Histology_Ross

Histology_Ross. In two general locations, surface epithelium and its un-derlying connective tissue are regarded as a functional unit called a membrane. The two types of membrane are mucus membrane and serous membrane. The term membrane as used here should not be confused with the biologic membranes of cells, nor should the designations mucus and serous be confused with the nature of the gland secretion as discussed above. Mucus membrane, also called mucosa, lines those cavities that connect with the outside of the body, namely, the alimentary canal, the respiratory tract, and the geni-tourinary tract. It consists of surface epithelium (with or without glands), a supporting connective tissue called the lamina propria, a basement membrane separating the epithelium from the lamina propria, and sometimes a layer of smooth muscle called the muscularis mucosae as the deepest layer. Serous membrane, also called serosa, lines the peri-toneal, pericardial, and pleural cavities. These cavities are usually


Histology_Ross

Histology_Ross. a layer of smooth muscle called the muscularis mucosae as the deepest layer. Serous membrane, also called serosa, lines the peri-toneal, pericardial, and pleural cavities. These cavities are usually described as closed cavities of the body, although in the female the peritoneal cavity communicates with the exterior via the genital tract. Structurally, the serosa con-sists of a lining epithelium, the mesothelium, a support-ing connective tissue, and a basement membrane between the two. Serous membranes do not contain glands, but the fluid on their surface is watery.


Histology_Ross

Histology_Ross.  FOLDER 5.5 Functional Considerations: Mucus and Serous Membranes FIGURE 5.42 • Autoradiograph of intestinal gland (crypt). Autoradiograph of crypts in the jejunum of a rabbit that had been injected with tritiated thymidine 8 hours before death and fixation. Nearly all of the epithelial cells in this replicative zone of the intestinal mucosa are labeled, indicating that they were synthesizing DNA at the time the animal was injected. 600. (Reprinted with permission from Parker FG, Barnes EN, Kaye GI. The pericryptal fibroblast sheath. IV. Replication, migration, and differentiation of the subepithelial fibroblasts of the crypt and villus of the rabbit jejunum. Gastroenterology 1974;67: 607–621.) trauma or acute toxic destruction is accommodated by active proliferation of undamaged liver cells. The liver tissue is essentially restored by the stimulated mitotic activity of healthy liver tissue.


Histology_Ross

Histology_Ross. Epithelium consists of a diverse group of cell types, each of which possesses specific functional characteristics. The cells that make up a given epithelium are arranged in close apposition with one another and typically are located at what may be described as the free surfaces of the body. Such free surfaces include the exterior of the body, the outer surface of many internal organs, and the lining of body cavities, tubes, and ducts. Epithelium is classified on the basis of the arrangement of the cells that it contains and their shape. If the cells are present in a single layer, they constitute a simple epithelium. If they are present in multiple layers, they constitute a stratified epithelium. The shape of the cells is typically described as squamous, if the cell is wider than it is tall; cuboidal, if its height and width are approximately the same; or columnar, where the cell is taller than it is wide.


Histology_Ross

Histology_Ross. Simple squamous epithelium, mesovarium, human, body. The mesothelial cells (MC) are recognized by their nuclei at this low H&E, x350; inset x875. magnification. Beneath the mesothelial cells is a thin layer of connective tissue (CT) and adipose cells (A). The inset reveals at higher magnification the This micrograph shows the surface epithelium of the mesovarnuclei (N) of the mesothelial cells. ium covered by mesothelium, a name given to the simple squamous epithelium that lines the internal cavities of the Simple squamous epithelium, mesentery, rat, silver impregnation, x350; inset x700.


Histology_Ross

Histology_Ross. ium covered by mesothelium, a name given to the simple squamous epithelium that lines the internal cavities of the Simple squamous epithelium, mesentery, rat, silver impregnation, x350; inset x700. This is an intermediate magnification of a whole mount of a piece of mesentery. The mesentery was placed on the slide and prepared for microscopic examination. The microscope was focused at the surface of the mesentery. By this method, the boundaries of the surface mesothelial cells are delineated as black lines by the precipitated silver. Note that the cells are in close apposition to one another and that they have a polygonal shape. The inset reveals several mesothelial cells, each of which exhibits a nucleus (N) that has a round, or oval profile. Because of the squamous shape of the mesothelial cells, the nuclei are not spherical, but rather are disc-like. Simple squamous epithelium, kidney, human, H&E, x350.


Histology_Ross

Histology_Ross. Simple squamous epithelium, kidney, human, H&E, x350. This micrograph shows a kidney renal corpuscle. The wall of the renal corpuscle, known as the parietal layer of Bowman’s capsule, is a spherical structure that consists of a simple squaous epithelium (SSE). The interior of the corpuscle Simple cuboidal epithelium, pancreas, human, H&E, x700. This photomicrograph shows two pancreatic ducts (PD) that are lined by a simple cuboidal epithelium. The duct cell nuclei (N) tend to be spherical, a feature consistent with the Simple cuboidal epithelium, lung, human, H&E, x175; inset x525.


Histology_Ross

Histology_Ross. Simple cuboidal epithelium, lung, human, H&E, x175; inset x525. This photomicrograph shows the epithelium of the smallest conducting bronchioles of the lung. The simple cuboidal epithelium consists of cuboidal cells (CC). The inset shows a higher contains a capillary network from which fluid is filtered to enter the urinary space (US) and then into the proximal convoluted tubule (PCT). Nuclei (N) of the squamous cells of the parietal layer of Bowman’s capsule are ovoid and appear to protrude slightly into the urinary space. The free surface of this simple squamous epithelium faces the urinary space, whereas the basal surface of the epithelial cells rests on a layer of connective tissue (CT). cuboidal shape of the cell. The free surface of the epithelial cells face the lumen of the duct and the basal surface rests on connective tissue (CT). Careful examination of the free surface of the epithelial cells reveals some of the terminal bars (TB) between adjacent cells.


Histology_Ross

Histology_Ross. magnification of the cuboidal cells (CC). Note the spherical nuclei. These are small cells with relatively little cytoplasm, thus the nuclei appear close to one another. The free surface of the epithelial cells face the airway (AW), whereas the basal surface of these cells rests on its basement membrane and underlying dense connective tissue (CT). Simple cuboidal epithelium, liver, human, H&E, x450; inset x950. This micrograph reveals cords of hepatocytes (H), simple cuboidal cells that make up the liver parenchyma. The hepatic epithelial cell cords are mostly separated from one another by blood sinusoids (S). The inset shows a higher magnification of a hepatic cell and reveals an unusual feature in that several surfaces of these cells posses a groove representing the free surface of the cell. Where the groove of one cell faces a groove of the adjacent cell, a small canal-like structure, the canaliculus (C), is formed. Bile is secreted from the cell into the canaliculus.


Histology_Ross

Histology_Ross. PLATE 1 KEY A, adipose tissue AW, airway C, canaliculus CC, cuboidal cells CT, connective tissue H, hepatocytes MC, mesothelial cells N, nucleus PCT, proximal convoluted tubule PD, pancreatic duct S, sinusoid SSE, simple squamous epithelium TB, terminal bar US, urinary space Simple epithelia are only one cell layer thick. They are characteristic of organ systems primarily concerned with transport, absorption, and secretion, such as the intestine, the vascular system, the digestive glands and other exocrine glands, and the kidney. Stratified epithelia have more than one layer and are typical of surfaces that are subject to frictional stress, such as skin, oral mucosa and esophagus, and vagina. Simple epithelium, exocrine pancreas, monkey, H&E ×450.


Histology_Ross

Histology_Ross. This shows three epithelial forms. In the circle is a well-oriented acinus, a functional group of secretory cells, each of which is pyramidal in shape. The secretory cells form a spherical or tubular structure. The free surface of the cells and the lumen are located in the center of the circle. The lumen is not evident here but is evident in a similar cell arrangement in the middle right image below (see circle). Because the height of the cells (the distance from the edge of the circle to the lumen) is greater than the width, the epithelium is simple columnar. The second epithelial type is represented by a small, longitudinally sectioned duct (arrows) extending across the field. It is composed of flattened cells (note the nuclear shape), and on this basis, the epithelium is simple squamous. Finally, there is a larger cross-sectioned duct (asterisk) into which the smaller duct enters. The nuclei of this larger duct tend to be round, and the cells tend to be square in profile. Thus,


Histology_Ross

Histology_Ross. Finally, there is a larger cross-sectioned duct (asterisk) into which the smaller duct enters. The nuclei of this larger duct tend to be round, and the cells tend to be square in profile. Thus, these duct cells are a simple cuboidal epithelium.


Histology_Ross

Histology_Ross. Simple cuboidal epithelium, kidney, human, of a simple cuboidal epithelium. The arrows point to the lateral cell H&E ×450. boundaries; note that cell width approximates cell height. The cross-sectioned structures marked with asterisks are another type of tubule; they are This section shows cross-sectioned tubules of several types. smaller in diameter but are also composed of a simple cuboidal epithelium. Those that are labeled with the arrows provide another example Simple columnar epithelium, colon, human, H&E ×350. The simple columnar epithelium of the colon shown here consists of a single layer of absorptive cells and mucus-secreting cells (goblet cells). The latter can be recognized by the light Pseudostratifed epithelium, trachea, monkey, H&E ×450.


Histology_Ross

Histology_Ross. Pseudostratifed epithelium, trachea, monkey, H&E ×450. In addition to the tall columnar cells (CC) in this columnar epithelium, there is a definite layer of basal cells (BC). The columnar cells, which contain elongate nuclei and possess cilia (C), extend from the surface to the basement membrane (clearly visible in the trachea as a thick, acellular, homogeneous region that is part of the connective tissue (CT )). The basal cells are interspersed Pseudostratifed epithelium, epididymis, human, H&E ×450.


Histology_Ross

Histology_Ross. Pseudostratifed epithelium, epididymis, human, H&E ×450. This is another example of pseudostratified columnar epithelium. Again, two layers of nuclei are evident, those of basal cells (BC) and those of columnar cells (CC). As in the previous example, however, although not evident, the columnar cells staining “goblet” (arrows) that contains the cell’s secretory product. The epithelium lines the lumen of the colon and extends down into the connective tissue to form the intestinal glands (GL). Both cell types are tall with their nuclei located at the base of the cell. The connective tissue (CT) contains numerous cells, many of which are lymphocytes and plasma cells.


Histology_Ross

Histology_Ross. between the columnar cells. Because all of the cells rest on the basement membrane, they are regarded as a single layer, as opposed to two discrete layers, one over the other. Because the epithelium appears to be stratified but is not, it is called pseudostratified columnar epithelium. The circle in the micrograph delineates a tracheal gland similar to the acinus in exocrine pancreas (circle). Note that the lumen of the gland is clearly visible and the cell boundaries are also evident. The gland epithelium is simple columnar. rest on the basement membrane; thus, the epithelium is pseudostratified. Note that where the epithelium is vertically oriented, on the right of the micrograph, there appear to be more nuclei, and the epithelium is thicker. This is a result of a tangential plane of section. As a rule, always examine the thinnest area of an epithelium to visualize its true organization.


