Biochemistry of connective tissue

Biochemistry of connective tissue

Composition of connective tissue Cells: Fibroblasts Chondroblasts Osteoblasts Adipocytes Keratinocytes Melanocytes Fixed macrophages Mast cells Lymphocytes Other cells Fibers: Collagen Reticular Elastic fibrs Ground substance: Proteoglycans (GAG) glycoproteins

Cells Fibroblasts - the most common resident cells in connective tissue. Responsible for normal development and wound healing. Fibroblasts from different regions display extensively differentiated patterns of gene expression which may guide differentiated patterns of tissue organization. Resting fibroblasts retain the ability to become active as during healing after injury. The substance of the scar is collagen Closely related cell types chondroblasts (cartilage) and osteoblast (bone)

Classification of connective tissues Connective tissue are classified according to the characteristics of their ground substance and the types of fibers found within the matrix Connective tissue proper - generalized form of connective tissue with all of the basic components (cells, fibers, ground substance) fibers collagen type I, III, elastin ground substance - background material within which all other connective tissue elements are embedded. ground substance consists mainly of water, complex of proteoglycans, with glycosaminoglycans (GAGs), and glycoproteins Major role of water is to provide a route for communication and transport (by diffusion) between tissues. Special connective tissue - very highly differentiated and localized forms (sharing many common features) bone, cartilage, lymphoid tissue, blood.

Connective tissue proper Generalized tissue. Fibroblasts are present in all connective tissue proper. Fibrocytes, adipocytes, and mesenchymal cells are fixed cells, which means they remain within the connective tissue. Other cells move in and out in response to chemical signals. Macrophages, mast cells, lymphocytes, plasma cells, and phagocytic cells are found in connective tissue proper but are actually part of the immune system.

Connective tissue proper Dense/loose connective tissue - depending on the proportion of fibers. Loose (areolar) connective tissue - lacks the massive fibrous reinforcement that characterizes dense connective tissue. Dense connective tissue - high density of extracellular fibers, relatively smaller proportions of ground substance and cells. dense collagen connective tissues - tendons, ligaments, organ sheaths (top priority is strength) dense elastic connective tissue (top priority is elasticity) the wall of the aorta, the elastic ligament of the spine (ligamentum flavum) Relatively large proportion of ground substance, cells or both (adipose tissue, lymphoid tissue)

Areolar connective tissue The most widespread connective tissue of the body. It is used to attach the skin to the underlying tissue. It fills the spaces between various organs. It surrounds and supports the blood vessels. The fibers run in all directions, form a loose network in the intercellular material collagen is predominant, some elastic fibers are also present. http://agi.seaford.k12.de.us/sites/dhammaker/anatomy/anatomy%20pics/forms/dispform.aspx?id=51

Adipose connective tissue Cells are characterized by a large internal fat droplet. The cytoplasm is reduced to a thin layer. Function - a storage site for lipids, protection of certain organs, insulating layer under the skin (regulation of temperature). http://www.kln.ac.lk/science/depts/zoology/index.php?option=com_content&view=article&id=43&itemid=10

Reticular connective tissue Reticular tissue is a network of reticular fibers of collagen type III that provides a supportive framework for soft organs. A mesh-like, supportive framework for soft organs such as lymphatic tissue, the spleen, and the liver. http://www.kln.ac.lk/science/depts/zoology/index.php?option=com_content&view=article&id=43&itemid=10

Dense (fibrous) regular connective tissue Characteristics - an abundance of fibres with fewer cells, also called fibrous or collagenous connective tissue, it displays greater resistance to stretching. Tissue fibers are parallel to each other, enhancing tensile strength and resistance to stretching in the direction of the fiber orientations. Dense regular elastic tissue contains elastin fibers in addition to collagen fibers, which allows the ligament to return to its original length after stretching. T Fibroblasts are the only cells visible, and are arranged in rows between the fibres.

Dense irregular connective tissue In dense irregular connective tissue, the direction of fibers is random. This arrangement gives the tissue greater strength in all directions and less strength in one particular direction. The dermis of the skin is an example of dense irregular connective tissue rich in collagen fibers. Dense irregular elastic tissues give arterial walls the strength and the ability to regain original shape after stretching http://www.kln.ac.lk/science/depts/zoology/index.php?option=com_content&view=article&id=43&itemid=10

Functions of connective tissue Most important function of connective tissues - support and connect other tissues connective tissue sheath that surrounds muscle cells the tendons that attach muscles to bones the skeleton that supports the positions of the body fibrous capsules and bones protect sensitive organs Other important functions: transport of fluid, nutrients, waste, and chemical messengers by specialized fluid connective tissues - blood and lymph. immunological defense from microorganisms that enter the body Additional functions reserve energy store (fat) heat generation (brown fat) haemopoiesis

