Extracellular Matrix

19.03.16 Saradbrata Mishra 119.03.16 Saradbrata Mishra
Moderater
Dr. Geetashree Mishra
EXTRACELLULAR MATRIX IN
HEALTH AND DISEASE
Presented By
Saradbrata Mishra

Discussion
• Definition
• Functions of ECM
• Forms of ECM
• Components of ECM
• Abnormalities in ECM in different diseased states
• Future therapeutic approaches by targetting ECM

DEFINITION
• Extracellular matrix (ECM) is a network of interstitial proteins
that constitutes a significant proportion of any tissue.

FUNCTIONS OF ECM
In addition to space filler other functions are
• Mechanical support- For cell anchorage and cell migration, and
maintenance of cell polarity.
• Control of cell proliferation- By acting as a depot of latent growth
factors, binding and displaying growth factors, and signalling
through cellular receptors.
• Scaffolding for tissue renewal- Integrity of ECM is critical for the
organized regeneration of tissues. So ECM disruption results in
defective tissue regeneration and repair (eg. Cirrhosis of Liver).
• Establishment of tissue microenvironments- Basement membrane
acts as boundary between epithelium and connective tissue, and is
also functional, eg in kidney BM forms part of the filtration
apparatus.
• Cellular interaction- For maintaining normal tissue architecture.

TWO BASIC STRUCTURAL FORMS OF ECM
1) Interstitial Matrix-
• Present in the spaces between cells in the connective tissue, and between
the parenchymal epithelium and underlying vascular and smooth muscle
structures.
• It is synthesized by mesenchymal cells (eg-fibroblast).
• Forms a three dimensional amorphous gel structure.
• Its major constituents are fibrillar & non fibrillar collagen, fibronectin, elastin,
proteoglycan, hyaluronate and others.
2) Basement Membrane-
• The interstitial matrix becomes highly organized around epithelial cells,
endothelial cells & smooth muscle cells, and forms the specialized BM.
• Synthesized by contribution of both underlying mesenchyme and overlying
epithelium.
• It is a porous structure forming a flat lamellar "chicken wire" mesh.
• Its major constiuents are
1. Collagen IV
2. Laminin 12
3. Entactin/Nidogen 2 isoforms
4.Sulfated proteoglycans such as Perlecan and Agrin

COMPONENTS OF ECM
Components of ECM are chatagorised into 3 groups of proteins;
1) Fibrous structural proteins- Collagens, Elastins & Fibrillin.
Confer tensile strength and recoil.
2) Water-hydrated gels- Proteogylcans & Hyaluronan.
Permit compressive resistance and lubrication.
3) Adhesive glycoproteins & receptor- Fibronectin, Laminin & Integrin
Connect ECM elements to one another and to
cells.
4) Water - 65%

• The most abundant protein in the body, making 25%-35% of all the
whole-body proteins.
• About 30 collagens type have been identified, some are unique to
specific cells and tissues.
• Collagen contributes to the stability of tissues and organs.
• It maintains their structural integrity.
• It has great tensile strength.
• Plays an important role in cell differentiation, polarity, movement.
• Plays an important role in tissue and organ development.
• Principal producers of collagen fibers are fibroblasts; epithelial and
smooth muscle cells also secrete their own type-IV collagen.
(1) COLLAGENS

COLLAGEN BIOSYNTHETIC PATHWAY

INTRACELLULAR
• mRNA attach to the rER, and protocollagen or precollagen-chains are
deposited in the cisternae; every 3rd amino acid is glycine.
• Proline and lysine residues within the chains are then hydroxylated in the ER to
form hydroxyproline and hydroxylysine.
• Core sugars (galactose and glucose) attach to the hydroxylysine residues in the
ER.
• Each chain is synthesized with an extra length of peptides known as registration
peptides, which ensure that the appropriate chains assemble in their correct
position in the resulting triple helical molecule called procollagen.
• Glycosylation may occur in the Golgi complex and procollagen is released into
the extracellular space by exocytosis.

