M14: Extracellular Matrix

Development Team

Paper Coordinator : Prof. Kuldeep K. Sharma

Department of Zoology, University of Jammu Principal Investigator : Prof. Neeta Sehgal

Department of Zoology, University of Delhi

Content Writer : Dr. Simran Jit

Miranda House, University of Delhi Content Reviewer : Prof. Rup Lal

Department of Zoology, University of Delhi .

Co-Principal Investigator : Prof. D.K. Singh

Department of Zoology, University of Delhi Paper : 15 Molecular Cell Biology

Module : 14 Cell-Cell Adhesion and Communication: Extracellular Matrix

Description of Module

Subject Name

ZOOLOGY

Paper Name

Zool 015: Molecular Cell Biology

Cell-Cell Adhesion and Communication

M14: Extracellular Matrix

Keywords

ECM, Collagen, Proteoglycans and Basal Lamina

Contents

1. Learning Outcomes 2. Introduction

3. Role and Importance of Extracellular Matrix 4. Molecular Components of ECM

5. Basal Lamina and Interstitial Matrix 6. Remodelling and Degradation of ECM 7. Dysregulation of ECM Components

8. Application of ECM and ECM like Components for Regenerative Medicine 9. Summary

1. Learning Outcomes

After studying this module, you shall be able to

• Understand the composition of Extra Cellular Matrix.

• Appreciate the dynamic role of ECM in shaping the environment of cell.

• Understand how a plethora of molecules interact with components of cell surface and

regulate cell division, signaling and migration.

2. Introduction

Cells in complex metazoans are cooperatively assembled into tissues to perform categorized functions and characteristic shapes and structure which are recognized as tissues. For instance, all the different cell types of vertebrates are organized into only a few classes of tissues: epithelial tissue, connective tissue, muscular tissue, nervous tissue, and blood. The entire lattice of organization of cells into tissues and further into organs is determined by cellular interactions mediated by a wide array of adhesive molecules.

Cells can be integrated into tissues by two basic types of interaction (Fig. 1):

a. Cell–cell adhesion: directly connect the adjacent cells through specialized integral membrane proteins called cell-adhesion molecules (CAMs). These proteins can be clustered into specialized cell junctions such as desmosomes.

b. Cell–matrix adhesion: plasma membrane via adhesion receptors is bound to components of the surrounding extracellular matrix (ECM), an intercellular space filled with3-D meshwork of proteins and polysaccharides secreted by the local cells.

Fig. 1: Summary of mechanisms used for cell-cell and cell-ECM adhesion. The junctional mechanisms in epithelial cells are specialized region of contact, while the non-junctional mechanisms (shown here in nonepithelial cells shows no such obvious specialized structure. A few proteins like integrins and cadherins can be involved in both non-junctional and junctional cell-cell (cadherins) and cell-matrix (integrins) contacts. The cadherins, integrins, and selectins act as transmembrane adhesion molecules and depend on extracellular divalent cations to function; for this reason, most cell-cell and cell-matrix contacts are divalent-cation-

dependent. A functional classification of cellular junction: A) OCCLUDING JUNCTIONS: 1. tight junctions (vertebrates only) and 2. septate junctions (invertebrates mainly); B) ANCHORING JUNCTIONS: Actin filament attachment sites 1. Cell-cell junctions (adherens junctions) and 2. Cell-matrix junctions (focal adhesions), Intermediate filament attachment sites 1. cell-cell junctions (desmosomes) and 2. cell-matrix junctions (hemidesmosomes); C) COMMUNICATING JUNCTIONS: 1. gap junctions, 2. chemical synapses and 3. plasmodesmata (plants only).

Source: Author.

These molecules mediate integration of cells into distinct and diverse tissues as well as conduct a two-way transfer of information between the exterior and the interior of cells. The adhesion molecules generally integral membrane proteins, can bind through their cytosolic domains to intracellular adapter proteins. These are further directly or indirectly linked to the cytoskeleton (actin or intermediate filaments) and intracellular signaling pathways. As a consequence, information is transferred by this macromolecule complex from the cell exterior into the intracellular environment. This junctional highway can also transfer information from the cell interior to its surrounding.

Since the time of divergence of plant and animal cell pre-dates the origin of multicellularity, the molecular means of integrating cells into tissues and organs must have evolved independently in both the lineages.

In the present chapter we will discuss the role and components of ECM and how cell–matrix adhesion helps in the organization of epithelial and non-epithelial tissues in animals.

