The Anatomy and Cell Biology of the Human Cornea, Limbus, Conjunctiva, and Adnexa
Ilene K. Gipson
Nancy C. Joyce
James D. Zieske
The cornea, conjunctiva, and intervening transition area known as the limbus comprise the tissues at the ocular surface. These three regions, shown diagrammatically and histologically in Figs. 1-1 and 1-2, have both structural and functional features in common. All three are covered by a stratified, squamous, nonkeratinizing epithelium at the surface of the eye that functions in innate defense at the ocular surface. These epithelia sit on a basement membrane and are connected through an identical anchoring complex to an underlying connective tissue stroma. Functionally, all three regions of the epithelium serve as the most important barrier to fluid loss and pathogen entrance, and they support the tear film by synthesizing membrane-associated and secreted mucins. The connective tissue of all three regions serves not only as a structural support but also as the conduit of fluids and nutrients, and it houses support cells that provide for maintenance of the matrix and overlying epithelium. The unique features of each region, however, indicate the special functional roles of the tissue zones. The cornea, because of its critically important functions of light refraction and transmittance, has accordingly received the most attention in studies of its structure, function, and pathology. Recently, more attention has been given to the surrounding limbal and conjunctival regions, which to some degree function as support tissues for the cornea.
Maintenance and support of the ocular surface are also provided by the adnexa (Figs. 1-1 and 1-3), which include the all-important eyelids that function to spread and mix the tear film and, thus, lubricate the ocular surface. Glands that secrete products for maintenance and defense of the ocular surface include the meibomian glands, the main lacrimal gland, the accessory lacrimal glands of Kraus and Wolfring, and the small glands of Zeiss and Moll, which empty into the space surrounding the eyelash cilia.
This chapter reviews aspects of anatomy and cell biology of, first, the cornea, followed by the limbus and the conjunctiva—highlighting the unique features of each—and, finally, major features of the anatomy and cell biology of the adnexa. More complete details of the gross anatomy of these ocular surface regions are available (1, 2, 3).
 FIGURE 1-1. Diagram demonstrating the ocular surface and adnexal tissues described in this chapter. The ocular surface tissues include the cornea, limbus, and conjunctiva; and the adnexa include the lids with eyelash cilia, the meibomian gland, the main lacrimal gland, the accessory lacrimal glands of Krause and Wolfring and the small glands, that empty into the cilia sheath—the glands of Zeiss and Moll.(see color image) |
 FIGURE 1-2. A: Histologic appearance of a full-thickness cross-section of human cornea shows the five- to seven-cell-layered epithelium, the three layers of the stroma, including Bowman’s layer (BL), the lamellar stroma with its resident keratocytes, which occupies over 90% of the corneal tissue (LS), and Descemet’s membrane (D), which is the thickened basement membrane of the corneal endothelium. The endothelium (E) is a low, cuboidal monolayer of cells that borders the anterior chamber. B: Light micrograph of anterior limbus showing the end of Bowman’s layer (arrow) at the juncture between cornea to the left and the first limbal blood vessel (BV) directly beneath the area of corneal epithelial stem cells. C: Micrograph of bulbar conjunctiva. Note numerous goblet cells (arrowheads) intercalated within the stratified epithelium as well as numerous blood vessels (BV) in the connective tissue. D: The junction (arrow) between nonkeratinized (left) and keratinized (right) epithelium of the epidermal tarsal and palpebral conjunctiva. Note numerous cells with the substantia propria below the epithelium. (Original magnifications: A, ×180; B, C, D, ×250.) |
CORNEA
The cornea is a tissue highly specialized to refract and transmit light. It is approximately 1 mm thick peripherally and 0.5 mm centrally. It comprises an outer stratified squamous nonkeratinized epithelium, an inner connective tissue stroma with resident keratocytes, and, bordering the anterior chamber, a low cuboidal endothelium (Fig. 1-2). Although this avascular tissue seems simple in composition, it is extraordinarily regular and precisely arranged. All three layers have a uniform and consistent arrangement throughout the tissue so that light is precisely bent and transmitted through to the lens and then to the retina.
Corneal Epithelium
The human corneal epithelium has five to seven cell layers and is 50 to 52 μg thick (1,4,5) (Fig. 1-4). It has several unique characteristics among other stratified squamous epithelia of the body. It is extraordinarily regular in thickness over the entire cornea and it has an absolutely smooth, wet, apical surface that serves as the major refractive surface of the eye. Its location on the surface of the translucent cornea requires that it be transparent. Unlike other epithelia of its class, it is specialized to exist over an avascular connective tissue. Protection of the vital refraction and “light passage” functions is provided by an extraordinarily dense sensory nerve system that can induce rapid response to
potential danger. In addition to its unique functions over the translucent cornea, the epithelium carries out “routine” housekeeping functions common to any epithelium that borders the external environment. These include provision of a barrier to fluid loss and pathogen entrance, and resistance to abrasive pressure. A barrier to fluid loss is provided by tight junctions surrounding lateral membranes of apical cells, and a pathogen barrier is provided by membrane-spanning mucins at the apical surface. Resistance to abrasion requires that cells of the epithelium be specialized for tight adherence to one another and to their underlying extracellular matrix. Finally, its position adjacent to the outside world requires that the epithelium have a rapid and highly developed ability to respond to wounding.
potential danger. In addition to its unique functions over the translucent cornea, the epithelium carries out “routine” housekeeping functions common to any epithelium that borders the external environment. These include provision of a barrier to fluid loss and pathogen entrance, and resistance to abrasive pressure. A barrier to fluid loss is provided by tight junctions surrounding lateral membranes of apical cells, and a pathogen barrier is provided by membrane-spanning mucins at the apical surface. Resistance to abrasion requires that cells of the epithelium be specialized for tight adherence to one another and to their underlying extracellular matrix. Finally, its position adjacent to the outside world requires that the epithelium have a rapid and highly developed ability to respond to wounding.