EXTRACELLULAR
• In the extracellular space, the enzyme procollagen peptidase
cleaves the registration peptides from procollagen, converting it to
tropocollagen.
• Catalyzed by lysyl oxidase(dependent on vitamin C), these become
aligned in staggered fashion to form collagen fibers, possibly under
the control of adjacent fiber-producing cells.
• The turnover of collagen is slowest in tendons, fastest in loose
connective tissue. Macrophages and neutrophils break down old
collagen, and replaced by fibroblasts.
• As humans age, extracellular collagen becomes increasingly cross-
linked, & turn-over slows down in connective tissue.

Contd....
• Prepro collagen → Procollagen → Tropocollagen → Fibrillar collagen.
• Collagens are composed of three separate polypeptide chains, braided into
a tripple helix structure.
• There are 3 helical α chains. They may be identical in some type of
collagens (Homotrimer) or different to each other in other types of collagens
(Heterotrimer)
Collagen polypeptide structure:
- G – X – A – G – A – A – G – Y – A – G – A – A – G – X – A – G – A –
– A – G – X – A – G – A – A – G – Y – A – G – A – A – G – X – A – G –
G - glycine, X - proline or hydroxyproline, Y – lysin or hydroxylysine, A –
amino acid
• Due to rod like fibril stacking & lateral crosslinking, fibrillar collagens have
marked tensile strength but do not have much elasticity.

Diversity of Collagens

(2) ELASTIN
• Elastin is a major protein component of tissues that require elasticity such as
arteries, lungs, cardiac valves, uterus, bladder, skin and elastic ligaments
and cartilage.
• Polypeptide chains are cross-linked together to form rubberlike, elastic
fibers. Each elastin molecule uncoils when the fiber is stretched and recoils
spontaneously as soon as the force is relaxed.
• 90% of all the amino acid residues of elastin are nonpolar (characterized by
a high index of hydrophobicity).
• Elastic fibres consist of a central core made of elastin, surrounded by a
peripheral network of microfibrils.
• Over time, tropoelastin accumulates within the bed of microfibrils.

Structure
•Elastin is more massively crosslinked than collagen and also have large
hydrophobic segments (unlike collagen) that forms globular configuration at rest.
•As stretch is exerted the hydrophobic domains are pulled open, but the cross-link
keep the tissue intact.
•Release of stretch tension allows the hydrophobic domains of the proteins to refold.
•Thus elastin has more elasticity and less tensile strength than that of collagen.

(3) FIBRILLIN
• Large glycoprotein secreted by EC fibroblast.
• Found commonly in zonular fibres of the lens, periosteum, arterial wall.
• In the elastic fibre the peripheral microfibrillar network that surrounds the
core consists largely of fibrillin.
• Types of fibrilin-
1.Fibrilin 1:- Major component of microfibril.
2.Fibrilin 2:- Have a role in early elastogenesis.
3.Fibrilin 3:- Mainly located in the brain.
4.Fibrilin 4:- Structure is similar to fibrilin 2.

BIOSYNTHETIC PATHWAY OF ELASTIC FIBRE

(4) PROTEOGLYCAN & HYALURONAN
• Proteoglycans are composed of a core protein to which glycosaminolycans
(GAGs) are attached. GAGs consist of repeating disaccharide subunits.
• The core proteins are attached to long haluronic acid polymer.

Contd....
• The highly negatively charged sulfated
sugars on the proteoglycan "bristles"
recruit sodium and water to generate a
viscous but compressible matrix.
• They have diverse role in regulating
connective tissue structure and
permeability (ie regulates movement of
molecules through matrix).
• They also serve as reservoir of growth factors (eg FGF & HGF),
they act as modulators of cell growth and differentiation.
• In joint cartilage they also provide layer of lubrication between bony
surfaces.
• Some are integral part of cell membrane & have roles in cell
proliferation, migration and adhesion.

