The Extracellular Matrix (ECM) Sashwati Roy, PhD Associate Professor Department of Surgery

At the end of this module, you will learn to: Learning Objectives At the end of this module, you will learn to: Describe the composition of the extracellular matrix and its function. Determine the role and significance of insoluble and soluble components of ECM the clinical implication of ECM in tissue repair

Extracellular matrix (ECM) Network of proteins and carbohydrates that binds cells together Supports and surrounds cells Regulates cells activities Lattice for cell movement Cells surrounded by spaces filled with extracellular matrix (Molecular Biology of the Cell. 4th edition. Alberts B, Johnson A, Lewis J, et al. New York: Garland Science; 2002)

ECM key functions Mechanical support Embryonic development Pathways for cellular migration Wound healing Management of growth factors The connective tissue underlying an epithelium This tissue contains a variety of cells and extracellular matrix components. The predominant cell type is the fibroblast, which secretes abundant extracellular matrix. (Molecular Biology of the Cell. 4th edition. Alberts B, Johnson A, Lewis J, et al. New York: Garland Science; 2002)

Class of macromolecules that form the ECM Insoluble components (fibrous proteins: Collagen, Elastic fibers, Fibronectin, Laminin Soluble components- polysaccharide chains - Proteoglycans, Hyaluronan, Adhesive glycoproteins

INSOLUBLE COMPONENTS Collagen Elastic fibers Fibronectin Laminin

Collagen Collagen is strong, resisting tensile forces (in abundance in the Achilles heel) Major insoluble fibrous protein of the extracellular matrix and connective tissue Most abundant protein in animals Made by fibroblasts and some epithelial cells

Collagen –structure Crystal Structure of The Collagen Triple Helix Model Pro- Pro-Gly103 04 Crystal structure of the collagen triple helix model [(Pro-Pro-Gly)10]3. Protein Sci. 2002 February; 11(2): 262–270. It is long, rigid structure in which three polypeptides are coiled around one another in a rope like manner. These polypeptides are called α-helix. The polypeptides are arranged in a triple helix.

Types of collagen Fibrillar Sheet forming Connecting Trans-membrane Forms structures such as tendon or cartilage Sheet forming Connecting Supports and organizes fibrous collagen Trans-membrane

Fibrillar Collagen Fibrillar collagen are components of the well-known striated fibrils The fibrillar collagen family includes types I-III, V and XI

Sheet forming collagen Polymerizes into sheets Forms basal membranes – collagen IV Collagen VIII – Descemet’s membrane of cornea

Connecting collagens Link fibrillar and sheet forming collagens to into networks and connect them to other structures Collagen VI – short helices interspersed with globular domains Align collagen I into parallel structures

Role of Collagen in Wound healing (Collagen is a key component of a healing wound) Collagens serve as a structural support Recent evidences indicate that collagen and collagen-derived fragments control many cellular functions, including cell shape and differentiation, migration, and synthesis of proteins

Collagen-based Wound Dressings are a number of different collagen dressings available. are purified in order to render it non-antigenic. can vary in concentration and type. may contain ingredients, that can enhance absorbency, flexibility, and comfort, and help maintain a moist wound environment. are meant to enhance the wound management aspects of the dressings. There are a number of different collagen dressings available, which employ a variety of carriers/combining agents such as gels, pastes, polymers, oxidized regenerated cellulose (ORC), and ethylene diamine tetraacetic acid (EDTA). The collagen within these products tends to be derived from bovine, porcine, equine, or avian sources, which is purified in order to render it nonantigenic. The collagen in a given collagen dressing can vary in concentration and type. Certain collagen dressings are comprised of Type I (native) collagen; whereas, other collagen dressings contain denatured collagen as well. A given collagen dressing may contain ingredients, such as alginates and cellulose derivatives that can enhance absorbency, flexibility, and comfort, and help maintain a moist wound environment. Collagen dressings have a variety of pore sizes and surface areas, as well. All of these attributes are meant to enhance the wound management aspects of the dressings.