 FIGURE 1-3. A: Low-power light micrographs of the lid margin showing the meibomian gland (M), a portion of the eyelash cilia (C), and its associated gland of Zeiss (Z). The juncture between conjunctival epithelium and epidermal epithelium is indicated by the arrow. B: Low-magnification micrograph of a section of human lacrimal gland. Acini are single cell layered. A duct (arrow) at D from the acini is shown running through connective tissue at lower left. (Original magnifications: A, ×120; B, ×220.) |
The cell layers of the epithelium include three to four outer, flattened squamous cells (squames); one to three layers of mid-epithelial cells, termed wing cells because they have lateral, thin, winglike extensions from a more rounded cell body; and a single layer of columnar basal cells (Fig. 1-4). The corneal epithelium, like all stratified squamous epithelia, is self-renewing and, in the cornea, complete turnover occurs in approximately 5 to 7 days (6). The basal cells are the mitotically active cells of the epithelium, and until recently, it was thought that after they undergo mitosis, one daughter cell remains on the basal lamina while the other moves into the suprabasal layers. More recent studies using bromodeoxyuridine to label dividing cells have shown that the two progeny of basal cell division move together toward the apical surface (7).
The basal cells of the epithelium adhere to their basement membrane and underlying stroma through an anchoring complex that is a series of linked structures, described later (8). The major component of the cytoplasm of the basal cells, as well as that of wing and squamous cells, is the intermediate filaments, composed of proteins known as keratins or cytokeratins (Fig. 1-5). Keratins are a complex family of approximately 30 proteins, each given designations of “K” plus a specific number. There are two classes of keratins—type I or acidic, and type II or neutral/basic. Intermediate filaments are formed by the pairing of type I and type II proteins. As cells of the corneal epithelium differentiate from the basal layer to the apical layer, two keratin pairs are sequentially expressed. K5 and K14 are expressed by basal cells, and K3 and K12 are expressed by all cells (9, 10, 11). One of these keratins, K12, a 64-kD protein, is cornea specific (12,13). Keratins in general constitute the major protein of the corneal epithelium.
The two other cytoskeletal filament types present in cells—actin filaments and microtubules—are also present in corneal epithelial cells. Actin filaments are distributed throughout the cytoplasm of cells of the corneal epithelium, but they are particularly prominent within and under the microplicae region of the apical cell membrane (14). Microtubules have not been studied extensively in human corneal epithelium, but they are prominent in mitotic cells, where they determine the plane of cell division and are involved in chromosome separation.
Perhaps in keeping with its requirement that the epithelium be transparent, all cell layers of the epithelium have, by comparison, a rather sparse accumulation of cytoplasmic organelles such as mitochondria, Golgi apparatus, and endoplasmic reticulum. In all cells, mitochondria and endoplasmic reticulum are sparsely distributed around the ectoplasm, and a prominent Golgi apparatus can usually be seen (Fig. 1-5). In basal cells, the apparatus is particularly obvious in the supranuclear position. In squamous cell layers, Golgi cisterna and small membrane-bound vesicles consistent in size and structure to Golgi-associated vesicles are prominent (Fig. 1-5). Also in keeping with maintenance of corneal epithelial transparency is the high expression level of transketolase, which has been proposed to be a corneal crystalline (15).
The apical membranes of basal cells and the entire membranes of wing and squamous cells are highly undulating and interdigitating (Fig. 1-5). Desmosomes are prominent cell—cell anchoring junctions along these cell borders (Fig. 1-5). Other cell-cell junctions in the corneal epithelium are gap junctions, which contain the gap junction protein connexin 43 (16), and, between lateral membranes of the apical cells, tight junctions containing Z01 protein (17) (Fig. 1-5). A more complete description of the cytoskeletal and molecular natures of cell-cell junctions in the corneal epithelium of humans and other species has been published (18).
 FIGURE 1-4. Low-magnification electron micrograph and light micrograph (inset) of the corneal epithelium and subjacent Bowman’s layer (BL). Note single layer of columnar basal cells, one to two layers of wing cells, and two to three layers of flattened squamous cells. (Original magnifications: main, ×4800; inset, ×750.) |
 FIGURE 1-5. Electron micrographs demonstrating aspects of the ultrastructure of the corneal epithelium of apical cells (A plus inset) and wing cells (B and C plus inset). A: Apical and lateral borders of several adjacent apical cells are evident. Note microplicae (MP), region of tight junction (TJ), presence of Golgi apparatus (G), Golgi vesicles (GV), and rough endoplasmic reticulum (RER). The inset demonstrates a filamentous glycocalyx on the surface of the microplicae. B: Electron micrograph demonstrates the elaborate interdigitation of membranes of adjacent cells, characteristic of wing and squamous cells. A mitochondrion (M), Golgi apparatus (G), and rough endoplasmic reticulum (RER) are present. C: Higher-magnification electron micrograph demonstrates that the cytoplasm of epithelial cells is rich in keratin filaments (KF). A, B, and C all show the presence of the cell-cell anchoring junctions known as desmosomes, which are present along interdigitating cell membranes. A high-magnification section through a desmosome is shown in the inset of C. Desmosomes of corneal epithelia appear similar to those of all other stratified squamous epithelium. (Original magnifications: A, ×21,000, inset, ×51,000; B, ×21,000; C, ×42,000, inset, ×164,000.) |
Specializations of the Corneal Epithelium at Its Apical Tear Film Surface
The outermost apical cell layer has microplicae, ridgelike folds that form regular undulations of the membrane as viewed in cross-section (Fig. 1-5). Scanning electron microscopy of the surface of the cornea demonstrates that apical cells scatter electrons to varying degrees (19,20). Cells that scatter fewer electrons have been termed dark cells; those that do so to a greater degree are called light cells. The number of surface microplicae correlates with the degree of scatter, with dark cells having the fewest per unit area (19). It has been hypothesized that the dark cells are the “oldest” cells at the ocular surface, indicating that they are about to desquamate (19,20). This undulating, specialized apical membrane exhibits a prominent filamentous glycocalyx (Figs. 1-5 and 1-6), which has been studied most extensively in guinea pig (21) and rat (22). The glycocalyx has been hypothesized to be intimately but loosely associated with the mucus of the tear film layer (21), and it has been further hypothesized to play a role in mucin and tear film spread over the surface of the eye (22,23).