Some Major Proteoglycan Family
Members

19.03.16 Saradbrata Mishra 23
• Syndecan is a cell surface proteoglycan.
• It has a transmembrane core protein with
an extracellular side chain and a
cytoplasmic tail.
• Extracellularly it binds to bFGF (released
from damaged ECM) & intracellularly with
actin cytoskeleton.
• Finally syndecan facilitates interaction with
cell surface receptor for FGF.
• Thus ECM regulates activity of basic
fibroblast growth factor (bFGF, FGF-2)
SYNDECAN

(5) GLYCOSAMINOGLYCAN
• Unbranched polysaccharide chains composed of repeating
disaccharide units.
• Disaccharide subunits are:
1. uronic acid
D-glucuronic acid or L-iduronic acid
2. amino sugar
N-acetyl glucosamin (GlcNAc) or
N-acetyl galactosamin (GalNAc)
• They are negatively charged under physiological conditions (due to
the occurrence of sulfate and uronic acid groups).

7 Types of GAGs
1. Hyaluronic acid-
The only non sulfated GAG.
Found in joint fluid, vitreous body, cartilage.
Important in wound healing.
2. Chondroitin sulfate-
Tensile strength to cartilage, tendons, ligaments & aortic wall.
3. Dermatan sulfate-
Widely distributed troughout the body, mostly skin and blood vessels.

Contd....
4. Heparin-
Heparin is mostly intracellular unlike rest of GAGs (e.g. in mast cells).
Involved in anticoagulation by binding factor IX, XI and AT-3.
Binds lipoprotein lipase in endothelial cell walls and puts them into
circulation.
5. Heparan sulfate-
Role in developmental processes, angiogenesis, blood coagulation and
tumor metastasis.
6. Keratan sulfate type 1
7. Keratan sulfate type 2
Present in cornea, cartilage and bone.
In CNS they have role in development and formation of glial scars.

(6) FIBRONECTIN
• Fibronectin is a large (450 kD) molecule, having 2 forms (extra & intra cellular
forms).
• Intracellular cytoskeleton will align with the extracellular fibronectin to detemine cell
shape.
• Fibronectins guide immune cells to wounded areas and thus promote wound healing.
• It has a crucial role in wound healing, tissue and plasma fibronectin provide the
scaffolding for subsequent ECM deposition, angiogenesis, and reepithelialization.
• In the plasma it links to fibrin and helps in blood clotting process.
• Helps in cell adhesion, growth, migration, and differentiation.
• During cell movement (as during embryogenesis), pathways of fibronectin guide cells
to their destinations.
• Fibronectin is also found in normal human saliva, which helps prevent colonization of
the oral cavity and pharynx by potentially pathogenic bacteria.

STRUCTURE OF FIBRONECTIN....
• Fibronectin exists as a protein dimer, consisting of two
nearly identical monomers linked by a pair of disulfide bonds
• Mainly 2 types
1) Soluble plasma fibronectin- Produced in the liver by
hepatocytes.
2) insoluble cellular fibronectin- It is a major component of
the extracellular matrix. It is secreted by
various cells, primarily fibroblasts.
• There are four fibronectin-binding domains, allowing
fibronectin to associate with other fibronectin molecules.
• The fibrin, heparan and collagen binding domains link it to
the ECM.
• The integrin binding domain binds to cell membrane by
membrane-spanning receptor – integrin through arginine-
glycine-aspartic acid (RGD) motifs.

(7) LAMININ
• Laminin is the most abundant glycoprotein in basement membrane; has
binding domains for both ECM and cell surface receptors.
• It is an 820-kD cross-shaped heterotrimer that connects cells to
underlying ECM components such as type IV collagen and heparan
sulfate.
• Laminins bind cells to the basal lamina of epithelial and connective
tissues, and to their surrounding muscle cells, fat cells, and Schwann
cells.
• The basal lamina serves as a structural support for tissues and as a
permeability barrier to regulate movement of both cell and molecules.
• It also modulates primordial germ cell migration during embryogenesis.

Contd....

