1.7 Molecular Structure

absent in extravascular sclera but was dramatically represented in vessel walls.

Scleral blood vessels showed the presence of collagen types IV, V, and VI, the GAGs heparan sulfate and chondroitin sulfate, and the glycoproteins Þbronectin and laminin in endothelial cell basement membranes.

1.6Biomechanics

By virtue of its poor distensibility, the sclera provides a stable viscoelastic system for the globe. This property appears to be dependent, at least in part, on the GAG water-binding properties: the higher the water-holding capacity of the GAG, the more distensible the sclera. The adult sclera exhibits a biphasic response to a sudden force: a rapid lengthening is followed by a slow stretching [62Ð64]. However, like most viscoelastic systems, the sclera stretches proportionately more with small pressure changes. The sclera stretches with initial elevations of intraocular pressure; therefore, small increases in intraocular volume at low pressure result in small increases in intraocular pressure. As the pressure increases, the resistance to further stretching also increases, and therefore small increases in intraocular volume at high pressures result in large increases in intraocular pressure. Following severe transient stretching of the sclera, as in acute glaucoma, scleral distensibility returns to the baseline levels prior to the increase of intraocular pressure.

Scleral distensibility is an important consideration when methods of intraocular pressure measurement are studied. The indentation method results in a signiÞcant increase of intraocular volume; the applanation method does not. In some eyes with increased scleral distensibility (e.g., as produced by inßammatory diseases, high myopia, or retinal detachment surgery), the indentation measurement method results in a false low reading because the sclera stretches to accommodate the pressure of the tonometer.

Scleral distensibility decreases with age [65]. This property appears to depend, at least in part, on the degree of hydration of connective tissue; highly hydrated tissues, such as embryonic skin

or fetal cornea, are highly distensible, whereas adult tissues become more rigid as their waterholding capacity decreases [66Ð69]. Posterior sclera is more distensible than anterior sclera, and the choroid is more distensible than the sclera; the latter helps explains why the choroid forms redundant folds in orbital or choroidal tumors, ocular hypotony, and subretinal neovascularization.

1.7Molecular Structure

Scleral connective tissue consists of cells and extracellular matrix. The cells, or Þbroblasts, play a critical role in the synthesis and organization of the matrix elements. The extracellular matrix is composed of Þbrillar proteins, such as collagen and elastin, and of amorphous ground substance, such as proteoglycans and glycoproteins. The speciÞc turnover rate of the scleral matrix by Þbroblasts and the degradative enzymes they secrete (collagenases, elastases, proteoglycanases, and glycoproteinases) is unknown, but collagen Þbrils have a slower turnover rate than proteoglycans [70]. Healing of scleral wounds, based on a delicate balance of Þbroblast matrix synthesis and enzyme matrix degradation, is a slow process, taking months or years, and the area of the wound can always be identiÞed histologically by the abrupt change in scleral collagen Þber orientation and disorganization that persists throughout the life of the individual [71].

1.7.1Collagen

Collagen types I, III, IV, V, VI, and VIII have been identiÞed in scleral tissue. Each collagen molecule is composed of three polypeptide a chains containing triple-helical and globular domain [72, 73]. The triple-helical regions have a repeating triplet amino acid sequence, summarized as (Gly- X-Y)n, where X and Y are often proline and hydroxyproline, respectively. The presence of glycine at every third residue, with the exception of short sequences at the ends of the chain, contributes to the triple-helical conformation.

Interchain hydrogen bonds, especially with the hydroxyl groups of hydroxyproline, stabilize the triple-helical structure.

Collagen biosynthesis by scleral Þbroblasts is a complex process consisting of several speciÞc intracellular steps. Each polypeptide pro-a chain is a distinct gene product [74Ð76]. The pro-a chains, assembled in the lumen of the rough endoplasmic reticulum, undergo hydroxylation of speciÞc proline and lysine residues by the action of prolyl-3-hydroxylase, prolyl-4-hydrox- ylase, and lysyl hydroxylase. Subsequent to hydroxylation, some of the hydroxylysine residues become glycosylated by the action of glycosyltransferases. After completion of the synthesis, the pro-a chain has two globular domains, the NH2 terminal and the COOH terminal. Following alignment of three polypeptide pro-a chains, interchain and intrachain disulÞde bonds form at the COOH-terminal propeptides, stabilizing and facilitating helix formation. Procollagen type I contains interchain disulÞde bonds within the COOH-terminal propeptides. Procollagen type III contains interchain disulÞde bonds within the NH2-terminal propeptides as well. DisulÞde bonds within the NH2-terminal propeptides form after helix formation [77]. The assembled triplehelical procollagen type I and III molecules are secreted into the extracellular space, where the terminal propeptides are proteolytically removed. The resulting molecules have a remarkable tendency for spontaneous formation of Þbrils [78].

1.7.2Elastin

Small but important amounts of the Þbrillar protein elastin are synthesized by scleral Þbroblasts as part of the extracellular matrix. Elastin is composed primarily of nonpolar hydrophobic amino acids, such as alanine, valine, isoleucine, and leucine, and contains little hydroxyproline and no hydroxylysine [79]. It also contains two unique amino acids, desmosine and isodesmosine, which serve to cross-link the polypeptide chains. Studies on genomic elastin clones from chick embryo aortas indicate that the rate of elastin synthesis is controlled at the level of transcription [73].

