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Extracellular Matrix


Cells of similar structure and function assemble to form structural and functional associations, known as tissues , in all multicellular organisms. Groups of these tissues are assembled in various organizational and functional arrangements into organs , which carry out functions of the body. The four basic tissue types are epithelium , connective tissue , muscle , and nervous tissue . Each of these tissues and their component cells possess specific, defined characteristics and traits, which are detailed in subsequent chapters. These traits include the cells themselves and the extracellular matrix ( ECM) , a complex of nonliving macromolecules that they manufacture and export into the extracellular space , which is the space between cells.

The extent of ECM located in the extracellular space varies with the particular tissue type. Epithelia, for instance, form sheets of cells with only a scant amount of ECM, whereas connective tissue is composed mostly of ECM, with a limited number of cells scattered throughout the matrix. Cells maintain their associations with the ECM by forming specialized junctions that hold them to the surrounding macromolecules. This chapter explores the nature of the ECM and its functions, not only as it relates to the tissues that house it but also its relationship with the cells contained within it. Although it was initially believed that the ECM forms merely the skeletal elements of the tissue in which it resides, it is now known that it has additional functions, such as:

  • Modifying the morphology and functions of cells

  • Modulating the survival of cells

  • Influencing the development of cells

  • Regulating the migration of cells

  • Directing mitotic activity of the cells

  • Forming junctional associations with cells

  • Providing a milieu for the immune defense of the body

  • Resisting forces of compression and tensile forces acting on tissues

The ECM of connective tissue proper, the most common connective tissue of the body, is composed of a hydrated gel-like ground substance with fibers embedded in it. Ground substance resists forces of compression, whereas fibers withstand tensile forces. The water of hydration permits the rapid exchange of nutrients and waste products carried by the extracellular fluid as it percolates through the ground substance ( Fig. 4.1 ).

Fig. 4.1
Schematic diagram of extracellular fluid flow. Fluid from the higher-pressure arterial ends of the capillary bed enters the connective tissue spaces and becomes what is known as extracellular fluid , which percolates through the ground substance. Some, but not all, of the extracellular fluid then reenters the blood circulatory system at the lower pressure venous end of the capillary bed and venules. The extracellular fluid that did not reenter the blood vascular system will enter the even lower-pressure lymphatic system, which will eventually deliver it to the blood vascular system.

Ground Substance

Ground substance is an amorphous, gel-like material composed of glycosaminoglycans, proteoglycans, and glycoproteins.

Extracellular fluid (derived from the fluid components of blood) percolates through ground substance , which is composed of glycosaminoglycans ( GAGs ), proteoglycans , and cell adhesive glycoproteins . These three families of macromolecules form various interactions with each other, with fibers, and with the cells of connective tissue and epithelium ( Fig. 4.2 ).

Fig. 4.2, Light micrograph of areolar connective tissue, displaying cells, collagen fibers (Co), elastic fibers (EF), and ground substance (GS). Observe that, in this very loose type of connective tissue, the fibers, although interwoven, present a relatively haphazard arrangement. This permits the stretching of the tissue in any direction. The cells of areolar connective tissue are principally of three types: fibroblast, macrophages, and mast cells. The extensive extracellular spaces are occupied by ground substance composed mainly of glycosaminoglycans and proteoglycans, a large component of which is aggrecan aggregate, a highly hydrated macromolecule. (×132)

Glycosaminoglycans

GAGs are negatively charged, long, rod-like chains of repeating disaccharides that have the capability of binding large quantities of water.

GAGs are long, inflexible, unbranched polysaccharides composed of chains of repeating disaccharide units. One of the two repeating disaccharides is always an amino sugar ( N -acetylglucosamine or N -acetylgalactosamine); the other one is typically a uronic acid (iduronic or glucuronic). GAGs are classified into 4 groups, depending on their core disaccharide constituents ( Table 4.1 ).

