentiating into myofibroblasts7, 8 which contain actin stress fibers and α-smooth muscle actin9. Versican is the main proteoglycan whose expression is upregulated by fibroblasts and 2  A version of this chapter will be submitted for publication. Pourmalek, S, and Roberts, C. Fibroblast cell morphology in 3-dimensional collagen-versican-hyaluronan matrix. 105  myofibroblasts at the site of injury. In the fibrotic lung, the early accumulation of versican occurs in association with proliferating and contractile myofibroblasts, prior to deposition of collagenous matrix in later stages of the disease10, 11. In atherosclerotic lesions (reviewed in12), versican is found in association with proliferating vascular smooth muscle cells13. The spatial and temporal association of large splice-variants of versican, namely V0 and V1, with proliferating and contracting myofibroblasts (as defined by ∝-SMA and collagen type-1 expression) has also been observed in all fibrotic lung diseases10. Studies suggest that growth factors that regulate smooth muscle cell type proliferation and migration, such as TGF-β14 and PDGF15, are also involved in upregulating  versican15, 16  and  hyaluronan17, 18  expression,  and  formation  of  versican-hyaluronan aggregates19. Versican is a member of hyalectan family of proteoglycans20,  21  which interact with  22  hyaluronan through their highly homologous globular N-terminus . Glycosaminoglycan chains, bound to the core of the molecule23-25, are responsible for many of the structural and functional properties of these proteoglycans.  Two distinct glycosaminoglycan  binding domains, GAG-α and GAG-β are unique to versican and bind 10-30 chondroitin sulfate chains. Splicing of this region also gives rise to 4 different isoforms of versican that are differentially expressed in different tissues. Studies to date suggest that different versican splice variants namely: V0 (containing GAG-α and GAG-β), V1 (containing GAG-β), V2 (GAG-α), and V326, 27 (devoid of both glycosaminoglycan binding regions), exhibit different functions in development and regeneration. In comparison, aggrecan which is found as a structural component in cartilage, contains the most GAG of approximately 100 chondroitin sulfate chains.  C-terminal (G3) globular domain of  lecticans, like the N-terminal domain, is also highly conserved. C-terminal domain of versican consists of two EGF repeats, a C-type lectin domain and complement regulatory protein (CRP)-like domain28.  The complex structure of versican has lead to the  discovery of multiple functions for this molecule in different tissues29, in development and disease30-36. As fibroblasts are normally embedded within a collagen-rich matrix in vivo, the use of three dimensional collagen and fibrin matrices have become more popular as models best 106  resembling the granulation tissue matrix37. Spatial signals in a 3D matrix can control cell morphology38 and gene expression, and research shows that transitioning between 2D and 3D matrices by mechanically flattening a 3D matrix39 or sandwiching cells between two 2D surfaces to mimic a 3D environment40 can induce such morphological changes, even though the same molecules and growth factors are present. Fibroblast cells allowed to interact with collagen matrices can penetrate into the substance of the matrix and become entangled with matrix fibrils41, 42. Achieving tensional homeostasis has been suggested as the main reason43, 44 for the observed changes in signaling and migration pattern of the cells39, 45-47, and the remodeling of the matrix5, 48-50 that follows. It is possible that in attached stiff collagen matrices, resistance of collagen fibrils to mechanical forces exerted by fibroblasts51 leads to increased intracellular tension, and differentiation of fibroblasts into α-smooth muscle expressing myofibroblasts5. In this study, we tested the hypothesis that fibroblast cell morphology is altered in a versican-hyaluronan, three-dimensional collagenous matrix. We investigated the roles of versican and hyaluronan in relieving tension, and their effects on myofibroblast cell morphology and proliferation, in an in vitro model of wound healing matrix. In order to investigate the influence of collagen gel matrix containing stimulating factors found in granulation tissue of healing wounds on the morphology of HFL1 human fetal lung fibroblast cells, we established a three-dimensional collagen-proteoglycan gel matrix system to study fibroblast cell behavior under 5 different conditions: buffer, hyaluronan, versican/hyaluronan, aggrecan/hyaluronan, and versican G3/hyaluronan, against a background of 3D collagen gel matrix. The experiment is illustrated in the schematic below (Figure 3.1).  107  12 H  24 H  48 H  72 H  Collagen + PBS Collagen + HA Collagen + HA + Versican Collagen + HA + G3 Collagen + HA + Aggrecan Figure 3.1 Illustration of 3D Collagen Experiment In short, low passage fibroblast cells were seeded in collagen gels under each of the five different conditions. Gels were submerged in serum free media, as I found in preliminary experiments that fetal calf serum contains versican fragments. Fetal calf serum also contains growth factors such as PDGF and TGFβ-1 that can stimulate versican expression. Fluorescent staining and immunofluorescent staining was used to study changes in cell and nuclear morphology, actin expression, and α-smooth muscle actin expression. Our study shows that fibroblast cell morphology is affected by the presence  108  of granulation tissue components, versican and hyaluronan, and that versican is capable of inducing α-smooth muscle actin expression and fibroblast differentiation into a myofibroblast phenotype.  3.2 Materials and Methods Expression and Purification of Versican – Human fetal lung (HFL1) fibroblast cells American Type Culture Collection (Manassas, VA) were cultured in 75-cm2 flasks (Sarstedt; Quebec, Canada) in Dulbecco’s Modified Eagle Medium (DMEM; Life Technologies, Inc., Gaithersburg, MD) supplemented with 10% (v/v) fetal bovine serum (FBS; HyClone, Logan, UT) to 80% confluence. Cells from two confluent 75-cm2 flasks were trypsinized and transferred to a 850-cm2 tissue culture roller bottle (Becton Dickinson) with 200ml of DMEM and incubated at 37˚C in a BELCO Biotechnology Roll-in incubator. Serum free conditioned medium from fibroblast cultures (CM) was collected and centrifuged at 1500 X g for 15 minutes to remove cellular debris. Then, Urea and salt concentrations in HFL1 conditioned media were adjusted to 7M Urea and 0.4M NaCl and loaded onto Q-Sepharose Fast Flow ion exchange resin (Amersham Biosciences, Piscataway, NJ) at approximately 1 litre CM per 5 mls resin. The column was washed with a 10 fold bed volume of 7M Urea, 0.4M NaCl, 0.1M NaOAc, pH 6.0 before elution with 7M Urea, 1.5M NaCl, 0.1M NaOAc, pH 6.0. Peak fractions were pooled and dialyzed against PBS (140mM NaCl, 2.7mM KCl, 10mM Na2HPO4, 1.8mM NaH2PO4, pH7.4) exhaustively, then flash frozen with liquid nitrogen and stored at -70˚C. Fractions were monitored for versican content by alcian blue (Sigma, St. Louis, MO) staining of SDS-PAGE gels52 and by Western blotting. Purified versican concentration was estimated using the dimethylmethylene blue (DMMB, Serva, Heidelberg) assay53 to quantify sulfated glycosaminoglycan using known concentrations of chondroitin sulfate C as standards (Seikagaku). The concentration of versican was estimated based on an average of 1.5 mg total proteoglycan per 1 mg sulfated glycosaminoglycan detected with an estimated concentration of 1.12 mg/ml or approximately 1.12 µM versican.  109  Generation of Recombinant Constructs (HisL, HisLC, HisG3) – Before ligating the His-tagged C-terminal construct cDNA to the expression vector pGYMXC, the PCR product was amplified using the pPCR-Script strategy. Briefly, cDNA amplified from PCR was purified using a QIAEX II Gel Extraction Kit (Qiagen, Mississauga, Ontario). The DNA fragment was ligated into the pPCR-Script Cam SK(+) plasmid (Stratagene, CA, USA). The ligation mixture contained 1µl of 10X reaction buffer, 1µl of Srf I restriction enzyme (5U), 1µl of T4DNA ligase (4U), 0.5µl of 10mM rATP, 3µl of the HisLC insert (190ng), and 1µl of the cloning vector (10ng) for a 100:1 molar ratio of insert to cloning vector. The mixture was diluted to 10 µl with ddH2O, gently mixed, and incubated at room temperature for 1 hr before heating at 65˚C for 10min. Two µl of the ligation mixture was used for heat shock transformation of supercompetent E. coli strain DH5α. The bacteria was transferred to 50µl of 2X YT media (1.0% w/v yeast extract, 1.6%w/v tryptone, 0.5% w/v NaCl, pH 7.5) and agitated at 275rpm for 30min at 37˚C before plating on LB agar plates containing 30µg/ml chloramphenicol. Plates were incubated for 16hrs at 37˚C. Colonies were selected and grown in 5ml of terrific broth media (1.2% w/v tryptone, 2.4% w/v yeast extract, 0.231% w/v KH2PO4, 1.254% w/v K2HPO4, 0.4% glycerol) with 30µg/ml chloramphenicol at 37˚C for 16hrs at 275rpm. The plasmid was isolated using the QIAprep Spin Miniprep Kit (Qiagen, Mississauga, Ontario). The insert was removed from the CAM SK(+) plasmid by Hind III and NheI restriction enzyme digestion and purified by QIAEX II Gel Extraction Kit (Qiagen, Mississauga, Ontario). The insert was ligated into the expression vector pGYMX using the HindIII and Nhe I sites. The ligation reaction mixture contained 2µl of 10X reaction buffer, 1µl T4 DNA ligase, 2 µl 10mM ATP, 2µl of the pGYMX expression vector (15ng), a 1:1 weight ratio of insert to cloning vector and diluted to 20µl with ddH2O. This was heated at 60˚C for 10min, placed on ice for 30min, and incubated at 16˚C for 16hrs. Two µl of the ligation mixture was used for heat shock transformation of supercompetent E.coli strain DH5α following the above protocol before plating on LB agar plates containing 100µg/ml ampicillin. Colonies were selected and grown in 5ml of terrific broth as described above. DNA was isolated using the QIAprep Spin Miniprep Kit (Qiagen, Mississauga, Ontario). The identity of the new HisLC pGYMX construct was confirmed by Hind III and Nhe I restriction enzyme digestion and by DNA sequencing  110  performed by Nucleic Acids-Protein Service (NAPS) at the University of British Columbia. Transformation, Expression and Purification of Engineered Versican C-terminal Domain Constructs (His-L, His-LC, and His-G3)– The pGYMX His-G3 expression vector was transformed into E.coli BL21(DE3) competent cells at a ratio of 1µl of cDNA into 50µl BL21 Gold E.coli cells. Briefly, the mixture was iced for about an hour before a 90 seconds heat shock at 42˚C. The mixture was iced for 2 minutes, and incubated at 37˚C for 30 min in shaker with 50µl of 2XYT media. The cells were then plated on Luria–Bertani (LB) agar plates containing 100µg/ml ampicillin. 5ml of superbroth (0.8% w/v yeast extract, 1.0%w/v tryptone, 0.5% w/v NaCl, 0.1% glycerol, pH 7.5) and incubated in 37˚C shaker for 24 hours. Next, another plate of agar was inoculated with a single colony and incubated at 37˚C for 16 hrs at 275rpm. Aliquots of this log phase seed culture were used to innoculate 3.5L of superbroth in a1:1000 (v/v) ratio with 100µg/ml ampicillin and incubated at 37˚C for 24hrs at 275rpm. Collected cells were washed with 500ml of NET buffer (100mM NaCl, 1mM EDTA, 20mM Tris-HCl, pH 8.0) then lysed in 250ml of lysis buffer (50mM NaCl, 1mM EDTA, 20mM Na2HPO4, 1mg/ml lysozyme, 1mM PMSF, pH 8.0) for 2hrs at 37˚C and 275rpm and sonicated with 5 sec bursts. The inclusion bodies were washed with 500ml of NET buffer then dissolved in a solubilization buffer (8M Urea, 10mM Tris-HCl, 100mM Na2HPO4, pH 8.0) for 16hrs at 4˚C. Dissolved inclusion bodies were centrifuged at 20000rpm for 1hr and purified using a 30ml Ni2+ charged chelating sepharose column (Amersham Pharmacia) equilibrated in column buffer (8M Urea, 0.5M NaCl, 20mM Na2HPO4, pH 7.4). The column was washed in succession with 10-fold bed volume of column buffer, column buffer with 1M NaCl, column buffer with 1M NaCl, pH 6.0, and again with column buffer. Proteins with non-specific interactions to the Ni-chelate column were pre-eluted by a 10-fold bed volume of column buffer with 200mM imidazole. His-LC fusion protein was then eluted by a 200mM to 1M imidazole gradient over a 10-fold bed. Fractions were analyzed by SDS-PAGE and stored at –20˚C. Refolding of HisG3 Recombinant Protein – Peak fractions obtained from the imidazole gradient were pooled and diluted 20-fold before dialysis in equal volume of  111  refolding buffer (18.2mM Na2CO3, 24mM NaHCO3, 125mM NaCl, 1:10 ration of 3mM Cysteine/Cystine, pH 10.0) with aeration at room temperature. Refolding buffer was changed every 2 hrs for 8 hrs before exhaustive dialysis with refolding buffer minus the redox pair of Cysteine/Cystine for complete removal of urea.  Because the pooled  fractions were diluted 20 fold before refolding, a 10ml Ni-chelate column equilibrated in 18.2mM Na2CO3, 24mM NaHCO3, 125mM NaCl, pH 10.0 was used to concentrate the diluted pool. His-LC was eluted with 18.2mM Na2CO3, 24mM NaHCO3, 125mM NaCl, 500mM imidazole, pH 10.0. Peak fractions were pooled and dialyzed against excess volume of 18.2mM Na2CO3, 24mM NaHCO3, 125mM NaCl, pH 10.0. The protein pool was aliquoted into 1ml fractions, flash frozen with liquid nitrogen, and stored at –70˚C. Electrophoretic Techniques – Samples in nonreducing sample buffer (125 mM TrisHCL, pH 6.8, 2.0% SDS, 2.0 M urea, 0.05% bromophenol blue) were separated on discontinuous SDS-PAGE gels with 4% (stacking) and 12.5% (separating) acrylamide. Stacking and separating gels were kept during staining and Western blotting to monitor high molecular weight versican aggregates within the stacking gel. Relative molecular mass (measured in kDa) was estimated based on mobility of molecular weight markers: HiMark Prestained (Invitrogen), MagicMark XP (Invitrogen) and Kaleidoscope Prestained (Bio-Rad, Hercules, CA). Western blotting was performed using the XCell II blot module (Invitrogen) to PVDF membrane (Millipore). Blocking was performed with a solution of 2.5% (w/v) bovine serum albumin, 20 mM Tris, 5 mM EDTA, 0.9%NaCl, and 0.3% (v/v) Tween 20. RP57 rabbit polyclonal antibody (British Biotech, 1mg/ml), raised against a peptide corresponding to the structural z binding S-loop in all MMP catalytic domain, was used to detect general MMP expression, 1:250 dilution. Antibodies were diluted in a solution of 2% (w/v) bovine serum albumin, 20 mM Tris, pH 7.5, 0.9 % NaCl and 0.05% (v/v) Tween 20. Highly cross adsorbed goat anti-rabbit horseradish peroxidase-conjugate (Bio-Rad) secondary antibody was diluted to 1:2000. Visualization of the peroxidase was performed with Enhanced Chemiluminescence Plus Western blotting reagents (Amersham Biosciences) and exposed to X-ray film (Kodak, New Haven, CT) or captured using the ChemiGenius-2 bio-imaging system and Gene Snap software (Perkin Elmer, Woodbridge, ON)  112  3D Collagen Cell Culture System – Human fetal lung fibroblasts (HFL1) were obtained from American Type Culture Collection (Manassas, VA) and grown in Dulbecco’s modified Eagle medium (DMEM) supplemented with 20 mM HEPES and 10% (v/v) Cosmic Calf Serum (Hyclone, Logan, UT) in cell culture flasks. Once cells were at 70% confluence, they were trypsinized, rinsed with serum-free media, and resuspended in PBS and kept on ice. Collagen solution was prepared following the manufacturer’s instructions for 3D Collagen Cell Culture System (Chemicon International, ECM675). Collagen solution was then mixed with either: PBS; PBS and 20µg/ml HA; 0.55mg/ml aggrecan and 20µg/ml HA; 0.50 mg/ml versican and 20µg/ml HA; or 0.53 mg/ml HisG3 and 20µg/ml HA. HFL cells, suspended in PBS in the previous step, were counted and mixed with this solution at a final concentration of 4x104 cells/ml. 20µl droplets of each solution were placed in the center of 8well chamber slides (Lab-Tek, Electron Microscopy Sciences, Hatfield, PA, USA) and incubated at 37ºC for up to 4 hours until the gels polymerized. Flipping plate upside down kept cells from attaching to the slide, ensuring their suspension in a 3D environment.  