 FIGURE 1-6. A: Electron micrograph of microplicae and glycocalyx (small arrow) on the surface of guinea pig conjunctiva. The membrane-associated mucins can be seen emanating from the tips of the microplicae (arrowheads) in the electron-dense glycocalyx. At least three different membrane-associated mucins are present in the human glycocalyx; these include MUC1, MUC4, and MUC16. The cytoplasmic domains of the mucins are believed to associate with actin filaments (large arrow), which extend toward the membrane where membrane-associated mucins insert. (From Nichols BA, Chiappino ML, Dawson CR. Demonstration of the mucous layer of the tear film by electron microscopy. Invest Ophthalmol Vis Sci 1985;26:464-473.) B: The immunohistochemical localization of one of the membrane-associated mucins, MUC16, is shown on a section of corneal epithelium. (Original magnifications: A, ×89,000; B, ×300.) |
Major components of the glycocalyx along the apical cell-tear film interface are membrane-associated mucins (Fig. 1-6). [For review, see Gipson and Argüeso (23) and Argüeso and Gipson (24).] Once thought to be secreted only from goblet cells, molecular techniques have demonstrated that mucins also are expressed by the surface cells of all wetsurfaced epithelia. On the corneal glycocalyx, at least three membrane-associated mucins are present, and they appear to be particularly prominent at the tips of the microplicae (Fig. 1-6). These mucins, designated MUC1, MUC4, and MUC16, have structural features in common, a short cytoplasmic domain, which may be associated with cytoskeletal proteins and the actin cytoskeleton in the cytoplasm of the microplicae, and an extended, 200- to 500-μm extracellular domain that is highly O-glycosylated. One of the membrane-spanning mucins, MUC16, carries carbohydrate recognized by an antibody designated H185, whose distribution at the ocular surface is altered in non-Sjögren’s dry eye (25, 26). Current concepts of the function of the membrane-associated mucins is that these highly glycosylated hydrophilic glycoproteins in the glycocalyx are responsible for maintenance of the tear fluid on the ocular surface and that their alteration leads to dry spots as stained by rose bengal (23). Studies of regulation of expression and glycosylation of these mucins will yield information relevant to treatment of drying, keratinizing ocular surface disease. To date, two compounds, dexamethasone (27) and retinoic acid, are known to regulate the expression of membrane-associated mucins MUC1 and MUC4, respectively (28).
Epithelial Anchorage to the Stroma
Basal cells (Fig. 1-7) adhere to their basement membrane and underlying connective tissue stroma by a series of linked structures termed collectively the anchoring complex (8). These structures and basement membrane are products of the basal cells of the epithelium. Figure 1-7 ultrastructurally and diagrammatically shows the components of the anchoring complex and their molecular constituents. The structural components include, on the cytoplasmic face of the basal cell membrane, an electron-dense region into which keratin filaments insert. This dense region is termed the hemidesmosome, and approximately 28% of the basal cell membrane is occupied by these junctions in central cornea (29). An integral membrane protein, α6β4-integrin, links the intracellular components of the hemidesmosome to the extracellular components of the basement membrane (30). On the opposite side of the basement membrane from the hemidesmosome, anchoring fibrils insert. These uniquely cross-banded anchoring fibrils have as a component type VII collagen (31). Type VII collagen has a globular domain and a helical domain (32). Groups of helical domains of molecules associate to form the cross-banded fibril; globular domains of type VII collagen associate within the basement membrane at hemidesmosome sites as well as distal from the basement membrane in the anterior 1 to 2 μm of Bowman’s layer, in small patches of basement membrane-appearing material termed anchoring plaques. Cross-banded anchoring fibrils thus extend from the basement membrane throughout the anterior 1 to 2 μg of Bowman’s layer, forming a complex network that is interwoven with the conventionally cross-banded type I and type V collagen fibrils. The network serves to hold the epithelium and its basement membrane to the stroma; this is clearly demonstrated in the human blistering disease, epidermolysis bullosa acquisita, where a defect in the genetic expression of type VII collagen causes a lack of anchoring fibrils. In this disease, all stratified epithelia are disadherent (33). An example demonstrating the function of the anchoring fibril network in corneal epithelial anchoring comes from a study of diabetic eyes. Diabetic patients have duplicated and thickened basement membranes, including the corneal epithelial basement membrane. During vitrectomy surgery, if the corneal epithelium is removed, the thickened basement membrane, which has within it pockets of anchoring fibrils no longer extending into anterior stroma, comes off with the epithelium (34). This does not occur in normal individuals. A study of the depth of penetration of the anchoring fibril network in diabetic corneas showed a significant decrease in depth compared with age-matched control subjects (35). These data indicate the importance of anchoring fibril network penetration into the anterior stroma in anchorage of the basement membrane and its epithelium.