3. Role and Importance of Extracellular Matrix

Extracellular matrix is a general term used for the complex meshwork of large proteins including collagens, fibronectins, laminins, and proteoglycans which acts as connective material to hold cells in a defined space required for the structure and function of a tissue. It

Extracellular Structures of Eukaryotic Cells Kind of

Organisms

Extracellular Structure

Structural Fibre

Components of Hydrated Matrix

Adhesive Molecules

Animals ECM Collagen and

Elastin

Proteoglycans Fibronectins and laminins Plants Cell wall Cellulose Hemicellulose

and extensins

Pectins

is secreted by local cells and organized into meshwork in close association with cell surface protein interaction. The proportions of these components can vary greatly and influence the development and physiology of the tissue type (Fig. 2).

A B C Fig. 2:(A) Blood, (B) Osseous tissue (C) Hyaline cartilage.

Source: https://en.wikipedia.org/wiki/Connective_tissue

The Extracellular Matrix performs multiple functions and can be the major constitute the mass of skin, bones, and tendons. Variations in composition are suited to perform different function such in blood in which the ECM is referred as plasma and is fibres are generally absent. On the other hand, it can be deposited with calcium salts to form solid structure as in bones or packed with fibres to provide tensile strength to the cartilage. Cells also protect the matrix and constantly modulate its properties and activity by synthesizing, trafficking, sequestering proteins. This dynamic nature of ECM controls all aspect of cell behaviour and development. This entire communication between the ECM and cells is facilitated by bidirectional signaling (Fig. 5) hence communicating changes in the ECM to the cytoskeleton.

For instance, the ECM in the basement membrane underlying the epidermis of the skin is a

thin layer that helps to organize the skin cells into a nearly-impenetrable barrier while ECM

proteins are organized into massive 3-dimensionalmatrix surrounding each chondrocyte in

cartilaginous tissue, thus enabling cartilage in our knee to withstand the repeated shock of the

footsteps. The matrix can become calcified to give strength to bones and teeth, or it can form

the transparent matrix of the cornea. At the interface between an epithelium and connective

tissue, the matrix forms a basal lamina, a multi-layered proteinaceous structure secreted by

the epithelium which is important in controlling cell behaviour. The basal lamina and a thick

reticular lamina (ECM secreted by another cell type) together form the basement membrane.

Fibroblasts, the flat bulk cells in most connective tissues secrete major ECM macromolecules. However, certain specialized cells of fibroblast family viz., chondroblasts which produce cartilage components and osteoblasts form bone.

Some of the important functions of the ECM include:

Functions of the ECM

1. General function of the ECM is to provide mechanical support to tissues by organizing cells into tissues and coordinating their cellular functions by modulating transmembrane adhesion receptors. Specialized ECM in cartilage acts as a compression buffer when tissues are subjected to stress.

2. Determining the biomechanical properties (stiffness/elasticity, porosity, shape) of the extracellular environment.

3. Provide positional information for controlling cellular polarity, survival, proliferation, differentiation, and fate, and thus embryonic and neonatal development and adult function and responses to the environment and to disease.

4. Serving as a barrier to movement or, providing tracks or lattice through or on which cells can migrate.

5. Store and release extracellular signaling molecules essential for controlled cell growth and differentiation.

6. Act as a reservoir of growth factors; and in some cases create an extracellular concentration gradient of the growth factor; serve as a co-receptor for the growth factor and facilitate binding of growth factor to the rector by serving as co-ligand.

7. ECM is dynamic and is constantly remodelled by enzymatic phosphorylation, sulfation and desulfation, cross-linking, cleavage by proteases and glycosidases, and oxidation influencing interactions of the cell with its microenvironment.

8. The process of cell movement and reorganization called morphogenesis is critically dependent on cell–matrix adhesion as well as cell–cell adhesion.

9. ECM homeostasis is also essential for wound healing and sustained imbalance synthesis and breakdown can result in life-threatening pathological conditions.

Hence the CAMs, adhesion receptors, and ECM molecule complex not only organize cell into diverse classes of tissues but also enable varying functions.

4. Molecular Components of ECM

Epithelial cells can be taken as example to understand various types of anchorage and

junctions. These cells are polarized having discrete apical, basal, lateral and often the plasma

membrane at the apex is drawn into microvilli. All epithelial cells in a sheet are connected to

one another and to the ECM by specialized junctions which been briefly summarized in the

Fig. 1.

Each anchoring junction requires a tri-molecular arrangement of adhesive proteins-adapter proteins-cytoskeletal filaments. Adhesive proteins in the plasma membrane of the cell establish connect between adjacent cell (through CAMs) or to the ECM (through adhesion receptors).

Epithelial cells organized as sheets are firmly attached to the underlying ECM via adhesion receptors called integrins which are a component of hemidesmosomes. This anchoring junction comprises of integral membrane proteins linked via cytoplasmic adapter proteins (e.g., plakins) to keratin-based intermediate filaments. The principal ECM adhesion receptor in epithelial hemidesmosomes is integrin α6β4. From 18 types of α subunit and 8 types of β subunit, 24 heterodimers combinations of integrins exist which bind to various ECM proteins.