Histology_Ross

Histology_Ross. Stratifed squamous epithelium, vagina, human, small, with little cytoplasm, and thus the nuclei appear closely packed. As H&E ×225. the cells become larger, they tend to flatten out, forming disc-like squames. Because the surface cells retain this shape, the epithelium is This is the stratified squamous epithelium of the vaginal called stratified squamous. wall. The deeper cells, particularly those of the basal layer, are KEY BC, basal cell C, cilia CC, columnar cell CT, connective tissue GL, intestinal gland arrows: upper left, duct composed of simple squamous epithelium; upper right, lateral boundaries of cuboidal tubule cells; middle left, mucus cups of goblet cells asterisk, duct or tubule of simple cuboidal epithelium


Histology_Ross

Histology_Ross. Tissues that resemble epithelia but lack the characteristic free surface are designated epithelioid tissues. This is the characteristic structure of the endocrine organs, which develop from typical epithelia but lose their connection to a surface during development. Stratifed epithelia, esophagus, monkey, H&E ×250. This part of the wall of the esophagus reveals two different epithelia. On the left is the lining epithelium of the esophagus. It is multilayered with squamous surface cells; therefore, it is a stratified squamous epithelium (SS). On the right is the duct of an esophageal gland cut in several planes. By examining a region where the plane of section is at a right angle to the surface, the true character of the epithelium becomes apparent. In this case, the epithelium consists of two cell layers with cuboidal surface cells; thus, it is stratified cuboidal epithelium (StCu).


Histology_Ross

Histology_Ross. Stratifed epithelia, skin, human, H&E ×450. (StCu) in two layers; the cells of the inner layer (the surface cells) appear more or less square. Because the epidermal surface cells are not included in This shows a portion of the duct of a sweat gland just before the field, the designation stratified squamous cannot be derived from the in- the duct enters the stratified squamous epithelium (SS) of formation offered by the micrograph. the skin. The dashed line traces the duct within the epidermis. This duct also consists of a stratified cuboidal epithelium PLATE 3 • STRATI FI E D E PITH E LIA AN D E PITH E LIOI D TI SS U E S KEY C, capillary CT, connective tissue En, endocrine cells Ex, exocrine cells IC, interstitial (Leydig) cells SCol, simple columnar epithelium SS, stratified squamous epithelium StCu, stratified cuboidal epithelium arrowhead, transition site of simple stratified epithelium to stratified cuboidal arrows, surface cuboidal cells asterisks, dome-shaped cells


Histology_Ross

Histology_Ross. Epithelial transition, anorectal junction, human, H&E ×300. The area shown here is the terminal part of the large intestine. The luminal epithelium on the left is typical simple columnar epithelium (SCol) of the colon. This epithelium undergoes an abrupt transition (arrowhead) to a stratified cuboidal epithelium (StCu) at the anal canal. Note the general cuboidal Transitional epithelium (urothelium), urinary bladder, monkey, H&E ×400. The epithelium of the urinary bladder is called transitional epithelium, a stratified epithelium that changes in appearance according to the degree of distension of the bladder. In the nondistended state, as here, it is about four or five cells deep. The surface cells are large and dome shaped (asterisks). The cells Epithelioid tissues, testis, monkey, H&E ×350.


Histology_Ross

Histology_Ross. Epithelioid tissues, testis, monkey, H&E ×350. This shows the intestinal (Leydig) cells of the testis (IC). These cells possess certain epithelial characteristics. They do not possess a free surface, however, nor do they develop from a surface; shape of most of the surface cells (arrows) and the underlying layers of cells. The simple columnar epithelium on the left is part of an intestinal gland that is continuous with the simple columnar epithelium at the intestinal luminal surface. The connective tissue (CT) at this site is heavily infiltrated with lymphocytes, giving it an appearance unlike the connective tissue of other specimens on this page.


Histology_Ross

Histology_Ross. immediately under the surface cells are pear shaped and slightly smaller. The deepest cells are the smallest, and their nuclei appear more crowded. When the bladder is distended, the superficial cells are stretched into squamous cells, and the epithelium is reduced in thickness to about three cells deep. The bladder wall usually contracts when it is removed, unless special steps are taken to preserve it in a distended state. Thus, its appearance is usually like that in Figure 4. instead, they develop from mesenchymal cells. They are referred to as epithelioid cells because they contact similar neighboring cells much the same as epithelial cells contact each other. Leydig cells are endocrine in nature. Epithelioid tissues, endocrine pancreas, human, H&E ×450.


Histology_Ross

Histology_Ross. Epithelioid tissues, endocrine pancreas, human, H&E ×450. Cells of the endocrine islet (of Langerhans) (En) of the pancreas also have an epithelioid arrangement. The cells are in contact but lack a free surface, although they have developed from an epithelial surface by invagination. In contrast, the surrounding alveoli of the exocrine pancreas (Ex), which developed from the same epithelial surface, are made up of cells with a free surface onto which the secretory product is discharged. Capillaries (C) are prominent in endocrine tissues. Similar examples of epithelioid tissue are seen in the adrenal and the parathyroid and pituitary glands, all of which are endocrine glands.


Histology_Ross

Histology_Ross. GENERAL STRUCTURE AND FUNCTION OF CONNECTIVE TISSUE / 158 EMBRYONIC CONNECTIVE TISSUE / 159 CONNECTIVE TISSUE PROPER / 160 CONNECTIVE TISSUE FIBERS / 161 Collagen Fibers and Fibrils / 161 Biosynthesis and Degradation of Collagen Fibers / 164 Reticular Fibers / 171 Elastic Fibers / 171 EXTRACELLULAR MATRIX / 173 CONNECTIVE TISSUE CELLS / 178 Fibroblasts and Myofibroblasts / 178 Macrophages / 181 Mast Cells / 182 Basophils / 187 Adipocytes / 187 Adult Stem Cells and Pericytes / 187 Lymphocytes, Plasma Cells, and Other Cells of the Immune System / 189 Folder 6.1 Clinical Correlation: Collagenopathies / 170 Folder 6.2 Clinical Correlation: Sun Exposure and Molecular Changes in Photoaged Skin / 173 Folder 6.3 Clinical Correlation: Role of Myofibroblasts in Wound Repair / 183 Folder 6.4 Functional Considerations: The Mononuclear Phagocytotic System / 185 Folder 6.5 Clinical Correlation: The Role of Mast Cells and Basophils in Allergic Reactions / 188


Histology_Ross

Histology_Ross. Connective tissue comprises a diverse group of cells within a tissue-specific extracellular matrix. In general, connective tissue consists of cells and an extracellular matrix (ECM). ECM includes structural (fibers) and specialized proteins that constitute the ground substance. Connective tissue forms a vast and continuous compartment throughout the body, bounded by the basal laminae of the various epithelia and by the basal or external laminae of muscle cells and nerve-supporting cells. Different types of connective tissue are responsible for a variety of functions.


Histology_Ross

Histology_Ross. The functions of the various connective tissues are reflected in the types of cells and fibers present within the tissue and the composition of the ground substance in the ECM. For example, in loose connective tissue, many cell types are present (Fig. 6.1). One type, the fibroblast, produces the extracellular fibers that serve a structural role in the tissue. Fibroblasts also produce and maintain the ground substance. Other cell types, such as lymphocytes, plasma cells, macrophages, and eosinophils, are associated with the body’s defense system; they function within the ECM of the tissue. In contrast, bone tissue, another form of connective tissue, contains only a single cell type, the osteocyte. This cell produces the fibers that make up the bulk of bone tissue. A unique feature of bone is that its fibers are organized in a specific pattern and become calcified to create the hardness associated with this tissue. Similarly, in tendons and ligaments, fibers are the prominent feature of


Histology_Ross

Histology_Ross. that its fibers are organized in a specific pattern and become calcified to create the hardness associated with this tissue. Similarly, in tendons and ligaments, fibers are the prominent feature of the tissue. These fibers are arranged in parallel array and are densely packed to impart maximum strength.


Histology_Ross

Histology_Ross. Classification of connective tissue is based on the composition and organization of its cellular and extracellular components and on its functions. Connective tissue encompasses a variety of tissues with differing functional properties but with certain common characteristics that allow them to be grouped together. For


Histology_Ross

Histology_Ross. FIGURE 6.1 • Loose connective tissue. a. Photomicrograph of a mesentery spread stained with Verhoeff’s hematoxylin to show nuclei and elastic fibers; it has been counterstained with safranin for identification of mast cell granules and with orange G for identification of other proteins (mainly collagen fibers). The elastic fibers appear as blue-black, thin, long, and branching threads without discernible beginnings or endings. Collagen fibers appear as orange-stained, long, straight profiles, and are considerably thicker than the elastic fibers. Most of the visible nuclei are presumed to be those of fibroblasts. Nuclei of other cell types (e.g., lymphocytes, plasma cells, and macrophages) are also present but are not identifiable. Mast cells are identified by the bright reddish granules within their cytoplasm. Note the presence of the small blood vessel filled with red blood cells. 150. b. Schematic diagram illustrating the components of loose connective tissue. Note the association


Histology_Ross

Histology_Ross. within their cytoplasm. Note the presence of the small blood vessel filled with red blood cells. 150. b. Schematic diagram illustrating the components of loose connective tissue. Note the association of different cell types with the surrounding extracellular matrix, which contains blood vessels and different types of fibers.


Histology_Ross

Histology_Ross. TABLE Classification of Connective Tissue6.1 Embryonic connective tissue Mesenchyme Mucous connective tissue Connective tissue proper Loose connective tissue Dense connective tissue Regular Irregular Specialized connective tissuea Cartilage (Chapter 7) Blood (Chapter 10) Bone (Chapter 8) Hemopoietic tissue (Chapter 10) Adipose tissue (Chapter 9) Lymphatic tissue (Chapter 14) aIn the past, the designations elastic tissue and reticular tissue have been listed as separate categories of specialized connective tissue. The tissues usually cited as examples of elastic tissue are certain ligaments associated with the spinal column and the tunica media of elastic arteries. The identifying feature of reticular tissue is the presence of reticular fibers and reticular cells together forming a three-dimensional stroma. Reticular tissue serves as the stroma for hemopoietic tissue (specifically the red bone marrow) and lymphatic tissue organs (lymph nodes and spleen, but not the thymus).


Histology_Ross

Histology_Ross. tissues, including subtypes. , the middle embryonic germ layer, gives rise to alall of the connective tissues of the body. An exception is head region, where specific progenitor cells are derived a primitive connective tissue referred to as mes(in the head region, it is sometimes called ectomes) is established in the early embryo. Maturation and tissue is classified into two subtypes: Mesenchyme is primarily found in the embryo. It contains small, spindle-shaped cells of relatively uniform appearance (Fig. 6.2a). Processes extend from these cells and contact similar processes of neighboring cells, forming a three-dimensional cellular network. Gap junctions are present where the processes make contact. The extracellular space is occupied by a viscous ground substance. Collagen (reticular) fibers are present; they are very fine and relatively sparse. The paucity of collagen fibers is consistent with the limited physical stress on the growing fetus.