 FIGURE 1-7. A: Electron micrograph of section through the epithelial-stromal junction region. The structures that are linked to form the epithelial anchoring complex can be seen. They include the hemidesmosome with keratin filaments (KF) inserting into the hemidesmosome plaque (HD). Extracellularly, anchoring filaments (AFL) can be seen in the lamina lucida zone of the basement membrane (BM). Anchoring fibrils (AFB) extend into the stroma at sites opposite the basement membrane from hemidesmosomes; anchoring fibrils insert into anchoring plaques (AP) distal from their insertion into the basement membrane. These plaques have the appearance of small bits of basement membrane. (Original magnification, ×65,000.) B: Diagram illustrating a three-dimensional view of the anchoring complex of the corneal epithelium. The column on the left lists the individual structures (underlined) of the linked complex with their known components underneath. The anchoring fibrils insert into the basement membrane opposite from hemidesmosomes. The cross-banded fibrils splay out among the collagen fibrils (CF), forming a three-dimensional network holding the epithelium tightly to the stroma. The anchoring fibrils terminate distally from the basement membrane in anchoring plaques. |
Corneal Stroma
Structural Zones
The human corneal stroma is the middle connective tissue layer, which, at a thickness of approximately 500 μm, forms the bulk of or approximately 90% of the thickness of the cornea (36) (Figs. 1-2, 1-8, and 1-9). It is unique among connective tissues in that it is the most highly organized and most transparent of any in the body. In addition to its function as a window to light passage, the stroma meshes with the surrounding scleral connective tissue to form a rigid framework for maintaining intraocular pressure and, thus, alignment of the optic pathway.
The stroma is arranged in three clearly defined layers of extracellular matrix (Fig. 1-2). These include, bordering the epithelium, the thin 8- to 10-μm Bowman’s layer, the middle lamellar stroma, which comprises by far the major portion of the stroma, and, adjacent to the endothelium, the 8- to 12-μm Descemet’s membrane, the thickened basement membrane secreted by the corneal endothelium.
Bowman’s Layer
Bowman’s layer is an acellular zone consisting of collagen fibrils and associated proteoglycans densely woven in a random manner into a felt-like matrix (Fig. 1-8). Individual collagen fibrils are approximately 20 to 30 μm in diameter (36). The layer stretches from limbus to limbus, tapering in thickness and ending at the limbus (Fig. 1-2). Based on ultrastructural criteria, Bowman’s has been described embryonically as being derived from stromal cells (36), but at 13 weeks of gestation, one can also see palisades of collagen fibrils emanating from the epithelial basement membrane (37), indicative of a potential epithelial contribution to Bowman’s assembly. The function of Bowman’s layer is not clear; some have hypothesized that it functions to form a smooth, rigid base for maintaining epithelial uniformity and, thus, appropriate refractive power. Others have proposed that the acellular zone is necessary to prevent close contact between epithelial and stromal cells. Such proximity might induce stromal cell “activation” and an inappropriate extracellular matrix assembly. Still others (38) have proposed, however, that Bowman’s layer is the result of the epithelial-stromal interactions, and that Bowman’s layer has no critical function. Most species of mammals do not have a Bowman’s layer and seemingly have appropriate refraction and no epithelial-stromal cell interaction problems. Thus, the question as to the function of Bowman’s layer remains unanswered.
Lamellar Stroma
The lamellar stroma is the major layer of the stroma and comprises lamellae formed from flattened bundles of collagen fibrils oriented in a parallel manner. These bundles, shown ultrastructurally in Fig. 1-8, number approximately 200 to 250 in the human cornea (36). Each bundle extends the width of the cornea, is 2 μm thick, and 9 to 260 μm wide (1). The lamellae in the posterior part of the stroma have a regular orthogonal layering—that is, bundles are at right angles to one another. In the anterior third of the stroma, the lamellae have a more oblique layering, and branching of lamellae in this superficial region has been described (1). Individual collagen fibrils in the bundles have a diameter of 27 to 35 nm and therefore are larger than those of Bowman’s layer (36). The fibril diameters of both Bowman’s and the lamellar stroma are extraordinarily uniform compared with those in other connective tissues.
Within lamellae of the human or bovine cornea, small bundles of microfibrils can be observed. Individual fibers in these bundles are 10 nm in diameter. These microfibrils are composed of fibrillin (39) and are identical in composition to the microfibrils of the zonular fibers, which extend between the ciliary body and the lens. The function of these microfibrillar bundles is unknown, but they are characteristic of most connective tissue.
The lamellar stroma is secreted and maintained by the stromal fibroblasts, commonly termed keratocytes. (Keratinocytes is the term classically given to individual cells of the stratified squamous epithelia, which occasionally leads to confusion in the literature.) These cells usually reside between lamellae (occasionally they are seen with processes in lamellae) and are very flat, with many long, attenuated processes extending from a central cell body in all directions. The tips of the processes touch processes of adjacent cells, forming gap junctions (40,41). Thus, cells of the stroma form a network of coupled cells. Segments of basement membrane can also be demonstrated along the keratocytes. The cytoplasm of the stromal fibroblast is rich in rough endoplasmic reticulum and Golgi apparatus, in keeping with its function as the synthesizer and maintenance cell of the stromal lamellae. Recent data suggest that the stroma also houses a relatively high number of bone marrow-derived cells (42).
