6. Connective Tissue

collagen molecule (tropocollagen)

measures about 300 nm long by 1.5 nm thick and has a head and a tail. consists of three polypeptides known as a chains. The a 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 a chains. A hydroxyproline or hydroxylysine frequently precedes each glycine in the chain, and a proline frequently follows each glycine in the chain. Along with proline and hydroxy- proline, 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. A collagen molecule may be homotrimeric (consisting of three identical a chains) or heterotrimeric (consisting of two or even three genetically distinct a chains).

Macrophages originate in the bone marrow as

monocytes, circulate in the bloodstream and then migrate into the connective tissue, where they mature into functional macrophages (see Chapter 10 VI E). Macrophages increase in number because of the activity of the macrophage colony-stimulating factor (M-CSF). In addition, the colony-forming unit monocyte (CFU-M) facilitates the mitosis and differentiation of monocytes to form macrophages.

NK lymphocytes are

non-T, non-B lymphocytes that express the CD16, CD56, and CD94 proteins not found on other lymphocytes.

Proteolytic degradation

occurs outside the cells through the activity of enzymes called matrix metal-loproteinases (MMPs).

Dupuytren's disease

palmar fibromatosis is characterized by the thickening of palmar aponeurosis, which leads to progressive flexion contracture of the fourth and fifth digits of the hand (Fig. F6.3.2). If scar tissue grows beyond boundaries of the original wound and does not regress, it is called a keloid. Its formation is more common among African Americans than other ethnic groups.

Neutrophils

phagocytose, kill, and digest bacteria at sites of acute inflammation. Pus is an accumulation of dead neutrophils, bacteria, extracellular fluid, and additional debris at an inflammatory site.

Loose connective tissue (areolar tissue)

possesses fewer fibers but more cells than dense connective tissue. a. This tissue is well vascularized, flexible, and not very resistant to stress. b. It is more abundant than dense connective tissue and is the connective tissue that fills in the spaces just deep to the skin.

Collagen fibers are degraded either by

proteolytic or phagocytic pathways. Excessive collagen degradation is observed in several diseases (e.g., degradation of cartilage collagen in rheumatoid arthritis or bone collagen in osteoporosis).

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 an orthogonal array is also found in the cornea of the eye and is responsible for its transparency.

Anaphylactic shock

results from the effects of powerful mediators released during an immediate hypersensitivity reaction following a second exposure to an allergen. 1. This reaction can occur within seconds or minutes after contact with an allergen. 2. Signs and symptoms include shortness of breath, decreasing blood pressure, and other signs and symptoms of shock. 3. Anaphylactic shock may be life-threatening if untreated.

Active fibroblasts are

spindle-shaped (fusiform) and contain well-developed rough endoplasmic reticulum (RER) and many Golgi complexes. Myosin is located throughout the cytoplasm, and actin and a-actinin are located at the cell periphery. Synthetically active, they produce procollagen, collagen, and elastic fibers and most of the ground substance as well as other components of the ECM.

Dense, regular connective tissue,

which contains fiber bundles and attenuated fibroblasts that are arranged in a uniform parallel fashion. (1 ) It is present only in tendons and ligaments. (2) This tissue may be collagenous or elastic.

fibrillogenesis involves extracellular events.

• As procollagen is secreted from the cell, it is converted into a mature collagen molecule by procollagen peptidases associated with the cell membrane, which cleave the uncoiled ends of the procollagen (Fig. 6.9). Serum levels of procollagen type I N-terminal propeptide (PINP) can be measured and used as indicators for collagen type I metabolism. Elevated level of PINP is indicative of increased production of collagen type I, which is associated with bone metastases in breast and prostate cancer. • The aggregated collagen molecules then align together to form the final collagen fibrils in a process known as fibrillogenesis. 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 accumulate and assemble (see Fig. 6.8). Within the cove, the collagen molecules align in rows and self-assemble longitudinally in a head-to-tail fashion. Th ey 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 fibrils. The fibrils further associate with each other to form larger collagen fibers, which on a per weight basis have the tensile strength comparable to that of steel. For example, collagen type I fiber of 1 mm in diameter can withstand a load of 10 to 40 kg before it breaks.