Components Structure Function Collagen Elastin Proteoglycans Hyaluronic acid Components of connective tissue Triple helical glycoprotein molecules rich glycine, proline hydroxyproline, hydroxylysine Elastic hydrophobic protein, forms of interaction with glycosylated protein Heterogenous long glycoasminoglycan chains covalently linked to core protein A very large, specialized, nonsulfated glycosaminoglycan Strength, support and structure for all tissues and organs Allows tissues and structures to expand and contract Moisture stores, shock absorption, sequestration of cytokines Provides a fluid environment for cell movement and differentiation and binds to cytokines Fibronectin Specialized adhesive glycoprotein Mediates cell-matrix adhesion Laminin Large complex adhesive glycoprotein Bind cells to type IV collagen and heparan sulfate

Collagen Three polypeptide chain units twist to form a triple-helix. The tropocollagen molecule has a length of approximately 300 nm and a diameter close to 1.5 nm. In the typical fibrillar collagens, only short terminal portions of the polypeptides (the telopeptides) are not triple helical.

Collagen degradation Interstitial collagenase cleavages a monomer of collagen in the position of the glycine 775 leucine/isoleucine 776 bond.

Matrix metalloproteinases (MMPs) Zn-dependent endopeptidases, proteolytic activity is preferentially targeted on extracellular matrix (ECM) and basal membranes. MMPs are involved in tissue degradation and remodeling of the ECM, both in physiological conditions, such as pathological. embryogensis, morfogenesis, angiogenesis, healing etc. Inflammatory diseases (arthritis), cardiovascular diseases, cancer They have a broad substrate specificity and are present in the tissues of vertebrates, invertebrates and plants.

Structure of MMP s Catalytic zinc (middle), coordinated with three histidin residues (pink), structural Zn (black and white). The structural zinc binds 1-3 Ca 2+ ions (depending on the type of MMP )

Classification of MMP s and their substrates (1) Collagenases Enzyme MMP# Substrate Collagenase-1 (fibroblast type) Collagenase-2 (neutrophile type) Collagenase-3 (breast cancer) MMP-1 MMP-8 MMP-13 collagens (I-III, VII, VIII, X), gelatine collagens (I-III, VII, VIII, X), gelatine, fibronectin collagens (I-IV, IX, X a XIV), gelatine, Collagenase-4 MMP-18 collagens I

Classification of MMP s and their substrates (2) Gelatinases Enzyme MMP# Substrate Gelatinase A MMP-2 collagens (IV, V,VII, X a XIV), gelatine, fibronectin, osteonectin, Gelatinase B MMP-9 collagens (IV, V, VII, X a XIV), gelatine, elastin, fibronectin, osteonectin

Classification of MMP s and their substrates (3) Stromelysins Enzym MMP# Substrát Stromelysin-1 MMP-3 collagens III-V a IX, gelatine, laminin, fibronectin, elastin, casein, osteonectin, Stromelysin-2 MMP-10 collagens III-V, gelatine, casein, elastin, MMP-1, MMP-8 Stromelysin-3 MMP-11 unknown (casein) Matrilysin MMP-7 collagens IV, X, gelatine, fibronectin, laminin, elastin, casein, transferrin

Inhibitors of MMP s TIMP s MMP s are inhibited by specific endogenous inhibitors tissue inhibitors of matrix metalloproteinasestimp s, inhibitors of metalloproteinases IMPS, a 2 -makroglobulin. TIMP s group of four different proteases TIMP-1, -2, -3 and -4. TIMP binds to the active site of enzyme by non-covalent bond, forms a complex with the catalytic zinc in a ratio of 1:1

Proteoglycans

Degradation of proteoglycans Glycosamino glycans (GAG) or glycoconjugates are degraded in lysosomes, a portion of the liberated monosaccharides are reused for glycoconjugate synthesis. Enzymes glycosidases that degrade carbohydrate chains have ph optima between 4.0 and 5.5. The endoglycosidases cleave internal glycosidic linkages of larger chains, yielding fragments that can then be degraded by exoglycosidases. Exoglycosidases cleave the glycosidic linkage of terminal sugars from the nonreducing end of the chain.