Once gels  polymerized, serum-free media was added to the cells and they were allowed to spread in the 37ºC incubator for either 12 hours, 24 hours, 48 hours, or 72 hours time points. Cytochemistry – At the end of each time point, cells were fixed in 4% paraformaldehyde in PBS for 15 minutes in 37°C incubator. In the first run, monoclonal antibody to α-smooth muscle actin (Clone 1A4, Sigma ImmunoChemicals, St. Louis, MO, USA) was used to establish the contractile physiology of the fibroblast cells, 1:400 dilution; α-mouse AlexaFluor-594, 1:2000. In the second run, Mitochondria were stained with 333nM MitoTracker Red CMXRos (Invitrogen M-7512, Life Technologies) for 20 minutes before being fixed, to follow apoptotic behavior54. In live cells, MitoTracker dye accumulates in the Mitochondria and due to its reactivity with the thiol groups of peptides and proteins, it is retained within the mitochondria during the fixing and permeabilization process. As a result, cells with compromised mitochondria such as those going into apoptosis will exhibit less fluorescent mitochondria under the microscope. Fixed cells were permeabilized with 0.1% Triton X-100 and PBS for 1 minute and washed with PBS before being blocked by freshly prepared 1% bovine serum albumin (BSA) solution in  113  PBS for 5 minutes. Cells were then incubated in Alexafluor-488 Phalloidin (Molecular Probes, Eugene, Oregon, USA) at 1:1000 dilution in 1% BSA/PBS for 30 minutes. After being washed with PBS, cells were incubated in Hoechst stain (33342, Invitrogen) at 1:2000 dilution in 1% BSA/PBS for 10 minutes. Cells were then washed with PBS and stored with ProLong Gold antifade reagent (Invitrogen, Molecular Probes) on a covered slide Fluorescent Light Microscopy – Fixed and stained cells were viewed using a Leica CTR fluorescent light microscope. Images were captured at 40X magnification, in layers of 0.75µm thickness using the multi-channel z-stack capture option using Q-Imagine Retiga Exi mounted camera, on monochrome setting, and OpenLab software (4.0.2). Emission wavelengths were set to best view each stained organelle: actin filaments were viewed at 520nm 55; nuclei at 456nm (DAPI), and mitochondria at 594nm (Texas Red). Once images were captured, each image was deconvoluted using the Nearest Neighbor DCI function to remove unfocussed light. All layered images taken of the same coordinates but at different points along the z-axis (height) were merged to create one 3-dimentional image of cells spread in the collagen matrix. The appropriate color of the stain was then applied to the image, and images captured under different emission wavelength of different organelles were merged into one composite.  3.3 Results Collagen solution was prepared following the manufacturer’s instructions for 3D Collagen Cell Culture (Chemicon International, ECM675) with a final collagen content of 80%. Based on the manufacturer’s fact sheet, ECM675 collagen solution is 99.9% pure native atelomeric avian collagen, with approximately 85% type I and 15% type III collagen protein. The Collagen solution was mixed with either: PBS; PBS and 20µg/ml HA; 0.55mg/ml aggrecan and 20µg/ml HA; 0.50 mg/ml versican and 20µg/ml HA; or 0.53 mg/ml HisG3 and 20µg/ml HA. HFL cells, suspended in PBS in the previous step, were counted and mixed with this solution at a final concentration of 4x104 cells/ml. 20µl droplets of each solution were placed in the center of 8-well chamber slides (Lab-Tek, Electron Microscopy Sciences, Hatfield, PA, USA) and incubated at 37ºC for up to  114  4 hours until the gels polymerized. The collagen gels containing fibroblast cells were subsequently submerged in serum-free media, and fixed at identified time points in preparation for staining and microscopy. The results presented here are representative of at least 3 different trials, with each trial containing at least three different replicas (excluding some of the replicas that did not polymerize properly, or were lost during the preparation process for fluorescence microscopy). 3.3.1 Physical properties and polymerization rate of collagen I matrix is influenced by versican and hyaluronan According to manufacturer’s instructions, the polymerization of collagen should initiate after 60 minutes once gels are transferred to a 37oC incubator. Our observation was that while 60 minutes was enough for PBS and hyaluronan containing droplets to polymerize, longer incubation periods were necessary to achieve polymerization in versican and aggrecan containing gels (3-4 hours).  These proteoglycans inhibited collagen  self-assembly. We found that versican and aggrecan containing gels were especially prone to release from the slide and floating in the media during media changes and preparation for fluorescent microscopy, while PBS containing gels were tightly attached to the bottom of the plate.  These results are not surprising as negatively charged  hyaluronan, and the sulfated glycosaminoglycan chains of aggrecan and versican are known to attract and retain water molecules. This lengthened the polymerization period and appeared to give the matrix altered hydrodynamic properties. 3.3.2 Fibroblast cell morphology is affected by the composition of pericellular matrix Fibroblast (HFL1) cells were grown in a three dimensional collagen matrix (Figure 3.2, 3.3) droplet containing either: phosphate buffered saline (PBS) as control; hyaluronan; versican and hyaluronan; or aggrecan and hyaluronan. Cells were incubated for 12, 24, 48 or 72 hours in serum free DMEM media, then fixed and stained with Alexafluor 488 Phalloidin (green, actin filaments), Hoechst stain (blue, DNA) and ∝-smooth muscle actin antibody (red), in preparation for cell microscopy.  115  Fibroblasts grown in 3D collagen matrix, with phosphate buffer saline added as control at 10% of total volume, spread out at a much slower rate (at 24h-48h) than cells in the presence of hyaluronan and versican (12h). In addition, these cells did not achieve a contractile myofibroblast morphology, with cellular projections into the matrix and cytoskeletal organization of actin fibers, even at longer incubation periods. In contrast, fibroblast cells in collagen gels containing hyaluronan spread rapidly (12h), form long extensions into the matrix, and have a highly organized cytoskeleton, characteristic of migrating and proliferating fibroblasts. Fibroblast cells in the presence of hyaluronan alone, however, seemed to express lower amounts of ∝-smooth muscle actin (∝-SMA, red immunostain) than those grown in the presence of versican and hyaluronan. Fibroblast cells grown in versican/hyaluronan containing gels showed extended morphology and expressed high levels of ∝-SMA (red immunostain), characteristics of myofibroblasts.  After 48h of incubation, cells in the presence of versican showed  apoptotic characteristics of cell and nucleus rounding, and compartmentalization of cell content. Fibroblasts grown in the presence of aggrecan and hyaluronan showed the most retracted morphology from the start of incubation, and throughout the incubation period. Cells in the presence of aggrecan did not spread, and were round and detached from the collagen matrix. Only with longer incubation periods a slightly more extended morphology was observed. Cells under all four conditions showed apoptotic characteristics of cell shrinkage and chromatin condensation at 72 hours incubation period. In these experiments, it appeared that fibroblasts grown in the presence of versican show apoptotic changes at a shorter incubation period (48 hours) than cells grown under the other three conditions. Nuclear fragmentation was also clearly visible in collagen matrices containing versican.  116  3.3.3 Fibroblast cell apoptosis at 72 hours of incubation is independent of collagen matrix composition Fibroblast (HFL1) cells were grown in a three dimensional collagen matrix droplet (Figure 3.4) containing either: phosphate buffer saline (PBS, control); hyaluronan; versican and hyaluronan; versican HisG3 construct and hyaluronan; or aggrecan and hyaluronan. Cells were incubated for 24, 48 or 72 hours in serum free DMEM media, then fixed and stained with Alexafluor 488 Phalloidin (green, actin filaments), Hoechst stain (blue, DNA) and Mitotracker dye CMX-Ros (red, mitochondrion), in preparation for cell microscopy. Similar to the previous set of experiments (Figure 3.2, 3.3), cells in presence of PBS and aggrecan showed limited ability to spread out in the 3D collagen matrix, and were in a retracted state. However, cells in the presence of versican, hyaluronan, and G3 domain of versican spread out and form long extensions into their matrix. Cells in the presence of versican seemed to exhibit a more contractile phenotype and were less flattened compared to cells grown in the presence of hyaluronan or the G3 domain (24 hours). Fibroblasts in versican-rich matrix also contained higher number of active mitochondria spread throughout the cell cytoplasm (24h, 48h). Fibroblasts grown under all conditions displayed apoptotic characteristics at 72 hours incubation. Cell shrinkage, chromatin condensation and fragmentation and formation of fragmented sacs containing cell content were visible in collagen matrices under all five conditions.  Reduced fluorescence in the mitochondria, due to reduced uptake of  MitoTraker Red dye, was also observed and could be interpreted as loss of mitochondrial membrane potential, a clear sign of the apoptotic state of a compromised cell.  119  3.4 Discussion In this study, we first assessed collagen matrix assembly in the presence of proteoglycan versican/aggrecan and glycosaminoglycan hyaluronan, through visual cues such as the rate of polymerization and resistance to separation from the slide. Our results point to versican and hyaluronan as factors in delayed collagen matrix assembly. We found that presence of versican and hyaluronan could delay collagen matrix polymerization and result in a weaker matrix, which is more prone to detachment from the slide and tearing during  media  changes.  Indeed,  it  is  well-known  that  proteoglycans  and  glycosaminoglycans of connective tissue matrix can influence fibrillogenesis and matrix architecture. Hyaluronan is an anionic polysaccharide that can bind to a large amount of water to form a viscous hydrated gel that gives connective tissue an ability to spread the pressure around and to resist compression loads57. A recent study of the effects of HA on collagen fiber assembly showed that although the spontaneously packing of collagen monomers was not affected by HA after a longer incubation period (48 h), the diameter of collagen increased significantly at the initial incubating time and reached a constant diameter after 6 h of incubation. The collagen fibers were thinner in diameter upon addition of HA before 24 h incubation period (as compared with the pure collagen) and then increased to the same range of diameter after 48 h incubation57. It is also possible that as an anionic polymer, HA could aggregate or adhere onto the positively charged regions of collagen and interfere with the interaction of collagen monomers and collagen fibrillogenesis.  Moreover, an examination of collagen formation in reconstituted  collagen matrices prepared from the PG/GAG components of interstitial mucosa which included hyaluronic acid and chondroitin sulfate, revealed that collagen fibrils had increased in density, but decreased in diameter and length, compared to those of matrices lacking the PG/GAG components58.  It was also reported that interstitial ECM  polymerized in absence of the PG/GAG components appeared similar to matrices prepared with type I collagen alone58.  Increasing the concentrations of extracted  PG/GAG components in the solution, however, resulted in a progressive increase in the nucleation of collagen fibril formation and a decrease in the rate of fibrillogenesis58. In this regard, versican could be functioning to inhibit collagen accumulation in fibrosis. 121  Next, we tested the hypothesis that versican and hyaluronan alter the morphology of fibroblast cells in a three-dimensional collagenous matrix. We found that fibroblast cells grown in the presence of HA formed well organized actin stress fibers not observed in cells grown in collagen gels alone. In addition to stress fibers, cells grown in the presence of both versican and HA upregulate their α-SMA expression, and seem to spread long dendritic extensions into their matrix. It is known that cells with prominent stress fibers are generally present during activated conditions such as wound repair and fibrosis41. Studies of cells in attached stiff collagen matrices have suggested that resistance of collagen fibrils to mechanical forces exerted by fibroblasts51 leads to increased intracellular tension, and differentiation of fibroblasts into α-smooth muscle expressing myofibroblasts5. Formation of stress fibers and large extensions also resembles the morphology of fibroblasts grown on a 2D culture plate59, which are under continuous tensional stress from the surface of culture plates that resist deformation. Unlike the final product of cell contraction in 3D collagen matrices, which leads to a relaxed matrix, cells in 2D are continuously under pressure and never relax. When placed back into a 3D matrix, the cells from the culture plate re-acquire an elongated spindle-shaped phenotype59. This activated myofibroblast phenotype is also observed with our fibroblast cell line which, when grown in planer culture dishes in the presence of serum, expresses large amounts of versican and proliferate until confluent. Based on all these observations, it seems that versican and hyaluronan are necessary components of the matrix in which activated myofibroblasts reside, as is observed with myofibroblasts in our 3D collagen matrix system. By examining cell morphology in a matrix that contains hyaluronan and the G3 domain of versican, we also tested the hypothesis (discussed in Chapter 2) that versican exerts its effects on cells through its C-terminal EGF modules. It has been suggested that versican can stimulate proliferation in some cells through the interaction of its C-terminal EGF modules with the cell surface EGF receptors60-62. We found that cells grown in the presence of G3 domain of versican and hyaluronan showed no difference in morphology to cells grown in the presence of hyaluronan alone. This observation lead us to believe that the whole versican molecule is necessary for versican to exert its influence on cells.  