Both α -and the β subunits of an integrin molecule contribute to the primary extracellular ligand binding site (Fig. 3). The ligand binding is facilitated by simultaneous binding of

Features of Anchoring Junctions

adhesion protein

Extracellular ligand

Intracellular cytoskeletal attachment

Intracellular adaptor proteins

Function

Cell-Cell

Adherens junction

Classical cadherins

Classical cadherins on neighbouring cell

Actin filament

α-catenin, β- catenin, plakoglobin (γ- catenin), p120- catenin, vinculin

Shape, tension, signaling,force transmission

Desmosomes Non-classical cadherins (desmoglein, desmocolin)

Desmoglein and

desmocolin on

neighbouring cell

Intermediate filaments

Plakoglobin (γ- catenin), plakophilin, desmoplakin

Strength,

durability,signaling

Cell-Matrix

Actin-linked cell-matrix junction

Integrin ECM proteins Actin filament

Talin, kindlin, vinculin. paxillin, focal adhesion kinase, numerous others

Shape, signaling, force transmission, cell movement

Hemidesmosome Integrin, type XVII collagen

ECM proteins Intermediate filaments

Plectin, BP230 Shape, rigidity,

signaling

divalent cations. The cytosolic regions of integrins interact with adapter proteins, which in turn linked to the actin cytoskeleton and to intracellular signaling molecule. Some integrins, however, interact with intermediate filaments.

Fig. 3: A) Cells interact with their ECM microenvironment via integrins, detecting both chemical and physical signals from the matrix. Integrins interpret this information and deliver it to the cell via large, multiprotein plasma membrane complexes. This becomes conveyed via cytoskeletal and signaling proteins to determine the function of both nuclei (gene expression and proliferation) and cytosol (cell shape and migration).

Source: Streuli 2016. DOI:10.1091/mbc.E15-06-0369.

B) The subunit structure of an integrin adhesion receptor. The globular head projects more than 20 nm from the lipid bilayer. The protein acts as a transmembrane linking the matrix protein to the cytoskeleton (via the indicated anchor proteins) inside the cell. The two subunits α and β are held together by noncovalent bonds.

Each α-subunit and β-subunit has a single transmembrane helix and, usually, a short unstructured cytoplasmic tail. The α subunit is produced as a single 140,000-dalton polypeptide chain and cleaved into a small transmembrane domain and a larger extracellular domain containing divalent-cation binding sites. The two domains are linked by a disulfide bond. Single divalent-cation-binding is present in the extracellular part of the β subunit. Binding of extracellular ligands to ectodomains of integrin lead to 'outside–in' activation while binding of the talin head to cytoplasmic β-integrin tails triggers 'inside–out' activation.

Source: Author.

These heterodimeric transmembrane glycoprotein receptors are expressed by a wide range of cells. Between the two subunits, α-subunit is responsible for ligand specificity while β interacts with the intracellular cytoskeletal material. Ligands for integrins include collagens, fibronectins, laminins, and other cellular receptors and intercellular receptors. The integrin family of receptors is classified into four different categories (Fig. 4):

1. RGD binding: recognize Arg-Gly-Asp sequence and are combinations of α5β1, α8β1,

αIIb0β3, and αvβ

2. Collagen binding: combinations of α1β1, α 2β1, α 10β1, and α 11β1 3. Laminin binding: combinations of α3β1, α 6β1, α 6β4, and α7β1

4. Leukocyte binding types of integrins: αEb7, α4β7, α4β1 with β2 being limited to leucocytes.

Fig. 4: Classification of integrin receptors based on their ligand binding ability. Four different categories are presented: (a) RGD binding, (b) collagen binding, (c) laminin binding and, (d) leukocyte binding. β1-subunit is known to heterodimerize with eleven different kinds of α-subunits. While most of α-subunits pair up with one kind of β-subunit, αv- and α4-subunits are the exception.

Source: Goswami 2013. http://dx.doi.org/10.4236/abc.2013.32028

The integrin receptors can conduct a unique bi-directional signal i.e., inside-out and outside-

in, making the ligand binding a highly regulated process (Fig. 5). As a result integrin play a

major role in a variety of biological processes maintaining homeostasis by transmitting

information required for survival, proliferation, and migration. Emphasizing that correct

communication with the ECM is needed to determine cell fate.

Fig. 5: The regulation of the extracellular binding activity of a cell's integrins from inside the cell. Binding of an extracellular signal (ligand) to integrin triggers a signaling cascade in the interior of cell. This cascade of reactions leads to activation of extracellular binding site on integrin making it receptive for cell adhesion.