Laminin β1 Laminin β2
kidneybasementmembrane

Laminins- Primordial germ cell migration

(8) INTEGRINS
• Also known as The Molecular Glue of Life.
• Integrins are a large family of transmembrane heterodimeric
glycoproteins (composed of α- and β-subunits) that allow cells to
attach to ECM constituents.
• The ECM constituents are laminin and fibronectin mostly.
• Most integrins are receptors for extracellular matrix proteins.
• Intracellularly it links with intracellular cytoskeleton & also with focal
adhesion complexes(e.g.vinculin, α actinin and talin) that can recruit
and activate tyrosine kinases, that ultimately trigger downstream
signaling cascades.

Structure
of integrin

Functions of Integrin
• Integrins on the surface of leukocytes are essential in mediating adhesion
and transmigration of leukocytes across endothelium at sites of
inflammation.
• By binding to vWF or fibrinogen,(GPIIb/IIIa) integrin play a critical role in
platelet aggregation.
• Intracellularly the signaling cascades can influence locomotion, proliferation,
survival, apoptosis, shape and differentiation of cells.
• Therefore over all 2 functions of integrins are
1) Attachment of the cell to the ECM.
2) Signal transduction from the ECM to the cell.

DISEASES
RELATED TO
DEFECT IN ECM
COMPONENTS

SCURVY
• Acquired disease of fibrillar
collagen
• Because of the abundance of
ascorbic acid in many foods, scurvy
has ceased to be a global problem.
• Secondary deficiency, particularly
among older individuals, chronic
alcoholics, people having erratic and
inadequate eating patterns, patients
undergoing peritoneal dialysis and
hemodialysis, infants who are
maintained on formulas of
evaporated milk, soldiers during war
periods.
llustration from Man-of-War by
Stephen Biesty

Contd....
• Vitamin C has function of activation of prolyl and lysyl hydroxylases from inactive
precursors, providing for hydroxylation of procollagen.

PROGRESSIVE SYSTEMIC
SCLEROSIS (SCLERODERMA)
• Systemic sclerosis is characterized by triad of:
(1) chronic inflammation thought to be the result of autoimmunity,
(2) Wide sread vascular damage and
(3) progressive interstitial and perivascular fibrosis in the skin
and multiple organs.
• Fibrosis may be the result of activation of fibroblasts by cytokines
produced by T cells, but what triggers T-cell responses is unknown.
• A limited form of this disease k/a CREST syndrome
1) Calcification of subcutaneous tissue
2) Raynaud's phenomenon
3) Esophageal dysmotility
4) Sclerodactyly
5) Telangiectasia

In SCLERODERMA multisystemic changes are as follows
• skin:- Increase in dermal collagen in the dermis, thinning of epidermis,
atrophy of dermal appendages, thickening of walls of dermal capillaries.
• git:- Rubber-hose like inflexibility of lower third of esophagus, GERD &
loss of microvilli in small intestine l/t malabsorption.
• joint:- Hypertrophy & hyperplasia of synovial soft tissue.
• muscles:- Inflammatory myositis
• kidney:- Intimal thickening of interlobular arteries, Htn & renal failure.
• lungs:- Interstitial fibrosis & pulmonary hpertension.
• heart:- Myocardial fibrosis & pericarditis with effusion.

OTHER COLLAGEN VASCULAR DISEASES
• Also called as connective tissue diseases.
• Immune system causes inflammation in collagen and nearby joints.
• Constitute a group of diseases such as
Systemic Lupus Erythematosus (SLE)
Rheumatoid Arthritis (RA)
Sjogren syndrome (SS)
Mixed Connective Tissue diseases (MCTD)
• All CVDs share some common signs and symptoms.
fatigue
muscle weakness
fever
body aches
joint pain
skin rash

OSTEOGENESIS IMPERFECTA (OI)
• Also known as brittle bone disease or Lobstein syndrome.
• Caused by deficiency of type I collagen. Most cases are caused by
mutations in the COL1A1 and COL1A2 genes,
• A congenital bone disorder characterized by brittle bones that are prone
to fracture.
• Also present with shorter height, neurological features including
communicating hydrocephalus, basilar invagination, and seizures, blue
sclerae, hearing loss etc.
• Eight types of OI can be distinguished.