Mast cells mediate immediate (type I) hypersensitivity reactions (anaphylactic reactions) as follows:

(1 ) After the first exposure to an allergen, plasma cells manufacture immunoglobulin E (IgE) antibodies, which bind to Fc receptors (FcERI receptors) on the surface of mast cells and basophils, causing these cells to become sensitized. Common antigens that may evoke this response include plant pollens, insect venoms, certain drugs, and foreign serum. (2) During the second exposure to the same allergen, the membrane-bound IgE binds the allergen. Subsequent cross-linking and clustering of the allergen-IgE complexes trigger degranulation of mast cells and the release of primary and secondary mediators (Figure 6.4 and Table 6.1; see Chapter 12).

Laminin

(140 to 400 kDa) 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 136).

Fibronectin

(250 to 280 kDa) is the most abundant glycoprotein in connective tissue. Fibronectins are dimer molecules formed from two similar peptides linked by disulfide bonds at a carboxy-terminus to form 50-nm-long 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.

Wharton's jelly mesenchymal stem cells

Some of the cells isolated from Wharton's jelly express significant amounts of mesenchymal stem cell markers and have the ability to differentiate under adequate conditions into osteocytes, chondrocytes, adipocytes, and neural-like cells.

Lipomas

are benign fatty tissue tumors usually found in subcutaneous connective tissues of the neck, back, and proximal regions of the limbs of middle-aged and elderly persons.

Elastic fibers

are coiled branching fibers 0.2 to 1.0 J.Lm in diameter that sometimes form loose networks. a. These fibers may be stretched up to 150% of their resting length. b. They consist of a central amorphous substance known as elastin that is surrounded by thin microfibrils. The latter are composed of fibrillin-1, which is bound to elastin by the protein fibulin-5 (see Chapter 4 III B). c. Elastic fibers require special staining to be observed by light microscopy.

Fibers

are long, slender protein polymers present in different proportions in different types of connective tissue collagen, reticular, and elastic

Basophils

are similar to mast cells in that they possess FceRI receptors; their granules house the same primary mediators; and the same secondary mediators are manufactured de novo from the phospholipids of their plasmalemma. They differ, however, in that they circulate via the bloodstream, whereas mast cells do not.

Lymphoid cells

arise from lymphoid stem cells during hemopoiesis (see Chapter 10 VI G). They are located throughout the body in the subepithelial connective tissue and accumulate in the respiratory system, gastrointestinal tract, and elsewhere in areas of chronic inflammation. (For more information concerning lymphoid cells, see Chapter 12 II.) a. T lymphocytes (T cells) initiate the cell-mediated immune response. b. B lymphocytes (B cells), following activation by an antigen, differentiate into plasma cells, which function in the humoral immune response. c. Natural killer (NK) cells lack the surface determinants characteristic of T and B lympho cytes but may display cytotoxic activity against tumor cells.

asthma

have difficulty breathing due to bronchospasms resulting from leukotrienes released in the lungs.

Hay fever

is characterized by nasal congestion caused by localized edema in the nasal mucosa. This edema results from the increased permeability of small blood vessels beca use of excessive release of histamine from mast cells in the nasal mucosa.

Brown adipose tissue

is composed of multilocular adipose cells, which contain many large mitochondria. (1 ) This tissue is capable of generating heat by uncoupling oxidative phosphorylation. Thermogenin, a transmembrane protein in mitochondria, causes the release of protons away from adenosine triphosphate synthesis, resulting in heat production. (2) This tissue is found in infants (also in hibernating animals) and is much reduced in adults.

MHC

major histocompatibility complex

Tenascin

(280 kDa/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 kDa) 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 seques- tering calcium and promoting calcifi cation of the ECM.

Multiplexins

(collagens with multiple triple-helix domains and interruptions) comprise collagen types XV and XVIII, which reside in the basement membrane zones.

Dense, irregular connective tissue

(most common), which contains fiber bundles that have no definite orientation. This tissue is characteristic of the dermis and capsules of many organs. contains mostly collagen fibers.

Collagen molecule biosynthesis involves a number of intra- cellular events.