Source: Author.

Assembly and function of the ECM is dependent on interactions with transmembrane adhesion receptors. Integrins are the principal class of receptors that mediate cell-matrix adhesions in epithelial sheets. These adhere to three types of the most abundant molecules in the extracellular matrix of all tissues:

Three types of molecules are abundant in ECM of all tissues.

1. Proteoglycans: highly viscous group of glycoproteins that protect the cells and bind a wide variety of extracellular molecules

2. Collagen: provide mechanical strength and resilience

3. Multiadhesive matrix proteins: bind and cross-link cell-surface adhesion receptors and other ECM components.

5. Basal Lamina and Interstitial Matrix

The extracellular matrix presents as two clearly identifiable structures:

1. Basal Lamina: a condensed matrix layer formed adjacent to epithelial cells, and other covering cell sheets (e.g. mesothelium), or muscle cells, adipocytes, etc. (Fig. 6).

2. Interstitial Matrix: a space-filling structure of huge variety that determines the main

characteristics of a given connective tissue.

Fig. 6: Model of basal lamina based on major components. A) Specific interactions of molecules form a 2- dimensional network of the basal lamina. Important components are the proteins type IV collagen, laminin, and nidogen, and the proteoglycan perlecan. B) Arrows indicate interactions between molecules that can bind directly to each other. The various isoforms of type IV collagen and laminin have a distinctive tissue distribution.

Source: Author.

The basal lamina is a thin meshwork of ECM molecules which separates the organized

groups of cells from adjacent connective tissue. For the epithelial cells it lies just underneath

the basal surface while for non-epithelial cells such as muscle cells it may surround the cell

entirely where it also protects during contraction and relaxation. The laminae helps the cells

to adhere to each other such as during the embryonic stages, plays an important role in tissue

regeneration, forming tissue compartments, permeability barriers. Basal lamina forms a tight

blood-brain barrier limiting the diffusion of molecules, and in the kidney, the lamina around

the glomerular blood vessels is unusually thickened (upto 100 nm) to serve as an effective

filtration unit (Fig. 7). Together, the basal lamina and the immediately adjacent collagen

network form a structure called the basement membrane (Fig. 8).

Fig. 7: Three types of organization of basal lamina. A) The Basal laminae (brown) completely surrounds the skeletal muscle cells; B) forms a 2-D sheet underlie the epithelia and C) are interposed between two cell sheets (as in the kidney glomerulus). In the kidney glomerulus, the basal lamina serves as the permeability barrier determining which molecules will pass into the urine from the blood.

Source: Author.

Fig. 8: Basal Lamina. Regeneration, repair and aggregation of epithelia.

Source: Author.

Components of the ECM are synthesized and secreted by the cells it supports. Four distinct

types of proteins are found in the ECM.As seen in Fig. 8 the basal lamina on one side anchors

the cells by adhesion receptors (integrins) while the other side is linked to the surrounding

connective tissue by a layer of fibers embedded in a proteoglycan-rich matrix.

Major Proteins of Basal Lamina

Collagen Type IV collagen:Type of collagenprimarily found in basal lamina.Structure comprises of N-terminus; middle helical region and globular C-terminus. Three trimeric molecules self- assemble along their helical region forming a two-dimensional network (Fig. 9,10).

 The largest and most prominent of the extracellular matrix proteins.

 Categorized into fibrillar (e.g. collagens I, II, III) and nonfibrillar (e.g. collagen IV) types.

 The fibrillar collagens twist together to form collagenous triple helix of either identical α chains (homotrimer) or different (heterotrimer) chains. These associate with other collagen monomers forming long fibers increasing the tensile strength.

 Contain a high proportion of hydroxylated amino acids, mostly prolines and lysines. This hydroxylation is necessary for the extensive hydrogen bonding that occurs between subunits and between monomers.

 The chains are active sites for covalent modification e.g., hydroxylation, glycosylation, oxidation and cross- linking.

 The helical pattern is possible because of a characteristic repeating sequence motif Gly-X-Y, where X is generally proline and hydroxyproline in position Y, and less often lysine and hydroxylysine.

 Type IV collagen is a principal structural component of all basal laminae and can bind to adhesion receptors, including some integrins.

 Type IV collagen are not cleaved after secretion and interact by their terminal domains can bind to multiple ligands in forming a meshwork.

 Long fibrillar, alpha-helical domains and globular domains can interact in different orientations to form flexible, multi layered network of sheets.

 Receptors for Type IV include certain integrins, discoidin domain receptors 1 and 2, glycoprotein VI (on platelets), leukocyte-associated Ig-like receptor-1, members of the mannose receptor family, and a modified form of the protein CD44.