Histology_Ross

Histology_Ross. FIGURE 6.2 • Embryonic connective tissue. a. Photomicrograph of mesenchymal tissue from a developing fetus stained with H&E. Although morphologically the mesenchymal cells appear as a homogeneous population, they give rise to cells that will differentiate into various cell types. Their cytoplasmic processes often give the cell a tapering or spindle appearance. The extracellular component of the tissue contains a sparse arrangement of reticular fibers and abundant ground substance. 480. b. Photomicrograph of Wharton’s jelly from the umbilical cord stained with H&E. Wharton’s jelly consists of a specialized, almost gelatinlike ground substance that occupies large intercellular spaces located between the spindle-shaped mesenchymal cells. 480.


Histology_Ross

Histology_Ross.  Mucous connective tissue is present in the umbilical cord. It consists of a specialized, almost gelatinlike ECM; its ground substance is frequently referred to as Wharton’s jelly. The spindle-shaped cells are widely separated and appear much like fibroblasts in the near-term umbilical cord (e.g., the cytoplasmic processes are thin and difficult to visualize in routine hematoxylin and eosin [H&E] preparation). Wharton’s jelly occupies large inter-cellular spaces located between thin, wispy collagen fibers (Fig. 6.2b). Connective tissues that belong to this category are divided into two general subtypes:  Loose connective tissue, sometimes called areolar tissue, and  dense connective tissue, which can be further subcategorized into two basic types based on the organization of its collagen fibers: dense irregular connective tissue and dense regular connective tissue. Loose connective tissue is characterized by loosely arranged fibers and abundant cells of various types.


Histology_Ross

Histology_Ross. Loose connective tissue is characterized by loosely arranged fibers and abundant cells of various types. Loose connective tissue is a cellular connective tissue with thin and relatively sparse collagen fibers (Fig. 6.3). The FIGURE 6.3 • Loose and dense irregular connective tissue. Photomicrograph comparing loose and dense irregular connective tissue from the mammary gland stained with Masson’s trichrome. In the center, loose connective tissue surrounds the glandular epithelium. The loose connective tissue is composed of a wispy arrangement of collagen fibers with many cells. Note the large number of nuclei visible at this low magnification. On the upper left and lower right of the figure is dense irregular connective tissue. In contrast, few nuclei are revealed in the dense connective tissue. However, collagen is considerably more abundant and is composed of very thick fibers. 100.


Histology_Ross

Histology_Ross. ground substance, however, is abundant; in fact, it occupies more volume than the fibers do. It has a viscous to gel-like consistency and plays an important role in the diffusion of oxygen and nutrients from the small vessels that course through this connective tissue as well as in the diffusion of carbon dioxide and metabolic wastes back to the vessels.


Histology_Ross

Histology_Ross. Loose connective tissue is primarily located beneath the epithelia that cover the body surfaces and line the internal surfaces of the body. It is also associated with the epithelium of glands and surrounds the smallest blood vessels (Plate 4, page 192). This tissue is thus the initial site where pathogenic agents such as bacteria that have breached an epithelial surface are challenged and destroyed by cells of the immune system. Most cell types in loose connective tissue are transient wandering cells that migrate from local blood vessels in response to specific stimuli. Loose connective tissue is, therefore, the site of inﬂammatory and immune reactions. During these reactions, loose connective tissue can swell considerably. In areas of the body where foreign substances are continually present, large populations of immune cells are maintained. For example, the lamina propria, the loose connective tissue of mucous membranes, such as those of the respiratory and alimentary systems,


Histology_Ross

Histology_Ross. present, large populations of immune cells are maintained. For example, the lamina propria, the loose connective tissue of mucous membranes, such as those of the respiratory and alimentary systems, contains large numbers of these cells.


Histology_Ross

Histology_Ross. Dense irregular connective tissue is characterized by abundant fibers and few cells.


Histology_Ross

Histology_Ross. Dense irregular connective tissue contains mostly collagen fibers. Cells are sparse and are typically of a single type, the fibroblast. This tissue also contains relatively little ground substance (Plate 4, page 192). Because of its high pro portion of collagen fibers, dense irregular connective tissue provides significant strength. Typically, the fibers are arranged in bundles oriented in various directions (thus, the term irregular) that can withstand stresses on organs or structures. Hollow organs (e.g., the intestinal tract) possess a distinct layer of dense irregular connective tissue called the submucosa in which the fiber bundles course in varying planes. This arrangement allows the organ to resist excessive stretching and distension. Similarly, skin contains a relatively thick layer of dense irregular connective tissue called the reticular layer (or deep layer) of the dermis. The reticular layer provides resistance to tearing as a consequence of stretching forces from


Histology_Ross

Histology_Ross. layer of dense irregular connective tissue called the reticular layer (or deep layer) of the dermis. The reticular layer provides resistance to tearing as a consequence of stretching forces from different directions.


Histology_Ross

Histology_Ross. Dense regular connective tissue is characterized by ordered and densely packed arrays of fibers and cells. Dense regular connective tissue is the main functional component of tendons, ligaments, and aponeuroses. As in dense irregular connective tissue, the fibers of dense regular connective tissue are the prominent feature, and there is little ECM. However, in dense regular connective tissue, the fibers are arranged in parallel array and are densely packed to provide maximum strength. The cells that produce and maintain the fibers are packed and aligned between fiber bundles.  Tendons are cordlike structures that attach muscle to bone. They consist of parallel bundles of collagen fibers.


Histology_Ross

Histology_Ross. Situated between these bundles are rows of fibroblasts called tendinocytes (Fig. 6.4 and Plate 5, page 194). Tendinocytes are surrounded by a specialized ECM that separates them from the load-bearing collagen fibrils. In H&E–stained cross sections of tendon, the tendinocytes appear stellate. In transmission electron micrograph (TEM) sections parallel to the long axis of tendons, the cytoplasmic projections of the cell are seen to lie be tween the fibers and appear as thin cytoplasmic sheets. In most H&E–stained longitudinal sections, however, tendinocytes appear only as rows of typically flattened basophilic nuclei. The cytoplasmic sheets that extend from the body of the tendinocytes are not usually evident in longitudinal H&E–stained sections because they blend in with the collagen fibers. The substance of the tendon is surrounded by a thin connective tissue capsule, the epitendineum, in which the collagen fibers are not nearly as orderly (Plate 5, page 194). Typically, the tendon is


Histology_Ross

Histology_Ross. The substance of the tendon is surrounded by a thin connective tissue capsule, the epitendineum, in which the collagen fibers are not nearly as orderly (Plate 5, page 194). Typically, the tendon is subdivided into fascicles by endotendineum, a connective tissue extension of the epitendineum. It contains the small blood vessels and nerves of the tendon.


Histology_Ross

Histology_Ross.  Ligaments, like tendons, consist of fibers and fibroblasts arranged in parallel. The fibers of ligaments, however, are less regularly arranged than those of tendons. Ligaments join bone to bone, which in some locations, such as in the spinal column, requires some elasticity. Although collagen is the major extracellular fiber of most ligaments, some of the ligaments associated with the spinal column (e.g., ligamenta flava) contain many more elastic fibers and fewer collagen fibers. These ligaments are called elastic ligaments.


Histology_Ross

Histology_Ross.  Aponeuroses resemble broad, flattened tendons. Instead of fibers lying in parallel arrays, the fibers of aponeuroses are arranged in multiple layers. The bundles of collagen fibers in one layer tend to be arranged at a 90 angle to those in the neighboring layers. The fibers within each of the layers are arranged in regular arrays; thus, aponeurosis is a dense regular connective tissue. This orthogonal array is also found in the cornea of the eye and is responsible for its transparency. Connective tissue fibers are of three principal types. Connective tissue fibers are present in varying amounts, depending on the structural needs or function of the connective tissue. Each type of fiber is produced by fibroblasts and is composed of protein consisting of long peptide chains. The types of connective tissue fibers are Collagen fibers are the most abundant type of connective tissue fiber.


Histology_Ross

Histology_Ross. Collagen fibers are the most abundant type of connective tissue fiber. Collagen fbers are the most abundant structural components of the connective tissue. They are flexible and have a


Histology_Ross

Histology_Ross. FIGURE 6.4 • Dense regular connective tissue—tendon. a. Electron micrograph of a tendon at low magnification, showing tendinocytes (fibroblasts) and their thin processes (arrows) lying between the collagen bundles. 1,600. b. A tendinocyte with prominent profiles of rough endoplasmic reticulum (rER) is shown at higher magnification. The collagen fibers (C) can be resolved as consisting of very tightly packed collagen fibrils. The arrows indicate processes of tendinocytes. 9,500. Inset. Photomicrograph of a tendon. Note the orderly and regular alignment of the bundles of collagen fibers. Tendinocytes are aligned in rows between the collagen fibers. 200. (Electron micrographs modified from Rhodin J. Histology. New York: Oxford University Press, 1974.) remarkably high tensile strength. In the light microscope, collagen fibers typically appear as wavy structures of variable width and indeterminate length. They stain readily with eosin and other acidic dyes. They can also be colored with


Histology_Ross

Histology_Ross. light microscope, collagen fibers typically appear as wavy structures of variable width and indeterminate length. They stain readily with eosin and other acidic dyes. They can also be colored with the dye aniline blue used in Mallory’s connective tissue stain or with the dye light green used in Masson’s stain.


Histology_Ross

Histology_Ross. When examined with the TEM, collagen fibers appear as bundles of fine, threadlike subunits. These subunits are collagen fbrils (Fig. 6.5). Within an individual fiber, the collagen fibrils are relatively uniform in diameter. In different locations and at different stages of development, however, the fibrils differ in size. In developing or immature tissues, the fibrils may be as small as 15 or 20 nm in diameter. In dense, regular connective tissue of tendons or other tissues that are subject to considerable stress, they may measure up to 300 nm in diameter. Collagen fibrils have a 68-nm banding pattern.


Histology_Ross

Histology_Ross. When collagen fibrils stained with osmium or other heavy metals are examined with the TEM, they exhibit a sequence of closely spaced transverse bands that repeat every 68 nm along the length of the fibril (Fig. 6.5, inset). This regular banding pattern can also be observed on the surface of the collagen fibrils when they are examined with the atomic force microscope (AFM; Fig. 6.6). This banding pattern reflects the fibril’s subunit structure, specifically the size and shape of the collagen molecule and the arrangement of the molecules that form the fibril (Fig. 6.7). The collagen molecule (formerly called tropocollagen) measures about 300 nm long by 1.5 nm thick and has a head and a tail. Within each fibril, the collagen molecules align head to tail in overlapping rows with a gap between the molecules in each row and a onequarter-molecule stagger between adjacent rows. These gaps are clearly visible with the AFM (see Fig. 6.6). The strength of the fibril is created by the covalent


Histology_Ross

Histology_Ross. the molecules in each row and a onequarter-molecule stagger between adjacent rows. These gaps are clearly visible with the AFM (see Fig. 6.6). The strength of the fibril is created by the covalent bonds between the collagen molecules of adjacent rows, not the head-to-tail attachment of the molecules in a row. The banding pattern observed with the TEM (see Fig. 6.5, inset) is caused largely by osmium deposition in the space between the heads and tails of the molecules in each row.