TABLE 4.1
Types of Glycosaminoglycans (GAGs)
GAG Molecular Mass (Da) Repeating Disaccharides Covalent Linkage to Protein Location in Body
Group I
Hyaluronic acid 10 7 –10 8 D-glucuronic acid-β-1,3- N -acetyl-D-glucosamine No Most connective tissue, synovial fluid, cartilage, dermis
Group II
Chondroitin 4-sulfate 10,000–30,000 D-Glucuronic acid-β-1,3- N -acetylgalactosamine-4-SO 4 Yes Cartilage, bone, cornea, blood vessels
Chondroitin 6-sulfate 10,000–30,000 D-Glucuronic acid-β-1,3- N -acetylgalactosamine-6-SO 4 Yes Cartilage, Wharton jelly, blood vessels
Dermatan sulfate 10,000–30,000 L-Iduronic acid-α-1,3- and N -acetylglucosamine-4-SO 4 Yes Heart valves, skin, blood vessels
Group III
Heparan sulfate 15,000–20,000 D-Glucuronic acid-β-1,3- N -acetyl-galactosamine
L-Iduronic acid-2-SO 4 -β-1,3-N-acetyl-D-galactosamine		 Yes Blood vessels, lung, basal lamina
Heparin (90%)
(10%)		 15,000–20,000 L-Iduronic acid-beta-1,4-sulfo-D-Glucosamine-6-SO 4
D-Glucuronic acid-β-1,4-N -acetylglucosamine-6-SO 4 		No Mast cell granule, liver, lung, skin
Group IV
Keratan sulfate I and II 10,000–30,000 Galactose-β-1,4- N -acetyl-D-glucosamine-6-SO 4 Yes Cornea (keratan sulfate I), cartilage (keratan sulfate II)

Because the amino sugar is usually sulfated and these sugars also have carboxyl groups projecting from them, they are negatively charged and, thus, attract cations, such as sodium (Na + ).

A high-sodium concentration in the ground substance attracts extracellular fluid, which (by hydrating the intercellular matrix) assists in the resistance to forces of compression. As these molecules come into close proximity to each other, their negative charges repel one another, which gives them a slippery texture, as evidenced by the slickness of mucus (such as the mucus of the nasal cavity), vitreous humor of the eye, and synovial fluid.

With the exception of hyaluronic acid, the major GAGs of ECM are sulfated, each consisting of fewer than 300 repeating disaccharide units (see Table 4.1 ). The sulfated GAGs include keratan sulfate , heparan sulfate , heparin , chondroitin 4-sulfate , chondroitin 6-sulfate , and dermatan sulfate . These GAGs are usually linked covalently to protein molecules to form proteoglycans. The only nonsulfated GAG, hyaluronic acid ( hyaluronan ), may have as many as 10,000 repeating disaccharide units. It is a very large macromolecule (up to 10,000 kDa) that does not form covalent links to protein molecules (although proteoglycans do become attached to it via link proteins). All GAGs are synthesized within the Golgi apparatus by resident enzymes except for hyaluronic acid, which is synthesized as a free linear polymer at the cytoplasmic face of the plasma membrane by hyaluronan synthases . These enzymes are integral membrane proteins that not only catalyze the polymerization but also facilitate the transfer of the newly formed macromolecule into the ECM. It has been suggested that hyaluronic acid also has intracellular functions. Some of the newly released hyaluronic acid is endocytosed by some cells, especially during the cell cycle, where it appears to have a role in maintaining space and modulating microtubular activities during metaphase and anaphase stages of mitosis, thus, facilitating chromosomal movements. Additional intracellular roles may involve the directing of intracellular trafficking and influencing intracytoplasmic- and intranuclear-specific kinases.

Proteoglycans

Proteoglycans constitute a family of macromolecules; each is composed of a protein core to which GAGs are covalently bonded.

When sulfated GAGs form covalent bonds with a protein core, they form a family of macromolecules known as proteoglycans , many of which occupy huge domains. These large structures look like a bottle brush, with the protein core resembling the wire stem and the various sulfated GAGs projecting from its surface in three-dimensional space, as do the bristles of the brush ( Fig. 4.3 ).

Fig. 4.3, Schematic diagram of the association of aggrecan molecules with collagen fibers. The inset displays a higher magnification of the aggrecan molecule, indicating the core protein of the proteoglycan molecule to which glycosaminoglycans are attached. The core protein is attached to the hyaluronic acid by link proteins.