 They can play critical roles in helping to assemble the ECM and in integrating cellular activity with the ECM.

Laminins Family of multi-adhesive, high molecular weight, cross-shaped proteins that interdigitate with Type IV collagen adhesion receptors form a fibrous meshwork (Fig. 11).

 Large heterotrimeric glycoprotein comprising α, β, and γ chains linked by di-sulfide linkages into asymmetrical cross-shaped structure.

 Principal basal lamina ligands of integrins.

 The 5 α, 3 β, and 3 γ chains are assembled into 16 laminin isoforms. The composition is describes as: laminin- 111 (α1β1γ1) or laminin-511 (α5β1γ1).

 Polymerization occurs by self-assembly oflaminin globular (LG) domains of adjacent globular domains.

 Bind to perlecan, nidogen, and two or more to laminin receptor proteins on the surface of cells through multiple functional domains.

 Dystroglycan is an important laminin receptor may also organize the assembly of the basal lamina.

 Play a crucial role in neural development, where they act as a guiding path along which certain axons extend to find their eventual synaptic targets.

Perlecan Also called basement membrane-specific heparan sulfate proteoglycan core protein (HSPG) or heparan sulfate proteoglycan 2 (HSPG2).Multiple domains help to bind and cross-link several ECM components and cell-surface molecules.

 Consists of a large multidomain core protein (∼470 kDa) to which polysaccharidesare covalently attached.

 The core protein is made up of multiple repeats of five distinct domains, including laminin-like LGdomains (3 copies), EGF-like domains (12 copies), and Ig domains (22 copies).

 Three types of covalent polysaccharide chains are present viz., N-linked chains, O-linked chains, and glycosaminoglycans (GAGs).

 Cross-link ECM components by binding to about a dozen of molecules such as laminin, nidogen/ entactin), cell-surface receptors, and polypeptide growth factors.

Nidogen (Entactin)

Polyvalent matrix binding protein(Fig. 12).

 Sulfated monomeric glycoproteins.

 Stabilizes the basal lamina by cross-linking ECM components.

 NID1and N1D2 are critical for organogenesis during the late development.

Fig. 9: Biosynthesis of a collagen fibril. Synthesis of procollagen α chains occurs in rough ER), followed by addition of asparagine-linked oligosaccharides to the C-terminal, prophydroxylation (certain prolines and lysines), other covalent modifications (galactose or galactose-glucose (hexagons) are attached to some hydroxylysines) and cis→ trans isomerization of prolines. These modifications enable self-assembly of propeptides to form trimers which are also covalently linked by disulfide bonds. The chaperone protein Hsp47 also helps to stabilize the helices or prevent premature aggregation of the trimers. The folded procollagens are transported to and through the Golgi complex and secreted, the N- and C-terminal propeptides are removed, and the trimers assemble into fibrils and are covalently cross-linked.

Source: Author

COLLAGEN

Fig. 10: A) Collagen Assembly. 1. Three collagen precursor chains are assembled in the ER lumen to form triple-helical procollagen molecules and transported to Golgi complex. 2 After secretion into the ECM, procollagen is converted to collagen by the enzyme procollagen peptidase. 3 The processed molecules of collagen, called the tropocollagen, then bind to each other and self-assemble into collagen fibrils. 4. The fibrils assemble into collagen fibers by lateral interactions. In striated collagen, the 67-nm repeat distance is created by packing together rows of collagen molecules in which each row is displaced by one-fourth the length of a single molecule.

B) Structure and assembly of Type IV collagen. The structure comprises of a small non-collagenous globular domain at the N-terminus and a large globular domain at the C-terminus. Nonhelical segments (introduce flexible kinks) interrupt the collagenous triple helix. Tetramer and other higher order structures are formed by lateral interactions between triple helical segments, head-to-head and tail-to-tail interactions between the globular domains. Multiple, unusual sulfilimine (–S=N–) or thioether bonds between hydroxylysine (or lysine) and methionine residues covalently cross-link some adjacent C-terminal domains and contribute to the stability of the network.

Source: Author

Beneath the basal lamina, the sheet like deposits of ECM, lies the connective tissue. Cells of

connective tissue secrete gel like polysaccharides and fibrous proteins that fill the interstitial

space and act as a compression buffer against the stress placed on the ECM. Collagen secreted by fibroblast is the most abundant fibrous protein in connective tissue. The fibronectins, a family of multi-adhesive matrix proteins, form their own distinct fibrils in the ECM of most connective tissues.

Major Proteins of Connective Tissue

Collagen Trimeric molecules that are often bundled together into fibers (fibrillar collagens) which help in tissue formation (Fig. 13).

 A minimum of 28 different types of collagens are known from vertebrates. These can be heterotrimeric or homotrimeric in composition.

