Biochemistry of Connective Tissue

Jana Novotna

Connective Tissue

Tissue that supports and binds other tissues. It consists of connective tissue cells, embedded in a large amount of extracellular matrix. Ubiquitous, found in between other tissues everywhere in the body.

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2.

3.

4.

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Wraps around and shield and protects organs Stores nutrients, maintaines a constant concentration of ions and water Internal support for organs As tendon and ligaments protects joints and attached muscles to bone and each other Runs through organ capsules and in deep layers of skin giving strength Regeneration potential for other tissues

Composition of connective tissue

Cells:

Fibroblasts Adipocytes Fixed macrophages Mast cells Lymphocytes Pigment cells 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 woud 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 – (bone)

chondroblasts

(cartillage) and

osteoblast

Types of Connective Tissue

Types of Connective Tissue

•

"

Ordinary " connective tissue

- generalized form of connective tissue with all of the basic components (cells, fibers, ground substance).

•

Ground substance

- background material within which all other connective tissue elements are embedded. • Ground substance consists mainly of water, complex of glycosaminoglycans (

GAGs

),

proteoglycans

, 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

•

Dense

/

loose

connective tissue - depending on the proportion of fibers.

•

Dense

connective tissue - high density of extracellular fibers, relatively smaller proportions of ground substance and cells.

– Dense

collagen

connective tissues (top priority is strength)

tendons

,

ligaments

,

organ sheaths

– Dense

elastic

connective tissue (top priority is elasticity)

– aorta,

the elastic ligament of the spine (

ligamentum flavum

) the wall of the •

Loose

(

areolar

) connective tissue - lacks the massive fibrous reinforcement that characterizes dense connective tissue. • Relatively large proportion of ground substance, cells or both (adipose tissue, lymphoid tissue)

Dense (Fibrous) Regular Connective tissue

• Characteristics - an

abundance of fibers

with

fewer cells

, also called fibrous or collagen connective tissue.

• The fibers are organized in a regular, parallel pattern.

• Fibroblasts are the only cells visible, and are arranged in rows between the fibers.

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.

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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).

Functions of Connective Tissue • Main functions: – Transport – Immunological defense – Mechanical support – Repair • Additional functions – Reserve energy store (fat) – Heat generation (brown fat) – Haemopoiesis

ECM Components and Function

Components

Collagen Elastin Proteoglycans Hyaluronic acid Fibronectin

Structure

Triple helical glycoprotein molecules rich glycine, proline hydroxyproline, hydroxylysine Stretchable hydrophobic protein interacting with glycosylated microfibrils Heterogenous long glycoasminoglycan chains covalently linked to core protein A very large, specialized, nonsulfated glycosaminoglycan Specialized adhesive glycoprotein

Function

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 Mediates cell-matrix adhesion Laminin Large complex adhesive glycoprotein Bind cells to type IV collagen and heparan sulfate

Collagen

• Three helical polypeptide units twist to form a

triple-helical collagen molecule

: a molecular "rope" which has some bending stiffness and does not undergo rotation. • 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, matrixins)

• 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)

Enzyme

Collagenase-1 (fibroblast type) Collagenase-2 (neutrophile type) Collagenase-3 (breast cancer) Collagenase-4

Collagenases

MMP#

MMP-1 MMP-8 MMP-13

Substrate

collagens (I-III, VII, VIII, X), gelatine collagens (I-III, VII, VIII, X), gelatine, fibronectin collagens (I-IV, IX, X a XIV), gelatine, MMP-18 collagens I

Classification of MMP ´s and their substrates (2)

Enzyme

Gelatinase A

Gelatinases

MMP#

MMP-2

Substrate

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)

Enzym

Stromelysin-1 Stromelysin-2 Stromelysin-3 Matrilysin

Stromelysins

MMP#

MMP-3 MMP-10 MMP-11 MMP-7

Substr át

collagens III-V a IX, gelatine, laminin, fibronectin, elastin, casein, osteonectin, collagens III-V, gelatine, casein, elastin, MMP-1, MMP-8 unknown (casein) 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

Syndecans and Glypicans - Cell Surface Proteoglycans

• The syndecan family contains four members, they are transmembrane heparan sulfate proteoglycans (HSPG).