Collagen-based wound dressings By: David Brett, Wounds, 2009

Collagen Degradation Proteinase resistant triple helix MMP-1 Proteinase resistant triple helix Cleaved by collagenase (MMP-1)

Matrix Metalloproteinases J. Cell. Mol. Med. Vol 9, No 2, 2005 pp. 267-285

"Tissue" Proteinases Epidermis Mesenchyme collagenase (MMP-1) stromelysin-1,2 (MMP-3,10) plasminogen activator (urokinase type) Mesenchyme stromelysin 1 (MMP-3) 72kDa gelatinase (MMP-2) 92kDa gelatinase (MMP-9) plasminogen activator (uPA)

MMP Biology MMPs secreted as zymogens Complex activation pathways Counteraction by Tissue Inhibitors of Metalloproteinases (TIMPs) Cell-associated MMPs and their target substrates that modulate cell motility.

MMP Activation in Epithelial Repair Injury exposes epithelia to a new ECM environment - collagen I/fibrin/fibronectin Migration on to these substrates requires Regprogramming of matrix receptors (integrins) Loss of intercellular contacts Activation of MMP pathways

Granulocyte Proteinases Serine proteinases elastase cathepsin G proteinase 3 azurocidin neutrophil collagenase (MMP-8) Macrophage proteinases collagenase (MMP-1) metalloelastase (MMP-12)

Modulators of Proteinase Activity Serpins Maspins TIMP's SLPI a-2 macroglobulin PAI-1

Insoluble components Collagen Elastic fibers Fibronectin Laminin

Elastic fibers A network of elastic fibers in the extracellular matrix of the tissue gives it the required resilience so that they can recoil after transient stretch Elastic fibers. These scanning electron micrographs show (A) a low-power view of a segment of a dog's aorta and (B) a high-power view of the dense network of longitudinally oriented elastic fibers in the outer layer of the same blood vessel.

Elastic fibers Elastin is unusually rich in proline and glycine is not glycosylated contains some hydroxy-proline has no hydroxylysine. Soluble tropoelastin (the biosynthetic precursor of elastin) The main component of elastic fibers is elastin, a highly hydrophobic protein (about 750 amino acids long). Like collagen, is unusually rich in proline and glycine but, unlike collagen, is not glycosylated and contains some hydroxy-proline but no hydroxylysine. Soluble tropoelastin (the biosynthetic precursor of elastin) is secreted into the extracellular space and assembled into elastic fibers close to the plasma membrane, generally in cell-surface in foldings.

Elastin The molecules are joined together by covalent bonds (red) to generate a cross-linked network. In this model, each elastin molecule in the network can expand and contract as a random coil, so that the entire assembly can stretch and recoil like a rubber band.

Elastin Rich in hydrophobic amino acids Synthesized as soluble tropoelastin Crosslinked by lysyl oxidase 768-residue hydrophobic protein Rich in hydrophobic amino acids Synthesized as soluble tropoelastin Crosslinked by lysyl oxidase Formation of desmosine and isodesmosine via Schiff base condensation of lysyl aldehydes Crosslinking results in incredibly insoluble and durable polymer

Diseases of connective Tissue Cutis laxa (CL), or elastolysis. is a group of rare connective tissue disorders in which the skin becomes inelastic and hangs loosely in folds. Marfan syndrome. a genetic disorder of the connective tissue. The syndrome is carried by the gene FBN1, which encodes the connective protein fibrillin-1. Ehlers–Danlos syndrome (EDS) is an inherited connective tissue disorder caused by a defect in the synthesis of collagen, specifically mutations in the COL5A and COL3A genes. Cutis laxa (CL), or elastolysis, is a rare, inherited or acquired connective tissue disorder in which the skin becomes inelastic and hangs loosely in folds).   Marfan syndrome. Marfan syndrome is a disorder of connective tissue which causes skeletal defects typically recognized in a tall, lanky person. A person with Marfan syndrome may exhibit long limbs and spider-like fingers, chest abnormalities, curvature of the spine and a particular set of facial features including a highly arched palate, and crowded teeth.  Ehlers–Danlos syndrome (EDS) is an inherited heterogeneous group of generalized connective tissue disorders caused by a defect in the synthesis of collagen, specifically mutations in the COL5A and COL3A genes. Major clinical manifestations include increased skin fragility, skin hyperextensibility, and joint hypermobility.