1. Collagen a chains are synthesized in the rER as long precursors containing large globular amino- and carboxy-terminus propeptides called pro-a chains (preprocollagen molecules). The newly synthesized polypeptides are simultaneously discharged into the cisternae of the rER, where intracellular processing begins. 2. Within the cisternae of the rER, a number of posttranslational modifications of the preprocollagen molecules occur, including the following: • The amino-terminus signal sequence is cleaved. • 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-a 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). • O-linked sugar groups are added to some hydroxylysine residues (glycosylation), and N-linked sugars are added to the two terminal positions. • The globular structure is formed at the carboxy- terminus, which is stabilized by disulfide bonds. Formation of this structure ensures the correct alignment of the three a chains during the formation of the triple helix. • A triple helix (beginning from the carboxy-terminus) is formed by three a chains, except at the terminals where the polypeptide chains remain uncoiled. • Intrachain and interchain hydrogen and disulfide bonds form that influence the shape of the molecule. • 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. 3. 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.

intracellular events of collagen biosynthesis

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

Preformed mediators found inside mast cell granules are the following:

1. Histamine is a biogenic amine that increases the permeability of small blood vessels, causing edema in the surrounding tissue and a skin reaction demonstrated by an itching sensation. 2. Heparin is a sulfated GAG that is an anticoagulant. 3. Serine proteases (tryptase and chymase). 4. Eosinophil chemotactic factor (ECF) and neutrophil chemotactic factor (NCF),

Newly synthesized mediators include the following:

1. Leukotriene C (LTC4) is released from the mast cell and then cleaved in the ECM, yielding two active leukotrienes—D (LTD4) and E (LTE4). Similar to histamine, leukotrienes trigger prolonged constriction of smooth muscle in the pulmonary airways, causing bronchospasm. The bronchoconstrictive effects of leukotrienes develop more slowly and last much longer than the effects of histamine. Bronchospasm caused by leukotrienes can be prevented by leukotriene receptor antagonists (blockers) but not by antihistaminic agents. The leukotriene receptor antagonists are among the most prescribed drugs for the management of asthma; they are used for both treatment and prevention of acute asthma attacks. 2. Tumor necrosis factor a (TNF-a) is a major cytokine produced by mast cells. It increases expression of adhesion molecules in endothelial cells and has antitumor effects. 3. Several interleukins (IL-4, -3 -5, -6, -8 and -16), growth factors (GM-CSF), and prostaglandin D2 (PGD2) are also released during mast cell activation. Th ese mediators are not stored in granules but are synthesized by the cell and released immediately into the ECM

extracellular events

11. Exocytosis of procollagen molecules 12. Cleavage of trimeric globular C- and helical N-procollagen domains by procollagen N- and 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

Collagen fibrils have a

68-nm banding pattern.

CONNECTIVE TISSUE

A. Structure. Connective tissue is formed primarily of extracellular matrix (ECM), consisting of ground substance and fibers, in which various connective tissue cells are embedded. B. Function. Connective tissue supports organs and cells, acts as a medium for exchange of nutrients and wastes between the blood and tissues, protects against microorganisms, repairs damaged tissues, and stores fat.

The absence of fibrillin microfibrils during elastogenesis results in the formation of elastin sheets or lamellae, as found in blood vessels.

Abnormal expression of the fibrillin 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 microfibrils. One of the consequences of the disease is abnormal elastic tissue. In addition, mutation in the emilin-1 gene locus shows alterations of the fi ne structure of elastic fibers and of cell morphology in the elastic arteries.

M1 macrophages

Activation by interferon y (IFN-y), tumor necrosis factor-a (TNF-a), or by bacterial lipopolysaccharide (LPS) creates the classically activated macrophage These macrophages have the capacity, through the production of nitric oxide (NO) and other intermediates, to destroy microorganisms at the site of inflammation. They also secrete interleukin (IL)-12, which acts on helper CD4 T lymphocytes. In turn, the helper T cells secrete IL-2, which stimulates the cytotoxic CD8 T lymphocytes to arrive at the site of inflammation. In summary, M1 macrophages elicit chronic inflammation and tissue injury. When macrophages encounter large foreign bodies, they may fuse to form a large cell with as many as 100 nuclei that engulfs the foreign body. These multinucleated cells are called foreign body giant cells (Langhans cells)

Collagen fibers

Although there are at least 25 different types of collagen, the most common collagen types in connective tissue proper are types I and Ill collagen (see Chapter 4, Table 4.3), both consisting of many closely packed tropocollagen fibrils. The diameter of individual type I collagen fibrils varies greatly (10-300 nm). These fibrils may aggregate and form cable-like structures up to several centimeters in length and display 67-nm periodicity (see Chapter 4 and Figure 4.4). a. Collagen fibers are produced in a two-stage process, involving both intracellular and extracellular events (see Chapter 4 and Figure 4.3). b. Collagen fibers have great tensile strength, which imparts both flexibility and strength to tissues containing them. c. Bone, skin, cartilage, tendon, and many other structures of the body contain collagen fibers.