• The syndecan family members are type I integral membrane proteins with homologous transmembrane and cytoplasmic domains.

• The glypican, another cell surface HSPG family, is composed of five members.

Syndecans and Glypicans - Cell Surface Proteoglycans

• The syndecans are involved in the differentiation process across the epithelial-mesenchymal axis, principally through their ability to bind growth factors and modulate their downstream signaling. • Core proteins of syndecans carry three to five heparan sulfate (HS) and chondroitin sulfate (CS) chains, which allow for interaction with a large variety of ligands (FGF, VEGF, TGF b , fibronectin and antithrombin-1).

• Syndecan1 is upregulated in multiple myeloma.

• High levels of shed syndecan1 in a patient's serum typically is correlated with poor prognosis.

Syndecans and Glypicans - Cell Surface Proteoglycans

Proposed mechanism of glypican as co-receptors which bind both the ligand (FGF2) and the receptor • Glypican family members are selectively expressed on different cell types (only glypican-1 present on vascular endothelial cells).

• Glypicans are critically involved in developmental morphogenesis, and have been implicated as regulators in several cell signaling pathways.

• Glypicans plays an important role in regulating the biological activity of fibroblast growth factors via HS GAG chains like syndecan.

Proteoglycan degradation

• • • • • Most glycoconjugates are degraded in lysosomes, and a portion of the liberated monosaccharides are reused for glycoconjugate synthesis Most of the

endo-

and

exoglycosidases

that degrade sugar chains and their modifications 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. The exoglycosidases recognize only one (occasionally two) monosaccharide together with its anomeric linkage.

• Loss of a lysosomal enzyme leads to accumulation of non-degraded material in tissues – „

lysosomal storage disease

“

mucopolysaccharidoses

Degradation of Heparan Sulfate Proteoglycan

Mukopolysacharidosis VI

(Marotaux-Lamy syndrom) deficiency of enzyme N-acetyl galaktosamin 4-sulfatase

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

• • • Mechanisms of dermal wounds healing: 1.

2.

3.

Connective tissue matrix deposition Contraction Epithelisation Primary Intension healing (simple wounds closed by sutures, tape or staples) – connective tissue matrix deposition (collagen, proteoglycans adhesive glycoproteins), formation of new ECM Wounds remain open – healing mainly by contraction (complex interaction between contractile fibroblasts – „myofibroblasts“ and matrix components).

The Healing Cascade

• • • • • • The healing cascade begins immediately following injury, when platelets come into contact with exposed collagen.

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

The Healing Cascade - Inflammatory Phase

• Next predominant cell marker are neutrophils (within 24 hours)  foreign material is removed (bacteria, damaged matrix components by fagocytosis.

• Next marker – mast cells vessels become leaky (vasodilatation) PDGF and TGF damaged matrix  tissue macrophages.

b  release of histamin causes speedy passage of mononuclear cells.

• Fixed tissue monocytes are activated (48 hour after injury) to • Tissue macrophages release which attract fibroblasts, smooth muscle cells.

• Tissue macrophages removing

The Healing Cascade - Proliferative Phase

• TGF b has a three-prolonged effect on extracellular deposition – It increases transcription of the genes for collagen, proteoglycans and fibronectin  increasing of ECM deposition – decreases the secretion of proteases (MMPs) – stimulates the protease inhibitors, TIMP Diegelmann R.F., EvansM.: Frontiers in Bioscience 9, 283-289, 2004

The Healing Cascade - Remodelling Phase

• Predominant cell type in the wound site – fibroblasts.

• Fibroblasts are attached to the provisional fibrin matrix – start to produce collagen. The first type synthesized - type III collagen (fine reticular fibers) and then is substituted for type I.

• Type I collagen is predominant type in the scar tissue.

Injury 1 4 Phase of wound healing 20 Closure ??

Haemostasis Inflammation Proliferation Remodelling •

Demadge vessels constrict to slow blood flow

•

Platelets aggregate

•

Bleeding

•

Leucocyte migrate into tissue to initiate inflammatory 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

•

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

•

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 TGF b .