 FIGURE 1-8. Micrographs depicting structural aspects of the corneal stroma. A: Light micrograph showing basal epithelial cells, subjacent Bowman’s layer, and the anterior lamellar stroma with its flattened and attenuated stromal fibroblasts, termed keratocytes. B: Electron micrograph of a region in Bowman’s layer demonstrating the random, felt-like interweaving of the collagen fibrils. C: Lower-power electron micrograph of the layered lamellar stroma. Note the presence of the keratocytes running between lamellae. D: Micrograph showing a segment of a flattened keratocyte. Rough endoplasmic reticulum (RER), a mitochondrion (M), and numerous pinocytic vesicles (PV) are present in the cytoplasm. (Original magnifications: A, ×300; B, ×31,000; C, ×4800; D, ×21,000.) |
 FIGURE 1-9. Micrographs of posterior corneal stroma, Descemet’s membrane (DM), and the corneal endothelium. The inset is a light micrograph section from a newborn with its comparatively thin Descemet’s membrane. The electron micrograph is of a section from an 18-year-old human. Note the two layers in Descemet’s membrane. The inner-banded layer was deposited by the endothelium during fetal life. The portion of the endothelial cell visible in the micrograph shows the presence of numerous mitochondria and an interdigitating lateral membrane (arrows). (Original magnifications: main, ×10,000; inset, ×300.) |
The mechanism by which these cells initially lay down the lamellae in their orthogonal pattern has long been a subject of interest. In the developing chick, Birk and Trelstad (43) have demonstrated that surface compartments with bundles of parallel collagen fibrils are present in fibroblasts. These compartments are oriented along the fibroblast axes, and the orthogonality of the cells is in register with that of the extracellular matrix.
Descemet’s Membrane
Descemet’s membrane is the basement membrane of the corneal endothelium. It is synthesized by the endothelium and assembled at the basal surface of the cell layer. At birth, the human Descemet’s membrane is approximately 3 μm wide, but, by late adulthood, it can measure up to 12 μm (36). Its accrual during life is comparable with the thickening of the other basement membranes of the body, including that of the corneal epithelium (44). Two distinct regions can be discerned in electron micrographs of Descemet’s membrane (Fig. 1-9). The anterior one half to one third, depending on the age of the individual, is the “fetal” or “oldest” layer of the membrane. This region displays 100- to 110-nm “long spacing” or a banded collagen pattern. The posterior layer is not banded and appears as amorphous matrix, like all other basement membranes. The mechanism by which this switch in synthesis and matrix organization takes place is not known. The gradual increase in thickness of the posterior layer with age suggests that either there is no degradation of its constituents or the rate of synthesis of constituents is greater than the degradation rate.
TABLE 1-1. CORNEAL EXTRACELLULAR MATRIX COMPONENTS | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Descemet’s membrane is composed of a number of proteins, including fibronectin (45), laminin (46), collagen types IV and VIII (47,48), and proteoglycans containing heparan sulfate, dermatan sulfate, or keratan sulfate (49).
Descemet’s membrane is unique among basement membranes, not so much in its composition, but in its thickness and regional variation in structure. Why this basement membrane is so thick remains an unanswered question. Basement membranes in general are believed to serve as substrates of epithelial cell layers, functioning in the filtering of solutes passing to and from the epithelia and serving as substrates that induce polarity and differentiation of the overlying epithelium.
Corneal Stromal Components
The stroma consists of three main groups of proteins, which include collagens, associated proteoglycans, and other glycoproteins (Table 1-1). All three groups of proteins contain covalently attached carbohydrates and can thus be called
glycoproteins. However, any glycoprotein with a collagenous domain is generally referred to as collagen. The remaining noncollagenous proteins in the stroma are subdivided into two major groups, glycoproteins and proteoglycans. Proteoglycans are a special class of glycoproteins that have glycosaminoglycan (GAG) side chains.
glycoproteins. However, any glycoprotein with a collagenous domain is generally referred to as collagen. The remaining noncollagenous proteins in the stroma are subdivided into two major groups, glycoproteins and proteoglycans. Proteoglycans are a special class of glycoproteins that have glycosaminoglycan (GAG) side chains.
Collagen
The most abundant group of proteins in the body is the collagens. In the cornea, collagen makes up 71% of the dry weight of the cornea (50,51). Collagen, along with proteoglycans, forms the scaffolding of many tissues, including cornea, cartilage, skin, and tendon. These two groups of proteins make up the majority of the extracellular matrix between the cells. In the cornea, collagen is present in the epithelial and endothelial basement membranes, the relatively unorganized fibrils of Bowman’s layer, and the lamellae of the stroma.
Although collagens are expressed in a variety of locations and exhibit various functions, they are defined by the common feature of containing one or more domains having glycine in every third position of the polypeptide chain (52). This feature results in the collagenous domains of the protein, forming a helical structure. This helix in turn is assembled with two other collagen molecules to form a triple helix, resulting in a rod-shaped macromolecule. The triple helices, as well as the individual collagen chains, are cross-linked to each other, resulting in a structure with incredible strength and resiliency (53,54).