OI Types....

Ehlers-Danlos Syndromes (EDS)
• EDSs comprise a clinically and genetically heterogeneous group of
disorders that result from some defect in the synthesis or structure of
fibrillar collagen.
• EDS is known to affect men and women of all racial and ethnic
backgrounds.
• There are six distinct types of EDS currently identified.
• Each type is thought to involve a unique defect in connective tissue,
although not all of the genes responsible for causing EDS have been
found.
• All share joint laxity, soft skin, easy bruising, and some systemic
manifestations.

EDS Types....

ALPORT SYNDROME (AS)
• It comes under the category of hereditary nephritis.
• Due to defect in collagen 4 synthesis (α3, α4 & α5 chain) (encoded on
both autosomes eg chromosomes 2 & 13 and X- chromosome.
• Among them most common is deletion in collagen 4 α5 chain (COL4A5).
• In 85% cases females typically presents with only hematuria.
• BUT males present with hematuria with a spectrum of s/s eg. nerve
deafness, lens dislocation, posterior cataracts, corneal dystrophy etc.
• Hematuria is asociated with red cell cast, later on proteinuria and at last
nephrotic syndrome developes.

AS Contd....
• As the disease progresses there is development of focal segmental and
global glomerulosclerosis, vascular sclerosis, tubular atrophy, and
interstitial fibrosis.
• 90% of males progress to ESRD and renal failure before 40 years od
age.
 Collagen IV defects can also cause stroke, hemorrhage, porencephaly.

EPIDERMOLYSIS BULLOSA
DYSTROPHICA (EBD)
• It is one of the entities of Non Inflammatory Blistering Disorders.
• It usually results from mutations in the COL7A1 gene, which encodes
type VII collagen, a major component of the basement membrane
anchoring fibrils.
• It may follow an autosomal dominant or autosomal recessive mode of
inheritance.
• At the sites of trauma blisters are formed in the upper dermis followed
by scarring.

CHONDRODYSPLASIAS
• The spondyloepiphyseal dysplasias (SEDs) and spondyloepimetaphyseal
dysplasias (SEMDs):- are a heterogeneous group of skeletal dysplasias
(dwarfing disorders) characterized by abnormal
epiphyses, with and without varying egrees of
metaphyseal irregularities, flattened
vertebral
bodies, and myopia.
Due to type- 2 collagen defect.
• Schmid metaphyseal chondrodysplasia.:- A type X collagen mutation.
• Stickler syndrome (hereditary progressive arthro-ophthalmopathy):-
A subtype of collagenopathies, types II and XI
Characterized by distinctive facial abnormalities, ocular problems,
hearing loss, and joint problems.
• Achondroplasia:- Due to mutation in fibroblast growth factor receptor 3.

SOME LOCALISED FORM OF
COLLAGEN DEPOSITION DEFECT
• Peyronie's disease:- Growth of abnormal collagen (Type I & III) in the penis.
• Beal's syndrome:- Contractures of hip, knee, elbows and ankle joints.
• Hypertrophic scar:- Accumulation of excessive amounts of collagen may
give rise to a raised scar.
• Keloid:- When the scar tissue grows beyond the boundaries of the
original wound and does not regress
• Loeys-Dietz syndrome:- Aneurysms in the aorta, often in children.

MARFAN SYNDROME
• Marfan syndrome results from an inherited (autosomal dominant) defect in
an extracellular glycoprotein fibrillin-1.
• Most of these are missense mutations that give rise to abnormal fibrillin-1.
• skeletal abnormalities:- Unusually tall with exceptionally long extremities;
pectus excavatum or a pigeon-breast deformity.
• ocular changes:- bilateral subluxation or dislocation of lens.
• Cardiovascular lesions:- MVP, dissecting aortic aneurysm.
• Clinical diagnosis of Marfan syndrome is currently based on the so called
“revised Ghent criteria.”

