Albert & Jakobiec's Principles & Practice of Ophthalmology, 3rd Edition

CHAPTER 192 - Cellular Mechanisms in the Trabecular Meshwork Affecting the Aqueous Humor Outflow Pathway

Beatrice Y.J.T. Yue

The trabecular meshwork (TM) is a specialized tissue located at the chamber angle neighboring the cornea (Fig. 192.1). It is believed to be the major site for regulation of the normal bulk flow of aqueous humor.^[1] This tissue is divided into the uveal meshwork, corneoscleral meshwork, and juxtacanalicular (JCT) regions (Fig. 192.2). In the uveal and corneoscleral meshworks, sheets of trabecular beams that contain lamellae made of connective tissue or extracellular matrix (ECM) materials are lined by TM cells (Fig. 192.3). In the JCT region, the cells reside relatively freely and are embedded in the connective tissue. In the Schlemm's canal (SC), there are endothelial cells also referred to as inner wall cells.

The aqueous humor flows through the TM and the SC into collector channels and aqueous veins. In the normal outflow homeostasis, a pressure gradient exists between the anterior chamber and the episcleral veins. It is likely that the pressure gradient and the resistance to aqueous outflow are altered in the various types of glaucoma. The outflow resistance is believed to locate largely in the JCT/SC area.^[2,3]

FIGURE 192.1 Cross-sectional representation of the anterior chamber of the eye. Note the TM and SC in relation to the cornea, lens, sclera, ciliary body, anterior chamber, and iris. Sizes are not to scale. Illustration by Lisa Birmingham and Adrienne Boutwell University of Illinois Board of Trustees 2006.

FIGURE 192.2 Schematic representation of the structures in the chamber angle. There are three regions in the TM: the JCT area, corneoscleral meshwork, and uveal meshwork. The JCT area is the area underlying SC. The beams of the uveal meshwork are adjacent to the anterior chamber. Sizes are not to scale. Illustration by Lisa Birmingham and Adrienne Boutwell University of Illinois Board of Trustees 2006.

FIGURE 192.3 A low magnification micrograph demonstrating the corneoscleral (CS) meshwork and JCT structure. The CS meshwork, underneath the SC and the JCT area, is composed of interconnecting sheets of trabecular beams (TB). Trabecular cells (TCs) line the beams. Cells (JC) in the JCT area are by contrast embedded in connective tissues. E, SC cells. Scale bar = 5 ?m. Modified from Ueda J, Yue BYJT: Distribution of myocilin and extracellular matrix components in the corneoscleral meshwork of human eyes. Invest Ophthalmol Vis Sci 2003; 44:4772-4779. Copyright 2003 Association for Research in Vision and Ophthalmology.

TM AND SC CELL PROFILES

Key Features

. TM cells are highly specialized, adaptive and multifunctional cells of neural crest origin . SC cells are pressure sensitive endothelial cells of vascular origin . TM and SC cells in situ are interconnected and are essential for maintenance of the normal aqueous humor outflow pathway

TM cells are of a unique type. They have the capacity to perform a variety of functions including: phagocytosis;^[4-6] migration;^[7] elaboration of metabolic,^[8] lysosomal,^[9] and matrix-degrading enzymes;^[10] and production of ECM elements.^[11-18] They also possess endothelial morphologic characteristics in culture; and show polarity in their basal junctions adhering to the culture dish and in their ECM deposition. TM cells incorporate acetylated low-density lipoprotein,^[19] as do vascular endothelial cells in culture. However, they do not stain for Factor VIII antigen, a characteristic marker for vascular endothelial cells. Neither do they form an endothelium as tight as that of cultured vascular endothelial cells. The monolayer formed by TM cells is more porous compared with other endothelia when evaluated in flow systems.^[20] TM cells also phagocytose more avidly than vascular endothelial cells. They release lysosomal enzymes on phagocytosis^[9] and have been speculated to have macrophage-like properties. Immunohistochemical studies have revealed that some cells in TM tissues express major histocompatibility complex class II molecules^[21] and some stain positively for macrophage and dendritic cell markers.^[22] In addition, a neural crest origin of TM cells has been indicated.^[23,24]