Histology_Ross

Histology_Ross. FIGURE 6.5 • Collagen fbrils in dense irregular connective tissue. Electron micrograph of dense irregular connective tissue from the capsule of the testis of a young male. The threadlike collagen fibrils are aggregated in some areas (X ) to form relatively thick bundles; in other areas, the fibrils are more dispersed. 9,500. Inset. A longitudinal array of collagen fibrils from the same specimen seen at higher magnification. Note the banding pattern. The spacing of the arrows indicates the 68-nm repeat pattern. 75,000. Each collagen molecule is a triple helix composed of three intertwined polypeptide chains. A single collagen molecule consists of three polypeptides known as chains. The chains intertwine, forming a right-handed triple helix (see Fig. 6.7d). Every third amino acid in the chain is a glycine molecule, except at the ends of the chains. A hydroxyproline or hydroxylysine frequently precedes each glycine in the chain, and a proline frequently


Histology_Ross

Histology_Ross. FIGURE 6.6 • Collagen fbrils in dense irregular connective tissue. This atomic force microscopic image of type I collagen fibrils in the connective tissue shows the banding pattern on the surface of collagen fibrils. Note the random orientation of collagen fibrils that overlie and crisscross each other in the connective tissue matrix. 65,000. (Courtesy of Dr. Gabriela Bagordo, JPK Instruments AG, Berlin, Germany.) follows each glycine in the chain. Along with proline and hydroxyproline, the glycine is essential for the triple-helix conformation (see Fig. 6.7e). Associated with the helix are sugar groups that are joined to hydroxylysyl residues. Because of these sugar groups, collagen is properly described as a glycoprotein.


Histology_Ross

Histology_Ross. The chains that constitute the helix are not all alike. They vary in size from 600 to 3,000 amino acids. To date, a. Fibril D 68 b. Packing of (0.6 D) 0.4 D c. Collagen molecule 300 nm (4.4 D) 1.5 nm diameter d. Triple helix 10.4 nm (0.15 D) e. Typical sequence 1 of 1 and 2 1.74 nm .87 nm FIGURE 6.7 • Diagram showing the molecular character of a type 1 collagen fbril in increasing order of structure. a. A collagen fibril displays periodic banding with a distance (D) of 68 nm between repeating bands. b. Each fibril is composed of staggered collagen molecules. c. Each molecule is about 300 nm long and 1.5 nm in diameter. d. The collagen molecule is a triple helix. e. The triple helix consists of three chains. Every third amino acid of the chain is a glycine. The X position following glycine is frequently a proline, and the Y position preceding the glycine is frequently a hydroxyproline.


Histology_Ross

Histology_Ross. at least 42 types of chains encoded by different genes have been identified and mapped to loci on several different chromosomes. As many as 28 different types of collagens have been categorized on the basis of the combinations of chains they contain. These various collagens are classified by Roman numerals I to XXVIII according to the chronology of their discovery. A collagen molecule may be homotrimeric (consisting of three identical chains) or heterotrimeric (consisting of two or even three genetically distinct chains).


Histology_Ross

Histology_Ross. For example, type I collagen found in loose and dense connective tissue is heterotrimeric. Two of the chains, identified as 1, are identical, and one, identified as 2, is different. Thus, in collagen nomenclature it is designated [ 1(I)]2 2(I) (Table 6.2). Type II collagen is homotrimeric and present in hyaline and elastic cartilage, where it occurs as very fine fibrils. The collagen molecules of type II collagen are composed of three identical chains. Because these chains differ from those of other collagens, type II collagen is designated [ 1(II)]3. Several classes of collagens are identified on the basis of their polymerization pattern. Most of the collagen molecules polymerize into supramolecular aggregates such as fibrils or networks, and they are divided into several subgroups on the basis of their structural or amino acid sequence similarities.


Histology_Ross

Histology_Ross.  Fibrillar collagens include types I, II, III, V, and XI collagen molecules. These types are characterized by uninterrupted glycine–proline–hydroxyproline repeats and aggregate to form 68-nm-banded fibrils (as diagramed in Fig. 6.7a).  Fibril-associated collagens with interrupted triple helixes (FACITs) have interruptions in their triple helixes that provide flexibility to the molecule. They are located on the surface of different fibrils and are represented by types IX, XII, XIV, XVI, XIX, XX, XXI, and XXII collagens. For instance, type IX collagen molecule binds and interacts with type II collagen in the cartilage at the intersections of the fibrils. It serves to stabilize this tissue by binding type II collagen fibrils with proteoglycans of the ECM.  Hexagonal network–forming collagens are represented by collagen types VIII and X.


Histology_Ross

Histology_Ross.  Hexagonal network–forming collagens are represented by collagen types VIII and X.  Transmembrane collagens are represented by types: XIII (found in the focal adhesions), XVII (found within the hemidesmosomes), XXIII (found in metastatic cancer cells), and XXV (a brain-specific collagen).  Multiplexins (collagens with multiple triple-helix domains and interruptions) comprise collagen types XV and XVIII, which reside in the basement membrane zones.  Basement membrane–forming collagens include type IV collagen, which is responsible for the collagen suprastructure in the basement membrane of epithelial cells (page 139), type VI collagen, which forms beaded filaments, and type VII collagen, which forms anchoring fibrils that attach the basement membrane to the ECM.


Histology_Ross

Histology_Ross. Table 6.2 lists the collagens that have been characterized to date (I to XXV), including their structural variations and some of the roles presently ascribed to them. Recently identified collagen types (XXVI to XXVIII) have not been fully characterized and are not included in the table. Biosynthesis and Degradation of Collagen Fibers Collagen fiber formation involves events that occur both within and outside the fibroblast. The production of fbrillar collagen (I, II, III, V, and XI) involves a series of events within the fibroblast that leads to production of procollagen, the precursor of the collagen molecule. These events take place in membrane-bounded organelles within the cell. Production of the actual fibril occurs outside the cell and involves enzymatic activity at the plasma membrane to produce the collagen molecule, followed by assembly of the molecules into fibrils in the ECM under guidance by the cell (Fig. 6.8).


Histology_Ross

Histology_Ross. TABLE Types of Collagen, Composition, Location, and Function 6.2 Type Compositiona Location Functions I [ 1(I)]2 2(I) Connective tissue of skin, bone, tendon, Provides resistance to force, ligaments, dentin, sclera, fascia, and tension, and stretch organ capsules (accounts for 90% of body collagen) II [ 1(II)]3 Cartilage (hyaline and elastic), Provides resistance to notochord, and intervertebral disk intermittent pressure III [ 1(III)]3 Prominent in loose connective Forms reticular fibers, arranged tissue and organs (uterus, liver, spleen, as a loose meshwork of thin kidney, lung, etc.); smooth muscle; fibers, provides a supportive endoneurium; blood vessels; and scaffolding for the specialized fetal skin cells of various organs and blood vessels. IV [ 1(IV)]2 2(IV) or Basal laminae of epithelia, kidney Provides support and filtration 3(IV) 4(IV) 5(IV) or glomeruli, and lens capsule barrier [ 5(IV)]2 6(IV) V [ 1(V)]2 2(V) or Distributed uniformly throughout Localized at the surface of


Histology_Ross

Histology_Ross. kidney Provides support and filtration 3(IV) 4(IV) 5(IV) or glomeruli, and lens capsule barrier [ 5(IV)]2 6(IV) V [ 1(V)]2 2(V) or Distributed uniformly throughout Localized at the surface of type I 1(V) 2(V) 3(V) connective tissue stroma; may be collagen fibrils along with type XII related to reticular network and XIV collagen to modulate biomechanical properties of the fibril VI [ 1(VI)]2 2(VI) or Forms part of the cartilage matrix Attaches the chondrocyte to the 1(VI) 2(VI) 3(VI) immediately surrounding the matrix; covalently bound to type I chondrocytes collagen fibrils VII [ 1(VII)]3 Present in anchoring fibrils of skin, Secures basal lamina to connective eye, uterus, and esophagus tissue fibers VIII [ 1(VIII)]2 2(VIII) Product of endothelial cells Facilitates movement of endothelial cells during angiogenesis IX 1(IX) 2(IX) 3(IX) Found in cartilage associated with Stabilizes network of cartilage type II collagen fibrils type II collagen fibers by interaction with proteoglycan


Histology_Ross

Histology_Ross. cells during angiogenesis IX 1(IX) 2(IX) 3(IX) Found in cartilage associated with Stabilizes network of cartilage type II collagen fibrils type II collagen fibers by interaction with proteoglycan molecules at their intersections X [ 1(X)]3 Produced by chondrocytes in the Contributes to the bone mineral-zone of hypertrophy of normal ization process by forming growth plate hexagonal lattices necessary to arrange types II, IX, and XI collagen within cartilage XI [ 1(XI)]2 2(XI) or Produced by chondrocytes; Regulates size of type II collagen 1(XI) 2(XI) 3(XI) associated with type II collagen fibrils, fibrils; it is essential for cohesive forms core of type I collagen fibrils properties of cartilage matrix XII [ 1(XII)]3 Isolated from skin and placenta; Localized at the surface of type I abundant in tissues in which collagen fibrils along with type V mechanical strain is high and XIV collagen to modulate biomechanical properties of the fibril XIII [ 1(XIII)]3 An unusual transmembrane


Histology_Ross

Histology_Ross. abundant in tissues in which collagen fibrils along with type V mechanical strain is high and XIV collagen to modulate biomechanical properties of the fibril XIII [ 1(XIII)]3 An unusual transmembrane collagen Associated with the basal lamina detected in bone, cartilage, intestine, along with type VII collagen skin, placenta, and striated muscles continued next page


Histology_Ross

Histology_Ross. TABLE Types of Collagen, Composition, Location, and Function (Cont.)6.2 XIV [ 1(XIV)]3 Isolated from placenta; also Localized at the surface of type I detected in the bone marrow collagen fibrils along with type V and XII collagen to modulate biomechanical properties of the fibril; has a strong cell–cell binding property XV [ 1(XV)]3 Present in tissues derived from Involved in adhesion of basal lamina mesenchyme; expressed in heart to the underlying connective tissue and skeletal muscles XVI [ 1(XVI)]3 Broad tissue distribution; associated Contributes to structural with fibroblasts and arterial smooth integrity of connective tissue muscle cells, but not associated with type I collagen fibrils XVII [ 1(XVII)]3 Another unusual transmembrane collagen Interacts with integrins to stabilize found in epithelial cell membranes hemidesmosome structure XVIII [ 1(XVIII)]3 Found in epithelial and vascular Represents a basement membrane basement membrane heparan sulfate proteoglycan thought to