 Types I, II, and III constitute 80-90% of the total fibrillary protein.

 Secretion and assembly of collagen is described in Fig. 9 and 10.

 Type I collagen fibers are organized into long fibres of great tensile strength; tendons usually can be stretched without breakage. Thus, allowing tendons which connect muscles to bones to withstand enormous force. Other minor fibrillar collagens, Type V and XI, co-assemble to further regulate structure and properties of Type I fibres.

 In tendons, all the Type I fibrils interlinked by microfibrils of Type VI collagen are oriented in the direction of the applied stress. The associated proteoglycans and Type VI collagen coat the surface.

 Type II collagen is the major collagen in cartilage. The thinner fibrils are oriented randomly and cross-linked to the viscous proteoglycan matrix by Type IX collagen. The globular N-terminal segment of Type IX collagen projects outwards from the fibril along with the chondroitin sulfate chain which is covalently linked to the chain at the flexible kink. These anchor the type II fibril to proteoglycans and other components of the matrix.

 Based on function and sequence homology collagens have categorized as (Kadler et al 2007):

i. Fibril-forming collagens: Type I, II, III, V, XXIV and XXVII found in tendon, ligament, cornea, bone, skin and vitreous humour provide tensile strength.

ii. Fibril-associated collagens with interrupted triple helices (FACITs): These connect fibrillar collagens to one another or to other ECM components. Type IX associated with Type II in cartilage and vitreous humour, Type XII, XIV and XVI are important members of the category.

iii. Network-forming collagens: Head to head interaction between Type IV are stabilized by methionine- lysine crosslinks help to form intricate filtering networks in the basement membrane. Type VII and X are associated with Descemet’s membrane and growth plate of cartilage respectively.

iv. Anchoring collagens: Type VII anchor basal lamina of epithelia of skin to underlying connective tissue.

v. Transmembrane collagens: Type II with a short cytoplasmic N-terminus and long helical ecto-domains function as adhesion receptors. Also include Type XIII and XXV which are found on several cell types.

vi. Endostatin-producing collagens: The anti-angiogenic peptide is produced by cleavage of Type XV and XVIII found in some basement membranes.

vii. Beaded-filament-forming collagen: Type VI monomers assemble into a bead on string microfibrils.

Found in many tissues and link cells.

Elastin Forms the amorphous core of elastic fibers (Fig. 15).

 Elastins are highly hydrophobic ECM proteins with flexible fibers rich in glycine and proline with total length of 750 amino acids. However, proline residues are not hydroxylated, and hydroxylysine is absent.

 Elastin is processed from tropoelastin is secreted into the extracellular space and assembled into elastic fibers close to the plasma membrane.

 After secretion, the tropoelastin molecules are crosslinked by covalent binding of interchain lysine residues resulting in a flexible and elastic network. Such a property is necessary for lungs, arteries, skin and intestines which continuously change shape. Elastin comprises nearly 50% of the total dry weight of aorta-the largest artery!

 Permit repeated stretching and recoiling of tissues because of their highly elastic core of cross-linked, amorphous elastin, which is surrounded by a network of microfibrils that help, assemble the fibers and regulate signaling mediated by TGF-β.

Proteoglycans and Glycoproteins containing one or more covalently bound GAG chains.

Glycosaminoglycans (GAGs) Specialized linear polysaccharide chains of specific repeating disaccharides that can be highly hydrated and confer diverse binding and physical properties (e.g., resistance to compression).

 Glycosaminoglycans (GAGs) are linear polymers of disaccharides that are often modified by sulfation.

 Proteoglycans are the membrane-associated or secreted core forming proteins that are covalently attached to one or more GAG chains.

 The protein core of proteoglycans is not as large as the fibrillar collagens but due to heavy glycosylation take up a massive volume.

 The sugars are generally are usually repeating disaccharide units of one is usually uronic or D-galactose; the other sugar is N-acetylglucosamine or N-acetylgalactosamine.

 Major groups are: hyaluronan, chondroitin sulfate and dermatan sulfate, heparan sulfate, and keratan sulfate.

 These carry a net negative charge thereby attracting positively charged sodium ions (Na+), which attracts water molecules via osmosis, keeping the ECM and resident cells hydrated.

 Transmebrane proteoglycans such as the syndecans facilitate cell-ECM interactions and help present certain external signaling molecules to their cell-surface receptors. Syndecan is an important modulator of fibroblast growth factor signaling.

 Hyaluronan, a highly hydrated GAG, is a major component of the ECM of migrating and proliferating cells.

Certain adhesion receptors bind hyaluronan to cells.