The presence of heterogeneous populations of cells in the TM has been implied by findings from several laboratories. Raviola^[25] described a cell type termed Schwalbe line's cells in the TM of Macaca mulatta. Cells that are immunoreactive to smooth muscle myosin have also been localized in the aqueous outflow pathway.^[26] In bovine eyes,^[27] cell populations with different immunoreactivity toward the cytoskeletal proteins vimentin and ?-smooth muscle actin were found in different regions of the anterior chamber angle. Cultures derived from bovine^[28] and human TM contained a cell population that expressed electrophysiological properties of contractile cells. Despite these findings, an alternative possibility cannot be ruled out, namely, that there is a single TM cell type existing in different states, expressing different cellular and physiologic properties while adjusting to the microenvironments. As an example, the expression of ?-smooth muscle actin has been shown to be inducible by transforming growth factor-?1 (TGF-?1) in cultured human and monkey TM cells.^[29] It is also known that other cell types, such as corneal endothelial, retinal pigment epithelial, and vascular smooth muscle cells, can change their morphologic characteristics and phenotypic expression depending on factors such as the makeup of the underlying ECM.^[30,31] The heterogeneous and homogeneous hypotheses thus await further research for clarification.

TM cells are essential for maintenance of the normal aqueous humor outflow system.^[32] Disturbances in the vitality and functional status by genetic predisposition, aging, or other insults, may result in obstruction of the aqueous outflow, leading to intraocular pressure (IOP) elevation and glaucomatous conditions. In vivo, TM cells have limited proliferative activity. Morphometric evaluations indicate that a continuous loss of TM cells occurs during adulthood.^[33] In patients with primary open-angle glaucoma (POAG) and pigmentary glaucoma, the cell loss and disruption of the endothelial covering are striking.^[34] Areas in which the trabecular beams are denuded of cells are associated with a major loss of outflow channels, which represents a possible mechanism for the decreased outflow facility in these conditions. Laser trabeculoplasty, known to increase outflow facility, is associated with increased trabecular cell replication.^[35]

A major advance in TM research is the development and maintenance of viable TM cells in culture. TM cells have been isolated, propagated, and maintained from bovine, calf, porcine, murine, monkey, and human eyes.^[4,32,36] These cells in culture have polygonal endothelial morphologic features with a growth pattern distinct from that of fibroblastic and corneal endothelial cultures. They lay down prominent basement membranes and deposit ECM materials, including fibronectin, laminin, and collagens, similar to those found in vivo. Apical villous projections and cell junctions are frequently seen. TM cells grow as a single layer in sparse cultures, although cell borders and cell extensions often overlap and cluster formation is commonly observed in confluent cultures. TM cells are also actively phagocytic in culture,^[9]and possess modified low-density lipoprotein receptors.^[19] TM cells have also been grown on Millipore filters^[20] for the measurement of hydraulic conductivity and physiologic evaluation of antiglaucoma drugs.

The growth of SC cells is critically dependent on proper dissection methods. Stamer et al^[37] have successfully isolated and cultured SC cells by cannulating the canal with a gelatin-coated suture. These cells are shown to be vascular in origin. They endocytose low-density lipoprotein and acetylated low-density lipoprotein and organize in the presence of Matrigel into multicellular tube-like structures. In situ, cells in the various regions of TM and those in SC appear to be interconnected by cell processes (Fig. 192.4).^[38] The SC/TM configuration is pressure sensitive.^[38] Interactions between SC and TM cells have also been suggested as TM cells were found to release cytokines, which upon binding to SC cells, increased the SC permeability.^[39]