122  We also found that cells in the aggrecan-rich matrix have rounded retracted morphology and do not show any cellular extensions into the matrix. It is generally believed that the bulky glycosaminoglycan chains of chondroitin sulfate proteoglycans are anti-adhesive and that they sterically inhibit cell surface receptors, such as integrins, to interact with their ligands in the matrix, such as collagen.  Considering one aggrecan molecule  contains about 7-8 times more glycosaminoglycan chains than versican, the difference in the number of sugar chains bound to the core of the protein may contribute to the difference in cell morphology observed in versican- versus aggrecan-rich matrices. That the physical properties of the polyanionic glycosaminoglycan chains is the major determinant in the roles of these proteoglycans in the matrix, is further supported by the different distribution pattern for versican and aggrecan in vivo during development and disease. For example, the difference in versican and aggrecan localization during the development of central nervous system has been attributed to their partially complementary roles63. 64  aggrecan  In developing cartilage, versican is gradually replaced by  suggesting a role for versican as a temporary scaffold for the developing  cartilage matrix65.  This signifies the role of versican and the contributions of its  glycosaminoglycan chains on the physical properties of the matrix and the cellular response in wound healing matrix. A number of mechanisms have been suggested for versican regulation of cellular proliferation66. Versican can maintain the integrity of the ECM by interacting with hyaluronan22.  Further to maintaining the architecture and viscoelastic properties of  tissues, HA modulates cell functions such as adhesion, migration and proliferation via interaction with specific cell surface receptors (such as CD44).  Hyaluronan-CD44  signaling can lead to actin cytoskeleton reorganization67-69 and cell proliferation70 in tumor cells, and it has been suggested that the formation of versican-hyaluronan complex at the cell surface may facilitate the migration and proliferation of smooth muscle cells19, 71. We have also shown that the degradation of hyaluronan and release of versican from the pericellular matrix of fibroblast cells can lead to altered nuclear morphology and cell death (Chapter 2). Versican’s chondroitin sulfate chains can also interact with and localize a variety of growth factors72, 73 and cytokines in the ECM, which can indirectly regulate cellular behavior74, 75. In this regard, proteoglycans are a matrix store of growth 123  factors and chemokines that can contribute to cell survival and proliferation. Studies of self-assembling76 HA molecules have shown that HA, in association with aggregating CSPG, increases the viscosity of the matrix 79, 80  through the CS chains  77, 78  and adds swelling pressure  . CSPG aggregation can potentially stiffen the HA network81  and influence cell behavior through mechanically coupled signaling82. Other studies of fibroblast cell behavior in 3-dimentional (3D) collagen matrices, which resemble the in vivo wound environment, provide convincing evidence that mechanical tension is a factor in transforming resting fibroblasts to contractile myofibroblasts that express αsmooth muscle actin6,  8, 37  .  Alpha-smooth muscle actin is increasingly seen as a  “mechano-sensitive” protein that localizes to stress fibers in response to mechanical strain83. In addition, versican’s ability to bind hyaluronan and form highly hydrated, supra-molecular aggregates 84, can contribute to swelling pressure and thus influence the mechanical properties of matrices such as blood vessel walls or remodeling tissue which are under pressure. This role for versican resembles the role of its family member, aggrecan, in resisting compressive forces put on cartilage. That is why we also compared the morphology of cells grown in aggrecan which, in comparison to versican, contains about 7-8 times more chondroitin sulfate chains. In the lungs, degradation of human airway smooth muscle-associated matrix is associated with decreased passive tension and alterations in smooth muscle contractility85. This is consistent with the role of this matrix in modulating smooth muscle contractility by resisting compression. The large size (>1000 kDa) and hydration capability of versican, may also sterically hinder the interaction of integrins (large family of cell adhesion molecules) with their cell surface receptors86, 87. In our experiments with lung fibroblast cells grown in 3D collagen matrices, cells under all conditions showed apoptotic behavior at 48 hours of incubation. Most cells went into apoptosis at 72 hours of incubation. As these cells were grown in serum free media, the lack of growth factors and absence of other extracellular stimulants, pertinent to their survival, could have caused cells to go into apoptosis. We chose to use serum-free media in order to maintain the separation in matrix components under each condition. As fibroblasts are capable of expressing versican, and they do so particularly faster under the  124  influence of growth factors found in serum, starving cells from serum could maintain the integrity of matrix for longer periods. Also, we have found versican fragments in serum which could influence the results. Our experience with fibroblast cells grown on plastic culture plates has been that trypsinized fibroblast cells require fetal calf serum to establish a viable cell culture (data not shown). Once cells have attached to the bottom of culture plate and start proliferating, and once they reach a certain cell density, they can stay alive after serum starvation. In order to avoid this problem in the future, the media could be enriched with certain serum components, or cell morphology could be investigated in shorter time periods. More experiments are needed to confirm the effects of serum starvation on lung fibroblasts in our 3D collagen gel system.  125  3.5 References 1.  Diegelmann RF, Evans MC: Wound healing: an overview of acute, fibrotic and delayed healing, Front Biosci 2004, 9:283-289  2.  Broughton G, 2nd, Janis JE, Attinger CE: Wound healing: an overview, Plast Reconstr Surg 2006, 117:1e-S-32e-S  3.  Gabbiani G: The myofibroblast in wound healing and fibrocontractive diseases, J Pathol 2003, 200:500-503  4.  Hinz B: Formation and function of the myofibroblast during tissue repair, J Invest Dermatol 2007, 127:526-537  5.  Tomasek JJ, Gabbiani G, Hinz B, Chaponnier C, Brown RA: Myofibroblasts and mechano-regulation of connective tissue remodelling, Nat Rev Mol Cell Biol 2002, 3:349-363  6.  Hinz B, Dugina V, Ballestrem C, Wehrle-Haller B, Chaponnier C: Alpha-smooth muscle actin is crucial for focal adhesion maturation in myofibroblasts, Mol Biol Cell 2003, 14:2508-2519  7.  Desmouliere A, Tuchweber B, Gabbiani G: Role of the myofibroblast differentiation during liver fibrosis, J Hepatol 1995, 22:61-64  8.  Hinz B, Gabbiani G: Mechanisms of force generation and transmission by myofibroblasts, Curr Opin Biotechnol 2003, 14:538-546  9.  Darby I, Skalli O, Gabbiani G: Alpha-smooth muscle actin is transiently expressed by myofibroblasts during experimental wound healing, Lab Invest 1990, 63:21-29  10.  Roberts CR: Versican in the Cell Biology of Pulmonary Fibrosis. Edited by Hari G. Garg PJR, and Charles A. Hales. New York, Marcel Dekker, Inc., 2002, p. pp. 191-212  11.  Bensadoun ES, Burke AK, Hogg JC, Roberts CR: Proteoglycans in granulomatous lung diseases, Eur Respir J 1997, 10:2731-2737  12.  Wight TN, Merrilees MJ: Proteoglycans in atherosclerosis and restenosis: key roles for versican, Circ Res 2004, 94:1158-1167  13.  Evanko SP, Angello JC, Wight TN: Formation of hyaluronan- and versican-rich pericellular matrix is required for proliferation and migration of vascular smooth muscle cells, Arterioscler Thromb Vasc Biol 1999, 19:1004-1013  14.  Howell JE, McAnulty RJ: TGF-beta: its role in asthma and therapeutic potential, Curr Drug Targets 2006, 7:547-565  15.  Schonherr E, Kinsella MG, Wight TN: Genistein selectively inhibits plateletderived growth factor-stimulated versican biosynthesis in monkey arterial smooth muscle cells, Arch Biochem Biophys 1997, 339:353-361  16.  Schonherr E, Jarvelainen HT, Sandell LJ, Wight TN: Effects of platelet-derived growth factor and transforming growth factor-beta 1 on the synthesis of a large 126  versican-like chondroitin sulfate proteoglycan by arterial smooth muscle cells, J Biol Chem 1991, 266:17640-17647 17.  Nikitovic D, Zafiropoulos A, Katonis P, Tsatsakis A, Theocharis AD, Karamanos NK, Tzanakakis GN: Transforming growth factor-beta as a key molecule triggering the expression of versican isoforms v0 and v1, hyaluronan synthase-2 and synthesis of hyaluronan in malignant osteosarcoma cells, IUBMB Life 2006, 58:47-53  18.  Papakonstantinou E, Karakiulakis G, Roth M, Block LH: Platelet-derived growth factor stimulates the secretion of hyaluronic acid by proliferating human vascular smooth muscle cells, Proc Natl Acad Sci U S A 1995, 92:9881-9885  19.  Evanko SP, Johnson PY, Braun KR, Underhill CB, Dudhia J, Wight TN: Plateletderived growth factor stimulates the formation of versican-hyaluronan aggregates and pericellular matrix expansion in arterial smooth muscle cells, Arch Biochem Biophys 2001, 394:29-38  20.  Iozzo RV: Matrix proteoglycans: from molecular design to cellular function, Annu Rev Biochem 1998, 67:609-652  21.  Yamaguchi Y: Lecticans: organizers of the brain extracellular matrix, Cell Mol Life Sci 2000, 57:276-289  22.  LeBaron RG, Zimmermann DR, Ruoslahti E: Hyaluronate binding properties of versican, J Biol Chem 1992, 267:10003-10010  23.  Dours-Zimmermann MT, Zimmermann DR: A novel glycosaminoglycan attachment domain identified in two alternative splice variants of human versican, J Biol Chem 1994, 269:32992-32998  24.  Ito K, Shinomura T, Zako M, Ujita M, Kimata K: Multiple forms of mouse PGM, a large chondroitin sulfate proteoglycan generated by alternative splicing, J Biol Chem 1995, 270:958-965  25.  Shinomura T, Nishida Y, Ito K, Kimata K: cDNA cloning of PG-M, a large chondroitin sulfate proteoglycan expressed during chondrogenesis in chick limb buds. Alternative spliced multiforms of PG-M and their relationships to versican, J Biol Chem 1993, 268:14461-14469  26.  Lemire JM, Merrilees MJ, Braun KR, Wight TN: Overexpression of the V3 variant of versican alters arterial smooth muscle cell adhesion, migration, and proliferation in vitro, J Cell Physiol 2002, 190:38-45  27.  Merrilees MJ, Lemire JM, Fischer JW, Kinsella MG, Braun KR, Clowes AW, Wight TN: Retrovirally mediated overexpression of versican v3 by arterial smooth muscle cells induces tropoelastin synthesis and elastic fiber formation in vitro and in neointima after vascular injury, Circ Res 2002, 90:481-487  28.  Zimmermann DR, Ruoslahti E: Multiple domains of the large fibroblast proteoglycan, versican, Embo J 1989, 8:2975-2981  127  29.  Bode-Lesniewska B, Dours-Zimmermann MT, Odermatt BF, Briner J, Heitz PU, Zimmermann DR: Distribution of the large aggregating proteoglycan versican in adult human tissues, J Histochem Cytochem 1996, 44:303-312  30.  Cattaruzza S, Schiappacassi M, Ljungberg-Rose A, Spessotto P, Perissinotto D, Morgelin M, Mucignat MT, Colombatti A, Perris R: Distribution of PGM/versican variants in human tissues and de novo expression of isoform V3 upon endothelial cell activation, migration, and neoangiogenesis in vitro, J Biol Chem 2002, 277:47626-47635  31.  Lemire JM, Braun KR, Maurel P, Kaplan ED, Schwartz SM, Wight TN: Versican/PG-M isoforms in vascular smooth muscle cells, Arterioscler Thromb Vasc Biol 1999, 19:1630-1639  32.  Zako M, Shinomura T, Miyaishi O, Iwaki M, Kimata K: Transient expression of PG-M/versican, a large chondroitin sulfate proteoglycan in developing chicken retina, J Neurochem 1997, 69:2155-2161  33.  Zimmermann DR, Dours-Zimmermann MT, Schubert M, Bruckner-Tuderman L: Versican is expressed in the proliferating zone in the epidermis and in association with the elastic network of the dermis, J Cell Biol 1994, 124:817-825  34.  Landolt RM, Vaughan L, Winterhalter KH, Zimmermann DR: Versican is selectively expressed in embryonic tissues that act as barriers to neural crest cell migration and axon outgrowth, Development 1995, 121:2303-2312  35.  Paulus W, Baur I, Dours-Zimmermann MT, Zimmermann DR: Differential expression of versican isoforms in brain tumors, J Neuropathol Exp Neurol 1996, 55:528-533  36.  Beggah AT, Dours-Zimmermann MT, Barras FM, Brosius A, Zimmermann DR, Zurn AD: Lesion-induced differential expression and cell association of Neurocan, Brevican, Versican V1 and V2 in the mouse dorsal root entry zone, Neuroscience 2005, 133:749-762  37.  Arora PD, Narani N, McCulloch CA: The compliance of collagen gels regulates transforming growth factor-beta induction of alpha-smooth muscle actin in fibroblasts, Am J Pathol 1999, 154:871-882  38.  Larsen M, Artym VV, Green JA, Yamada KM: The matrix reorganized: extracellular matrix remodeling and integrin signaling, Curr Opin Cell Biol 2006, 18:463-471  39.  Beningo KA, Dembo M, Wang YL: Responses of fibroblasts to anchorage of dorsal extracellular matrix receptors, Proc Natl Acad Sci U S A 2004, 101:1802418029  40.  Cukierman E, Pankov R, Stevens DR, Yamada KM: Taking cell-matrix adhesions to the third dimension, Science 2001, 294:1708-1712  41.  Rhee S, Grinnell F: Fibroblast mechanics in 3D collagen matrices, Adv Drug Deliv Rev 2007, 59:1299-1305  128  42.  Jiang H, Grinnell F: Cell-matrix entanglement and mechanical anchorage of fibroblasts in three-dimensional collagen matrices, Mol Biol Cell 2005, 16:50705076  43.  Petroll WM, Vishwanath M, Ma L: Corneal fibroblasts respond rapidly to changes in local mechanical stress, Invest Ophthalmol Vis Sci 2004, 45:3466-3474  44.  Brown RA, Prajapati R, McGrouther DA, Yannas IV, Eastwood M: Tensional homeostasis in dermal fibroblasts: mechanical responses to mechanical loading in three-dimensional substrates, J Cell Physiol 1998, 175:323-332  45.  Wozniak MA, Modzelewska K, Kwong L, Keely PJ: Focal adhesion regulation of cell behavior, Biochim Biophys Acta 2004, 1692:103-119  46.  Cukierman E, Pankov R, Yamada KM: Cell interactions with three-dimensional matrices, Curr Opin Cell Biol 2002, 14:633-639  47.  