Histology_Ross

Histology_Ross. found in epithelial cell membranes hemidesmosome structure XVIII [ 1(XVIII)]3 Found in epithelial and vascular Represents a basement membrane basement membrane heparan sulfate proteoglycan thought to inhibit endothelial cell proliferation and angiogenesis XIX [ 1(XIX)]3 Discovered from the sequence of Pronounced vascular and stromal human rhabdomyosarcoma cDNA; interaction suggests involvement in present in fibroblasts and liver angiogenesis XX [ 1(XX)]3 Discovered from chick embryonic Binds to the surface of other tissue; also in corneal epithelium, collagen fibrils sternal cartilage, and tendons XXI [ 1(XXI)]3 Found in human gingiva, heart and Plays a role in maintaining three-skeletal muscle, and other tissues dimensional architecture of dense containing type I collagen fibrils connective tissues XXII [α1(XXII)]3 Found in myotendinous junction, Belongs to FACIT family skeletal and heart muscle, articular Expressed at tissue junctions cartilage–synovial fluid junction, at In skin,


Histology_Ross

Histology_Ross. tissues XXII [α1(XXII)]3 Found in myotendinous junction, Belongs to FACIT family skeletal and heart muscle, articular Expressed at tissue junctions cartilage–synovial fluid junction, at In skin, influences epithelial-the border between hair follicle mesenchymal interactions during hair and dermis follicle morphogenesis and cycling XXIII [ 3(XXIII)]3 Discovered in metastatic tumor cells Transmembrane collagen Also expressed in heart, retina, Interacts with ECM proteins (collagen and metastatic prostate cancer cells XIII and XXV, fibronectin, heparin) Increased expression in patient with metastatic prostate cancer XXIV [ 1(XXIV)]3 Found co-expressed with type I Fibrillarlike collagen collagen in the developing bone Regarded as an ancient molecule that and eye regulates type I collagen fibrillogenesis in bone and eye during fetal development XXV [ 1(XXV)]3 A brain-specific transmembrane collagen Binds to fibrillized β-amyloid peptide Discovered in amyloid of amyloid plaques in


Histology_Ross

Histology_Ross. fibrillogenesis in bone and eye during fetal development XXV [ 1(XXV)]3 A brain-specific transmembrane collagen Binds to fibrillized β-amyloid peptide Discovered in amyloid of amyloid plaques in Alzheimer’s plaques in brains of patients disease with Alzheimer’s disease Overexpressed in neurons aEach collagen molecule is composed of three polypeptide chains intertwined in a helical configuration. The Roman numerals in the parentheses in the Composition column indicate that the chains have a distinctive structure that differs from the chains with different numerals. Thus, collagen type I has two identical 1 chains and one 2 chain; collagen type II has three identical 1 chains. fibrillar collagen; FACITs; basement membrane–forming collagen; hexagonal network–forming collagen; transmembrane collagens; multiplexins Type Compositiona Location Functions


Histology_Ross

Histology_Ross. Formation of mRNA in the nucleus Initiation of synthesis of pro–α chains with signal sequences by ribosomes Synthesis of pro–α chains on the rER Hydroxylation of proline and lysine residues (vitamin C required) and cleavage of signal sequence from pro-α-chain Glycosylation of specific hydroxylysyl residues in the rER Formation of procollagen triple helix molecules from a C terminus toward the N terminus in a zipper-like manner 1. 2. 3. 4. 5. 6. Stabalization of the triple helix by formation of intraand interchain hydrogen and disulfide bounds and chaparone proteins (e.g., hsp-47) Transport of procollagan molecules to Golgi apparatus Packaging of procollagen molecules by Golgi into secretory vesicles Movement of vesicles to plasma membrane, assisted by molecular motor proteins associated with microtubules 7. 8. 9. 10. 11. Exocytosis of procollagen molecules 12. Cleavage of trimeric globular Cand helical N-procollagen domains by procollagen Nand C-proteinases 13. Polymerization


Histology_Ross

Histology_Ross. with microtubules 7. 8. 9. 10. 11. Exocytosis of procollagen molecules 12. Cleavage of trimeric globular Cand helical N-procollagen domains by procollagen Nand C-proteinases 13. Polymerization (self-assembly) of collagen molecules into collagen fibrils (in cove of fibroblast) with development of covalent cross-linking 14. Incorporation of other collagens (e.g, type V, FACITs, etc.) into collagen fibrils


Histology_Ross

Histology_Ross. FIGURE 6.8  Collagen biosynthesis. Schematic representation of the biosynthetic events and organelles participating in collagen synthesis. Bold numbers correspond to the events numbered in collagen biosynthesis listed at the bottom. Collagen molecule biosynthesis involves a number of intracellular events.


Histology_Ross

Histology_Ross. Collagen molecule biosynthesis involves a number of intracellular events. The steps in biosynthesis of almost all fibrillar collagens are similar, but type I collagen has been studied in most detail. In general, the synthetic pathway for collagen molecules is similar to other constitutive secretory pathways used by the cell. The unique features of collagen biosynthesis are expressed in multiple posttranslational processing steps that are required to prepare the molecule for the extracellular assembly process. Thus, we see the following:  Collagen chains are synthesized in the rER as long precursors containing large globular aminoand carboxyterminus propeptides called pro– chains (preprocollagen molecules). The newly synthesized polypeptides are simultaneously discharged into the cisternae of the rER, where intracellular processing begins.  Within the cisternae of the rER a number of posttranslational modifications of the preprocollagen molecules occur, including the following: 1.


Histology_Ross

Histology_Ross.  Within the cisternae of the rER a number of posttranslational modifications of the preprocollagen molecules occur, including the following: 1. The amino-terminus signal sequence is cleaved. 2. Proline and lysine residues are hydroxylated while the polypeptides are still in the nonhelical conformation. Ascorbic acid (vitamin C) is a required cofactor for the addition of hydroxyl groups to proline and lysine residues in pro– chains by the enzymes prolylhydroxylase and lysylhydroxylase; without hydroxylation of proline and lysine residues, the hydrogen bonds essential to the final structure of the collagen molecule cannot form. This explains why wounds fail to heal and bone formation is impaired in scurvy (vitamin C deficiency). 3. O-linked sugar groups are added to some hydroxy -lysine residues (glycosylation) and N-linked sugars are added to the two terminal positions. 4.


Histology_Ross

Histology_Ross. 3. O-linked sugar groups are added to some hydroxy -lysine residues (glycosylation) and N-linked sugars are added to the two terminal positions. 4. The globular structure is formed at the carboxyterminus, which is stabilized by disulfide bonds. Formation of this structure ensures the correct alignment of the three chains during the formation of the triple helix. 5. A triple helix (beginning from the carboxy-terminus) is formed by three chains, except at the terminals where the polypeptide chains remain uncoiled. 6. Intrachain and interchain hydrogen and disulfide bonds form that influence the shape of the molecule. 7. The triple-helix molecule is stabilized by the binding of the chaperone protein hsp47, which also prevents the premature aggregation of the trimers within the cell. The resultant molecule is procollagen.


Histology_Ross

Histology_Ross.  The folded procollagen molecules pass to the Golgi apparatus and begin to associate into small bundles. This bundling is achieved by the lateral associations between uncoiled terminals of the procollagen molecules. Free and small aggregates of procollagen molecules are packaged into secretory vesicles and transported to the cell surface. Formation of collagen fibrils (fibrillogenesis) involves extracellular events.  As procollagen is secreted from the cell, it is converted into a mature collagen molecule by procollagen peptidase associated with the cell membrane, which cleaves the uncoiled ends of the procollagen (Fig. 6.9).


Histology_Ross

Histology_Ross.  The aggregated collagen molecules then align together to form the final collagen fbrils in a process known as fbrillogenesis. The cell controls the orderly array of the newly formed fibrils by directing the secretory vesicles to a localized surface site for discharge. The cell simultaneously creates specialized collagen assembly sites called coves. These invaginations of the cell surface allow molecules to concentrate where assembly will SS FIGURE 6.9 • Cleavage of the pro-collagen molecule. Illustration showing the procollagen molecule with Nand C termini. Small curved arrows in the upper part of the illustration show C-terminal where terminals are split from the propeptide procollagen molecule to form the collagen (tropocollagen) molecule. On the C terminus of the molecule, the sugar subunit is GlcNac (N acetylglucosamine) attached to mannose (Mann). (Adapted with permission from Prockop DJ, Kivirikko


Histology_Ross

Histology_Ross. On the C terminus of the molecule, the sugar subunit is GlcNac (N acetylglucosamine) attached to mannose (Mann). (Adapted with permission from Prockop DJ, Kivirikko KI, Tuderman L, Guzman NA. The (1.5 nm) biosynthesis of collagen and its disorders (first of two parts). N Engl J Med 1979;301:13–23. Copyright © 1979. Massachusetts Medical Society. All rights reserved.) FIGURE 6.10 • Type I collagen fbril. The type I collagen fibril contains small amounts of other collagen types such as types II, III, V, and XI. Note that the core of the fibril contains collagen types V and XI, which help initiate the assembly of the type I fibril.


Histology_Ross

Histology_Ross. occur (see Fig. 6.8). Within the cove, the collagen molecules align in rows and self-assemble longitudinally in head-to-tail fashion. They also aggregate laterally in a quarter-staggered pattern (see Fig. 6.7). The collagen molecules are then cross-linked by covalent bonds that are formed between the lysine and hydroxylysine aldehyde groups. Collagen biogenesis results in the formation of highly organized polymers called fbrils. Collagen fibrils often consist of more than one type of collagen.


Histology_Ross

Histology_Ross. Collagen fibrils often consist of more than one type of collagen. Usually different types of fibrillar collagens assemble into fibrils composed of more than one type of collagen molecule. For example, type I collagen fbrils often contain small amounts of types II, III, V, and XI. Current studies indicate that assembly of type I collagen fibrils is proceeded by formation of a fibrillar core containing type V and type XI molecules. Subsequently, type I collagen molecules are deposited and polymerized on the surface of the fibrillar core (Fig. 6.10). In addition, small amounts of type II and III collagen molecules are incorporated into type I collagen fibrils. Collagen types V and XI are important regulators of fibrillogenesis. They control the thickness of type I fibrils by limiting the deposition of collagen molecules after the fibril has reached the desired diameter.


Histology_Ross

Histology_Ross. Fully mature collagen fibers are usually associated with the FACIT family of collagen molecules that reside on their surfaces. For example, type I fibrils are associated with type XII and type XIV collagens. These collagens contribute to the three-dimensional organization of fibers within the ECM. Type II collagen fibrils, which are abundant within the cartilage, are usually smaller in diameter than type I fibrils. However, these fibrils are also associated with type IX collagen (another member of the FACIT subgroup). Collagen type IX resides on the surface of the type II fibril and anchors it to proteoglycans and other components of the cartilaginous ECM (Fig. 6.11). Collagen molecules are synthesized by various types of connective tissue and epithelial cells.