The collagen type is determined by the polypeptide chains (termed α chains) present in the triple helix. The various α chains are individual gene products and thus have differing amino acid sequences. Each collagen type consists of three α chains. These chains can be identical or different gene products. Based on α-chain composition, there are 21 types of collagen in human tissues (53,54). Type I collagen is the most abundant collagen in the body and is found in bone, tendon, cornea, and skin. It contains two α1 type I chains and one α2 chain. The amount of collagenous domain (the portion of the polypeptide chain having glycine in every third position) varies tremendously between the different α chains. This results in a wide variation in the structure and function of the resulting collagen. For example, collagen types I, II, III, V, and VI are almost entirely formed by collagenous domains. In electron micrographs of corneal stroma and other tissues, they appear as classic banded collagen fibrils. In contrast, collagen type IV has numerous small noncollagenous domains interspersed between relatively short collagenous domains. In electron micrographs, type IV collagen appears as an amorphous matrix. As another example, type VI collagen has a beaded appearance in electron micrographs. This results from a structure that includes a single, large, uninterrupted collagenous domain in the middle of the chain that is flanked by globular domains at the ends. Collagens can be divided into several subfamilies based on their structure and organization (53,55). Fibrillar collagens (types I to III, V, and XI) have the same general structure and participate in the formation of cross-banded fibrils. The other collagen subfamilies have interruptions in their collagenous domains and are classified as nonfibrillar collagens. These subfamilies include: (a) basement membrane (type IV) collagen, which forms networks; (b) type VI, which forms beaded filaments; (c) type VII, which forms anchoring fibrils and is involved in anchorage of the basement membrane; (d) short-chain collagens, types VIII and X; (e) fibril-associated collagens with interrupted triple helices (FACIT), which include types IX, XII, XIV, XX, and XXI; (f) FACIT-related collagens, types XVI and XIX; (g) membrane collagens (types XIII and XVII); and (h) multiplexins, derived from multiple triple-helical domains and interruptions (types XV and XVIII). Type XVII is also known as bullous pemphigoid antigen and is a component of hemidesmosomes, and type XVIII can be cleaved to form the angiogenesis inhibitor endostatin. Of the 21 collagens identified in human tissues, at least 11 are present in adult mammalian cornea, including types I, III, IV, V, VI, VII, VIII, XII, XVII, XVIII, and XX (55, 56, 57) (Table 1-2). Spliced variants of some of the collagens are also present.
TABLE 1-2. COLLAGENS PRESENT IN CORNEA | ||||||||||||||||||||||||||
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Proteoglycans
The second major group of proteins found in the stroma is the proteoglycans (58). They consist of a core protein containing one or more GAG side chains. The size and percentage of GAG can vary considerably in different proteoglycans. At one extreme is aggrecan, the major proteoglycan of cartilage, which has a core protein of approximately 220 kD and 100 to 150 GAG side chains of approximately 25,000 kD. Thus, it is composed of at least 90% GAG and has a molecular weight of over one million daltons. At the other extreme is decorin, a major corneal proteoglycan, which has a 40-kD molecular weight core protein with one GAG side chain of approximately 50 kD. Thus, it is only 55% GAG and has a molecular weight of under 100 kD.
GAGs, also known as mucopolysaccharides or acid mucopolysaccharides, are characterized by linear polymers of repeating disaccharide units. The disaccharide typically consists of a hexosamine (D-glucosamine or D-galactosamine) plus an uronic acid (D-glucuronic acid or L-iduronic acid). As a result, the GAGs are highly charged, resulting from the presence of many carboxyl [—COOH] and sulfate [—SO4] groups.
The proteoglycans were originally named based on their GAG side chains because these were characterized long before their core proteins were. For example, chondroitin sulfate proteoglycans contain the GAG chondroitin sulfate. At least 30 different proteoglycans have been cloned (58). To date, no relationship between the type of GAG side chain on the core and any particular structural domain on the core protein has been found. Also, in contrast to the collagens, there is no amino acid sequence that is common to all proteoglycan core proteins.
Corneal Proteoglycans. The presence of GAGs in the stroma was first demonstrated with metachromatic toluidine blue and periodic acid-Schiff (PAS) staining (59). Subsequent biochemical analysis of the stromal GAGs has shown that approximately 65% of the corneal GAG is keratan sulfate, whereas 30% is chondroitin/dermatan sulfate. The two major chondroitin/dermatan sulfate proteoglycans in the cornea have been identified as decorin and biglycan. The major keratan sulfate core proteins have been identified as lumican, keratocan, mimecan/osteoglycan, and fibromodulin. Of the known proteoglycans present in the stroma, most are present in other tissues. Only keratocan appears to be specific to the cornea (60). All of the stromal proteoglycans belong to the family of the small leucine-rich proteoglycans (SLRPs). This gene family is defined by the following characteristics: (a) a molecular mass of 32 to 39 kD; (b) a centrally located leucine-rich domain, repeated seven to nine times, with the sequence -L-X-X-LX-L-X-X-N-X-L/I, where L is leucine, N is asparagine, I is isoleucine, and X is any other amino acid; and (c) cysteine residues in the carboxyl- and amino-terminal domains. The spacing and position of the leucine-rich repeats and the cysteine residues are highly conserved in all members of this gene family. The SLRPs bind to the fibrillar collagens and are thought to regulate spacing of the collagens. The cornea also contains a heparan sulfate proteoglycan, perlecan, which is localized in the epithelial basement membrane.
Glycoproteins
The third group of proteins that make up the corneal stroma are the glycoproteins, not included in the collagen or proteoglycan categories (Table 1-1). Glycoproteins are proteins containing one or more sugars covalently bound to the polypeptide chain. The sugar side chains most commonly contain several sugars (oligosaccharides), but may also contain only one or two (disaccharides) sugars. Compared with proteoglycans or mucins, the amount of sugar is low compared with the amount of protein. Also, unlike proteoglycans, there is no serially repeating sugar unit. The carbohydrate side chains in glycoproteins contain certain characteristic sugars, including D-galactose, D-mannose, L-fucose, D-xylose, N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, and sialic acid. Some of the corneal glycoproteins localized in the epithelium and/or Descemet’s membrane are laminin, fibronectin, and entactin/nidogen (Table 1-1). Interestingly, over half of the total amount of soluble glycoproteins in bovine stroma consists of serum proteins (61), including albumin, gamma globulin, transferrin, and α-lipoprotein. This suggests that many of the corneal stromal glycoproteins are derived from other sources.