Insoluble components Collagen Elastic fibers Fibronectin Laminin

Fibronectin a dimer composed of two very large subunits joined by disulfide bonds at one end Each subunit is folded into a series of functionally distinct domains All forms of fibronectin are encoded Fibronectin binds integrins. Fibronectin, a high-molecular weight (~440kDa) glycoprotein, is a dimer composed of two very large subunits joined by disulfide bonds at one end. Each subunit is folded into a series of functionally distinct domains separated by regions of flexible polypeptide chain. All forms of fibronectin are encoded by a single large gene that contains about 50 exons of similar size. Fibronectin binds to membrane-spanning receptor proteins called integrins.

The structure of a fibronectin dimer Molecular Biology of the Cell. 4th edition. Alberts B, Johnson A, Lewis J, et al. New York: Garland Science; 2002) (A) Electron micrographs of individual fibronectin dimer molecules shadowed with platinum; red arrows mark the C-termini. (B) The two polypeptide chains are similar but generally not identical (being made from the same gene but from differently spliced mRNAs). They are joined by two disulfide bonds near the C-termini. Each chain is almost 2500 amino acids long and is folded into five or six domains connected by flexible polypeptide segments. Individual domains are specialized for binding to a particular molecule or to a cell, as indicated for five of the domains. (C) The three-dimensional structure of two type III fibronectin repeats as determined by x-ray crystallography. The type III repeat is the main repeating module in fibronectin. Both the Arg-Gly-Asp (RGD) and the “synergy” sequences shown in red form part of the major cell-binding site (shown blue in B).

Fibronectin secreted primarily by fibroblasts cell adhesion, growth, migration, and differentiation important for wound healing cancer and fibrosis Fibronectin is secreted primarily by fibroblasts as a soluble protein dimer and is then assembled into an insoluble matrix in a complex cell-mediated process. Fibronectin plays a major role in cell adhesion, growth, migration, and differentiation, Fibronectin is important for wound healing. Along with fibrin, plasma fibronectin is deposited at the site of injury, forming a blood clot that stops bleeding and protects the underlying tissue Altered fibronectin expression, degradation, and organization has been associated with a number of pathologies, including cancer and fibrosis.

Insoluble components Collagen Elastic fibers Fibronectin Laminin

Laminin Molecular Biology of the Cell. 4th edition. Alberts B, Johnson A, Lewis J, et al. New York: Garland Science; 2002) Laminin-1 (classical laminin) is a large, flexible protein composed of three very long polypeptide chains (α, β, and γ) arranged in the shape of an asymmetric cross and held together by disulfide bonds The structure of laminin A) The subunits of a laminin-1 molecule. This multidomain glycoprotein is composed of three polypeptides (α, β, and γ) that are disulfide-bonded into an asymmetric crosslike structure. Each of the polypeptide chains is more than 1500 amino acids long. Five types of α chains, three types of β chains, and three types of γ chains are known; in principle, they can assemble to form 45 (5 × 3 × 3) laminin isoforms. Several such isoforms have been found, each with a characteristic tissue distribution. (B) Electron micrographs of laminin molecules shadowed with platinum.

Laminin modulation during wound repair Reduced laminin-322 levels has been associated with poor keratinocyte migration Hemidesmosomes bind to laminin in the basal lamina During wound healing, laminin-5 is expressed by keratinocytes The epidermal growth factor-like repeats in laminin are examples of matrikines. Laminin-322 is deposited as a part of a provisional matrix during wound healing. Reduced laminin-322 levels has been associated with poor keratinocyte migration The keratinocyte’s primary anchoring contacts are hemidesmosomes, which bind to laminin in the basal lamina by way of a6b4 integrins and have intracellular links with the keratin cytoskeletal network. During wound healing, laminin-5 is expressed by keratinocytes at the leading edge of the dermal-epidermal junction. The epidermal growth factor-like repeats in laminin are examples of matrikines. Matrikines are peptides liberated by partial proteolysis of extracellular matrix macromolecules.