Proteoglycans range from about 50,000 Da ( decorin and betaglycan ) to as large as 3 million Da ( aggrecan ). When the protein cores of proteoglycans, manufactured on the rough endoplasmic reticulum (RER), reach the Golgi apparatus, resident enzymes there covalently bind bridge tetrasaccharides (a series of four saccharides) to its serine side chains. Then, the GAG is assembled by the addition of sugars one at a time. Sulfation, catalyzed by sulfotransferases and epimerization (rearrangement of various groups around the carbon atoms of the sugar units), also occurs in the Golgi apparatus.

Many proteoglycans, especially aggrecan , a macromolecule present in cartilage and connective tissue proper, attach to hyaluronic acid (see Fig. 4.3 ). The mode of attachment involves a noncovalent ionic interaction between the sugar groups of the hyaluronic acid and the core protein of the proteoglycan molecule. The connection is reinforced by small link proteins that form bonds with both the core protein of aggrecan and the sugar groups of hyaluronic acid. Because hyaluronic acid may be as much as 20 μm in length, the result of this association is an aggrecan composite that occupies a very large volume and may have a molecular mass as large as several hundred million daltons. This immense molecule is responsible for the gel state of the ECM and acts as a barrier to fast diffusion of aqueous deposits, as when one observes the slow disappearance of an aqueous bubble after its subdermal injection.

Clinical Correlations

Many pathogenic bacteria, such as Staphylococcus aureus , secrete hyaluronidase , an enzyme that cleaves hyaluronic acid into numerous small fragments, thus converting the gel state of the ECM to a sol (liquid) state. The consequence of this reaction is to permit the rapid spread of the bacteria through the connective tissue spaces. This is the case in the condition known as necrotizing fasciitis , when methicillin-resistant Staphylococcus aureus , frequently in combination with other microorganisms, such as Streptococcus pyogenes and/or one of the Clostridium species, enters the connective tissue spaces through a wound and destroys the gel-like state of the connective tissue, permitting rapid spread of the infection. Most of the patients afflicted by necrotizing fasciitis are older, immunosuppressed, or diabetic. Others afflicted have chronic diseases or are abusers of alcohol, tobacco, or drugs. However, about 25% to 30% of patients are healthy and have no predisposing factors in their medical history. If the condition is discovered and diagnosed early enough in the infection process and extensive debridement is performed along with the administration of appropriate antibiotic therapy, the patient’s prognosis is very good. Contrary to popular belief, necrotizing fasciitis is not a new disease; its symptoms have been described over 2500 years ago in the fifth century BCE by Hippocrates.

Functions of Proteoglycans

By occupying a large volume, proteoglycans resist compression and retard the rapid movement of microorganisms and metastatic cells. However, in the same fashion, they facilitate normal cellular locomotion by permitting migrating cells to move into the space that these hydrated macromolecules occupied. Proteoglycans, in association with the basal lamina, form molecular filters of varying pore sizes and charge distributions that selectively screen and retard macromolecules as they pass through them.

Proteoglycans also possess binding sites for certain signaling molecules, whereby they can either prevent them from reaching their destinations or they can enhance the function of signaling molecules by concentrating them in a specific location near their targets. Proteoglycans, such as decorins, assist in the formation of collagen fibers; skins of mice that cannot produce decorins or those that produce defective decorins have reduced tensile strength.

Some proteoglycans, such as syndecans , instead of being released into the ECM remain attached to the cell membrane. The core proteins of syndecans act as transmembrane proteins and are attached to the actin filaments of the cytoskeleton. Their extracellular moieties bind to components of the ECM, thus permitting the cell to become attached to macromolecular components of the matrix. In addition, syndecans of fibroblasts function as coreceptors because they bind fibroblast growth factor and present it to cell membrane fibroblast growth factor receptors in their vicinity.

Cell Adhesive Glycoproteins (Glycoproteins)

Cell adhesive glycoproteins have binding sites for several components of the ECM, as well as for integrin molecules of the cell membrane that facilitate the attachment of cells to the ECM.