Histology_Ross

Histology_Ross. Collagen molecules are synthesized by various types of connective tissue and epithelial cells. Collagen molecules are largely synthesized by connective tissue cells. These cells include fibroblasts in a variety of tissues (e.g., chondrocytes in cartilage, osteoblasts in bone, and pericytes in blood vessels). In addition, the collagen molecules of basement membrane (see page 139) are produced by epithelial cells. The synthesis of collagen is regulated by complex interactions among growth factors, hormones, and cytokines. For example, transforming growth factor (TGF-) and platelet-derived growth factor (PDGF) stimulate collagen synthesis by fibroblasts, whereas steroid hormones (glucocorticoids) inhibit its synthesis.


Histology_Ross

Histology_Ross. FIGURE 6.11 • Type II collagen fbril. This diagram illustrates the interaction between type II collagen fibrils and type IX collagen molecules in the cartilaginous matrix. Collagen type IX provides the link between the collagen fibrils and GAG molecules, which stabilizes the network of cartilage fibers. Collagen fibers are degraded either by proteolytic or phagocytic pathways.


Histology_Ross

Histology_Ross. All proteins in the body are being continually degraded and resynthesized. These processes allow tissues to grow and to undergo remodeling. Initial fragmentation of insoluble collagen molecules occurs through mechanical wear, the action of free radicals, or proteinase cleavage. Further degradation is continued by specific enzymes called proteinases. The resulting colla gen fragments are then phagocytosed by cells and degraded by their lysosomal enzymes. Excessive collagen degradation is observed in several diseases (e.g., degradation of cartilage collagen in rheumatoid arthritis or bone collagen in osteoporosis). Secreted collagen molecules are degraded mainly by two different pathways:  Proteolytic degradation occurs outside the cells through the activity of enzymes called matrix metalloproteinases (MMPs). These enzymes are synthesized and secreted into the ECM by a variety of connective tissue cells (fibroblasts, chondrocytes, monocytes, neutrophils, and macrophages), some


Histology_Ross

Histology_Ross. metalloproteinases (MMPs). These enzymes are synthesized and secreted into the ECM by a variety of connective tissue cells (fibroblasts, chondrocytes, monocytes, neutrophils, and macrophages), some epithelial cells (keratinocytes in the epidermis), and cancer cells. The MMPs include collagenases (which degrade type I, II, III, and X collagens); gelatinases (which degrade most types of denatured collagens, laminin, fibronectin, and elastin); stromelysins (which degrade proteoglycans, fibronectin, and denatured collagens); matrilysins (which degrade type IV collagen and proteoglycans); membrane-type MMPs (which are produced by cancer cells and have a potent pericellular fibrinolytic activity); and macrophage metalloelastases (which degrade elastin, type IV collagen, and laminin).


Histology_Ross

Histology_Ross. In general, triple-helical undenatured forms of collagen molecules are resistant to degradation by MMPs. In contrast, damaged or denatured collagen (gelatin) is degraded by many MMPs, with gelatinases playing the prominent role. MMP activity can be specifically inhibited by tissue inhibitors of metalloproteinases (TIMPs). Because MMPs are secreted by invasive (migrating) cancer cells, researchers are investigating synthetic therapeutic agents that inhibit the activity of MMPs to control the spread of cancer cells.  Phagocytotic degradation occurs intracellularly and involves macrophages to remove components of the ECM. Fibroblasts are also capable of phagocytosing and degrading collagen fibrils within the lysosomes of the cell. FOLDER 6.1 Clinical Correlation: Collagenopathies


Histology_Ross

Histology_Ross. FOLDER 6.1 Clinical Correlation: Collagenopathies The important role of collagens in the body can be illus-trated by collagenopathies (collagen diseases), which are caused by a deficit or abnormality in the production of collagens. In the future, gene therapy could potentially be used either to control deposition of faulty collagen or to reverse the disease process caused by the mutated genes. specific collagens. Most collagenopathies are attributed to The following table lists the most common collagenopathies mutations in genes encoding the chains in the various that occur in humans.


Histology_Ross

Histology_Ross. The Most Common Collagenopathies in Humans Type of Collagen Disease Symptoms I Osteogenesis imperfecta Repeated fractures after minor trauma, brittle bones, abnormal teeth, thin skin, weak tendons, blue sclerae, progressive hearing loss II Kniest dysplasia; Short stature, restricted joint mobility, ocular changes Achondrogenesis, type 2 leading to blindness, wide metaphyses, and joint abnormality seen in radiographs III Ehlers-Danlos type IV Hypermobility of joints of digits, pale thin skin, severe bruisability, early morbidity and mortality, resulting from rupture of vessels and internal organs IV Alport’s syndrome Hematuria resulting from structural changes in the glomerular basement membrane of the kidney, progressive hearing loss, and ocular lesions VII Kindler’s syndrome Severe blistering and scarring of the skin after minor trauma, resulting from absence of anchoring fibrils IX Multiple ephiphyseal dysplasia Skeletal deformations resulting from impaired (MED) endochondral


Histology_Ross

Histology_Ross. blistering and scarring of the skin after minor trauma, resulting from absence of anchoring fibrils IX Multiple ephiphyseal dysplasia Skeletal deformations resulting from impaired (MED) endochondral ossification and dysplasia (MED), premature degenerative joint disease X Schmid metaphysal Skeletal deformations characterized by modifications chondrodysplasia of the vertebral bodies and chondrodysplasia metaphyses of the long bone XI Weissenbacher-Zweymuller Similar clinical features to type II collagenopathies syndrome Stickler’s syndrome in addition to craniofacial and skeletal deformations, (includes also additional mutations severe myopia, retinal detachment, and progressive of type II collagen gene) hearing loss XVII Generalized atrophic benign Blistering skin disease with mechanically induced epidermolysis bullosa (GABEB) dermal–epidermal separation, epidermolysis bullosa resulting from faulty hemidesmosomes, skin atrophy, nail dystrophy, and alopecia


Histology_Ross

Histology_Ross. Reticular fibers provide a supporting framework for the cellular constituents of various tissues and organs. Reticular fbers and collagen type I fibers share a prominent feature. They both consist of collagen fibrils. Unlike collagen fibers, however, reticular fibers are composed of type III collagen. The individual fibrils that constitute a reticular fiber exhibit a 68-nm banding pattern (the same as the fibrils of type I collagen). The fibrils have a narrow diameter (about 20 nm), exhibit a branching pattern, and typically do not bundle to form thick fibers.


Histology_Ross

Histology_Ross. In routinely stained H&E preparations, reticular fibers cannot be identified positively. When visualized in the light microscope with special techniques, reticular fibers have a threadlike appearance. Because they contain a greater relative number of sugar groups than collagen fibers, reticular fibers are readily displayed by means of the periodic acid–Schiff (PAS) reaction. They are also revealed with special silver-staining procedures such as the Gomori and Wilder methods. After silver treatment, the fibers appear black; thus, they are said to be argyrophilic (Fig. 6.12). The thicker collagen fibers in such preparations are colored brown. FIGURE 6.12 • Reticular fbers in the lymph node. Photo-micrograph of a lymph node silver preparation showing the connective tissue capsule at the top and a trabecula extending from it at the left. The reticular fibers (arrows) form an irregular anastomosing network. 650.


Histology_Ross

Histology_Ross. Reticular fibers are named for their arrangement in a mesh-like pattern or network.


Histology_Ross

Histology_Ross. In loose connective tissue, networks of reticular fbers are found at the boundary of connective tissue and epithelium, as well as surrounding adipocytes, small blood vessels, nerves, and muscle cells. They are also found in embryonic tissues. The prevalence of reticular fibers is an indicator of tissue maturity. They are prominent in the initial stages of wound healing and scar tissue formation, where they provide early mechanical strength to the newly synthesized ECM. As embryonic development or wound healing progresses, reticular fibers are gradually replaced by the stronger type I collagen fibers. Reticular fibers also function as a supporting stroma in hemopoietic and lymphatic tissues (but not in the thymus). In these tissues, a special cell type, the reticular cell, produces the collagen of the reticular fiber. This cell maintains a unique relationship to the fiber. It surrounds the fiber with its cytoplasm, thus isolating the fiber from other tissue components.


Histology_Ross

Histology_Ross. In most other locations, reticular fibers are produced by fibroblasts. Important exceptions to this general rule include the endoneurium of peripheral nerves, where Schwann cells secrete reticular fibers, tunica media of blood vessels, and muscularis of the alimentary canal, where smooth muscle cells secrete reticular and other collagen fibers. Elastic fibers allow tissues to respond to stretch and distension. Elastic fbers are typically thinner than collagen fibers and are arranged in a branching pattern to form a three-dimensional network. The fibers are interwoven with collagen fibers to limit the distensibility of the tissue and prevent tearing from excessive stretching (Plate 6, page 196).


Histology_Ross

Histology_Ross. Elastic fibers stain with eosin but not well, so they cannot always be distinguished from collagen fibers in routine H&E preparations. Because elastic fibers become somewhat refractile with certain fixatives, they may be distinguished from collagen fibers in specimens stained with H&E when they display this characteristic. Elastic fibers can also be selectively stained with special dyes such as orcein or resorcin-fuchsin, as shown in Figure 6.13. The elastic property of the elastin molecule is related to its unusual polypeptide backbone, which causes random coiling.


Histology_Ross

Histology_Ross. The elastic property of the elastin molecule is related to its unusual polypeptide backbone, which causes random coiling. Elastic fibers are produced by many of the same cells that produce collagen and reticular fibers, particularly fibroblasts and smooth muscle cells. In contrast to collagen fibers, however, elastic fibers are composed of two structural components: A central core of elastin and a surrounding network of fbrillin microfbrils.  Elastin (72 kilodaltons) is a protein that, like collagen, is rich in proline and glycine. Unlike collagen, it is poor in hydroxyproline and completely lacks hydroxylysine. The random distribution of glycines makes the elastin molecule


Histology_Ross

Histology_Ross. FIGURE 6.13 • Collagen and elastic fbers. Photomicrograph of a mesentery spread stained with resorcin-fuchsin. The mesentery is very thin, and the microscope can be focused through the entire thickness of the tissue. The delicate threadlike branching strands are the elastic fibers (E). Collagen fibers (C) are also evident. They are much thicker; although they cross one another, they do not branch. 200.


Histology_Ross

Histology_Ross. hydrophobic and allows for random coiling of its fibers. This permits elastic fibers to “slide” over one another or to be stretched and then recoil to their original state. Elastin also contains desmosine and isodesmosine, two large amino acids unique to elastin, which are responsible for the covalent bonding of elastin molecules to one another. These covalent bonds link four elastin molecules into either desmosine or isodesmosine cross-links (Fig. 6.14). Elastin forms fibers of variable thicknesses, or lamellar layers (as in elastic arteries). Elastin is encoded by one of the largest genes in the human genome. The elastin gene consists of 28 kilobases, but less than 10% of the kilobases carry the sequence that encodes elastin.