T lymphocytes are characterized by the presence of the

CD2, CD3, CD5, and CD7 marker proteins and the T -cell receptors (TCRs).

B lymphocytes are characterized by the presence of

CD9, CD19, and CD20 proteins and attached immu- noglobulins IgM and IgD.

hyaluronan (hyaluronic acid)

Extremely long, non-sulfated GAG to which proteoglycans attach to form proteoglycan aggregates

Role of Myofibroblasts in Wound Repair

Granulation tissue, a specialized type of tissue characteristic of the repair process. Usually by day 5 after injury, the fully developed granulation tissue bridges the incision gap. It is composed mainly of large numbers of small vessels, fibroblasts, and myofibroblasts, and variable numbers of other inflammatory cells. Migrating fibroblasts exert tractional forces on the ECM, reorganizing it along lines of stress. Under the influence of growth factors such as TGF -a1 and mechanical forces, fibroblasts undergo differentiation into myofibroblasts. This process can be visualized by monitoring the synthesis of a-SMA. This type of actin is not present in the cytoplasm of fibroblasts (Fig. F6.3.1). The myofibroblasts generate and maintain steady contractile force (similar to that of smooth muscle cells) that causes shortening of the connective tissue fibers and wound closure. At the same time, myofibroblasts synthesize and lay down collagen fibers and other ECM components that are responsible for tissue remodeling. During the second week of wound healing, the amount of cells in tissue undergoing repair decreases; most of the myofibroblasts undergo apoptosis and disappear, resulting in a connective tissue scar that has very few cellular elements.

Function of reticular fibers

In loose connective tissue, networks of reticular fibers 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 with the fiber. It surrounds the fiber with its cytoplasm, thus isolating the fiber from other tissue components. 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.

Mallory trichrome's

In the light microscope, collagen fibers typically appear as wavy structures of variable width and indeterminate length. Th ey stain readily with eosin and other acidic dyes. Th ey can also be colored with the dye aniline blue used in Mallory trichrome's connec- tive tissue stain or with the dye light green used in Masson trichrome's stain.

They are antigen presenting cells, in that they phagocytose antigens, break them down into epitopes, and, using

MHC I or MHC II molecules, place the epitopes on their cell surface to present it to immunocompetent T cells (see Chapter 12 Section III). For historical reasons, macrophages have different names in various regions of the body (e.g., Kupffer cells in the liver, dust cells in the lungs, osteoclasts in bone, Langer hans cells in skin, microglia in the central nervous system). Macrophages display Fc£RI receptors as well as receptors for complement.

tissue inhibitors of metalloproteinases (TIMPs)

MMP activity can be specifi cally inhibited by_______ 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

White adipose tissue is also an endocrine organ that produces a number of hormones known asadipokines

Some of these hormones, leptin, adiponectin, resistin, and retinol binding protein-4, are manufactured by adipocytes, whereas other hormones/factors, namely tumor necrosis factor-a and interleukin-6, are produced by the macrophages that reside in adipose tissue of obese individuals. It has been reported that obesity is always accompanied by chronic inflammation, and inflammatory agents released in response to inflammation-damaged tissue elicit recruitment of macrophages into the connective tissue stroma among the enlarged adipocytes.

Connective tissue cells include many types with different functions.

Some originate locally and remain in the connective tissue (fixed cells), whereas others originate elsewhere and remain only temporarily in connective tissue (transient cells) (Figure 6.1). Fixed connective tissue cells include fibroblasts, pericytes, adipose cells, mast cells, and fixed macrophages. Transient connective tis sue cells include certain macrophages, lymphocytes, plasma cells, neutrophils, eosinophils, and basophils.