Degradation of proteoglycans

Genetic disorders of GAGs metabolism Several genetically inherited diseases - the lysosomal storage diseases, result from defects in the lysosomal enzymes responsible for the metabolism of complex membraneassociated GAGs. These specific diseases are termed the mucopolysaccharidoses (MPS). MPS are chronic progressive disorders lead to severe psychomotor retardation, the clinical spectrum of disorders can vary widely within one enzyme deficiency. The disorders affect childern many of whom suffer and die within several years after birth.

Normal and pathological response to injury Wound disruption of normal anatomical structure and function Healing complex and dynamic process resulting in restoration of anatomical continuity and function. Four basic response following an injury: Regeneration Exact Replacement Normal Repaire Reestablished Eqilibrium Tissue Injury Excessive Healing Fibrosis and Contractures Deficient Healing Chronic Ulcers

Possible responses following tissue injury Wound healing process of repair after skin inury. The healing process is mediated by local wound factors and systemic mediators. Phases of normal wound healing: 1. Inflammatory phase 2. Proliferative phase or new tissue formation (neoangiogenesis, re-epithelization) 3. Tissue remodeling (remodeling of extracellular matrix)

Schematic representation of different stages of wound repair. A: 12 24 h after injury the wounded area is filled with a blood clot. Neutrophils have invaded into the clot. B: at days 3 7 after injury, the majority of neutrophils have undergone apoptosis. Instead, macrophages are abundant in the wound tissue at this stage of repair. Endothelial cells migrate into the clot; they proliferate and form new blood vessels. Fibroblasts migrate into the wound tissue, where they proliferate and deposit extracellular matrix. The new tissue is called granulation tissue. Keratinocytes proliferate at the wound edge and migrate down the injured dermis and above the provisional matrix. C: 1 2 wk after injury the wound is completely filled with granulation tissue. Fibroblasts have transformed into myofibroblasts, leading to wound contraction and collagen deposition. The wound is completely covered with a neoepidermis. SABINE WERNER, and RICHARD GROSE Physiol Rev 2003;83:835-870 2003 by American Physiological Society

The healing cascade The healing cascade begins immediately following injury, when platelets come into contact with exposed collagen. Platelet aggregation clotting factor release deposition of fibrin clot. Fibrin clot provisional matrix, platelets release cytokines and growth factors that initiate the healing response. Two most important factors are PDGF and TGF-b PDGF chemotaxis of neutrophils, macrophages, smooth muscle cells and fibroblasts TGF-b signal for macrophages to secrete cytokines (FGF, PDGF, TNF-a, Il-1) that modulate expression of collagen and collagenase in fibroblasts rapid deposition of new connective tissue Diegelmann R.F., EvansM.: Frontiers in Bioscience 9, 283-289, 2004

Inflammatory Phase Predominant cells are neutrophils (within 24 hours) foreign material is removed (bacteria, damaged matrix components by fagocytosis. Mast cells release histamin (vasodilatation), vessels become leaky speedy passage of mononuclear cells. Fixed tissue monocytes are activated (48 hour after injury) to tissue macrophages. Tissue macrophages release PDGF and TGF-b which attract fibroblasts, smooth muscle cells and removing damaged matrix

Multiple functions of TGF-β during wound healing. TGF-β is released in large amounts from platelets. In the healing wound, it is produced by leukocytes, macrophages, fibroblasts, and keratinocytes and acts on these cells to stimulate infiltration of inflammatory cells, fibroplasia, matrix deposition, and angiogenesis.. SABINE WERNER, and RICHARD GROSE Physiol Rev 2003;83:835-870 2003 by American Physiological Society

Proliferative phase - new tissue formation Fibroblasts (predominant cells) are attached to the provisional fibrin matrix start to produce collagen. The prolifatiteve phase consist of neoangiogenesis, granulation tissue formation, extracellular matrix deposition and re-epithelization TGF-b released by platelets, macrophages and T lymphocytes become critical signal TGF-b has a three-prolonged effect on extracellular deposition increases transcription of the genes for collagen, proteoglycans and fibronectin increasing of ECM deposition decreases the secretion of proteases (MMPs) stimulates the protease inhibitors, TIMPs

Remodelling phase The last stage of healing may last from weeks to one year Granulation tissue is remodeling The first collagen type synthesized in granular tissue is type III (fine reticular fibers) and then is replaced by stronger type I. Keratinocytes, fibroblasts, macrophages and neutrophiles produce MMPs degradation of granulation tissue