Cell adhesive glycoproteins are large macromolecules that have several domains, at least one of which usually binds to cell surface proteins called integrins , one to collagen fibers, and one to proteoglycans. In this manner, adhesive glycoproteins not only assist cells to adhere to the extracellular matrix but also aid in fastening the various components of tissues to each other. The major types of adhesive glycoproteins are fibronectin, laminin, entactin, tenascin, chondronectin, and osteonectin ( Table 4.2 ).

TABLE 4.2
The Major Types of Cell Adhesive Glycoproteins
Glycoprotein Size (Da) Location Function
Fibronectin 440,000 Connective tissue Assists cells in binding to the extracellular matrix
Laminin 950,000 Basal laminae and external laminae Binds cells to basal lamina and external lamina
Entactin 150,000 Basal laminae and external laminae Binds laminin to type IV collagen
Tenascin 250,000–300,000 Embryonic connective tissue Assists cells in binding to the extracellular matrix during their migration
Chondronectin 40,000 Cartilage Facilitates the binding of cartilage cells to their matrix
Osteonectin 40,000 Bone Facilitates the binding of bone cells to their matrix; assists in bone matrix mineralization

Fibronectin is a large, V-shaped dimer about 440,000 Da in molecular weight composed of two similar polypeptide subunits that are attached to each other at their carboxyl ends by disulfide bonds. Each subunit has binding sites for various extracellular components (e.g., collagen, heparin, heparan sulfate, and hyaluronic acid) and for specific fibronectin receptors ( integrins ), of the cell membrane. Fibronectin is produced mainly by connective tissue cells known as fibroblasts . The actin components of the cytoskeleton of these cells and their associated myosin counterparts interact, placing tension on their plasmalemma. The integrin molecules relay the tensile forces to the newly exocytosed fibronectin molecules, stretching them just enough to expose hidden binding sites that permit fibronectins to bind to each other, thus forming the fibronectin matrix.

Fibronectin is also present in blood as plasma fibronectin , where it facilitates wound healing, phagocytosis, and coagulation. Fibronectin may be temporarily attached to the plasma membrane as cell-surface fibronectin . In the embryo, fibronectin marks migratory pathways for cells so that the migrating cells of the developing organism can reach their destination.

Laminin is a very large glycoprotein (950,000 Da) composed of three large polypeptide chains: A, B 1 , and B 2 . The B chains wrap around the A chain, forming a cross-like pattern, held in position by disulfide bonds at the point where the three chains diverge from each other, thereby forming the two arms and head of the cross-like pattern. There are at least 15 different types of laminins, depending on the amino acid composition of the three chains. The location of laminin is almost strictly limited to basal laminae (and external laminae); therefore, this glycoprotein has binding sites for heparan sulfate, type IV collagen, entactin, and the cell membrane.

Clinical Correlations

In nephritic syndrome , the presence of an abnormal laminin results in the inability of the proximal tubules of nephrons from preventing proteins from entering urine. The symptoms of this condition include swollen ankles, feet, and the region of the eyes; reduced appetite; foamy urine; fatigue; and weight gain. Diagnosing of the condition is done by urine and blood tests to look for proteinurea, as well as hypoalbuminemia.

Entactin , a sulfated glycoprotein (also known as nidogen ) is about 150,000 Da in weight. It binds to the laminin molecule where the three short arms of that molecule meet each other. Entactin also binds to type IV collagen, thus facilitating the binding of laminin to the collagen meshwork.

Tenascin is a large glycoprotein (250,000–300,000 Da) composed of six polypeptide chains held together by disulfide bonds. It resembles an insect whose six legs project radially from a central body and has binding sites for the transmembrane proteoglycan syndecan and for fibronectin. Tenascin’s distribution is usually limited to embryonic tissue, where it marks migratory pathways for specific cells.

Chondronectin and osteonectin (about 40,000 Da) are similar to fibronectin. The former has binding sites for type II collagen, chondroitin sulfates, hyaluronic acid, and integrins of chondroblasts and chondrocytes. Osteonectin possesses domains for type I collagen, proteoglycans, and integrins of osteoblasts and osteocytes. In addition, it may facilitate the binding of calcium hydroxyapatite crystals to type I collagen in bone.

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