TEM of Myofibroblasts

The cell exhibits some features of a fibroblast, such as areas with a moderate amount of rER. Compare with Figure 6.20. Other areas, however, contain aggregates of thin filaments and cytoplasmic densities (arrows), features that are characteristic of smooth muscle cells. The arrowheads indicate longitudinal profiles of collagen fibrils. x11,000. displays typical characteristics of the fibroblast along with characteristics of smooth muscle cells. In addition to rER and Golgi profiles, the myofibroblast contains bundles of longitudinally disposed actin filaments and dense bodies similar to those observed in smooth muscle cells (Fig. 6.21). As in the smooth muscle cell, the nucleus often shows an undulating surface profile, a phenomenon associated with cell contraction. The myofibroblast differs from the smooth muscle cell in that it lacks a surrounding basal lamina (smooth muscle cells are surrounded by a basal or external lamina). Also, it usually exists as an isolated cell, although its processes may contact the processes of other myofibroblasts. Such points of contact exhibit gap junctions, indicating intercellular communication.

Type I collagen fibril.

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

GAGs (glycosaminoglycans)

These molecules represent long-chain unbranched polysaccharides composed of repeating disaccharide units. The disaccharide units contain one of two modified sugars—N-acetylgalactosamine (GalNAc) or N-acetylglucosamine (GlcNAc)—and a uronic acid such as glucuronate or iduronate. 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

Irisin

When an individual is doing physical exercises, his/her skeletal muscle cells release the hormone irisin that not only converts certain unilocular adipocytes into multilocular adipocytes but also enhances the maintenance of neuromuscular synapses.

M2 macrophage

When the infl ammatory stimulus is removed from the site of tissue injury, the body switches into a repair mode that includes the removal of cell debris, the synthesis of components of new ECM, and the revascularization of the injured tissue. During this period, macrophages are activated by cytokines, such as IL-4, -5, -10, or -13. These types of cells are called alternatively activated macrophages. They secrete IL-4 to promote differentiation of B lymphocytes into plasma cells and vascular endothelial growth factor (VEGF) to stimulate angiogenesis. M2 macrophages also secrete ECM components (e.g., fibronectin and other multiadhesive glycoproteins) and work toward resolution of inflammation. They promote wound repair due to their anti-inflammatory, proliferative, and angiogenic activities. M2 macrophages are also efficient at combating parasitic infections (i.e., schistosomiasis). In addition to their beneficial activities, M2 macrophages are involved in pathogenesis of allergy and asthma.

Retinol-binding protein-4 is

also produced by unilocular adipocytes, and it has been demonstrated to elevate insulin resistance, inflammation, and fatty liver disease with a concomitant increase in glucose output by hepatocytes.

Plasma cells

are antibody-manufacturing cells that arise from activated B lymphocytes and are responsible for humoral immunity. a. These ovoid cells contain an eccentric nucleus possessing clumps of heterochromatin, which appear to be arranged in a wheel-spoke fashion. b. Their cytoplasm is deeply basophilic because of an abundance of RER. c. A prominent area adjacent to the nucleus appears pale and contains the Golgi complex (negative Golgi image). d. They are most abundant at wound entry sites or in areas of chronic inflammation.

Tendons

are cord-like structures that attach muscle to bone. They consist of parallel bundles of collagen fibers. Situated between these bundles are rows of fibroblasts called tendinocytes (Fig. 6.4 and Plate 5, page 190). Tendinocytes are surrounded by a specialized ECM that separates them from the loadbearing collagen fibrils. In H&E-stained cross-sections of the tendon, the tendinocytes appear stellate. In transmission electron micrograph (TEM) sections parallel to the long axis of tendons, the cytoplasmic projections of the cell lie between 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 190). 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

Pericytes (adventitial cells; perivascular cells)

are derived from embryonic mesenchymal cells and may retain a pluripotential role. a. They possess characteristics of endothelial cells as well as smooth muscle cells because they contain actin, myosin, and tropomyosin, suggesting that they may function in contraction where they assist to modify blood flow through capillaries. b. They are smaller than fibroblasts, are located mostly along capillaries, and are completely enveloped by their own basal lamina, which is continuous with the basal lamina of the capillary endothelium. c. During blood vessel formation and repair, they may differentiate into fibroblasts, smooth muscle cells, as well as endothelial cells of blood vessel walls.