Injury Phase of wound healing Closure 1 4 20?? Haemostasis Inflammation Proliferation Remodelling Demadge vessels constrict to slow blood flow Platelets aggregate Bleeding Leucocyte migrate into tissue to initiate inflammatory process Neutrophils secrete chemicals to kill bacteria Macrophages engulf and digest foreign particles and necrotic debrits Macrophages release angiogenic substances to stimulate capillary growth and the granulation process Fibroblasts proliferate in the wound and secrete glycoproteins and collagen Epidermal cells migrate from wound edge Granulation tissue is formed from macrophages, fibroblasts and new capillaries Fibroblast secrete collagen to strengthen wound Wound remodelling occurs to reorganize fibers Wound contracts increasing tissue integrity Epidermal cells grow over connective tissue to close wound

Chronological order of wound healing

Mechanisms of dermal wounds healing - local factors in the wound microenvironment High metabolic activity in the wound site increasing demand for oxygen and nutrients. Local factors - low ph, reduced oxygen tension, increased lactate initiate the release of factors activating angiogenesis (vasculogenesis, neovascularisation). Epidermal cells, fibroblasts, macrophages and vascular endothelial cells produce mediator iniating angiogenesis - vasoactive endothelial growth factor (VEGF ), basic fibroblast growth factor (bfgf) and TGFb.

Mechanisms of dermal wounds healing - local factors in the wound microenvironment In the wound microenvironment prevails lower ph, lower oxygen pressure, increased lactate production stimulation of the release of factors stimulating angiogenesis and neovascularization (VEGF, bfgf a TGFb) produced by epidermal cells, fibroblasts, macrophages and vascular endothelial cells. Signaling pathway for angiogenesis low oxygen tension, low ph expression of nuclear transcription factor hypoxia-inducible factor (HIF) by vascular endothelial cells. The HIF binds to specific DNA sequences for VEGF expression. Formation of new blood vessels increased po 2, oxygen binds to blocking activity of HIF Synthesis of VEGF is decreased.

Chronic wound (chronic ulcer) In acute wounds, there is a precise balance between production and degradation of collagen and other extracellular matrix molecules. Chronic wounds often remain in the inflammatory stage for too long. Ischemia is an important factor in the formation and persistence of wounds - causes tissue to become inflamed and cells to release factors that attract neutrophils (cytokines, interleukins, chemokines, leukotrienes, complement components).

Chronic wound (chronic ulcer) A significant biological marker appears to be excessive infiltration by neutrophils, the over-abundant neutrophil infiltration is responsible for the chronic inflammation. The neutrophils release significant amounts of proteases collagenase (matrix metalloproteinase-8) responsible for destruction of the connective tissue matrix elastase capable of destroying important healing factors - PDGF and TGF-ß excess matrix metalloproteinases may also cause wounds to become chronic Excessive reactive oxygen species (ROS) - another marker of chronic ulcers further damage of the cells and prevent cell proliferation and wound closure.

Chronic wound (chronic ulcer) One of the principal feature of wound healing is formation of new small blood vessels at the site of injury. Endothelial progenitor cells (EPCs) from bone marrow in a process vasculogenesis form new vessels. EPCs are mobilized circulation repair site. The cascade of vasculogenesis begins when ischemic tissue releases VEGF (Vasoactive Endothelial Growth Factor). VEGF goes to the bone marrow, activates NO synthase to produce NO the mobilization of bone marrow EPCs to the circulation

Healthy wound: NO synthase activation (bone marrow) by VEGF (released by keratinocytes, fibroblasts, epithelial cells, macrophages) increased NO mobilization of bone marrow EPCs (endothelial progenitor cells) to the circulation. SDF-1a (stromal cell-derived factor 1-a) promotes the homing of EPCs to the site of injury neovascularization. Diabetic wound: NOS activation is impaired limitation of EPC mobilization (less than 50%), impaired neovasulogenesis (formation of small blood vessels) and impaired wound healing.

Fibrosis Fibrosis can be defined as the replacement of the normal structural elements of the tissue by distorted, non-functional and excessive accumulation of scar tissue. A clinical example of fibrosis - keloids, hypertrophic scars in the skin. Fibroblasts isolated from keloids produce about 2 to 3 times more collagen compared to fibroblasts isolated from normal skin in the same patients. Keloids have increased expression of TGFß and also an upregulation of receptors for TGFß.

Fibrosis Hypertrophic scars are characterized by excessive accumulation of scar collagen. Very significant biological marker that distinguishes keloids from hypertrophic scars the absence of myofibroblasts in keloids and an abundance of these contractile cells in hypertrophic scars. Most conditions of fibrosis are characterized by an increased density of mast cells. Diseases characterized by fibrotic processes - scleroderma, Crohn s disease, liver cirrhosis, lung fibrosis, atherosclerosis.