Corneal Collagen Fibrillogenesis
One of the unique features of the corneal stroma is the regular alignment of collagen fibrils. This alignment is crucial to the maintenance of transparency of the cornea as well as to the strength of the tissue. Another unique feature is the consistent diameter of the collagen fibrils (27 to 35 nm), with all other tissues exhibiting fibrils of a much larger and more heterogeneous diameter. Subsequently, the regulation of corneal fibril diameter has been of great interest to many corneal cell biologists. The synthesis of collagen molecules and their association into fibrils requires a series of events that are common to all fibrillar collagens. These events have been extensively reviewed elsewhere (53,54,62). These processes include hydroxylation and glycosylation of the individual molecules and then proteolytic processing and cross-linking. In part, the relatively small diameter of the corneal collagen fibers is the result of the elevated levels of type V collagen. In most other tissues, including tendon and sclera, type V collagen represents only 2% to 5% of the total collagen. However, in the cornea, type V makes up 15% to 20% of the total. In a series of elegant experiments, it has been demonstrated that type V and type I collagen form heterotypic fibrils in the cornea, and that the size of the fibril is regulated by the amount of type V collagen present. Experimental data suggested that type V collagen can lead to a reduction of the diameter of the fibril by over 50% compared with a fibril composed entirely of type I collagen [reviewed in Birk (62)]. The interaction of the two collagens does not completely explain the small size of the corneal fibrils, however. The size also depends on the association of the fibrils with proteoglycans. Decorin, lumican, keratocan, and fibromodulin are all known to associate with collagen fibrils. Studies using purified native collagen have shown that both decorin and lumican inhibit fibril diameter growth (63). This inhibition is due to properties of the core protein of these proteoglycans because enzymatic removal of the GAGs from the core protein does not alter the inhibitory activity. This suggests that the core
proteins of lumican and decorin bind to collagen and limit the size of the fibril. The importance of proteoglycans in corneal stromal fibril formation is demonstrated in mice lacking lumican, which have corneal opacities as a result of irregular fibril size and spacing (64), and also in humans who have mutations in keratocan. This mutation results in corneal flattening (65).
proteins of lumican and decorin bind to collagen and limit the size of the fibril. The importance of proteoglycans in corneal stromal fibril formation is demonstrated in mice lacking lumican, which have corneal opacities as a result of irregular fibril size and spacing (64), and also in humans who have mutations in keratocan. This mutation results in corneal flattening (65).
Wound Repair
For many years, stromal and epithelial wound repair have been generally examined and discussed as separate events. However, since the early 1990s, it has become increasingly clear that even the simplest epithelial wound results in the death of the subjacent keratocytes. Loss of keratocytes has been observed in corneal debridements, incision wounds, penetrating keratoplasty, photorefractive keratectomy (PRK), and laser in situ keratomileusis (LASIK). It therefore appears that almost all corneal wound repair involves both an epithelial and a stromal component.
Because of the difficulty in the use of human corneas for experimental studies, there are few direct studies that have examined corneal epithelial wound repair in humans. It is assumed that human corneas respond in a manner similar to that in animal models, but this has not been substantiated to any great extent. In animal models, corneal epithelial wound healing is a complex process that can be roughly divided into three overlapping phases. In the first phase, the epithelial cells flatten, elongate, and migrate as an intact sheet to cover the wound. To migrate, the hemidesmosomes, which normally attach the epithelial cells to the basement membrane, are disrupted, and dynamic anchoring structures termed focal contacts are formed. In the second phase of epithelial healing, cells distal to the original wound, including the limbal and peripheral corneal epithelium, undergo cell proliferation to repopulate the wound area. Stratification and differentiation of the epithelium follow cell proliferation. In the third phase, the hemidesmosomes are reformed and, depending on the original wound, basement membrane and the extracellular matrix are resynthesized and reassembled. Numerous proteins and signaling pathways have been postulated to be involved in these processes in animal models, and readers are referred to a number of reviews (66, 67, 68, 69, 70, 71). Two proteins, epidermal growth factor and fibronectin, found to promote epithelial healing in animal models, have been tested in human clinical trials for therapeutic effects. However, the results have been inconclusive, with some trials reporting a positive effect on healing rates, but others showing no beneficial effect.
Stromal wound healing can also be considered to occur in phases [reviewed in Zieske (71)]. In the first phase, the keratocytes adjacent to the area of epithelial damage undergo apoptosis, leaving a zone devoid of cells. This cell death has been postulated to be the result of the wounded epithelium, as well as due to unknown components in the tear film (72). In the second phase of stromal repair, the keratocytes immediately adjacent to the area of cell death enter the cell cycle and proliferate. This proliferation occurs 24 to 48 hours after wounding in both a rat and rabbit model (73,74). As part of the second phase, the keratocytes undergo a phenotype transformation (and are termed fibroblasts) and migrate into the wound area. This migration takes up to a week in animal models and may be even slower in human corneas. The third phase involves the transformation of fibroblasts into myofibroblasts. These cells express elevated levels of smooth muscle actin and are involved in contracting the wound. The extent of myofibroblast formation depends on the type of wound and the extent of interaction with the epithelium. In general, gaping wounds and wounds that destroy the basement membrane (e.g., PRK) result in greater myofibroblast formation than wounds that leave the basement membrane intact or disrupt it minimally (e.g., LASIK). In addition, larger wounds appear to generate a larger number of myofibroblasts than smaller ones. Myofibroblast formation in a rabbit model of PRK peaked at 1 month after wounding (74). The last phase of stromal healing involves the remodeling of the stroma and is also greatly dependent on the original wound. Wounds, such as those caused by PRK, that are horizontal to the corneal surface usually heal with minimal scarring. In this type of wound, the myofibroblasts regress and the basement membrane is reformed. However, in humans, this may require a year or more. In contrast, gaping or incisional wounds stimulate the synthesis of collagens not normally present in the cornea (type III) and other abnormal extracellular matrix materials. This results in improper collagen fibril formation and spacing, and corneal scarring. This type of wound can require many years of remodeling for the cornea to regain its normal optical quality. In humans, one of the complications after PRK is a resultant haze seen immediately subjacent to the ablated area. This appears to be the result of the deposition of extracellular matrix materials, and also to the wound-healing stromal cells themselves. It has recently been demonstrated that myofibroblasts localize beneath the area of laser ablation and that the reflectivity of these cells is a major cause of light scattering (75).