Reticular fibers

are extremely thin (0.5-2.0 J.Lm) in diameter and are composed primarily of type Ill collagen; they have higher carbohydrate content than other collagen fibers. a. Type III collagen fibers constitute the architectural framework of certain organs and glands. b. Because of their high carbohydrate content, they stain black with silver salts.

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 (brain-specific collagen).

Multilocular adipose cells

are smaller than unilocular adipose cells, and the fat is stored as many small fat droplets, and thus the spherical nucleus is centrally located.

Liposarcomas

are the most common malignant adipose tumors. Although liposarcomas are most frequently located in the leg and/or the retroperitoneal tissues, they are not restricted to these sites. Liposarcomas are difficult to diagnose since they resemble lipomas. Adipocytes of the malignant tissue resemble either unilocular or multilocular adipocytes. Presently, chromo some markers are used to differentially diagnose liposarcomas.

Macrophages

are the principal phagocytosing cells of connective tissue. They are responsible for removing large particulate matter and assisting in the immune response. They also secrete substances that function in wound healing.

Granulocytes

are white blood cells that possess cytoplasmic granules and arise from myeloid stem cells during hemopoiesis. At sites of inflammation, they leave the bloodstream and enter the loose connective tissue, where they perform their specific functions (see Chapter 10 II B 2 a).

Fibroblasts

arise from mesenchymal cells and are the predominant cells in connective tissue proper (Figure 6.2). They often possess an oval nucleus with two or more nucleoli. Fibroblasts seldom undergo mitosis except in wound healing. They may differentiate into other cell types under certain conditions undergo mitosis only during wound healing. However, under certain conditions, fibroblasts may differentiate into adipose cells, or during fibrocartilage formation, they may differentiate into chondrocytes. In addition, under pathological conditions, fibroblasts may differentiate into osteoblasts.

Adipose cells (adipocytes)

arise from mesenchymal cells and perhaps from fibroblasts. Adipogenesis occurs pre- and postnatally, and its rate is reduced in aging. Adipocytes do not normally undergo cell division because they are fully differentiated cells. However, they do increase in number in early neonatal life. There is debate about normal proliferation of adipocytes beyond 2 years after birth, although in obese individuals the cells not only become enlarged but also form new fat cells. They are surrounded by a basal lamina and are responsible for the synthesis, storage, and release of fat. There are two types of fat cells, unilocular and multilocular

Mast cells

arise from myeloid stem cells in the bone marrow and usually reside near small blood vessels. Although they share many structural and functional characteristics with basophils, they develop from different precursors and are not related. a. These cells are one of the largest cells of connective tissue proper. They possess a central spherical nucleus; their cytoplasm is filled with coarse, deeply stained metachromatic granules; their contents (known as primary mediators) are listed in Table 6.1. b. Their surfaces are folded, and in electron micrographs, they have a well-developed Golgi complex, scant RER, and many dense lamellated granules. Two populations of mast cells exist. Connective tissue mast cells possess secretory granules containing heparin. The other population, the smaller mucosal mast cells whose secretory granules contain chondroitin sulfate, is located in the mucosa of the alimentary canal and of the respiratory tract. Interestingly, there are no mast cells in the central nervous system presumably to prevent the possibility of an inflammatory response. c. When mast cells become activated during a type I hypersensitivity reaction (see next item), phospholipids of their cell membranes can be converted into arachidonic acid by the enzyme phospholipase A2• Arachidonic acid is, in turn, converted into secondary mediators (e.g., leukotrienes and prostaglandins; see Table 6.1)

Eosinophils

bind to antigen-antibody complexes on the surface of parasites (e.g., helminths) and then release cytotoxins that damage the parasites. (1 ) They are most prevalent at sites of chronic or allergic inflammation. (2) Eosinophils are attracted by eosinophil chemotactic factor (ECF), which is secreted by mast cells and basophils, to sites of allergic inflammation. There, eosinophils release enzymes that cleave histamine and leukotriene C, thus moderating the allergic reaction. (3) These cells also phagocytose antibody-antigen complexes.

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).

Reticular tissue

consists mostly of a network of branched reticular fibers (type Ill collagen) (Figure 6.5). a. This tissue invests liver sinusoids, smooth muscle cells, and fat cells and forms the stroma of lymphatic organs, bone marrow, and endocrine glands. b. It also forms the reticular lamina of basement membranes.