1.7.3Proteoglycans

Proteoglycans are complex molecules, synthesized by scleral Þbroblasts and consisting of a core protein of varying length to which GAG chains are covalently linked. GAGs are longchain, unbranched, linear polymers of repeating disaccharide units. One constituent of the unit is an N-acetylated amino sugar, which may or may not be sulfated, and the other is a uronic acid. The high-molecular-weight proteoglycan molecule is composed of 1 or 2 to more than 100 GAG chains with a potential of giving more than 10,000 negatively charged groups per proteoglycan molecule [80, 81]. During synthesis, assembly of the protein core and initiation of the GAG chain occur together in the rough endoplasmic reticulum. One or two different types of GAG chains attach to the core protein at one end and radiate from it in a bottle-brush conÞguration [80].

At least four types of GAGs have been detected in scleral tissue [61]. Dermatan sulfate, chondroitin sulfate, heparan sulfate, and hyaluronic acid largely compose the amorphous ground substance present in the intercellular and interÞbrillar spaces of the sclera. Dermatan sulfate consists of sulfated N-acetylgalactosamine and two different types of uronic acid, glucuronic acid and iduronic acid; chondroitin sulfate consists of sulfated N-acetylgalactosamine and glucuronic acid; heparan sulfate consists of sulfated N-acetylglucosamine and two different types of uronic acid, glucuronic acid and iduronic acid; hyaluronic acid consists of N-acetylglucosamine and glucuronic acid (it is not linked to a core protein and lacks a sulfate group).

Proteoglycans interact with collagen, determining the organization and size of collagen Þbrils [82Ð85]. They also interact with glycoproteins, such as Þbronectin. Most of these interactions are mediated by the GAG component, although some are mediated by the core protein of proteoglycan [86, 87]. The decrease in collagen Þbril arrangement and increase in collagen Þbril size in the area of transition from cornea to sclera coincide with the disappearance of keratan sulfate and the appearance of highly sulfated galactosaminoglycans, such as dermatan sulfate

and chondroitin sulfate [60]. Proteoglycans maintain the proper anatomical structure of the collagen Þbrils and protect them from attack [70]. Proteoglycans also function as modulators of growth factors, such as Þbroblast growth factor or transforming growth factor [82].

1.7.4Glycoproteins

Although collagen, elastin, and proteoglycans are technically glycosylated proteins, the term glycoprotein is primarily used for molecules composed of oligosaccharide with a mannose core N-glycosidically linked to asparagine. The glycoprotein Þbronectin has been detected in sclera as part of the amorphous ground substance [58].

Fibronectin is a high-molecular-weight molecule synthesized by scleral Þbroblasts. It consists of two similar subunits joined near their COOH termini by disulÞde bonds [88]. Each subunit can be divided into a number of globular domains that have speciÞc binding characteristics. There are binding sites for Þbrin, heparin, bacteria, collagen, DNA, cell membranes, and a variety of other macromolecules. Fibronectin is thought to be important in the organization of the pericellular and intercellular matrix by its ability to bind to collagen, Þbroblasts, and GAGs [89, 90]. Antibodies directed against the collagen-binding domain of Þbronectin have been shown to inhibit collagen Þbril deposition [91]. Fibronectin has also been found to play a role in host defense, presumably by its ability to interact with C1q component of complement, Þbrin, bacteria, and DNA [92Ð94]. Characterization of Þbronectin cDNA clones indicates that only a single Þbronectin gene exists; but multiple forms of cellular Þbronectin are generated by alternative splicing of mRNA [95Ð97]. These alternatively spliced forms appear to have differential functions in embryogenesis, defense, wound healing, and homeostatic cell maintenance.

Laminin, a glycoprotein found in basement membranes, consists of three polypeptide chainsÑA(440 kDa), and B1 and B2 (each 220 kDa)Ñlinked via disulÞde bonds to form an asymmetric cross-structure [98]. Laminin

possesses multiple functional sites that mediate its interactions with cells, such as endothelial cells, and with other extracellular matrix components, such as GAGs, nonintegrin proteins, and integrins [99]. Cells and extracellular matrix proteins interact with laminin via speciÞc surface receptors. Laminin participates in the promotion of cell adhesion, growth, migration, and differentiation, as well as assembly of basement membranes [100].

The fact that laminin is the Þrst extracellular matrix protein to appear in development emphasizes its importance in the intricate process of tissue organization [98].

1.7.5Matrix-Degrading Enzymes

Collagenase, elastase, proteoglycanase, and glycoproteinase are enzymes capable of degrading the matrix components. Some of these enzymes are synthesized by the scleral Þbroblasts themselves, whereas others are secreted by inßammatory cells, such as neutrophils and macrophages.

Collagenase degrades cross-linked type I and type II collagen Þbrils by attacking the collagen molecule at one speciÞc locus one-quarter of the distance from the COOH-terminal. Two fragments, TCA and TCB, three-quarters and onequarter of the collagen molecule, respectively, are generated. These fragments denature spontaneously at temperatures greater than 33¡C, are phagocytosed, and become susceptible of further attack in the lysosomes by proteinases, such as cathepsins B and N. Collagen degradation in normal and inßamed tissues depends on the balance between the collagenase and its inhibitors [101]. The protein core of the proteoglycans must be broken by proteoglycanases for the collagenase to come into contact with the underlying collagen [70].

Elastase is a powerful proteinase that, unlike collagenase, lacks speciÞcity. It degrades not only elastin, but also other components of the extracellular matrix, such as collagen and proteoglycans. The neutrophil and macrophage elastases may be important in degrading elastin in inßammatory reactions [102].