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 TGF b) – produced by epidermal cells, fibroblasts, macrophages and vascular endothelial cells.

• Signaling pathway for angiogenesis   low oxygen tension, low pH expression of nuclear transcription factor

factor

“ (

HIF

) by vascular endothelial cells.

– „

hypoxia-inducible

• The HIF binds to specific DNA sequences for VEGF expression.

• Formation of new blood vessels  blocking activity of HIF increased pO 2 , oxygen binds to • 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 NO .

VEGF

(Vasoactive Endothelial Growth Factor).

• VEGF goes to the bone marrow, activates NO synthase to produce

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-1 a (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 same patients.

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 • Keloids have increased expression of TGFß and also an up regulation 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.

Diseases caused by collagen gene mutation

• More than 1000 mutation have been characterized in 29 collagen genes (

COL1A1, COL1A2, COL 2A1, COL3A1, COL4A5

,

COL 7A1

a

COL8A2

).

• Most of the mutation are single base substitutions for obligatory glycine to a bigger amino acid, alter folding of triple helix structure.

• Bone disorders – osteogenesis imperfecta

COL1A2)

, some form of osteoporosis , (OI) (

COL1A1

and • Cartillage disorders – chondrodisplasias (

COL2A1

), some form of osteoarthritis (

COL11A1

,

COL11A2

), intervertebral discs.

• Ehlers-Danlos syndrome (

COL5A1

and

COL5A2

,

COL3A1

) – a heterogenous group, several subtypes – joint and skin changes, the rupture of arteries.

• Other mutation – duplication, insertion, complex rearrangements.

Congenital disorders of connective tissue collagen

Osteogenesis imperfecta

- predominantly autosomal dominant inheritance, defects in genes for collagen type I (COL1A1 or COL1A2).

• Characterized by brittle bones that are prone to fractures.

• Eight subtypes of OI can be distinguished. • The severity of the disease varies and depends on the specific type - the most serious forms  multiple fractures in the prenatal period or even death of fetus is the most severe form, the least severe forms can also be very discreet in adulthood and fractures is only slightly more often compared to the normal population.

Congenital disorders of connective tissue collagen

Ehlers-Danlos syndrome

- inherited disorder with different presentations. EDS is caused by a defect in the structure, production, or processing of collagen (COL3A1) or proteins that interact with collagen, such as mutations in the type V collagen (COL5A2) genes.

• EDS typically affects the joints, skin, and blood vessels.

• Major and typical signs and symptoms - hyper-flexible joints, fragile skin that tears easily, arterial rupture

Congenital disorders of connective tissue - collagen

Stickler syndrome -

hereditary progressive arthro-ophthalmopathy • Group of genetic disorders , a subtype of collagenopathy of types II and XI (COL2A1, COL11A1 and COL 11A2).

• Syndrome is characterized by distinctive facial abnormalities, ocular problems, hearing loss, and joint problems.

• • Individuals with Stickler syndrome experience a range of signs and symptoms - some people have no signs and symptoms; others have affected joints, hearing, eye and have cleft abnormalities.

Alport syndrome

or

hereditary nephritis -

Genetic disorder characterized by glomerulonephritis, kidney disease, and hearing loss, can also affect the eyes, causing eye abnormalities including cataracts.

• Alport syndrome is caused by mutation in COL4A3, COL4A4 and COL4A5.

• • • • • • •

Congenital disorders of connective tissue

Marfan syndrome

TGF β. – genetic disorder caused by the misfolding of fibrillin-1 and contributes to cell signaling activity by binding to and sequestering The mutated fibrilin binds poorly to TGF β, which results in an accumulation of excess TGF β in the lungs, heart valves, and aorta.

In humans diseases affect the cardiovascular, skeletal and ocular systems.

The most serious manifestations involve defects of the heart valves, and aorta, which may lead to early death if not properly managed. The syndrome also may affect the lungs, eyes, the skeleton.

People with Marfan syndrome tend to be unusually tall, with long limbs and long, thin fingers and toes.

Syndrome has a variable clinical presentation, ranging from mild to severe systemic disease.