Unilocular adipose cells

contain a single large fat droplet. To accommodate the large fat droplet, the cytoplasm and nucleus are squeezed into a thin rim around the cell's periphery. (1 ) These cells have plasmalemma receptors for insulin, growth hormone, norepinephrine, and glucocorticoids to control the uptake and release of free fatty acids and triglycerides. (2) They are surrounded by a basal lamina and are responsible for the synthesis, storage, and release of fat.

Dense connective tissue

contains more fibers but fewer cells than loose connective tissue. It is classified by the orientation of its fiber bundles into two types

Emilin-1

elastin microfibril interface-located protein, 106 kDa) is another glycoprotein found at the elastin- fibrillin microfibril interface that most likely regulates the deposition of elastin during the formation of fibers. Both elastin-associated fibrillin microfibrils and emilin-1 play a major role in regulating elastogenesis.

When stimulated, macrophages may fuse to

form foreign body giant cells. These multinucleated cells surround and phagocytose large foreign bodies.

OBESITY

has been recently reclassified as a disease. Individuals are said to be obese if their adipose tissue buildup is great enough that their body mass index is greater than 30 kg/m^2 with a resultant decrease in life span and an increase in the pos sibility of acquiring a group of health problems referred to as metabolic syndrome. Insulin resistance, type II diabetes mellitus, fatty liver disease, and cardiovascular disease are commonly included under the rubric of metabolic syndrome. Obesity occurs as either hypertrophic obesity, characterized by an increase in adipose cell size resulting from increased fat storage (adult-onset), or hypercellular (hyperplastic) obesity, characterized by an increase in the number of adipose cells that begins in childhood and is usually lifelong. A genetic basis for obesity is the result of mutations in the gene for the hormone leptin or in the genes for leptin receptors, thus preventing the production of leptin or producing an inactive form of the hormone or producing inactive leptin receptors. Since leptin functions in the regulation of appetite, persons affected by either of these two conditions possess an insatiable appetite, bringing about unrestrained weight gain. The volume of each adipocyte and the number of adipocytes a re greater in obese than in thin individuals; therefore, the blood leptin levels of obese individuals may be as much as seven times greater than in thin individuals.

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.

Basement membrane-forming collagens

include type IV collagen, which is responsible for the collagen suprastructure in the basement membrane of epithelial cells (page 136); type VI collagen, which forms beaded filaments; and type VII collagen, which forms anchoring fibrils that attach the basement membrane to the ECM.

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).

Ground substance

is a colorless, transparent, gel-like material in which the cells and fibers of connective tissue are embedded. 1. It is a complex mixture of glycosaminoglycans, proteoglycans, and glycoproteins (see Chapter 4). 2. Ground substance serves as a lubricant, helps to prevent an invasion of tissues by foreign agents, and resists forces of compression.

Fibrillin-1

is a glycoprotein that forms fine microfibrils measuring 10 to 12 nm in diameter. During the early stages of elastogenesis, fibrillin 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 microfi brils.

Mucous tissue (Wharton jelly)

is a loose connective tissue that is the main constituent of the umbilical cord. It consists of a jellylike matrix with some collagen fibers in which large stellate fibroblasts are embedded.

Edema

is a pathologic process resulting in an increased volume of tissue fluid. Edema may be caused by a venous obstruction or decreased venous blood flow (as in congestive heart failure), increased capillary permeability (due to injury), starvation, excessive release of hista mine, and obstruction of lymphatic vessels. Edema that is responsive to localized pressure (i.e., depressions persist after the release of pres sure) is called pitting edema.

Resistin

is a peptide hormone that is manufactured by adipocytes in rodents but by macrophages in humans. It is suggested that this hormone induces insulin resistance, resulting in hyperglycemia and type II diabetes mellitus in obese patients. Additionally, resistin contributes to the inflammatory response, a common finding in insulin resistance, obesity, and type II diabetes mellitus.

Adiponectin

is a protein hormone produced only by unilocular adipocytes. This hormone increases insulin sensitivity, therefore, facilitates the uptake of glucose and the oxidation of fatty acids in skeletal muscle cells, and decreases gluconeogenesis and glucose release by hepatocytes. Thus, adiponectin regulates the body's energy metabolism and facilitates weight loss. The blood levels of this hormone are greater in thin than in obese individuals.