Even-Ram S, Yamada KM: Cell migration in 3D matrix, Curr Opin Cell Biol 2005, 17:524-532  48.  Grinnell F: Fibroblast biology in three-dimensional collagen matrices, Trends Cell Biol 2003, 13:264-269  49.  Carlson MA, Longaker MT: The fibroblast-populated collagen matrix as a model of wound healing: a review of the evidence, Wound Repair Regen 2004, 12:134147  50.  Eastwood M, McGrouther DA, Brown RA: Fibroblast responses to mechanical forces, Proc Inst Mech Eng [H] 1998, 212:85-92  51.  Tamariz E, Grinnell F: Modulation of fibroblast morphology and adhesion during collagen matrix remodeling, Mol Biol Cell 2002, 13:3915-3929  52.  Krueger RC, Jr., Schwartz NB: An improved method of sequential alcian blue and ammoniacal silver staining of chondroitin sulfate proteoglycan in polyacrylamide gels, Anal Biochem 1987, 167:295-300  53.  Farndale RW, Buttle DJ, Barrett AJ: Improved quantitation and discrimination of sulphated glycosaminoglycans by use of dimethylmethylene blue, Biochim Biophys Acta 1986, 883:173-177  54.  Poot M, Zhang YZ, Kramer JA, Wells KS, Jones LJ, Hanzel DK, Lugade AG, Singer VL, Haugland RP: Analysis of mitochondrial morphology and function with novel fixable fluorescent stains, J Histochem Cytochem 1996, 44:1363-1372  55.  Fitch MT, Silver J: CNS injury, glial scars, and inflammation: Inhibitory extracellular matrices and regeneration failure, Exp Neurol 2007,  56.  Faggian J, Fosang AJ, Zieba M, Wallace MJ, Hooper SB: Changes in versican and chondroitin sulfate proteoglycans during structural development of the lung, Am J Physiol Regul Integr Comp Physiol 2007, 293:R784-792  57.  Kuo SM, Wang YJ, Niu GC, Lu HE, Chang SJ: Influences of hyaluronan on type II collagen fibrillogenesis in vitro, J Mater Sci Mater Med 2008, 19:1235-1241  129  58.  Brightman AO, Rajwa BP, Sturgis JE, McCallister ME, Robinson JP, VoytikHarbin SL: Time-lapse confocal reflection microscopy of collagen fibrillogenesis and extracellular matrix assembly in vitro, Biopolymers 2000, 54:222-234  59.  Berrier AL, Yamada KM: Cell-matrix adhesion, J Cell Physiol 2007, 213:565-573  60.  Zhang Y, Cao L, Yang BL, Yang BB: The G3 domain of versican enhances cell proliferation via epidermial growth factor-like motifs, J Biol Chem 1998, 273:21342-21351  61.  Yang BL, Yang BB, Erwin M, Ang LC, Finkelstein J, Yee AJ: Versican G3 domain enhances cellular adhesion and proliferation of bovine intervertebral disc cells cultured in vitro, Life Sci 2003, 73:3399-3413  62.  Wu Y, Chen L, Cao L, Sheng W, Yang BB: Overexpression of the C-terminal PG-M/versican domain impairs growth of tumor cells by intervening in the interaction between epidermal growth factor receptor and beta1-integrin, J Cell Sci 2004, 117:2227-2237  63.  Popp S, Andersen JS, Maurel P, Margolis RU: Localization of aggrecan and versican in the developing rat central nervous system, Dev Dyn 2003, 227:143149  64.  Kamiya N, Watanabe H, Habuchi H, Takagi H, Shinomura T, Shimizu K, Kimata K: Versican/PG-M regulates chondrogenesis as an extracellular matrix molecule crucial for mesenchymal condensation, J Biol Chem 2006, 281:2390-2400  65.  Matsumoto K, Kamiya N, Suwan K, Atsumi F, Shimizu K, Shinomura T, Yamada Y, Kimata K, Watanabe H: Versican/PG-M aggregates in cartilage: identification and characterization, J Biol Chem 2006,  66.  Cattaruzza S, Schiappacassi M, Kimata K, Colombatti A, Perris R: The globular domains of PG-M/versican modulate the proliferation-apoptosis equilibrium and invasive capabilities of tumor cells, Faseb J 2004, 18:779-781  67.  Bourguignon LY, Zhu H, Shao L, Zhu D, Chen YW: Rho-kinase (ROK) promotes CD44v(3,8-10)-ankyrin interaction and tumor cell migration in metastatic breast cancer cells, Cell Motil Cytoskeleton 1999, 43:269-287  68.  Zhu D, Bourguignon LY: Interaction between CD44 and the repeat domain of ankyrin promotes hyaluronic acid-mediated ovarian tumor cell migration, J Cell Physiol 2000, 183:182-195  69.  Oliferenko S, Kaverina I, Small JV, Huber LA: Hyaluronic acid (HA) binding to CD44 activates Rac1 and induces lamellipodia outgrowth, J Cell Biol 2000, 148:1159-1164  70.  Bourguignon LY, Zhu H, Chu A, Iida N, Zhang L, Hung MC: Interaction between the adhesion receptor, CD44, and the oncogene product, p185HER2, promotes human ovarian tumor cell activation, J Biol Chem 1997, 272:27913-27918  71.  Ogawa H, Oohashi T, Sata M, Bekku Y, Hirohata S, Nakamura K, Yonezawa T, Kusachi S, Shiratori Y, Ninomiya Y: Lp3/Hapln3, a novel link protein that co-  130  localizes with versican and is coordinately up-regulated by platelet-derived growth factor in arterial smooth muscle cells, Matrix Biol 2004, 23:287-298 72.  Hirose J, Kawashima H, Yoshie O, Tashiro K, Miyasaka M: Versican interacts with chemokines and modulates cellular responses, J Biol Chem 2001, 276:52285234  73.  Kawashima H, Hirose M, Hirose J, Nagakubo D, Plaas AH, Miyasaka M: Binding of a large chondroitin sulfate/dermatan sulfate proteoglycan, versican, to Lselectin, P-selectin, and CD44, J Biol Chem 2000, 275:35448-35456  74.  Ruoslahti E, Yamaguchi Y: Proteoglycans as modulators of growth factor activities, Cell 1991, 64:867-869  75.  Kinsella MG, Bressler SL, Wight TN: The regulated synthesis of versican, decorin, and biglycan: extracellular matrix proteoglycans that influence cellular phenotype, Crit Rev Eukaryot Gene Expr 2004, 14:203-234  76.  Scott JE, Cummings C, Brass A, Chen Y: Secondary and tertiary structures of hyaluronan in aqueous solution, investigated by rotary shadowing-electron microscopy and computer simulation. Hyaluronan is a very efficient networkforming polymer, Biochem J 1991, 274 (Pt 3):699-705  77.  Mow VC, Mak AF, Lai WM, Rosenberg LC, Tang LH: Viscoelastic properties of proteoglycan subunits and aggregates in varying solution concentrations, J Biomech 1984, 17:325-338  78.  Soby L, Jamieson AM, Blackwell J, Choi HU, Rosenberg LC: Viscoelastic and rheological properties of concentrated solutions of proteoglycan subunit and proteoglycan aggregate, Biopolymers 1990, 29:1587-1592  79.  Knudson W, Bartnik E, Knudson CB: Assembly of pericellular matrices by COS7 cells transfected with CD44 lymphocyte-homing receptor genes, Proc Natl Acad Sci U S A 1993, 90:4003-4007  80.  Lee GM, Johnstone B, Jacobson K, Caterson B: The dynamic structure of the pericellular matrix on living cells, J Cell Biol 1993, 123:1899-1907  81.  Morgelin M, Paulsson M, Heinegard D, Aebi U, Engel J: Evidence of a defined spatial arrangement of hyaluronate in the central filament of cartilage proteoglycan aggregates, Biochem J 1995, 307 (Pt 2):595-601  82.  Ingber DE: Cellular tensegrity: defining new rules of biological design that govern the cytoskeleton, J Cell Sci 1993, 104 (Pt 3):613-627  83.  Goffin JM, Pittet P, Csucs G, Lussi JW, Meister JJ, Hinz B: Focal adhesion size controls tension-dependent recruitment of alpha-smooth muscle actin to stress fibers, J Cell Biol 2006, 172:259-268  84.  Roughley PJ, Lee ER: Cartilage proteoglycans: structure and potential functions, Microsc Res Tech 1994, 28:385-397  131  85.  Bramley AM, Roberts CR, Schellenberg RR: Collagenase increases shortening of human bronchial smooth muscle in vitro, Am J Respir Crit Care Med 1995, 152:1513-1517  86.  Yamagata M, Kimata K: Repression of a malignant cell-substratum adhesion phenotype by inhibiting the production of the anti-adhesive proteoglycan, PGM/versican, J Cell Sci 1994, 107 (Pt 9):2581-2590  87.  Yamagata M, Saga S, Kato M, Bernfield M, Kimata K: Selective distributions of proteoglycans and their ligands in pericellular matrix of cultured fibroblasts. Implications for their roles in cell-substratum adhesion, J Cell Sci 1993, 106 (Pt 1):55-65  132  4. Versican Degradation by Macrophage Matrix Metalloproteinases MMP-7, MMP-12, and MMP-2 3 4.1 Introduction Fibrosis, or abnormal wound healing, is marked by excessive deposition of provisional matrix by persisting myofibroblasts1.  In many forms of human lung fibrosis,  proliferating myofibroblasts alters the normal structure and function of the lung by crossing the basal membrane and depositing a provisional matrix in the alveolar spaces at the site of injury2, 3.  We were first to identify versican, a large chondroitin sulfate  proteoglycan, as an abundant yet transient component of this provisional matrix in many different types of fibrotic lung disease4, 5. Versican is a member of hyalectan family of proteoglycans which interact with hyaluronan (HA) through their highly homologous globular N-terminus. Versican also interacts with a variety of molecules in the extracellular matrix (ECM) through its glycosaminoglycan (GAG) chains, bound to the core of the molecule.  Versican’s  globular C-terminal (G3) domain consists of two EGF repeats, a C-type lectin domain and complement regulatory protein (CRP)-like domain.  The complex structure of  versican6 has lead to the discovery of multiple functions for this molecule in different tissues, in development and disease. Versican is distributed widely in adult human tissues7-10. In the fibrotic lungs, the early accumulation of versican occurs in association with proliferating myofibroblasts prior to deposition of collagenous matrix characteristic of the later stages of the disease11,  12  .  Versican may promote cellular proliferation through several mechanisms13. The N-terminal domain of versican is implicated in smooth muscle cell proliferation and migration through formation of a pericellular matrix, rich in versican and hyaluronan14, 15. Versican’s chondroitin sulfate chains interact with a variety of growth factors16, 17 and cytokines in the ECM, which can indirectly regulate cellular behavior18,  19  . Versican  3  A version of this chapter will be submitted for publication. Pourmalek, S, Overall, C, and Roberts, C. Versican degradation by macrophage matrix metalloproteinases MMP-7, MMP-12, and MMP-2. 133  C-terminal domain can also stabilize the matrix and cell-matrix interactions and influence cell morphology through disulfide bond formation with the G3 domain of other versican molecules  20  , and interaction with a number of other ligands found in the matrix of  proliferating cells. Due to the effects of versican on fibroblast cell proliferation and survival, and the role that versican synthesizing myofibroblasts play in wound healing, the significance of versican degradation and ECM remodeling in wound healing has been the subject of many studies. Matrix degrading metalloproteinases are a major group of matrix remodeling enzymes, implicated in the wound healing process21-24. The expression of several matrix metalloproteinases (MMPs), namely MMP-725 26 and MMP-1227 increases in pulmonary fibrotic diseases as a result of injury.  In this study, we localize the  expression of these enzymes to macrophages that accumulate at the site of versican-rich lesions in the later stages of pulmonary fibrotic diseases. We also present our original data on the sensitivity of versican to these macrophage metalloproteinase, and compare the cleavage pattern of versican by MMP-7, MMP-12 and MMP-2. The results of our study supports our hypothesis that macrophage metalloproteinases are involved in removal of the versican-rich fibroproliferative lesions by cleaving and degrading versican, removal of which is associated with fibroblast cell apoptosis.  4.2 Materials and Methods Patient Samples – Lung tissues used in this study were obtained from the University of British Columbia St Paul's Hospital Pulmonary Research Laboratory tissue registry as previously described4, 5. Lung biopsy tissues were obtained at diagnostic biopsy of patients with a clinical diagnosis of idiopathic pulmonary fibrosis, and were entered into the study following histologic diagnoses of bronchiolitis obliterans organizing pneumonia (BOOP) or usual interstitial pneumonia (UIP). Age-matched control tissues were obtained from normal-appearing lung tissue, obtained from lung lobes that were resected from individuals with small localized tumors, as previously described4. Tissues from 6 UIP, 6 BOOP and 6 control patients were studied. From some of the control patients it was possible to obtain small samples of unfixed lung tissue for biochemical studies; these were flash-frozen in liquid nitrogen and stored at - 80°C until analysis. 134  Histology – Lung tissues were fixed overnight in 10% neutral buffered formalin, dehydrated, embedded in paraffin and serially sectioned at a thickness of 5µm. Sections were stained with hematoxylin and eosin to visualize overall architecture, alcian blue to localize glycosaminoglycans and picrosirius red to localize collagen as previously described4. Immunohistochemistry – Sections were deparaffinized and hydrated in Trisbuffered saline (TBS) for 5 minutes before being immersed in freshly prepared 0.6% hydrogen peroxide in methanol for 40 minutes to block endogenous peroxidase activity. Sections were blocked with 10% normal goat serum in 2% BSA for 4 hours. The following primary antibodies were used: mouse monoclonal anti-versican C-terminal domain, 2B128,  29  (Seikagaku, Tokyo, Japan), dilution 1:400; rabbit polyclonal  anti-versican30 used as previously described4, 5, dilution 1:500; rhMMP-7 monoclonal antibody (MAB9071, R&D Systems, USA, 0.5mg/ml) dilution 1:50; hMMP-12 mouse monoclonal antibody (R&D systems, MN, USA), dilution 1:40 or 25µg/ml; MMP-2 antibody (a kind gift from Dr. Chris Overall; 79µg/ml), dilution 1:50. Antibodies were diluted in 2% bovine serum albumin (BSA) in TBS and washed 4 times in TBS. Antibody labeling was visualized with the Vectastain Universal Elite ABC kit (Vector Laboratories, CA, USA) and DAB (3,3’-diaminobenzidine) as substrate (Vector Laboratories, CA, USA) according to the manufacturers instructions. Sections were counterstained with Gill's Haematoxylin. Negative controls were treated identically with the inclusion of non-immune IgG at the same concentration or with omission of primary antibody. For each antibody and detection system, conditions were established that allowed use of purified antibody or serum at concentrations that generated no staining with non-immune IgG or serum at equivalent concentrations. All sections that were to be compared were processed and stained concurrently. After staining, sections were mounted in Histochoice mounting medium (Amresco, OH, USA). Expression and Purification of Versican – Human fetal lung (HFL1) fibroblast cells American Type Culture Collection (Manassas, VA) were cultured in 75-cm2 flasks (Sarstedt; Quebec, Canada) in Dulbecco’s Modified Eagle Medium (DMEM; Life Technologies, Inc., Gaithersburg, MD) supplemented with 10% (v/v) fetal bovine serum  135  (FBS; HyClone, UT, USA) to 80% confluence. Cells from two confluent 75-cm2 flasks were trypsinized and transferred to a 850-cm2 tissue culture roller bottle (Becton Dickinson) with 200ml of DMEM and incubated at 37˚C in a BELCO Biotechnology Roll-in incubator. Serum free conditioned medium from fibroblast cultures (CM) was collected and centrifuged at 1500 X g for 15 minutes to remove cellular debris. Then, Urea and salt concentrations in HFL1 