DISEASES RELATED TO LAMININ
• Progeria:- (early onset of aging), is possibly due to a defective laminin.
• A rare form of congenital muscular dystrophy:-
Due to deficiency in Laminin α2 ( mutation in LAMA 2 gene).
Presents with proximal muscle weakness leukoencephalopathy, dilated
cardiomyopathy, conduction defects, epilepsy etc.
• Junctional Epidermolysis bullosa:-
Due to defect in laminin 5 & 6
A skin condition characterized by blister formation within the lamina lucida of
the basement membrane zone.
3 types have been described 1) JEB with pyloric atresia
2) Herlitz type (lethal during infancy)
3) Non-Herlitz type
• Pierson syndrome:-
Caused by mutations in LAMB2, the gene encoding the basement
membrane
protein laminin β2
Congenital nephrotic syndrome accompanied by ocular and neurological defects.

Clinical relevance of INTEGRIN
• In Bordetella pertussis infection (whooping cough):- The pathogen
inactivate G-proteins (linked to integrin) & renders the host's leukocytes
incapable of transducing signals. Patients exhibit an impairment in
leukocyte transmigration.
• GPIIb/IIIa integrin inhibitor is now approved in many countries for
symptomatic treatment of acute coronary symptoms.
• Leukocyte adhesion deficiency:- LAD1 is most common; due to
deficiency of the β-2 integrin subunit (CD18).
Presents as recurrent bacterial infections, neutrophilia, delay in
umbilical cord sloughing etc.
• Glanzmann Thrombasthenia:- Due to defect in β-3 subunit of integrin.

clinical relavance of other ECM components
• Enzyme collagenase, secreted by Clostridium perfringens causes
breakdown of collagen matrix & helps bacterial invasion.
• Similarly Hyaluronidase an enzyme secreted by some bacteria(Staphylo/
Strepto/ Pneumococci), which breaks the hyaluronan & helps with their
invasion of tissues.
• Heparan sulphate serve as cellular receptor for a number of viruses
including the respiratory syncytial virus.
• In many kinds of cancer, cells unable to make fibronectins loose shape and
detach from the ECM to become malignant.
• Fibronectin Glomerulopathy:- deposition of protein fibronectin in the
glomeruli; l/t proteinuria, renal tubular acidosis, hematuria, along with
hypertension eventually resulting in renal failure.

RESEARCH ON THERAPEUTICS
BY TARGETTING ECM
• Integrins represent potential drug targets for cancer therapy because of
its role in Epithelial-Msenchymal Transition.
• human gene therapy of stem cells have achieved limited success
partly because of low gene transfer efficiency. Recent studies show
Fragments of fibronectin can be used as a ligand to retrovirus in gene
transfer.
• Fibronectin promotes VEGF-induced CD34+ cell differentiation into
endothelial cells; have clinical potential for endothelialization of
prosthetic vascular grafts and revascularization of injured myocardium.

Contd....
• LAMC2 (a laminin components) overexpression appears to drive
tumorigenesis; thus a potential therapeutic anticancer target for
inhibiting tumorigenesis. Furthermore, elevated serum levels of
LAMC2 in cancer patients might be a diagnostic biomarker.
• Crucial role of β1-integrin in mammary tumor progression, in
particular in its metastatic phase, and are of great clinical interest.
• Elastin-like polypeptide is a new drug delivery technology that is
complementary and synergistic to current drug delivery modalities in
cancer chemotherapy.

SUMMARY
• Components of ECM fall into 3 groups;
1) Fibrous structural proteins:- Provides strength & elasticity
2) Water hydrated gels:- Provides compressive resistance & lubrication
3) Adhesive glycoprotein & adhesion receptor:- Connects ECM elements
to cells.
• ECM acts as a mechanical support, control cell proloferation, scaffolding
for tissue renewal and cellular interaction.
• In many autoimmune and genetically inherited diseases the basic
pathology lies in different ECM components.
• There is a wide area of research for treatment of many incurable diseases
including cancers by targetting various ECM constituents.

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