 Large proteoglycan aggregates containing a central hyaluronan molecule noncovalently bound to the core proteins of proteoglycan molecules (e.g., aggrecan) contribute to the ability of the matrix to resist compression forces. However, a rapid diffusion of nutrients, metabolites, and hormones between the blood and the tissue cells is permitted.

 Proteoglycans can function both as a substrate for cells to attach to while the heavy hydration shell acts as an effective barrier to signals from other cells. This is useful during development when there is a great deal of cell migration, and there needs to be ways to segregate cells both by attracting them and repelling them.

 Bind various secreted signal molecules, for example heparan sulfate binds to fibroblast growth factors (FGFs) to conduct cell proliferating signal to the cell.

 Regulatory role of proteoglycans is also exerted to other secreted proteins (kinases) by immobilization;

sterically block the activity of protein or promoting degradation. The matrix can also sequester the protein and creating a concentration gradient.

Multi-adhesive proteins Large multidomain proteins often comprising many copies (“repeats”) of a few distinctive domains that bind to and cross-link a variety of adhesion receptors and ECM components (Fig. 14, 16).

 Fibronectins (Fn) are important multi-adhesive matrix glycoproteins. These abundant proteins principally bind integrins along with several other ECM components like collagens, proteoglycans enabling anchoring the cells to the matrix.

 Role of fibronectins is extended to cell adhesion, growth, migration, and differentiation. The two types are:

i. Soluble plasma fibronectin (cold-insoluble globulin or CIg): major protein component of blood produced in the liver by hepatocytes.

ii. Insoluble cellular fibronectin: major component of the extracellular matrix.

 The ECM components initiate the process of wound healing by playing a chemotactic or stimulatory role and acts as scaffold during tissue repair. At the site of injury plasma Fn accumulate in the wound during first 24 hours and quickly polymerize. The multivalencies used to interact with different cells via integrin receptors specially the platelets. This stimulates the migration and adhesion of fibroblasts, keratinocytes, and endothelial cells. Several growth factors are released in this “entrapment”. Other cell types such as neutrophils, monocytes/macrophages, smooth muscle cells, endothelial cells, and fibroblasts are recruited to initiate the inflammatory response.

 Structure is a dimer composed of two similar, large subunits joined by disulfide bonds at their C-termini.

Internally the chain is folded into multiple functionally distinct domains which are separated by regions of flexible polypeptide chain.

 Each domain is built upon of smaller modules which are serially repeated and usually encoded by a separate exon. Based on sequence similarity the domains are classified as type I, II, and III fibronectins.

 Type III fibronectin repeat is the major module about 90 amino acids long and occurs at least 15 times in each subunit.

 Fibronectins attach the cells ECM components particularly fibrillar collagens and heparan sulfate proteoglycans, and to adhesion receptors such as integrins thereby influencing the shape and movement of cells and the organization of the cytoskeleton. This can also be viewed as how cells shape their surrounding ECM by regulating their receptor-mediated attachments to fibronectin and other ECM components.

6. Remodelling and Degradation of ECM

The components of ECM are critical for cell survival and functioning and these must be recycled and degraded by carefully regulated mechanisms. A variety of zinc dependent metalloproteases execute the degradation of different components of the ECM. These are synthesized in inactive form, often requiring cleavage for activation. Based on their target of degradation these are often named as collagenases, gelatinases, elastases and aggrecanases.

These can be secreted into the ECM or remain as integral part of the cell membranes as a trans-membrane protein or as covalently linked molecules to the membrane itself.

ECM metalloproteases are categorized in three major subgroups based on the enzymes’

structures:

i. matrix metalloproteases (MMPs)

ii. disintegrin and metalloproteinases (ADAMs)

iii. ADAMs with thrombospondin motifs (ADAMTSs).

The substrates for these also include non-ECM components such as adhesion receptors. For , ADAMs are also involved in cleaving extracellular domains from integral membrane proteins.

The activity of these metalloproteases is controlled by protein inhibitors called TIMPs (tissue inhibitors of metalloproteinases) and RECK (reversion-inducing–cysteine-rich protein). Apart from their inhibitory action on metalloproteinases, some of the regulators also have their own cell-surface receptors concerned with other independent functions.

7. Dysregulation of ECM Components

Increased ECM breakdown ECM breakdown by abnormally high levels of heart-specific MMP1 expression result in appropriate collagen loss and diminished contractility leading to tissue destruction (Fig. 17).

Excessive ECM production and imbalanced degradation can result in fibrosis which can be as severe to cause organ failure such as in liver cirrhosis. In fibroblast, the TGFβ pathway is the most effective inducer of the ECM gene expression. It has been shown by Bailey et al 2013 that in liver, interleukin-33 (IL-33) promotes the expansion of resident innate lymphoid cells to produce IL-13 indirectly enhances ECM production by stimulating hepatic stellate cells.