conditioned media were adjusted to 7M Urea and 0.4M NaCl and loaded onto Q-Sepharose Fast Flow ion exchange resin (Amersham Biosciences, NJ, USA) at approximately 1 litre CM per 5 mls resin. The column was washed with a 10 fold bed volume of 7M Urea, 0.4M NaCl, 0.1M NaOAc, pH 6.0 before elution with 7M Urea, 1.5M NaCl, 0.1M NaOAc, pH 6.0. Peak fractions were pooled and dialyzed against PBS (140mM NaCl, 2.7mM KCl, 10mM Na2HPO4, 1.8mM NaH2PO4, pH7.4) exhaustively, then flash frozen with liquid nitrogen and stored at -70˚C. Fractions were monitored for versican content by alcian blue (Sigma, MO, USA) staining of SDSPAGE gels31 and by Western blotting. Purified versican concentration was estimated using the dimethylmethylene blue (DMMB) assay (Serva, Heidelberg)32 to quantify sulfated glycosaminoglycan using known concentrations of chondroitin sulfate C as standards (Seikagaku, Japan). This is possible considering versican V0 and V1 are the primary mRNA splice variants expressed in proliferating HFL1 cultures. The concentration of versican was estimated based on an average of 1.5 mg total proteoglycan per 1 mg sulfated glycosaminoglycan detected with a resultant concentration of 1.12 mg/ml or approximately 1.12 µM versican. Generation of Recombinant Constructs (HisL, HisLC, HisG3) – Before ligating the His-tagged C-terminal construct cDNA to the expression vector pGYMXC, the PCR product was amplified using the pPCR-Script strategy. Briefly, cDNA amplified from PCR was purified using a QIAEX II Gel Extraction Kit (Qiagen, Mississauga, Ontario). The DNA fragment was ligated into the pPCR-Script Cam SK(+) plasmid (Stratagene, CA, USA). The ligation mixture contained 1µl of 10X reaction buffer, 1µl of Srf I restriction enzyme (5U), 1µl of T4DNA ligase (4U), 0.5µl of 10mM rATP, 3µl of the HisLC insert (190ng), and 1µl of the cloning vector (10ng) for a 100:1 molar ratio of insert to cloning vector. The mixture was diluted to 10 µl with ddH2O, gently mixed, and incubated at room temperature for 1 hr before heating at 65˚C for 10min. Two µl of the 136  ligation mixture was used for heat shock transformation of supercompetent E. coli strain DH5α. The bacteria was transferred to 50µl of 2X YT media (1.0% w/v yeast extract, 1.6%w/v tryptone, 0.5% w/v NaCl, pH 7.5) and agitated at 275rpm for 30min at 37˚C before plating on LB agar plates containing 30µg/ml chloramphenicol. Plates were incubated for 16hrs at 37˚C. Colonies were selected and grown in 5ml of terrific broth media (1.2% w/v tryptone, 2.4% w/v yeast extract, 0.231% w/v KH2PO4, 1.254% w/v K2HPO4, 0.4% glycerol) with 30µg/ml chloramphenicol at 37˚C for 16hrs at 275rpm. The plasmid was isolated using the QIAprep Spin Miniprep Kit (Qiagen, Mississauga, Ontario). The insert was removed from the CAM SK(+) plasmid by Hind III and NheI restriction enzyme digestion and purified by QIAEX II Gel Extraction Kit (Qiagen, Mississauga, Ontario). The identity of the new HisLC pGYMX construct was confirmed by Hind III and Nhe I restriction enzyme digestion and by DNA sequencing performed by Nucleic Acids-Protein Service (NAPS) at the University of British Columbia. Transformation, Expression and Purification of Engineered Versican C-terminal Domain Constructs (His-L, His-LC, and His-G3)– The pGYMX His-G3 expression vector was transformed into E.coli BL21(DE3) competent cells at a ratio of 1µl of cDNA into 50µl BL21 Gold E.coli cells. Briefly, the mixture was iced for about an hour before a 90 seconds heat shock at 42˚C. The mixture was iced for 2 minutes, and incubated at 37˚C for 30 min in shaker with 50µl of 2XYT media. The cells were then plated on Luria–Bertani (LB) agar plates containing 100µg/ml ampicillin. 5ml of superbroth (0.8% w/v yeast extract, 1.0%w/v tryptone, 0.5% w/v NaCl, 0.1% glycerol, pH 7.5) and incubated in 37˚C shaker for 24 hours. Next, another plate of agar was inoculated with a single colony and incubated at 37˚C for 16 hrs at 275rpm. Aliquots of this log phase seed culture were used to innoculate 3.5L of superbroth in a1:1000 (v/v) ratio with 100µg/ml ampicillin and incubated at 37˚C for 24hrs at 275rpm. Collected cells were washed with 500ml of NET buffer (100mM NaCl, 1mM EDTA, 20mM Tris-HCl, pH 8.0) then lysed in 250ml of lysis buffer (50mM NaCl, 1mM EDTA, 20mM Na2HPO4, 1mg/ml lysozyme, 1mM PMSF, pH 8.0) for 2hrs at 37˚C and 275rpm and sonicated with 5 sec bursts. The inclusion bodies were washed with 500ml of NET buffer then dissolved in a solubilization buffer (8M Urea, 10mM Tris-HCl, 100mM Na2HPO4, pH 8.0) for 16hrs at  137  4˚C. Dissolved inclusion bodies were centrifuged at 20000rpm for 1hr and purified using a 30ml Ni2+- charged chelating sepharose column (Amersham Pharmacia) equilibrated in column buffer (8M Urea, 0.5M NaCl, 20mM Na2HPO4, pH 7.4). The column was washed in succession with 10-fold bed volume of column buffer, column buffer with 1M NaCl, column buffer with 1M NaCl, pH 6.0, and again with column buffer. Proteins with non-specific interactions to the Ni-chelate column were pre-eluted by a 10-fold bed volume of column buffer with 200mM imidazole. His-LC fusion protein was then eluted by a 200mM to 1M imidazole gradient over a 10-fold bed. Fractions were analyzed by SDS-PAGE and stored at –20˚C. Refolding of HisG3 Recombinant Protein – Peak fractions obtained from the imidazole gradient were pooled and diluted 20-fold before dialysis in equal volume of refolding buffer (18.2mM Na2CO3, 24mM NaHCO3, 125mM NaCl, 1:10 ration of 3mM Cysteine/Cystine, pH 10.0) with aeration at room temperature. Refolding buffer was changed every 2 hrs for 8 hrs before exhaustive dialysis with refolding buffer minus the redox pair of Cysteine/Cystine for complete removal of urea.  Because the pooled  fractions were diluted 20 fold before refolding, a 10ml Ni-chelate column equilibrated in 18.2mM Na2CO3, 24mM NaHCO3, 125mM NaCl, pH 10.0 was used to concentrate the diluted pool. His-LC was eluted with 18.2mM Na2CO3, 24mM NaHCO3, 125mM NaCl, 500mM imidazole, pH 10.0. Peak fractions were pooled and dialyzed against excess volume of 18.2mM Na2CO3, 24mM NaHCO3, 125mM NaCl, pH 10.0. The protein pool was aliquoted into 1ml fractions, flash frozen with liquid nitrogen, and stored at –70˚C. Electrophoretic Techniques – Samples in nonreducing sample buffer (125 mM TrisHCL, pH 6.8, 2.0% SDS, 2.0 M urea, 0.05% bromophenol blue) were separated on discontinuous SDS-PAGE gels with 4% (stacking) and 12.5% (separating) acrylamide. Stacking and separating gels were kept during staining and Western blotting to monitor high molecular weight versican aggregates within the stacking gel. Relative molecular mass (measured in kDa) was estimated based on mobility of molecular weight markers: HiMark Prestained (Invitrogen), MagicMark XP (Invitrogen) and Kaleidoscope Prestained (Bio-Rad, Hercules, CA). Western blotting was performed using the XCell II blot module (Invitrogen) to PVDF membrane (Millipore). Blocking was performed with  138  a solution of 2.5% (w/v) bovine serum albumin, 20 mM Tris, 5 mM EDTA, 0.9%NaCl, and 0.3% (v/v) Tween 20. Anti-versican 2B1(Seikagaku Corporation, Tokyo, Japan), 1:500 dilution, was used for detection of Versican at an epitope near the C-terminal domain; and anti-G3 antibody (LC2, in-house antibody), 1:10,000 dilution, for detection of the C-terminal domain of Versican. Antibodies were diluted in a solution of 2% (w/v) bovine serum albumin, 20 mM Tris, pH 7.5, 0.9 % NaCl and 0.05% (v/v) Tween 20. Highly cross adsorbed goat anti-mouse horseradish peroxidase-conjugate (Bio-Rad) and highly cross adsorbed goat anti-rabbit horseradish peroxidase-conjugate (Bio-Rad) secondary antibodies were diluted to 1:5000. Visualization of the peroxidase was performed with Enhanced Chemiluminescence Plus Western blotting reagents (Amersham Biosciences) and exposed to X-ray film (Kodak, New Haven, CT, USA) or captured using the ChemiGenius-2 bio-imaging system and Gene Snap software (Perkin Elmer, Woodbridge, ON, Canada). Versican and HisG3 Degradation with MMPs – Versican and HisG3 were incubated with selected MMPs (MMP-2 (kindly provided by Dr. Chris Overall, Faculty of Dentistry, University of British Columbia), MMP-7 (USB Corporation, Cleveland, Ohio), and MMP-12 (provided by Dr. Richard Dean, University of British Columbia) at equal substrate:enzyme ratio (1:0, 1:2.5, 1:5, 1:10, 1:20, 1:40, 1:80) diluted in the respective buffers for 24 hours at 37°C. Detection was by silver staining and Western blotting with appropriate versican antibodies. Enzyme digestion was stopped by the addition of EDTA for a final concentration of 10mM.  HisG3, purified in our  laboratories, was incubated with expressed and purified human MMP-7 at substrate:enzyme ratio of 1:20, at 37°C for 24h. Sequencing of HisG3 fragment cleaved by MMP-7 – MMP-7 digested HisG3 fragments were first concentrated by Microcon YM-3 concentrators (Millipore, Ontario, Canada). Concentrated samples were then analyzed by 15% SDS-PAGE and transferred to PVDF membrane. The membrane was stained with 40% methanol, 1% Acetic Acid, and 0.1% R-250 for 15min, destained 3 times in 50% methanol, for 5min periods. The highly stained band at around 26kDa was sequenced by N-terminal sequencing at NAPS center at UBC (Vancouver, Canada).  139  4.3 Results 4.3.1 Macrophages surround versican-rich lesions in BOOP Patient samples exhibiting the pathological pattern bronchiolitis obliterans organizing pneumonia (BOOP) showed characteristic intraluminal buds composed of loose connective tissue adjacent to a thickened interstitium (Fig 4.1). Staining of the matrix and the cells involved in this type of pulmonary fibrosis revealed fibroblast cell proliferation in areas of intensive versican staining (Fig 4.1, A). Alcian blue stain, typically used to stain glycosaminoglycans, also stained the intraluminal buds signaling the presence of a matrix rich in versican decorated with chondroitin sulfate chains (Fig 4.1, B and C). This is consistent with our previous demonstration that the glycosaminoglycan is chondroitin sulfate, that versican is the predominant proteoglycan within intraluminal buds and that the histochemical glycosaminoglycan staining and versican staining are highly congruent4. Another pathological characteristic of BOOP was the accumulation of macrophage cells around the fibroproliferative lesions in the late stages of the disease (Fig 4.1, D). 4.3.2 Macrophages surrounding versican-rich lesions express high levels of MMPs Macrophages surround versican-rich lesions in later stages of human pulmonary fibrosis and  their  accumulation  is  concomitant  with  versican  degradation.  An  immunohistochemical examination of tissue from pulmonary fibrotic lung showed increased expression of several MMPs by macrophages around versican-rich lesions. Population of macrophage cells surrounding versican-rich lesions were higher in Fibrotic lung samples (Fig 4.1, F H J L) than Normal lung tissue (Fig 4.1, E G I K). The levels of MMP-7, MMP-12 and MMP-2 expression were also higher in Fibrotic lung compared to Normal lungs or the control with no antibody staining against matrix metalloproteinases (Fig 4.1, L). Strongest staining of these metalloproteinases is observed in association with the macrophage cells, which suggests a role in the changes that take place in the matrix subsequent to the arrival of macrophages.  140  4.3.3 Versican degradation assays by macrophage metalloproteinases The macrophage matrix metalloproteinases (MMPs), MMP-7 (matrilysin), MMP-12 (macrophage metalloelastase), and MMP-2 (gelatinase), were chosen as candidate macrophage enzymes that were localized to the resolving phase of wound healing in the lung. Considering versican slowly disappears in the final stages of wound healing and this phase is concurrent with macrophage co-localization around versican-rich lesions in a rat BOOP model of the lung (data not shown), sensitivity of versican to these enzymes were examined by silver staining and detected by western blotting.  Versican was  expressed by Human Lung Fibroblast (HFL1) cell culture, and purified using a Nickel column. The purified Versican was incubated with each of the chosen MMPs at 37°C at a range of ratios for 24h. We also examined the degradation pattern of the C-terminal construct of versican (G3 domain). Histidine tagged G3 was expressed by transfected E-coli cells and purified, and used in degradation assays with MMPs.  2B1 mouse  monoclonal antibody, which recognizes an epitope in the C-terminal domain  of  versican29 was used in all cases. 4.3.4 MMP-7 degrades versican at multiple sites The MMP-7 degradation of versican resulted in a drastic change in intact versican concentration with increase in enzyme concentration as detected by silver stain and western blot (Figure 4.2A and 4.2B respectively). High molecular weight intact versican molecules were trapped at the top of the stacking gel (Figure 4.2, Lane 1). With the introduction of a small amount of MMP-7 enzyme (1:80 enzyme : substrate), the large fragmented versican moved further down the stacking gel (Figure 4.2, Lane 2). As the concentration of MMP-7 enzyme increased, fragments of different sizes appeared sequentially, from larger than 120 kDa to about 20 kDa (Figure 4.2, Lanes 2-7). The degradation products were better observed in the western blot, recognized by the 2B1 mouse monoclonal antibody, than the silver-stained gel. However, both methods clearly illustrated the ability of MMP-7 enzyme to cleave versican at multiple sites.  142  4.3.5 Recombinant human MMP-12 degrades C-terminal domain of versican Our data showed, for the first time, the susceptibility of 2B1 epitope of versican (near the C-terminal domain) to degradation by macrophage metalloelastase (Figure 4.3A). From the western blot analysis of the degradation pattern, a rapid disappearance of versican antibody (2B1) signal was observed.  From the silver stain, we inferred that high  molecular weight versican is not prone to cleavage by the enzyme. Even though low molecular weight fragments appeared as the enzyme concentration increased (Figure 4.3B), there seemed to be little effect on the concentration of high molecular weight versican trapped at the top of the stacking gel.  144  4.3.6 MMP-2 degradation of versican is limited Our western blot and silver stain analysis (Figure 4.4A and 4.4B respectively) of versican degradation by recombinant MMP-2 showed a pattern that might be expected from selective degradation. There seemed to be limited cleavage of versican into smaller fragments that appeared with the lowest concentration of enzyme (Lane 2) when compared to the intact versican (Lane 1). Interestingly, as the enzyme concentration increased, the degradation pattern did not change. The same pattern was observed in silver stain (40kDa – stacking gel) and western blot (as a smear in the stacking gel) from lane 2-7.  