Soluble components of extracellular matrix polysaccharide chains of the class called glycosaminoglycans (GAG), which are usually found covalently linked to protein in the form of proteoglycans

Glycosaminoglycan (GAG) Glycosaminoglycans (GAGs) are unbranched polysaccharide chains composed of repeating disaccharide units. They are called GAGs because one of the two sugars in the repeating disaccharide is always an amino sugar (N-acetylglucosamine or N-acetylgalactosamine), which in most cases is sulfated. Molecular Biology of the Cell. 4th edition. Alberts B, Johnson A, Lewis J, et al. New York: Garland Science; 2002) Figure. The repeating disaccharide sequence of a dermatan sulfate glycosaminoglycan (GAG) chain.

Glycosaminoglycan (GAG) Hyaluronan chondroitin sulfate and dermatan sulfate heparan sulfate keratan sulfate Four main groups of GAGs are distinguished according to their sugars, the type of linkage between the sugars, and the number and location of sulfate groups.

Hyaluronan is a polysaccharide The relative dimensions and volumes occupied by various proteins, a glycogen granule, and a single hydrated molecule of hyaluronan are shown. Hyaluronan is a polysaccharide 25,000 subunits Made directly outside the cell Most common No SO3 Joint fluid, wound healing, development Source of cell migration Degraded by hyaluronidase Hyaluronan is thought to have a role in resisting compressive forces in tissues and joints. It is also important as a space filler during embryonic development, where it can be used to force a change in the shape of a structure, as a small quantity expands with water to occupy a large volume

Proteoglycans Molecular Biology of the Cell. 4th edition. Alberts B, Johnson A, Lewis J, et al. New York: Garland Science; 2002) Are molecule consisting of one or more glycosaminoglycan (GAG) chains attached to a core protein. Proteoglycans are usually easily distinguished from other glycoproteins by the nature, quantity, and arrangement of their sugar side chains. By definition, at least one of the sugar side chains of a proteoglycan must be a GAG The linkage between a GAG chain and its core protein in a proteoglycan molecule. A specific link tetrasaccharide is first assembled on a serine side chain. In most cases, it is unclear how the particular serine is selected, but it seems that a specific local conformation of the polypeptide chain, rather than a specific linear sequence of amino acids, is recognized. The rest of the GAG chain, consisting mainly of a repeating disaccharide unit, is then synthesized, with one sugar being added at a time. In chondroitin sulfate, the disaccharide is composed of D-glucuronic acid and N-acetyl-D-galactosamine; in heparan sulfate, it is D-glucosamine (or L-iduronic acid) and N-acetyl-D-glucosamine; in keratan sulfate, it is D-galactose and N-acetyl-D-glucosamine.

Examples of proteoglycans The proteoglycan aggrecan: a major component of cartilage, has a mass of about 3 × 106 daltons with over 100 GAG chains. The proteoglycans decorin: is secreted by fibroblasts and has a single GAG chain Examples of a small (decorin) and a large (aggrecan) proteoglycan found in the extracellular matrix. These two proteoglycans are compared with a typical secreted glycoprotein molecule, pancreatic ribonuclease B. All three are drawn to scale. Molecular Biology of the Cell. 4th edition. Alberts B, Johnson A, Lewis J, et al. New York: Garland Science; 2002)

SUMMARY ECM are network of proteins and carbohydrates that binds cells together. ECM is required for mechanical support, embryonic development, pathways for cellular migration, wound healing and management of growth factors ECM is composed of insoluble and soluble macromolecules Insoluble components involve: collagen, elastic fibers, fibronectin and laminin The soluble components involve polysaccharide chains of the class called glycosaminoglycans (GAG), which are usually found covalently linked to protein in the form of proteoglycans The inheritable ECM disorders in human are Marfan syndrome, Ehlers-Danlos syndrome, venous malformation etc. Other ECM disorders include cancer, fibrosis, scleroderma etc,

Learning Resources 1. The Molecular and Cellular Biology of Wound Repair, edited by R.A.F. Clark, Plenum Press, NY 2. The Extracellular Matrix of Animals. Molecular Biology of the Cell. 4th edition. New York: Garland Science; 2002.

ECM Quiz

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