Fibrosis is further supported by simultaneous collagen accumulation and downregulation of metalloproteases. Recent studies have also indicated fibrotic ECM to stimulate fibroblasts to further increase ECM production (Parker et al 2014).

Overexpression of collagen IV enhances cell survival suggesting the role of ECM as promoter of tumour progression.

ECM can promote cancer pathogenesis in a number of ways such as byfunctioning as a

physical barrier to chemotherapy and by blocking monoclonal antibodies. It can also form

migration tracks regulating the interaction of immune cells with cancer cells. ECM stiffness

i.e., resistance to degradation is increased through integrin signalling inducting lysyl oxidase

(LOX) and LOX-like 2 (LOXL2) that invasion and that crosslink collagen. Limited

breakdown of ECM proteins can produce peptides that diffuse and act as potent ligands for

growth stimulating receptors or angiogenesis. Imbalanced and erroneous generation of bioactive ECM fragments can regulate transcriptional pathways to increase MMP expression.

Fig. 17: Dysregulation of ECM Components leads to pathological conditions. A) Over-degradation of the matrix triggered due to tissue injury or inflammation by matrix metalloproteinases (MMPs) and ADAMTS can result in osteoarthritis.B)On the other hand increased production of ECM components can also be initiated by chronic inflammation or tissue injury. The cascade starts from production of transforming growth factor-β (TGFβ), connective tissue growth factor (CTGF) and interleukin-13 (IL-13) leading to a stimulatory effect on producer cells resulting in pathological fibrosis and increasing the chances of cancer. The excess ECM exerts a positive feedback loop on fibroblast to synthesize more of ECM components.

Source: Author.

8. Application of ECM and ECM like Components for Regenerative Medicine

Stem cell-based remedies and tissue engineering applications are envisioned as regenerative strategies for the replacement, remodelling, regenerating damaged tissues or by inducing activation of body’s self-healing ability.

The 3-D microenvironment in which the cell resides and functions has proposed ECM and

ECM like components as target molecules for biomaterials design and fabrication, especially

when aiming for implant or test system generation.

Simplest of the application includes decellularization by removal of cells and hence the antigens reducing the chances of inflammatory reaction have been successfully used for burn therapy, and plastic surgery. However, a major limitation is the disruption of crucial structural tissue elements specially elastin and depletion of important connectors, proteoglycans (PGs) and glycosaminoglycans (GAGs).

ECM proteins are important determinants of cell behaviour and have been used as biomaterials for producing functional scaffolds of choice. ECM components are coated over the surface of polymeric surface to enhance cell adhesion and biocompatibility. Another example is of using Type IV collagen coatings induced pluripotent stem (iPS) cells to differentiate into functional cells of the cardiovascular and hematopoietic lineage (Ma et al 2015).

Cell sheet technique is an interesting approach to generate ECM-based biomaterials in which the cells under defined in vitro conditions are instructed to produce their own ECM, resulting in a scaffold-like biomaterial (Fig. 18). These have been tested at clinical level trials for delivering cells, drugs as well as proteins to the site of injury in order to initiate tissue regeneration (Matsuura et al 2015).

Fig. 18: Fibroblast-derived ECM sheets. (A) Fibroblast-produced, cell and ECM containing sheets in a 6-well plate. (B) ECM sheet after decellularization in phosphate buffer saline.

Source: S. Hinderer et al. Advanced Drug Delivery Reviews 97 (2016) 260–269

9. Summary

 The extracellular matrix (ECM) provides a 3-dimensional microenvironment affecting cellular function and behaviors through a bi-directional signal transduction via cell surface.

 Based on location and composition the ECM can be categorized: i) the interstitial connective tissue matrix, which surrounds cells and provides structural scaffolding for tissues; ii) and the basement membrane, which is a specialized form of ECM that separates the epithelium from the surrounding stroma.

 The ECM is a highly complex network of chemical, physical and biological components that vary in composition from tissue to tissue and organ to organ.

 It functions as a reservoir for biochemical signals controlling cellular function and undergoes constant remodelling by balancing its synthesis and degradation by a variety of enzymes (e.g., matrix metalloproteinases).

 Major molecular components include glycosaminoglycans and fibrous proteins (e.g., collagen, elastin, fibronectin and laminin), which self-assemble into nanofibrillar supramolecular networks that fill the extracellular space between cells.

Fig. 19: Demonstration of extracellular matrix components in groups. These ECM types consist of a group of three different molecular components.

Source: Derya M, Yılmaz I, Aytekin M (2014). The Role of Extracellular Matrix in Lung Diseases. Biol Med 6:

200. doi: 10.4172/0974-8369.1000200