146  4.3.7 Versican C-terminal (G3 domain) is cleaved by MMP-7 From the western blot degradation pattern of intact versican by MMP-7 (Figure 4.2 A), the presence of several degraded protein bands (lanes 4 – 7; 20 – 40 kDa) suggest that Cterminal domain of versican may be prone to proteolytic degradation by this enzyme. The degradation pattern of HisG3 construct of versican with MMP-7 on the PVDF membrane immunoblotted with anti-G3 antibody is shown (Figure 4.5). A small degraded fragment, approximately 26kDa in size, appeared with the introduction of smallest amount of enzyme (Figure 4.5, lanes 2-5). The cleavage site of this fragment was determined by N-terminal sequencing. The product was sequenced to the histidine 3204 – leucine 3205 site in the lectin domain of the C-terminal of versican. Our reported cleavage site is comparable to the cleavage sites reported for other substrates of this enzyme, with either leucine or isoleucine amino acids at the P1’ site34. HisG3 monomers (~30kDa), dimers (~60kDa) and aggregates (at the top of the gel) started to disappear with higher concentrations of MMP-7 (Figure 4.5, lanes 4 and 5).  148  4.4 Discussion The basic science of wound healing and fibrosis are covered in a number of recent reviews35-37. The highly regulated process of normal wound healing is initiated at the site of injury by blood clotting and inflammation, followed by fibroblast migration and proliferation in a provisional matrix. The final stage of wound healing is characterized by matrix resolution and remodeling into a collagenous matrix, and by myofibroblast apoptosis.  In the pathological process of fibrosis, excessive matrix deposition by  proliferating myofibroblasts in the remodeling process leads to scarring and abnormal organ function. The balance between ECM deposition and degradation is integral to a normal wound healing process. Versican is abundant in fibroproliferative lesions in the fibrotic lung, and is associated with proliferating myofibroblasts in the early stages of remodeling4, 5. Versican promotion of cell proliferation in other matrices is well established13-15,  38  .  Histochemical studies in our laboratory have shown that versican is a transient component of the provisional matrix4, 5, and it is known that versican is absent from the collagenous matrix at later stages of the disease course39, 40. The mechanism of removal of versican from the healing matrix, however, is unknown. Immunohistochemical data presented in this study show that macrophages, identified by anti-CD-68 antibody staining, surround versican-rich fibroproliferative cores and even penetrate some of these lesions in the fibrotic lung (Figure 4.1). Our data also shows that expression of MMP-2, MMP-7, and MMP-12 is upregulated in the macrophages that accumulate at the site of lesions in the later stages of pulmonary fibrotic diseases (Figure 4.1), suggesting that a substrate for these proteinases lies within these lesions. Matrix metalloproteinases are key regulators of all stages of wound healing including inflammation, re-epithelialization, and matrix remodeling41-43, as it is the case in the lungs. It is shown that expression of MMP-7 and MMP-12 increases in asbestos-induced lung injury in mice and it is suggested that these metalloproteinases promote fibrosis through effects on inflammation26.  Also, matrilysin expression increases in airway  epithelial cells and alveolar type II cells in cystic fibrosis and facilitates 150  re-epithelialization24. We are the first, however, to show an increased expression of MMP-7, MMP-12, and MMP-2 in tissue repair.  We also make a strong case for  metalloproteinase involvement in proteolytic cleavage and degradation of versican (Figures 4.2-4.5) in the remodeling matrix. It is known that matrilysin is constitutively expressed in the epithelium of peribronchial glands and conducting airways in normal lung. Up-regulation of matrilysin after injury, however, suggests a role for this metalloproteinase in injury-mediated responses of the lung. Matrilysin (MMP-7) expression increased in pulmonary fibrosis  25  . Matrilysin  knockout-mice were dramatically protected from pulmonary fibrosis in response to intratracheal bleomycin 25. Other studies suggest that versican may be a substrate for this enzyme. It has been shown that versican is present at the site of MMP-7 expression by lipid laden macrophages in atherosclerotic tissues44. Versican has also been detected in the same areas as MMP-7 in actinic damage45. Our biochemical data shows that versican is a substrate for macrophage metalloproteinases, MMP-7 (Figure 4.2), MMP-2 (Figure 4.3), and MMP-12 (Figure 4.4), and that the versican degradation pattern by each of these three metalloproteinases is unique. MMP-7 seems to cleave intact versican at multiple sites as seen in both the immunoblot (Figure 4.2A) and silver stained gel (Figure 4.2B). MMP-7 is known to be an effective enzyme in degrading the extracellular matrix, and due to lack of a hemopexin domain characteristic of other MMPs, it has multiple cleavage site activities. This seemingly indiscriminate degradation pattern points to a possible role for MMP-7 in clearing versican from the provisional matrix. MMP-12 is upregulated in the airways of rats with allergic bronchial asthma, and is mainly expressed in airway epithelia and alveolar macrophages  27  . Degradation of high  molecular weight versican with MMP-12 resembles its degradation pattern by MMP-7 when only the immunoblots are compared (Figure 4.3A). A closer look at the silver stained gels (Figure 4.3B), however, leads one to postulate that MMP-12 activity is more focused on the G3-domain of versican. Considering the immunoblots are prepared by an antibody that recognizes a site near the C-terminal domain of versican29, and that the large intact versican is still apparent at the top of the silverstained gel, it is possible that MMP-12 cleaves the 2B1 epitope near the G3 domain which not only reduces the  151  molecular weight of the intact versican slightly, as seen in the silver-stained gel, but also appears to completely degrade versican, as detected by the western blot. Versican degradation pattern by MMP-2 is quite distinguishable from that of MMP-7 and MMP-12. Limited and what seems like selective cleavage of versican by MMP-2 is observed both by the immunoblot (Figure 4.4A) and the silver stained gel (Figure 4.4B). It is important to note that active site titration of MMP-2 with TIMP-2 has shown this enzyme to be fully active at the time of incubation with substrate (data not shown). MMP-7 and MMP-12 were already in their active form and did not require activation. Thus, the same number of active enzyme per substrate was used in all cases. The presence of high molecular weight intact versican at top of the stacking gel, and appearance of few low molecular weight bands with increasing enzyme concentration may point to a different role played by MMP-2, as compared to MMP-7 and MMP-12. MMP-2 may cleave versican to produce molecules that regulate cell behavior or to destabilize the matrix, though this hypothesis needs to be studied further. From the results of versican degradation by macrophage enzymes MMP-7 and MMP-12, it is clear that versican C-terminal domain is susceptible to degradation by macrophage MMPs investigated in this study. The activity of MMP-7 on the C-terminal domain of versican was further investigated by N-terminal sequencing of one of the fragments produced from cleavage of HisG3 construct. The product corresponded to the histidine 3204 - leucine 3205 site in the lectin domain of the C-terminal of versican, and the sequence was comparable to other cleavage sites reported for this enzyme with either leucine or isoleucine amino acids at the P1’ site.  The significance of this specific  cleavage is discussed below. Versican degradation could result in destabilization of a hyaluronan- and versican-rich pericellular matrix, the structural integrity of which is required for proliferation of myofibroblast cells4, 5, 14, 15. Degradation of versican may also lead to release of growth factors bound to its chondroitin sulfate chains and perhaps leave them more vulnerable to cleavage by the pool of MMPs present at the site of repair. The degradation of G3 domain of versican by MMP-7 and MMP-12 is also physiologically significant  152  considering its roles in stabilizing cell-matrix interactions20 and modulating cell proliferation13, 46. Our results are supported by other studies that point to the colocalization of MMPs with versican, a substrate for the enzymes, in other matrices. For example, the amount of immunoreactivity for versican, identified as a substrate for MMP-7, was increased in sundamaged skin, and versican was detected in the same areas as MMP-745. In atherosclerotic lesions, matrilysin expressed by lipid-laden macrophages localized to areas of versican deposition and could cleave proteoglycan versican, in vitro, possibly disrupting the structural integrity of the plaques44. Versican isolated from rabbit lung is cleaved by purified MMP-2 in a limited fashion47, comparable to the data that we have presented here using versican expressed by human lung fibroblasts. Together, these results make a strong case for the involvement of MMP-2, MMP-7, and MMP-12 in the breakdown of versican in later stages of wound healing process. It has been suggested that reduced growth factor expression and increased matrix turnover could be responsible for myofibroblast cell apoptosis48. It has been shown that MMP activity enhances myofibroblast apoptosis in lungs49. We have uncovered the degradation pattern of versican by three matrix metalloproteinases expressed by macrophages surrounding the fibrotic lesions in human lung diseases. Although the association between cell apoptosis and degradation of versican is circumstantial, and does not imply causality, better understanding of the molecular mechanism of matrix resolution in pathological conditions such as fibrosis can lead to the development of new therapies for the treatment of these diseases.  153  4.5 References 1.  Darby IA, Hewitson TD: Fibroblast differentiation in wound healing and fibrosis, Int Rev Cytol 2007, 257:143-179  2.  Fukuda Y, Ferrans VJ, Schoenberger CI, Rennard SI, Crystal RG: Patterns of pulmonary structural remodeling after experimental paraquat toxicity. The morphogenesis of intraalveolar fibrosis, Am J Pathol 1985, 118:452-475  3.  Basset F, Ferrans VJ, Soler P, Takemura T, Fukuda Y, Crystal RG: Intraluminal fibrosis in interstitial lung disorders, Am J Pathol 1986, 122:443-461  4.  Bensadoun ES, Burke AK, Hogg JC, Roberts CR: Proteoglycan deposition in pulmonary fibrosis, Am J Respir Crit Care Med 1996, 154:1819-1828  5.  Bensadoun ES, Burke AK, Hogg JC, Roberts CR: Proteoglycans in granulomatous lung diseases, Eur Respir J 1997, 10:2731-2737  6.  Zimmermann DR, Ruoslahti E: Multiple domains of the large fibroblast proteoglycan, versican, Embo J 1989, 8:2975-2981  7.  Bode-Lesniewska B, Dours-Zimmermann MT, Odermatt BF, Briner J, Heitz PU, Zimmermann DR: Distribution of the large aggregating proteoglycan versican in adult human tissues, J Histochem Cytochem 1996, 44:303-312  8.  Zako M, Shinomura T, Ujita M, Ito K, Kimata K: Expression of PG-M(V3), an alternatively spliced form of PG-M without a chondroitin sulfate attachment in region in mouse and human tissues, J Biol Chem 1995, 270:3914-3918  9.  Zimmermann DR, Dours-Zimmermann MT, Schubert M, Bruckner-Tuderman L, Heitz PU: (Expression of the extracellular matrix proteoglycan, versican, in human skin), Verh Dtsch Ges Pathol 1994, 78:481-484  10.  Cattaruzza S, Schiappacassi M, Ljungberg-Rose A, Spessotto P, Perissinotto D, Morgelin M, Mucignat MT, Colombatti A, Perris R: Distribution of PG-M/versican variants in human tissues and de novo expression of isoform V3 upon endothelial cell activation, migration, and neoangiogenesis in vitro, J Biol Chem 2002, 277:47626-47635  11.  Roberts CR: Versican in the Cell Biology of Pulmonary Fibrosis. Edited by Hari G. Garg PJR, and Charles A. Hales. New York, Marcel Dekker, Inc., 2002, p. pp. 191212  12.  Roberts CR, Burke AK: Remodelling of the extracellular matrix in asthma: proteoglycan synthesis and degradation, Can Respir J 1998, 5:48-50  13.  Cattaruzza S, Schiappacassi M, Kimata K, Colombatti A, Perris R: The globular domains of PG-M/versican modulate the proliferation-apoptosis equilibrium and invasive capabilities of tumor cells, Faseb J 2004, 18:779-781  154  14.  Evanko SP, Angello JC, Wight TN: Formation of hyaluronan- and versican-rich pericellular matrix is required for proliferation and migration of vascular smooth muscle cells, Arterioscler Thromb Vasc Biol 1999, 19:1004-1013  15.  Yang BL, Zhang Y, Cao L, Yang BB: Cell adhesion and proliferation mediated through the G1 domain of versican, J Cell Biochem 1999, 72:210-220  16.  Hirose J, Kawashima H, Yoshie O, Tashiro K, Miyasaka M: Versican interacts with chemokines and modulates cellular responses, J Biol Chem 2001, 276:5228-5234  17.  Kawashima H, Hirose M, Hirose J, Nagakubo D, Plaas AH, Miyasaka M: Binding of a large chondroitin sulfate/dermatan sulfate proteoglycan, versican, to L-selectin, P-selectin, and CD44, J Biol Chem 2000, 275:35448-35456  18.  Ruoslahti E, Yamaguchi Y: Proteoglycans as modulators of growth factor activities, Cell 1991, 64:867-869  19.  Kinsella MG, Bressler SL, Wight TN: The regulated synthesis of versican, decorin, and biglycan: extracellular matrix proteoglycans that influence cellular phenotype, Crit Rev Eukaryot Gene Expr 2004, 14:203-234  20.  Chen L, Yang BL, Wu Y, Yee A, Yang BB: G3 domains of aggrecan and PGM/versican form intermolecular disulfide bonds that stabilize cell-matrix interaction, Biochemistry 2003, 42:8332-8341  21.  Parks WC, Shapiro SD: Matrix metalloproteinases in lung biology, Respir Res 2001, 2:10-19  22.  Manuel JA, Gawronska-Kozak B: Matrix metalloproteinase 9 (MMP-9) is upregulated during scarless wound healing in athymic nude mice, Matrix Biol 2006, 25:505-514  23.  Hsu JY, McKeon R, Goussev S, Werb Z, Lee JU, Trivedi A, Noble-Haeusslein LJ: Matrix metalloproteinase-2 facilitates wound healing events that promote functional recovery after spinal cord injury, J Neurosci 2006, 26:9841-9850  24.  Dunsmore SE, Saarialho-Kere UK, Roby JD, Wilson CL, Matrisian LM, Welgus HG, Parks WC: Matrilysin expression and function in airway epithelium, J Clin Invest 1998, 102:1321-1331  25.  Zuo F, Kaminski N, Eugui E, Allard J, Yakhini Z, Ben-Dor A, Lollini L, Morris D, Kim Y, DeLustro B, Sheppard D, Pardo A, Selman M, Heller RA: Gene expression analysis reveals matrilysin as a key regulator of pulmonary fibrosis in mice and humans, Proc Natl Acad Sci U S A 2002, 99:6292-6297  26.  Tan RJ, Fattman CL, Niehouse LM, Tobolewski JM, Hanford LE, Li Q, Monzon FA, Parks WC, Oury TD: Matrix metalloproteinases promote inflammation and fibrosis in asbestos-induced lung injury in mice, Am J Respir Cell Mol Biol 2006, 35:289-297  27.  Chiba Y, Yu Y, Sakai H, Misawa M: Increase in the expression of matrix metalloproteinase-12 in the airways of rats with allergic bronchial asthma, Biol Pharm Bull 2007, 30:318-323  155  28.  