Histology_Ross

Histology_Ross.  Fibrillin-1 (350 kilodaltons) is a glycoprotein that forms fine microfibrils measuring 10 to 12 nm in diameter. During the early stages of elastogenesis, fibrillin-1 microfibrils are used as substrates for the assembly of elastic fibers. The microfibrils are formed first; elastin material is then deposited on the surface of the microfibrils. Elastin-associated fbrillin microfbrils play a major role in organizing elastin into fibers. The absence of fibrillin


Histology_Ross

Histology_Ross. FIGURE 6.14 • Diagram of elastin molecules and their interaction. a. Elastin molecules are shown joined by covalent bonding between desmosine and isodesmosine (purple) to form a cross-linked network. Inset shows enlargement of the elastin molecule in its individual and random-coiled conformation with the covalent bond formed by desmosine. b. The effect of stretching is shown. When the force is withdrawn, the network reverts to the relaxed state as in panel a. (Modified with permission from Alberts B, et al. Essential Cell Biology, p. 153. Copyright 1997. Routledge, Inc., part of The Taylor & Francis Group.) microfibrils during elastogenesis results in the formation of elastin sheets or lamellae, as found in blood vessels. Abnormal expression of the fbrillin gene (FBN1) is linked to Marfan’s syndrome, a complex, autosomal dominant, connective tissue disorder. Immunoﬂuorescence of a skin biopsy specimen from a person with this syndrome shows an absence of elastin-associated fibrillin


Histology_Ross

Histology_Ross. syndrome, a complex, autosomal dominant, connective tissue disorder. Immunoﬂuorescence of a skin biopsy specimen from a person with this syndrome shows an absence of elastin-associated fibrillin microfibrils. One of the consequences of the disease is abnormal elastic tissue.


Histology_Ross

Histology_Ross. With both the TEM and SEM, elastin appears as an amorphous structure of low electron density. In contrast, the fibrillin microfibrils are electron dense and are readily apparent even within the elastin matrix (Fig. 6.15). In mature fibers, the fibrillin microfibrils are located within the elastic fiber and at its periphery. The presence of microfibrils within the fiber is associated with the growth process; thus, as the fiber is formed and thickens, the microfibrils become entrapped within the newly deposited elastin.  FOLDER 6.2 Clinical Correlation: Sun Exposure and Molecular Changes in Photoaged Skin


Histology_Ross

Histology_Ross. Chronological aging of the skin is a complex process that is associated with functional and structural changes within the stratified squamous epithelium (epidermis) as well as the underlying connective tissue of the dermis. When these changes are intensified by prolonged exposure to solar or ultraviolet (UV) radiation, the process is referred to as photoaging. Chronic sun exposure ages the skin at an accelerated rate, especially in exposed areas of the body such as the face, neck, dorsal surface of the hands, and fore-arms. Clinical signs associated with photoaging include dyspigmentation, freckles, deep wrinkles, increased laxity, and increased risk for cutaneous cancers. The most prominent changes in the dermis of pho-toaged skin are associated with connective tissue fibers. Decreased production of type I and type III collagen fibers is observed in normal aged skin; however, these changes are more pronounced in sun-exposed regions. Sunlight exposure affects collagen biogenesis by


Histology_Ross

Histology_Ross. production of type I and type III collagen fibers is observed in normal aged skin; however, these changes are more pronounced in sun-exposed regions. Sunlight exposure affects collagen biogenesis by altering the cross-linking that occurs between collagen molecules during fibrillogenesis (page 168). These alterations result in for-mation of collagen fibers with abnormal stability and decreased resistance to enzymatic degradation. The overall number of elastic fibers also decreases with age; however, in photoaged skin, the number of abnormally thick and nonfunctional elastic fibers increases. Recent studies of fbrillin microfbrils from photoaged skin reveal that the microfibrillar network is affected by solar radiation. Excessive sun exposure causes the fibrillin microfibrils to undergo extensive changes. They became sparse and trun-cated, leading to the formation of aberrant nonfunctional elastic fibers ECM that finally degenerate into homogenous and amorphous elastin-containing


Histology_Ross

Histology_Ross. extensive changes. They became sparse and trun-cated, leading to the formation of aberrant nonfunctional elastic fibers ECM that finally degenerate into homogenous and amorphous elastin-containing masses. Photoaging is also characterized by abnormal degrada-tion of the connective tissue matrix associated with accu-mulation of nonfunctional matrix components. Fibroblasts and neutrophils residing in radiation-damaged areas of the skin secrete matrix metalloproteinases (MMP-1 and -9), elastases, and other proteases (cathepsin G). These enzymes are modulated by tissue inhibitors of metallopro-teinases (TIMPs) that protect extracellular proteins from endogenous degradation. In photoaged skin, TIMP levels are significantly reduced, which further contributes to photodamage of the skin. The best strategies to prevent photodamage caused by solar and UV radiation is the use of physical and chemical sunscreens to prevent UV penetration into skin. Other methods are also used in treating damaged


Histology_Ross

Histology_Ross. strategies to prevent photodamage caused by solar and UV radiation is the use of physical and chemical sunscreens to prevent UV penetration into skin. Other methods are also used in treating damaged skin. These include reducing skin inflammatory reactions with anti-inflammatory medications, inhibiting activities of elastase and other MMPs to prevent degradation of ECM, and stim-ulating natural or applying synthetic inhibitors of MMPs activities to control destruction of connective tissue ECM.


Histology_Ross

Histology_Ross. Elastic material is a major extracellular substance in vertebral ligaments, larynx, and elastic arteries. In elastic ligaments, the elastic material consists of thick fibers interspersed with collagen fibers. Examples of this material are found in the ligamenta flava of the vertebral column and the ligamentum nuchae of the neck. Finer fibers are present in elastic ligaments of the vocal folds of the larynx. In elastic arteries, the elastic material is in the form of fenestrated lamellae, sheets of elastin with gaps or openings. The lamellae are arranged in concentric layers between layers of smooth muscle cells. Like the collagen fibers in the tunica media of blood vessel walls, the elastic material of arteries is produced by smooth muscle cells, not by fibroblasts. In contrast to elastic fibers, microfibrils are not found in the lamellae. Only the amorphous elastin component is seen in electron micrographs. Elastin is synthesized by fibroblasts and vascular smooth muscle cells.


Histology_Ross

Histology_Ross. Elastin is synthesized by fibroblasts and vascular smooth muscle cells. As noted, elastic fibers are produced by fibroblasts or smooth muscle cells within the walls of the vessels. Elastin synthesis parallels collagen production; in fact, both processes can occur simultaneously in a cell. The orderly modification and assembly of procollagen and proelastin, as well as the synthesis of other connective tissue components, are controlled by signal sequences that are incorporated into the beginning of the polypeptide chains of each of the molecules.


Histology_Ross

Histology_Ross. Signal sequences can be compared to airline tags on luggage. Just as the tags ensure that baggage moves correctly from one aircraft to another at airports, so signal peptides ensure that the components of procollagen and proelastin remain separate and properly identified as they pass through the organelles of the cell. During this transit, a series of synthetic events and posttranslational modifications occur before the polypeptides ultimately arrive at their proper destination.


Histology_Ross

Histology_Ross. The extracellular matrix (ECM) is a complex and intricate structural network that surrounds and supports cells within the connective tissue. As previously mentioned, it contains a variety of fibers such as collagen and elastic fbers that are formed from different types of structural proteins. In addition, the ECM contains a variety of proteoglycans (e.g., aggrecan, syndecan); multiadhesive glycoproteins (such as fibronectin and laminin); and glycosaminoglycans (e.g., dermatan sulfate, keratan sulfate, hyaluronan). The last three groups of molecules constitute the ground substance.


Histology_Ross

Histology_Ross. FIGURE 6.15 • a. Electron micrograph of an elastic fber. The elastin (E) of the fiber has a relatively amorphous appearance. The fibrillin microfibrils (arrows) are present at the periphery and within the substance of the fiber. A number of collagen fibrils (C) are also present in this electron micrograph. 40,000. b. Scanning electron micrograph of an elastic fber. This scanning electron micrograph of the human dens irregular connective tissue from the dermis shows structure of elastic fiber (E) and illustrate it’s relative size in comparison to surrounding collagen fibrils (C). Note the presence of small fibrillin microfibrils (arrows) at the surface of elastic fiber 40,000. (Courtesy of Douglas R. Keene)


Histology_Ross

Histology_Ross. R. Keene) All molecules found in the ECM share common domains, and the function of the ECM relies largely on the interactions between these molecules. Each connective tissue cell secretes a different ratio of ECM molecules that contribute to the formation of many different architectural arrangements; therefore, the ECM possesses specific mechanical and biochemical properties characteristic for the tissue in which it is present. For instance, the properties of the ECM in loose connective tissue are different from those of the ECM in cartilage or bone. The extracellular matrix not only provides mechanical and structural support for tissue but also influences extracellular communication.


Histology_Ross

Histology_Ross. The ECM provides mechanical and structural support as well as tensile strength for the tissue. It also functions as a biochemical barrier and plays a role in regulating metabolic functions of the cells surrounded by the matrix. The ECM anchors cells within tissues through cell-to-ECM adhesion molecules and provides pathways for cell migration (e.g., during wound repair). Recent studies indicate that the ECM exerts a regulatory effect on embryonic development and cell differentiation. The matrix is capable of binding and retaining growth factors, which in turn modulate cell growth. With the aid of cell adhesion molecules, the ECM also influences the transmission of information across the plasma membrane of the connective tissue cells. Thus, the current view of ECM components (fibers and ground substance molecules) is that they form a dynamic and interactive system that informs cells about the biochemical and mechanical changes in their extracellular environment.


Histology_Ross

Histology_Ross. Ground substance is the part of the extracellular matrix that occupies the spaces between the cells and fibers; it consists of glycosaminoglycans (GAGs), proteoglycans, and multiadhesive glycoprotein.


Histology_Ross

Histology_Ross. Ground substance is a viscous, clear substance with a slippery feel and high water content. In the light microscope, ground substance appears amorphous in sections of tissue preserved by freeze-drying or in frozen sections stained with basic dyes or by the PAS method. In routine H&E preparations, ground substance is always lost because of its extraction during fixation and dehydration of the tissue. The result is an empty background; only cells and fibers are evident. Thus, in most histologic preparations, the appearance of ground substance—or its lack of appearance—belies its functional importance. Ground substance consists predominately of three groups of molecules: Proteoglycans, very large macromolecules composed of a core protein; glycosaminoglycan molecules (GAGs), which are covalently bound to the proteoglycans; and multiadhesive glycoproteins. The size and structure of the three groups of molecules vary enormously.