Recent studies suggest that the stroma contains a relatively high number of immature bone marrow-derived cells in addition to the keratocytes (42). To date, almost all studies of stromal wound healing have focused on the keratocytes and their wound-healing phenotypes. Thus, it is unknown if the immature bone marrow-derived cells play a role in the repair process.
Provisional Matrix
Although it is sometimes assumed that the corneal epithelium migrates over the basement membrane or underlying extracellular matrix component after a wound, this may not be correct. Indeed, a variety of matrix components are
synthesized by the epithelium in response to a wound [reviewed in Zieske (71)] (Table 1-1). These include laminin-5, entactin, collagen IV, and perlecan, which are normally present in the basement membrane. In addition, components not normally present are synthesized and deposited, including lumican, fibrin, and an unprocessed form of laminin-5. These components appear to be present in all types of wounds and make up a wound-healing surface that has, in the past, been termed a pseudomembrane, but is more properly termed a provisional matrix. Epithelial cells actually migrate on this provisional matrix after wounding, and several lines of evidence in animal models suggest that the matrix influences wound healing [reviewed in Zieske (71)]. For example, corneal epithelial cells in culture migrate at a faster rate on unprocessed laminin-5 than on the processed form seen in unwounded corneas. Stromal wound healing also appears to involve a provisional matrix. A number of extracellular matrix components not normally seen in the stroma have been localized after wounding, including fibronectin, fibrin, tenascin, collagen types IV and VII, and laminin-1 (Table 1-1). In addition, keratan sulfate proteoglycan levels decrease whereas chondroitin sulfate levels increase in the wounded stroma. In an experimental model, fibronectin and chondroitin sulfate stimulate fibroblast migration into a matrix. Thus, the provisional matrix may promote migration of fibroblasts into the wound area.
synthesized by the epithelium in response to a wound [reviewed in Zieske (71)] (Table 1-1). These include laminin-5, entactin, collagen IV, and perlecan, which are normally present in the basement membrane. In addition, components not normally present are synthesized and deposited, including lumican, fibrin, and an unprocessed form of laminin-5. These components appear to be present in all types of wounds and make up a wound-healing surface that has, in the past, been termed a pseudomembrane, but is more properly termed a provisional matrix. Epithelial cells actually migrate on this provisional matrix after wounding, and several lines of evidence in animal models suggest that the matrix influences wound healing [reviewed in Zieske (71)]. For example, corneal epithelial cells in culture migrate at a faster rate on unprocessed laminin-5 than on the processed form seen in unwounded corneas. Stromal wound healing also appears to involve a provisional matrix. A number of extracellular matrix components not normally seen in the stroma have been localized after wounding, including fibronectin, fibrin, tenascin, collagen types IV and VII, and laminin-1 (Table 1-1). In addition, keratan sulfate proteoglycan levels decrease whereas chondroitin sulfate levels increase in the wounded stroma. In an experimental model, fibronectin and chondroitin sulfate stimulate fibroblast migration into a matrix. Thus, the provisional matrix may promote migration of fibroblasts into the wound area.
Corneal Endothelium
Ultrastructure
Corneal endothelium is the single layer of cells forming a boundary between the corneal stroma and anterior chamber (Figs. 1-2 and 1-9). The endothelial monolayer from young individuals consists of polygonal cells, 4 to 6 μm thick, with a diameter of approximately 20 μm (76). The posterior (apical) cell surface contains numerous microvilli (77), whereas the lateral and basal plasma membranes are extensively interdigitated (78,79). Both of these types of membrane folding provide for increased surface area, and the interdigitations between neighboring cells provide a means for maintaining strong cell-cell contacts. A circumferential band of actin filaments, located toward the apical aspect of the cells, helps maintain cell shape (80,81). Ultrastructural studies of corneal endothelial cells reveal the presence of abundant mitochondria, indicating that these cells are highly metabolically active (Fig. 1-9). Extensive rough and smooth endoplasmic reticulum, as well as a distinct Golgi apparatus, provide evidence of significant protein synthesis. The apical aspect of the lateral membranes contain focal, rather than “beltlike,” tight junctions (maculae occludentes) (82,83). Corneal endothelial cells express occludin, a tight junctional protein located in the lateral plasma membrane, and ZO-1, a member of a submembranous cytoplasmic complex associated with tight junctions (84,85). Ultrastructurally, endothelial cells form gap junctions, with typical connexin structure between cells. These junctions are located on the lateral plasma membranes anterior to the tight junctions (78,86,87) and are sites of dye transfer and electrical coupling between cells (88). Corneal endothelial cells express the gap junction protein, connexin-43 (85). Anchoring junctions mediate close contact between the plasma membranes of adjacent cells and the underlying actin filament network, thereby strengthening cell-cell associations. N-cadherin and α-, β-, and γ-catenin (plakoglobin) are among the constituents of the anchoring junction complex in corneal endothelial cells (89,90). The basal (anteriormost) aspect of corneal endothelial cells rests on Descemet’s membrane, the thick basement membrane that is secreted by the endothelium. The nature of structural specializations that anchor endothelial cells to Descemet’s membrane is unclear, although focal areas of increased electron density suggest the presence of anchoring plaques (86). Proteins expressed in corneal endothelial cells that are known to facilitate normal cell—substrate anchoring include vinculin (91), talin (92), β3-integrin (85), and α-v, β5-integrin (93).
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