Isogai Z, Aspberg A, Keene DR, Ono RN, Reinhardt DP, Sakai LY: Versican interacts with fibrillin-1 and links extracellular microfibrils to other connective tissue networks, J Biol Chem 2002, 277:4565-4572  29.  Isogai Z, Shinomura T, Yamakawa N, Takeuchi J, Tsuji T, Heinegard D, Kimata K: 2B1 antigen characteristically expressed on extracellular matrices of human malignant tumors is a large chondroitin sulfate proteoglycan, PG-M/versican, Cancer Res 1996, 56:3902-3908  30.  LeBaron RG, Zimmermann DR, Ruoslahti E: Hyaluronate binding properties of versican, J Biol Chem 1992, 267:10003-10010  31.  Krueger RC, Jr., Schwartz NB: An improved method of sequential alcian blue and ammoniacal silver staining of chondroitin sulfate proteoglycan in polyacrylamide gels, Anal Biochem 1987, 167:295-300  32.  Farndale RW, Buttle DJ, Barrett AJ: Improved quantitation and discrimination of sulphated glycosaminoglycans by use of dimethylmethylene blue, Biochim Biophys Acta 1986, 883:173-177  34.  Remy L, Trespeuch C, Bachy S, Scoazec JY, Rousselle P: Matrilysin 1 influences colon carcinoma cell migration by cleavage of the laminin-5 beta3 chain, Cancer Res 2006, 66:11228-11237  35.  Diegelmann RF, Evans MC: Wound healing: an overview of acute, fibrotic and delayed healing, Front Biosci 2004, 9:283-289  36.  Broughton G, 2nd, Janis JE, Attinger CE: The basic science of wound healing, Plast Reconstr Surg 2006, 117:12S-34S  37.  Kisseleva T, Brenner DA: Mechanisms of fibrogenesis, Exp Biol Med (Maywood) 2008, 233:109-122  38.  Zimmermann DR, Dours-Zimmermann MT, Schubert M, Bruckner-Tuderman L: Versican is expressed in the proliferating zone in the epidermis and in association with the elastic network of the dermis, J Cell Biol 1994, 124:817-825  39.  Raghu G, Striker LJ, Hudson LD, Striker GE: Extracellular matrix in normal and fibrotic human lungs, Am Rev Respir Dis 1985, 131:281-289  40.  Saldiva PH, Delmonte VC, de Carvalho CR, Kairalla RA, Auler Junior JO: Histochemical evaluation of lung collagen content in acute and chronic interstitial diseases, Chest 1989, 95:953-957  41.  Gill SE, Parks WC: Metalloproteinases and their inhibitors: Regulators of wound healing, Int J Biochem Cell Biol 2007,  42.  Lemaitre V, D'Armiento J: Matrix metalloproteinases in development and disease, Birth Defects Res C Embryo Today 2006, 78:1-10  43.  Ravanti L, Kahari VM: Matrix metalloproteinases in wound repair (review), Int J Mol Med 2000, 6:391-407  44.  Halpert I, Sires UI, Roby JD, Potter-Perigo S, Wight TN, Shapiro SD, Welgus HG, Wickline SA, Parks WC: Matrilysin is expressed by lipid-laden macrophages at 156  sites of potential rupture in atherosclerotic lesions and localizes to areas of versican deposition, a proteoglycan substrate for the enzyme, Proc Natl Acad Sci U S A 1996, 93:9748-9753 45.  Saarialho-Kere U, Kerkela E, Jeskanen L, Hasan T, Pierce R, Starcher B, Raudasoja R, Ranki A, Oikarinen A, Vaalamo M: Accumulation of matrilysin (MMP-7) and macrophage metalloelastase (MMP-12) in actinic damage, J Invest Dermatol 1999, 113:664-672  46.  Zhang Y, Cao L, Yang BL, Yang BB: The G3 domain of versican enhances cell proliferation via epidermial growth factor-like motifs, J Biol Chem 1998, 273:21342-21351  47.  Passi A, Negrini D, Albertini R, Miserocchi G, De Luca G: The sensitivity of versican from rabbit lung to gelatinase A (MMP-2) and B (MMP-9) and its involvement in the development of hydraulic lung edema, FEBS Lett 1999, 456:9396  48.  Darby IA, Bisucci T, Pittet B, Garbin S, Gabbiani G, Desmouliere A: Skin flapinduced regression of granulation tissue correlates with reduced growth factor and increased metalloproteinase expression, J Pathol 2002, 197:117-127  49.  Mizuno S, Matsumoto K, Li MY, Nakamura T: HGF reduces advancing lung fibrosis in mice: a potential role for MMP-dependent myofibroblast apoptosis, Faseb J 2005, 19:580-582  157  5. CONCLUSIONS AND FUTURE DIRECTIONS This study centers around wound healing and fibrosis in the lungs. Replacement of normal lung architecture with collagenous matrix causes decreased lung air space volume and obstruction of gas exchange, which can result in considerable loss of lung function and ultimately respiratory failure and death. Persistence and magnitude of initial injury, multiple modes of tissue injury and slower than normal clot resolution are all possible causes that may lead to pulmonary fibrosis instead of normal wound healing. Whatever the cause, however, heavy proteoglycan deposition in association with proliferating myofibroblasts is central to persistence of active lesions in most fibrotic lung diseases. My thesis is largely focused on the role of versican in the transient granulation tissue and subsequent matrix maturation and remodeling. Studies in our laboratory have shown a clear association between versican and proliferating and migrating contractile myofibroblasts in fibroproliferative lesions of fibrosis in the lungs. We now know that deposition of large amounts of versican and hyaluronan in association with active fibroproliferative lesions at the early remodeling stages is a constant feature of all of major fibrotic lung diseases.  Macrophages accumulate around versican-rich  fibroproliferative lesions in the later stages of wound healing, and have been implicated in the resolution of these lesions. The main hypotheses tested in this thesis were: 1.  Versican interacts with ligands on the surfaces of fibroblasts and macrophages (Chapter 2);  2.  The interaction of versican with the cell surface promotes fibroblast cell proliferation (Chapter 3); and  3.  Versican is removed from the versican-rich fibroproliferative lesions by the action of macrophage matrix metalloproteinases (Chapter 4).  The use of 3D collagen matrix, containing a versican-hyaluronan mixture, as an in vitro model of wound healing was also tested (Chapter 3).  158  5.1 Summary and Significance of Results 5.1.1 Generation of Versican C-terminal Constructs to Study Versican Interactions with the Cell Surface In order to identify macrophage and fibroblast versican-binding cell surface ligands (Chapter 1), we expressed and purified C-terminal constructs of versican from transformed E.coli cells. Use of versican constructs simplifies the design of experiments and interpretation of data compared to the full-length proteoglycan. The negatively charged glycosaminoglycan chains prevent versican from penetrating SDS-gels, make it difficult to transfer onto a membrane for use in western blotting experiments, and render N-terminal sequencing and mass spectrometry ineffective. An added benefit to using constructs is that the interaction of versican’s ligands can be narrowed down to specific domains of the protein. We used biotinylated constructs of the C-terminal domain of versican as baits in far-western blotting experiments with cell membrane fraction of fibroblasts and macrophages. We identified versican and versican fragments as the main ligands for HisG3 construct.  Magnetic beads coated with versican’s C-terminal construct and  incubated with cultured fibroblast cells aggregated together and localized mainly to the surfaces of fibroblast cells, possibly through interacting with cell-surface versican C-terminal domain. Next, we examined whether versican is held at the fibroblast cell surface mainly through its interaction with cell surface hyaluronan. As hyaluronan was gradually degraded from the fibroblast cell surface with hyaluronidase, versican was released until almost no versican staining was observed at the cell surface. We also observed that the release of hyaluronan/versican complex from the cell surface resulted in morphological changes in the nuclei, and fibroblast cell death. These results are consistent with versican-versican1 and versican-hyaluronan2-5 interactions at the cell surface found in other studies.  Against a background of  versican-versican and versican-hyaluronan interactions, with these cell types, we were unable to find evidence for other major receptor-ligand interactions. A shortcoming of using protein domain constructs in determining ligand-receptor interactions is the possibility that the construct is not properly folded into its natural tertiary structure. This 159  may be due to the absence of sequences in the full length protein that may assist in folding of the construct. In addition, new sequences which would be hidden in the full length protein may be revealed and may result in abnormal ligand-receptor interactions. Our findings, however, support previous research, and suggest that versican is held at fibroblast cell surface predominantly through its interactions with hyaluronan, and that formation of this pericellular matrix is essential for the maintenance of fibroblast cell phenotype. 5.1.2 Three-Dimensional Proteoglycan-Glycosaminoglycan-Collagen Gel Matrix as a Model of Wound Healing Matrix Three dimensional (3D) collagen or fibrin matrices, containing cultured fibroblasts, have become popular as in vitro models of wound contraction6. Lack of any direct interactions between fibroblast cell surface proteins and versican C-terminal constructs lead us to believe that versican may exert its effects on the cells either as a structural molecule in the matrix (through its interaction with hyaluronan and other matrix proteins), or based on the biochemical properties of its glycosaminoglycan chains. We investigated the biological significance of versican and its glycosaminoglycan chains on fibroblast cells in a three dimensional collagen matrix which contained hyaluronan and either versican or aggrecan. We found that fibroblast cells grown in the presence of HA formed well-organized actin stress fibers and focal adhesions not observed in cells grown in collagen gels alone. In addition to stress fibers, cells grown in the presence of both versican and HA upregulate their α-SMA expression, and seem to have spread dendritic extensions in addition to prominent stress fibers and focal adhesions. It is known that cells with prominent stress fibers and focal adhesions are only present during activated conditions such as wound repair and fibrosis7. We also found that cells in the aggrecan-rich matrix have rounded retracted morphology and do not show any cellular extensions into the matrix.  Considering one aggrecan molecule contains about 7-8 times more  glycosaminoglycan chains than versican, the difference in the number of polysaccharide chains bound to the core of the protein may contribute to the difference in cell morphology observed in versican- versus aggrecan-rich matrices.  Other studies of  160  fibroblast cell behavior in fibronectin-collagen matrices8 confirm that the composition of the matrix is critical for regulating cell phenotype. Several mechanisms have been suggested for the differentiation of fibroblasts to myofibroblasts.  Combined effects of growth factors, such as TGF-β19-11 and  PDGF 12, 13, 14, and mechanical tension, in particular, have been the focus of multiple reviews in recent years7, 15, 16. Although it is unclear how mechanical stress and TGF-β1 signaling converge to promote increased α-SMA expression and myofibroblast differentiation, one possibility may be the expression of matrix proteins downstream of both growth factor and mechanical tension stimulation, namely versican and hyaluronan. Based on our results, it seems that versican and hyaluronan are necessary components of the matrix in which activated myofibroblasts reside, as is observed with myofibroblasts in our 3D collagen matrix system. However, it is important to note that fibroblasts are not a homogeneous population and phenotypically diverse populations of fibroblasts can differ in growth rate  17, 18  and cytoskeletal arrangement19. Also, migrating fibroblasts can  change collagen concentrations and remodel the matrix throughout the experiment20, which may explain the apoptotic behavior of our fibroblasts after 3 days of incubation. 5.1.3 Matrix Metalloproteinase Degradation of Versican Once the process of wound contraction by differentiated myofibroblasts is completed, contractile myofibroblasts disappear from the scar21 through the process of apoptosis22. Although it is well established that regression of granulation tissue occurs by apoptosis23-25, factors and mechanisms that lead to myofibroblast apoptosis are not well understood. As myofibroblast apoptosis occurs concomitantly with granulation tissue degradation in vivo; and as granulation tissue proteoglycans, such as versican are essential for myofibroblast cell proliferation, the role of proteoglycan degradation in myofibroblast apoptosis were investigated. Immunohistochemical data presented in this study show that macrophages, identified by anti-CD-68 antibody staining, surround versican-rich fibroproliferative cores and even penetrate some of these lesions in the fibrotic lung (Figure 4.1). Our data also shows that expression of MMP-2, MMP-7, and MMP-12 is upregulated in the macrophages that  161  accumulate at the site of lesions in the later stages of pulmonary fibrotic diseases (Figure 4.1), suggesting that a substrate for these proteinases lies within these lesions. Other studies also point to the colocalization of MMP-7 with versican in atherosclerotic lesions26 and sun-damaged skin27. MMP-7 seems to cleave intact versican at multiple sites, and this extensive degradation pattern points to a possible role for MMP-7 in clearing versican from the provisional matrix. Degradation of high molecular weight versican with MMP-12 resembles its degradation pattern by MMP-7, however, it seems MMP-12 activity is more focused on the G3-domain of versican. Versican degradation pattern by MMP-2 is quite distinguishable from that of MMP-7 and MMP-12. Limited cleavage of versican by MMP-2 was observed, although same number of active enzyme per substrate was used in all cases. This difference in degradation pattern may point to a different role played by MMP-2, as compared to MMP-7 and MMP-12. Limited proteolysis of versican by MMP-2 yields large fragments that might be expected to influence cell behavior or to destabilize the matrix, though this hypothesis needs to be studied further. Versican degradation could result in destabilization of a hyaluronan- and versican-rich pericellular matrix, structural integrity of which is required for proliferation of myofibroblast cells2,  28-30  . Degradation of versican may also lead to release of growth  factors bound to its chondroitin sulfate chains and perhaps leave them more vulnerable to cleavage by the pool of MMPs present at the site of repair. The degradation of G3 domain of versican by MMP-7 and MMP-12 is also physiologically significant considering its roles in stabilizing cell-matrix interactions1 and modulating cell proliferation31,  32  . It has been suggested that reduced growth factor expression and  increased matrix turnover could be responsible for myofibroblast cell apoptosis33. It has been shown that MMP activity enhances myofibroblast apoptosis in lungs34. MMP substrate specificity and compartmentalization