Histology_Ross

Histology_Ross. GAGs are responsible for the physical properties of ground substance. The GAGs are the most abundant heteropolysaccharide components of ground substance. These molecules represent long-chain unbranched polysaccharides composed of repeating disaccharide units. The disaccharide units contain either of two modified sugars—N-acetylgalactosamine (GalNAc) or N-acetylglucosamine (GlcNAc)—and a uronic acid such as glucuronate or iduronate. GAGs (except hyaluronan) are synthesized by connective tissue cells as a covalent, posttranslational modification of proteins called proteoglycans. For example, heparin is formed by enzymatic cleavage of heparan sulfate; dermatan sulfate is similarly modified from chondroitin sulfate.


Histology_Ross

Histology_Ross. GAGs are highly negatively charged because of the sulfate and carboxyl groups located on many of the sugars, hence their propensity for staining with basic dyes. The high density of the negative charge (polyanions) also attracts water, forming a hydrated gel. The gel-like composition of ground substance permits rapid diffusion of water-soluble molecules. At the same time, the rigidity of the GAGs provides a structural framework for the cells. GAGs are located primarily within the ground substance as well as on the surface of cells within the ECM. On the basis of differences in specific sugar residues, the nature of their linkages, and the degree of their sulfation, a family of seven distinct GAGs is recognized. They are listed and partially characterized in Table 6.3. Hyaluronan is always present in the extracellular matrix as a free carbohydrate chain.


Histology_Ross

Histology_Ross. Hyaluronan is always present in the extracellular matrix as a free carbohydrate chain. The GAG hyaluronan (hyaluronic acid) deserves special note because it differs from the other GAGs in several respects. It is an exceedingly long, rigid molecule composed of a carbohydrate chain of thousands of sugars rather than the several hundred or fewer sugars found in other GAGs. Hyaluronan polymers are very large (100 to 10,000 kilodaltons) and can displace a large volume of water. They are synthesized by enzymes on the cell surface; therefore, they are not posttranslationally modified like all other GAGs. Hyaluronan is also unique among the GAGs in that it does not contain any sulfate.


Histology_Ross

Histology_Ross. Each hyaluronan molecule is always present in the form of a free carbohydrate chain; in other words, it is not covalently bound to protein, so it does not form proteoglycans. By means of special link proteins, however, proteoglycans indirectly bind to hyaluronan, forming giant macromolecules called proteoglycan aggregates (Fig. 6.16). These molecules are abundant in the ground substance of cartilage. The pressure, or turgor, that occurs in these giant hydrophilic proteoglycan aggregates accounts for the ability of cartilage to resist compression without inhibiting ﬂexibility, making them excellent shock absorbers.


Histology_Ross

Histology_Ross. Another important function of hyaluronan is to immobilize certain molecules in the desired location of the ECM. For instance, ECM contains binding sites for several growth factors, such as TGF-. The binding of growth factors to proteoglycans may cause either their local aggregation or dispersion, which in turn either inhibits or enhances the movement of migrating macromolecules, microorganisms, or metastatic cancer cells in the extracellular environment. In addition, hyaluronan molecules act as efficient insulators,


Histology_Ross

Histology_Ross. TABLE Glycosaminoglycans6.3 Molecular Disaccharide Name Weight (kDa) Composition Localization Function Hyaluronan 100–10,000 D-Glucuronic acid Synovial fluid, vitreous Large polymers of N-acetylglucosamine humor, ECM of connective hyaluronan can displace tissues a large volume of water. Thus this polymer is excellent lubricant and shock absorber Chondroitin 25 D-Glucuronic acid 4-sulfate N-acetylgalactosamine Chondroitin sulfates and 4-sulfate hyaluronan are fundamental components of aggrecan found in articular cartilage. Aggrecan confers on articular cartilage shock-Chondroitin 25 D-Glucuronic acid absorbing properties 6-sulfate N-acetylgalactosamine 6-sulfate Dermatan 35 L-Iduronic acid Skin, blood vessels, Dermatan sulfate proteogly-sulfate N-acetylgalactosamine heart valves cans have been implicated 4-sulfate in cardiovascular disease, tumorigenesis, infection, wound repair, fibrosis, and as a modulator in cell behavior Keratan 10 Galactose or galactose Bone, cartilage, cornea


Histology_Ross

Histology_Ross. been implicated 4-sulfate in cardiovascular disease, tumorigenesis, infection, wound repair, fibrosis, and as a modulator in cell behavior Keratan 10 Galactose or galactose Bone, cartilage, cornea Keratan sulfate proteoglycans sulfate 6-sulfate N-acetylgluco-function in cellular recognition samine 6-sulfate of protein ligands, axonal guidance, cell motility, corneal transparency, and embryo implantation Heparan 15 Glucuronic acid Basal lamina, normal Facilitates interactions with sulfate or L-iduronic acid component of cell fibroblastic growth factor 2-sulfate N-sulfamyl-surface (FGF) and its receptor glucosamine or N-acetyl-glucosamine Heparin 40 Glucuronic acid Limited to granules of Functions as an anticoagulant, or L-iduronic acid mast cells and facilitates interactions with 2-sulfate N-sulfamyl-basophiles FGF and its receptor glucosamine or N-acetylglucosamine 6-sulfate Cartilage, bone, heart valves kDa, kilodaltons.


Histology_Ross

Histology_Ross. because other macromolecules have difficulty diffusing through the dense hyaluronan network. With this property, hyaluronan (and other polysaccharides) regulates the distribution and transport of plasma proteins within the connective tissue. Proteoglycans are composed of GAGs covalently attached to core proteins.


Histology_Ross

Histology_Ross. Proteoglycans are composed of GAGs covalently attached to core proteins. The majority of GAGs in the connective tissue are linked to core proteins, forming proteoglycans. The GAGs extend perpendicularly from the core in a brushlike structure. The linkage of GAGs to the protein core involves a specific trisaccharide composed of two galactose residues and a xylulose residue. The trisaccharide linker is coupled through an O-glycosidic bond to the protein core that is rich in serine and threonine residues, allowing multiple GAG attachments. Proteoglycans are remarkable for their diversity (Fig. 6.17). The number of GAGs attached to the protein core varies from 1 (i.e., decorin) to more than 200 (i.e., aggrecan). A core protein may have identical GAGs attached to it (as in the


Histology_Ross

Histology_Ross. FIGURE 6.16 • Proteoglycan structure. This schematic drawing shows, on the right, a proteoglycan monomer and its relationship to the hyaluronan molecule as represented in the ground substance of cartilage. The proteoglycan monomer is composed of a core protein to which GAGs are covalently bound. The proteoglycan monomer consists of different numbers of GAGs joined to the core protein. The end of the core protein of the proteoglycan monomer interacts with a link protein, which attaches the monomer into the hyaluronan forming the proteoglycan aggregate. On the left, hyaluronan molecules forming linear aggregates, each with many proteoglycan monomers, are interwoven with a network of collagen fibrils. case of fibroglycan or versican) or different GAG molecules (as in the case of aggrecan or syndecan).


Histology_Ross

Histology_Ross. case of fibroglycan or versican) or different GAG molecules (as in the case of aggrecan or syndecan). Proteoglycans are found in the ground substance of all connective tissues and also as membrane-bound molecules on the surface of many cell types. Transmembrane proteoglycans such as syndecan link cells to ECM molecules (Fig. 6.17). For instance, syndecan is expressed two different times on the surface of B lymphocytes. Syndecan molecules are first expressed during early development when lymphocytes are attached to the matrix protein of the bone marrow as they undergo differentiation. The loss of expression of this proteoglycan coincides with the release of the B lymphocyte into the circulation. The second time the B lymphocyte expresses syndecan is during its differentiation into a plasma cell within the connective tissue. Syndecan anchors the plasma cell to the ECM proteins of the connective tissue.


Histology_Ross

Histology_Ross. Aggrecan is another important extracellular proteoglycan. Its molecules are noncovalently bound to the long molecule of hyaluronan (like bristles to the backbone in a bottle brush); this binding is facilitated by linking proteins. To each aggrecan core protein multiple chains of chondroitin sulfate and keratan sulfate are covalently attached through the trisaccharide linker. The most common proteoglycans are summarized in Table 6.4. Multiadhesive glycoproteins play an important role in stabilizing the ECM and linking it to cell surfaces.


Histology_Ross

Histology_Ross. Multiadhesive glycoproteins play an important role in stabilizing the ECM and linking it to cell surfaces. Multiadhesive glycoproteins represent a small but important group of proteins residing in the ECM. They are multidomain and multifunctional molecules that play an important role in stabilizing the ECM and linking it to the cell surface. They possess binding sites for a variety of ECM proteins such as collagens, proteoglycans, and GAGs; they also interact with cell-surface receptors such as integrin and laminin receptors (Fig. 6.18). Multiadhesive glycoproteins regulate and modulate functions of the ECM related to cell movement and cell migration as well as stimulate cell proliferation and differentiation. Among the best characterized multiadhesive glycoproteins are the following:  Fibronectin (250 to 280 kilodaltons) is the most abun dant glycoprotein in connective tissue. Fibronectins are


Histology_Ross

Histology_Ross. FIGURE 6.17 • Common proteoglycan monomers of the connective tissue matrix. Note the diversity of proteoglycan molecules; the number of GAGs attached to the protein core varies from one in decorin to more than 200 in aggrecan. Note also that versican has identical GAG molecules (chondroitin sulfate) attached to the core molecule whereas aggrecan has a mixture of chondroitin sulfate and keratan sulfate attached to the core protein. Syndecan is a transmembrane proteoglycan that attaches the cell membrane to the extracellular matrix.


Histology_Ross

Histology_Ross. by disulfide bonds at a carboxy terminus to form 50-nmlong arms (see Fig. 6.18). Each molecule contains several binding domains that interact with different ECM molecules (e.g., heparan sulfate; collagen types I, II, and III; fibrin; hyaluronan; and fibronectin) and integrin, a cell-surface receptor. Binding to a cell-surface receptor activates fibronectin, which then assembles into fibrils. Fibronectin plays an important role in cell attachment to the ECM. At least 20 different fibronectin molecules have been identified to date.  Laminin (140 to 400 kilodaltons) is present in basal and external laminae. It possesses binding sites for collagen type IV molecules, heparan sulfate, heparin, entactin, laminin, and the laminin receptor on the cell surface. The process of basal lamina assembly and the role of the laminin in this process are described in Chapter 5 (see page 138).


Histology_Ross

Histology_Ross.  Tenascin (280 kilodaltons/monomer) appears during embryogenesis, but its synthesis is switched off in mature tissues. It reappears during wound healing and is also found within musculotendinous junctions and malignant tumors. Tenascin is a disulfide-linked dimer molecule that consists of six chains joined at their amino-terminus (see Fig. 6.18). It has binding sites for fibrinogen, heparin, and EGF-like growth factors; thus, it participates in cell attachment to the ECM.  Osteopontin (44 kilodaltons) is present in the ECM of bone. It binds to osteoclasts and attaches them to the underlying bone surface. Osteopontin plays an important role in sequestering calcium and promoting calcification of the ECM. Important multiadhesive glycoproteins found in the ECM of connective tissue are summarized in Table 6.5. Connective tissue cells can be resident or wandering.