Leptin

is a protein hormone, produced primarily by adipocytes, which inhibits appetite for extended periods of time by binding to the appetite increasing neuropeptide Y, thus suppressing the area of the hypothalamus that control feeding, and induces the regions of the hypothalamus that controls the disbursement of energy. Additionally, leptin also induces the synthesis and release of the appetite repressing neuropeptide a-melanocyte-stimulating hormone. Individuals who have mutations in the leptin gene or in the receptors for leptin exhibit uncontrollable hunger and become obese as a result. The volume of each adipocyte and the number of adipocytes are greater in obese than in thin individuals; therefore, the blood leptin levels of obese individuals may be as much as seven times greater than those of thin individuals.

Elastin

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 hydrophobic and allows for the 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.

Interleukin-6 (IL-6)

is also manufactured and released by macrophages in the connective tissue interstices of unilocular adipose tissue of obese individuals. Similar to tumor necrosis factor-a interleukin-6 increases insulin resistance but also enhances skeletal muscle cells' ability to take up glucose and oxidize fatty acids.

Myofibroblasts

is an elongated, spindly connective tissue cell not readily identifiable in routine H&E preparations. It is characterized by the presence of bundles of actin filament with associated actin motor proteins such as nonmuscle myosin (page 59). Expression of the a-smooth muscle actin (a-SMA; actin isoform found in the vascular smooth muscles) in myofibroblasts is regulated by TGF-b1. The actin bundles transverse the cell cytoplasm originating and terminating on the opposite sites of the plasma membrane. The site of actin fibers attachment to the plasma membrane also serves as a cell-to-ECM anchoring junction and is called fibronexus. It resembles focal adhesion found in the epithelial cells (page 141). Th is arrangement is the basis of a mechanotransduction system in which force that is generated by the contraction of intracellular actin bundles is transmitted to the ECM.

Elastic tissue

is composed of coarse, branching elastic fibers with a sparse network of collagen fibers and some fibroblasts filling the interstitial spaces. It is present in the dermis, lungs, elastic cartilage, and elastic ligaments and in large (conducting) blood vessels, where it forms fenestrated sheaths.

White adipose tissue

is composed of unilocular adipose cells. (1 ) This tissue constitutes nearly all of the adult adipose tissue throughout the body. White adipose tissue controls the body's fatty acid homeostasis by accumulating free fatty acids during an excess of caloric ingestion and releasing free fatty acids during a scarcity of caloric intake. (2) It stores and releases lipids as follows: (a) Adipose cells synthesize the enzyme lipoprotein lipase, which is transferred to the luminal aspect of the capillary endothelium. (b) Dietary fat is transported to adipose tissue as very-low-density lipoproteins and chylomicrons. Lipoprotein lipase then hydrolyzes these substances into fatty acids and glycerol. (c) The free fatty acids enter the adipose cells, where they are re-esterified and stored as triglycerides (in fat droplets). Adipose cells also synthesize fatty acids from glucose. (d) Lipid storage is stimulated by insulin, which increases the rate of synthesis of lipoprotein lipase and the uptake of glucose by adipose cells. (e) The release of lipids is affected by neural impulses and/or adrenaline. Stored triglycerides are hydrolyzed by hormone-sensitive lipase, which is activated by cyclic adenosine monophosphate. The free fatty acids are released into the ECM and then enter the capillary lumen.

Mesenchymal tissue

is found only in embryos. It consists of a gel-like amorphous matrix containing only a few scattered reticular fibers, in which star-shaped, pale-staining mesenchymal cells are embedded. Mitotic figures are often observed in these pluripotential cells.

Tumor necrosis factor-a (TNF-a)

is manufactured by macrophages that reside in adipose tissues of obese individuals and is believed to be the principal cause of insulin resistance. Additionally, TNF-a also depresses liver cell's ability to oxidize fatty acids.

Adipose tissue

is the primary site for storage of energy (in the form oftriglycerides) and has a rich neurovascular supply. It acts as an insulator, as a body cushion by filling in certain spaces, and as a shock absorber. Insulin and prostaglandins assist in the control of lipid storage in adipose tissue.

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., ligaments Flava) contain many more elastic fibers and fewer collagen fibers. Th ese ligaments are called elastic ligaments.