studies also support our versican degradation results. It has been suggested that MMP-7 is a more potent proteoglycanase than MMP-3 or MMP-926, and macrophage metalloelastase (MMP-12) is the most elastolytic enzyme of the MMP family35 capable of efficiently degrading fibronectin and chondroitin sulfate chains of proteoglycans36. Also, MMP-2 binds to αvβ3 integrin37  162  whose expression is increased in human lung fibroblasts by TGF-β138, and MMP-7 binds to cell surface proteoglycans39. The following model is proposed: i. In an effort to relieve tensile forces of the wound and in response to PDGF and TGF-β1, fibroblast cells up-regulate the expression of versican and hyaluronan among other granulation tissue components. ii. Formation of versican-hyaluronan complexes at the cell surface increases local pressure on the cells and leads to myofibroblast contraction of the matrix. iii. Versican provides a suitable environment for myofibroblast contraction, a necessary step in relief of external tension and return of the provisional matrix to a normal state. iv. Macrophages which surround the versican-rich lesions in the final stage of wound healing release matrix metalloproteinases, such as MMP-7 and MMP-12, which degrade versican. v. Fibroblast cells may ‘sense’ the drop in pressure in the matrix and go into apoptosis concomitant with versican removal from the matrix. vi. This leaves a collagenous matrix which resembles the normal tissue architecture. The following diagram is a schematic representation of our new hypothesis which incorporates our new findings into the current understanding of the mechanism of wound healing. Studies of the 3D models suggest that mechanical tension in anchored matrices leads to intracellular tension and formation of stress fibers as the fibroblast cells differentiate first into proto-myofibroblasts with organized stress fibers and then into myofibroblasts with α-SMA decorated stress fibers40. Once the gels are contracted or released from their support (free-floating gels), cells go into a quiescent state41, 42 and the process of apoptosis begins43. In vivo, this drop in mechanical tension could be due to the degradation of versican by macrophage matrix metalloproteinases. The stiffness of the provisional matrix of wounds at different stages of wound healing has estimated through a number of different studies (reviewed in 15). Tension in newly synthesized granulation  163  tissue is comparable with that of the newly polymerized collagen gels at 10-100 Pa44. Gradual increase in tension induces the formation of stress fibers20,45 which for fibroblasts  grown  on  soft  two-dimensional  culture  substrates  occurs  at  46, 47  3,000-6,000 Pa  . The expression of α-SMA in stress fibers occurs at around  20,000 Pa as demonstrated for contractile wound granulation tissue and for myofibroblasts cultured on elastic substrates48. Matrix stiffness of greater than 50,000 Pa has been measured for mature granulation tissue and other fibrotic tissues48.  164  Resting Fibroblasts in Relaxed Matrix  Matrix Pressure 10-100 Pa  Collagen Versican HA  TGF-β PDGF HA  Slow Decrease in Pressure Apoptotic Myofibroblast in Collagenous Matrix  Protomyofibroblast with low levels of intracellular stress fibers (green) and α-SMA (red)  Versican is Degraded by MMPs  MMP-7 MMP-12 MMP-2 Versican  TGF-β PDGF HA Versican  Contractile Myofibroblast with high levels of intracellular stress fibers (green) and α-SMA (red) [Merge: yellow]  Matrix Pressure 3000-6000 Pa  Matrix Pressure 15,000-30,000 Pa  Macrophage  Fiber Under Remodeling  PDGF  Organized Collagen Fiber  TGF-β  Hyaluronan (HA)  Matrix Metalloproteinases (MMP)  Versican  Figure 5.1 Schematic of Wound Healing  165  5.2 Future Studies Future studies in our laboratories could involve our newly established in vitro model of wound  healing  in  which  fibroblasts  are  grown  in  a  3-dimensional  collagen-versican-hyaluronan matrix. Cell morphology could be videotaped on a live stream to evade the effects that fixing and permeabilizing cells could have on cell morphology, and also to observe cell contraction, migration, and proliferation. The stiffness of the matrix could also be measured at different time points. As experiments in this study were performed in serum free media and in the absence of any growth factors, the effects of different growth factors and serum could be studied on cells embedded in collagen matrix. In terms of investigating protein expression patterns by cells under different conditions, the changes in gene expression can be evaluated by comparing the mRNA content of cells grown in the presence or absence of versican. Genechip arrays for gene expression analysis are provided by a number of companies, including Affymetrix, which provides whole genome arrays, and SuperArray Biocsience, which produces focused microarrays. Another method for evaluating changes in gene expression is “stable isotope labeling with amino acids in cell culture” (SILAC)49. This method relies on the incorporation of amino acids that have stable isotopic nuclei, such as deuterium 2H,  13  C,  15  N. The genes that show significant altered expression will then be  evaluated for their physiological relevance to fibroblast and macrophage cell morphology in wound healing.  166  5.3 References 1.  Chen L, Yang BL, Wu Y, Yee A, Yang BB: G3 domains of aggrecan and PGM/versican form intermolecular disulfide bonds that stabilize cell-matrix interaction, Biochemistry 2003, 42:8332-8341  2.  Evanko SP, Angello JC, Wight TN: Formation of hyaluronan- and versican-rich pericellular matrix is required for proliferation and migration of vascular smooth muscle cells, Arterioscler Thromb Vasc Biol 1999, 19:1004-1013  3.  LeBaron RG, Zimmermann DR, Ruoslahti E: Hyaluronate binding properties of versican, J Biol Chem 1992, 267:10003-10010  4.  Evanko SP, Johnson PY, Braun KR, Underhill CB, Dudhia J, Wight TN: Plateletderived growth factor stimulates the formation of versican-hyaluronan aggregates and pericellular matrix expansion in arterial smooth muscle cells, Arch Biochem Biophys 2001, 394:29-38  5.  Ogawa H, Oohashi T, Sata M, Bekku Y, Hirohata S, Nakamura K, Yonezawa T, Kusachi S, Shiratori Y, Ninomiya Y: Lp3/Hapln3, a novel link protein that colocalizes with versican and is coordinately up-regulated by platelet-derived growth factor in arterial smooth muscle cells, Matrix Biol 2004, 23:287-298  6.  Bell E, Ivarsson B, Merrill C: Production of a tissue-like structure by contraction of collagen lattices by human fibroblasts of different proliferative potential in vitro, Proc Natl Acad Sci U S A 1979, 76:1274-1278  7.  Rhee S, Grinnell F: Fibroblast mechanics in 3D collagen matrices, Adv Drug Deliv Rev 2007, 59:1299-1305  8.  Greiling D, Clark RA: Fibronectin provides a conduit for fibroblast transmigration from collagenous stroma into fibrin clot provisional matrix, J Cell Sci 1997, 110 (Pt 7):861-870  9.  Montesano R, Orci L: Transforming growth factor beta stimulates collagen-matrix contraction by fibroblasts: implications for wound healing, Proc Natl Acad Sci U S A 1988, 85:4894-4897  10.  Finesmith TH, Broadley KN, Davidson JM: Fibroblasts from wounds of different stages of repair vary in their ability to contract a collagen gel in response to growth factors, J Cell Physiol 1990, 144:99-107  11.  Fukamizu H, Grinnell F: Spatial organization of extracellular matrix and fibroblast activity: effects of serum, transforming growth factor beta, and fibronectin, Exp Cell Res 1990, 190:276-282  12.  Clark RA, Folkvord JM, Hart CE, Murray MJ, McPherson JM: Platelet isoforms of platelet-derived growth factor stimulate fibroblasts to contract collagen matrices, J Clin Invest 1989, 84:1036-1040  167  13.  Gullberg D, Tingstrom A, Thuresson AC, Olsson L, Terracio L, Borg TK, Rubin K: Beta 1 integrin-mediated collagen gel contraction is stimulated by PDGF, Exp Cell Res 1990, 186:264-272  14.  Tingstrom A, Reuterdahl C, Lindahl P, Heldin CH, Rubin K: Expression of platelet-derived growth factor-beta receptors on human fibroblasts. Regulation by recombinant platelet-derived growth factor-BB, IL-1, and tumor necrosis factoralpha, J Immunol 1992, 148:546-554  15.  Hinz B: Formation and function of the myofibroblast during tissue repair, J Invest Dermatol 2007, 127:526-537  16.  Kisseleva T, Brenner DA: Mechanisms of fibrogenesis, Exp Biol Med (Maywood) 2008, 233:109-122  17.  Raghu G, Chen YY, Rusch V, Rabinovitch PS: Differential proliferation of fibroblasts cultured from normal and fibrotic human lungs, Am Rev Respir Dis 1988, 138:703-708  18.  Uhal BD, Ramos C, Joshi I, Bifero A, Pardo A, Selman M: Cell size, cell cycle, and alpha-smooth muscle actin expression by primary human lung fibroblasts, Am J Physiol 1998, 275:L998-L1005  19.  Akamine A, Raghu G, Narayanan AS: Human lung fibroblast subpopulations with different C1q binding and functional properties, Am J Respir Cell Mol Biol 1992, 6:382-389  20.  Tamariz E, Grinnell F: Modulation of fibroblast morphology and adhesion during collagen matrix remodeling, Mol Biol Cell 2002, 13:3915-3929  21.  Darby I, Skalli O, Gabbiani G: Alpha-smooth muscle actin is transiently expressed by myofibroblasts during experimental wound healing, Lab Invest 1990, 63:21-29  22.  Desmouliere A, Redard M, Darby I, Gabbiani G: Apoptosis mediates the decrease in cellularity during the transition between granulation tissue and scar, Am J Pathol 1995, 146:56-66  23.  Skalli O, and Gabbiani, G.: The biology of the myofibroblast in relationship to wound contraction and fibrocontractive disease. Edited by Henson RAFCaPM. New York, Plenum, 1988, p. pp. 373-402  24.  Rudolph R, Berg, J. V., and Ehrlich, H. P.: Wound contraction and scar contracture. Edited by I. K. Cohen RFD, and W. J. Lindbald. Philadelphia, Saunders, 1992, p. pp. 96-114  25.  Ronnov-Jessen L, Petersen OW: Induction of alpha-smooth muscle actin by transforming growth factor-beta 1 in quiescent human breast gland fibroblasts. Implications for myofibroblast generation in breast neoplasia, Lab Invest 1993, 68:696-707  26.  Halpert I, Sires UI, Roby JD, Potter-Perigo S, Wight TN, Shapiro SD, Welgus HG, Wickline SA, Parks WC: Matrilysin is expressed by lipid-laden macrophages at sites of potential rupture in atherosclerotic lesions and localizes to areas of versican  168  deposition, a proteoglycan substrate for the enzyme, Proc Natl Acad Sci U S A 1996, 93:9748-9753 27.  Saarialho-Kere U, Kerkela E, Jeskanen L, Hasan T, Pierce R, Starcher B, Raudasoja R, Ranki A, Oikarinen A, Vaalamo M: Accumulation of matrilysin (MMP-7) and macrophage metalloelastase (MMP-12) in actinic damage, J Invest Dermatol 1999, 113:664-672  28.  Bensadoun ES, Burke AK, Hogg JC, Roberts CR: Proteoglycan deposition in pulmonary fibrosis, Am J Respir Crit Care Med 1996, 154:1819-1828  29.  Bensadoun ES, Burke AK, Hogg JC, Roberts CR: Proteoglycans in granulomatous lung diseases, Eur Respir J 1997, 10:2731-2737  30.  Yang BL, Zhang Y, Cao L, Yang BB: Cell adhesion and proliferation mediated through the G1 domain of versican, J Cell Biochem 1999, 72:210-220  31.  Zhang Y, Cao L, Yang BL, Yang BB: The G3 domain of versican enhances cell proliferation via epidermial growth factor-like motifs, J Biol Chem 1998, 273:21342-21351  32.  Cattaruzza S, Schiappacassi M, Kimata K, Colombatti A, Perris R: The globular domains of PG-M/versican modulate the proliferation-apoptosis equilibrium and invasive capabilities of tumor cells, Faseb J 2004, 18:779-781  33.  Darby IA, Bisucci T, Pittet B, Garbin S, Gabbiani G, Desmouliere A: Skin flapinduced regression of granulation tissue correlates with reduced growth factor and increased metalloproteinase expression, J Pathol 2002, 197:117-127  34.  Mizuno S, Matsumoto K, Li MY, Nakamura T: HGF reduces advancing lung fibrosis in mice: a potential role for MMP-dependent myofibroblast apoptosis, Faseb J 2005, 19:580-582  35.  Shapiro SD: Elastolytic metalloproteinases produced by human mononuclear phagocytes. Potential roles in destructive lung disease, Am J Respir Crit Care Med 1994, 150:S160-164  36.  Gronski TJ, Jr., Martin RL, Kobayashi DK, Walsh BC, Holman MC, Huber M, Van Wart HE, Shapiro SD: Hydrolysis of a broad spectrum of extracellular matrix proteins by human macrophage elastase, J Biol Chem 1997, 272:12189-12194  37.  Brooks PC, Stromblad S, Sanders LC, von Schalscha TL, Aimes RT, StetlerStevenson WG, Quigley JP, Cheresh DA: Localization of matrix metalloproteinase MMP-2 to the surface of invasive cells by interaction with integrin alpha v beta 3, Cell 1996, 85:683-693  38.  Pechkovsky DV, Scaffidi AK, Hackett TL, Ballard J, Shaheen F, Thompson PJ, Thannickal VJ, Knight DA: Transforming growth factor beta1 induces alphavbeta3 integrin expression in human lung fibroblasts via a beta3 integrin-, c-Src-, and p38 MAPK-dependent pathway, J Biol Chem 2008, 283:12898-12908  39.  Yu WH, Woessner JF, Jr.: Heparan sulfate proteoglycans as extracellular docking molecules for matrilysin (matrix metalloproteinase 7), J Biol Chem 2000, 275:4183-4191 169  40.  Tomasek JJ, Gabbiani G, Hinz B, Chaponnier C, Brown RA: Myofibroblasts and mechano-regulation of connective tissue remodelling, Nat Rev Mol Cell Biol 2002, 3:349-363  41.  Grinnell F, Zhu M, Carlson MA, Abrams JM: Release of mechanical tension triggers apoptosis of human fibroblasts in a model of regressing granulation tissue, Exp Cell Res 1999, 248:608-619  42.  Fringer J, Grinnell F: Fibroblast quiescence in floating or released collagen matrices: contribution of the ERK signaling pathway and actin cytoskeletal organization, J Biol Chem 2001, 276:31047-31052  43.  Fluck J, Querfeld C, Cremer A, Niland S, Krieg T, Sollberg S: Normal human primary fibroblasts undergo apoptosis in three-dimensional contractile collagen gels, J Invest Dermatol 1998, 110:153-157  44.  Kaufman LJ, Brangwynne CP, Kasza KE, Filippidi E, Gordon VD, Deisboeck TS, Weitz DA: Glioma expansion in collagen I matrices: analyzing collagen concentration-dependent growth and motility patterns, Biophys J 2005, 89:635-650  45.  Marenzana M, Wilson-Jones N, Mudera V, Brown RA: The origins and regulation of tissue tension: identification of collagen tension-fixation process in vitro, Exp Cell Res 2006, 312:423-433  46.  Discher DE, Janmey P, Wang YL: Tissue cells feel and respond to the stiffness of their substrate, Science 2005, 310:1139-1143  47.  Yeung T, Georges PC, Flanagan LA, Marg B, Ortiz M, Funaki M, Zahir N, Ming W, Weaver V, Janmey PA: Effects of substrate stiffness on cell morphology, cytoskeletal structure, and adhesion, Cell Motil Cytoskeleton 2005, 60:24-34  48.  Goffin JM, Pittet P, Csucs G, Lussi JW, Meister JJ, Hinz B: Focal adhesion size controls tension-dependent recruitment of alpha-smooth muscle actin to stress fibers, J Cell Biol 2006, 172:259-268  49.  Brack SS, Silacci M, Birchler M, Neri D: Tumor-targeting properties of novel antibodies specific to the large isoform of tenascin-C, Clin Cancer Res 2006, 12:3200-3208  170  

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