FIGURE 192.4 (a) Mechanism of SC endothelium (SCE) attachment to underlying trabecular lamellae. Cytoplasmic processes of SCE attachment to juxtacanalicular cell (JC) processes. Juxtacanalicular cell processes (JCC) in turn attach to trabecular lamellae (TL). Intertrabecular cell processes rather than collagen attachments limit excursions of adjacent trabecular lamellae. IOP-induced tissue loading (hollow arrows). (b) Region of scanning electron micrograph in (c) (white square). Numerous cytoplasmic processes arise from JCT cells creating extensive attachments to the undersurface of SCE. (d) Appearance and relationships of SCE to JCT cells and TM when IOP is low. (e) Appearance and relationships at physiologic IOP. SCE attachments to the underlying TM modulate SCE distention into SC. When IOP progressively increases, structural elements responsible for resistance to pressure respond by configuration changes as illustrated by the configuration from (d) to (e). Configuration changes illustrated in transition from (d) to (e) modulate in response to IOP-induced tissue loading, place resistance to aqueous outflow to SCE. The JCT space enlarges, ECM material density reduces and cell processes (CP) restraining the inner wall endothelium change from a parallel to perpendicular configuration. Signs of pressure-induced cell stresses are present at cell process origins where cytoplasm and nuclei of both JCT cells and SCE undergo deformation toward respective processes; cytoplasm and nuclei of SCE undergo elongation and attenuation. Evidence of SC endothelial cell tethering by ECM material is absent. Trabecular lamellae, which participate in modulating and restraining SCE distention into SC progressively separate from one another. (a) From Johnstone MA: The morphology of the aqueous outflow channels. In: Drance SM, ed. Glaucoma: applied pharmacology in medical treatment. New York: Grune & Stratton; 1984. Copyright 1984 Grune & Stratton. (b and c) From Johnstone MA: The aqueous outflow system as a mechanical pump: evidence from examination of tissue and aqueous movement in human and non-human primates. J Glaucoma 2004; 13: 421-438. Copyright 2004 Lippincott Williams & Wilkins. (d and e) From Johnstone MA: Pressure-dependent changes in nuclei, and the process origins of the endothelial cells lining Schlemm's canal. Invest Ophthalmol Vis Sci 1979; 18:44-51. Copyright 2003 Association for Research in Vision and Ophthalmology.

TM cells from patients with POAG are more difficult to propagate in vitro than those from nonglaucomatous sources. The limited growth of cells from POAG patients is probably related to the cell damage observed in diseased conditions, evidenced by decreased TM cell numbers and ECM changes. A TM cell strain derived from a glaucoma patient has been transformed with SV-40 T-antigen into an immortalized cell line.^[40] Characterization and pharmacologic studies have been performed.^[41] However, the adequacy of these cells to be used for specific types of studies needs to be carefully evaluated.^[42]

While a substantial amount of useful information has been obtained from cell culture studies, limitations of in vitro systems and the inherent difficulties in making in vivo correlations do exist. To bridge the gap between the in vitro and clinical observations, one sound approach is to begin with cultured cell observations, proceed to organ culture and animal experiments, and conclude with histologic and other evaluations of human eyes. In this regard, a perfusion organ culture system, developed by Johnson and Tschumper^[43] that allows extended physiologic, morphologic, and molecular investigations of various cellular mechanisms^[44,45] on the intact outflow pathway has proved particularly novel and useful.

The cell culture system nevertheless does offer exciting prospects for glaucoma research, including determination of the fundamental properties of TM cells, assessment of the roles of these cells and their activities in the maintenance of the aqueous humor outflow pathway, and evaluation of new drugs for glaucoma therapy. What follows are the discussions of cellular mechanisms in the TM system that may be relevant to the aqueous humor outflow pathway. While discussed under separate headings, the mechanisms that include the ECM composition and modulation, cell adhesion, cytoskeletal structures, contractility, volume regulation, and intracellular signaling events are all interconnected, directly or indirectly. Effects or influences of aqueous humor components, glucocorticoid, myocilin overexpression or mutations, and other stress-inducing conditions are also described.

ECM COMPOSITION, TURNOVER, AND MODULATION

Key Features

. The ECM in the TM is dynamic; made up of macromolecules including collagens, proteoglycans and glycoproteins . The ECM in the TM is modulated by cytokines and exogenous stimuli . Accumulation of SD plaques and long spacing collagens is observed in glaucoma . Matrix metalloproteinases are important regulators of IOP homeostasis

The ECM components in the TM are also factors thought to be essential for maintenance of the normal aqueous humor outflow.^[36,46,47] In the eyes obtained from POAG patients, excessive, abnormal accumulation of ECM materials has been noted in the TM by electron microscopic, histologic, and immunologic studies.^[46,47]

The ECM produced by the cells is an intricate network composed of an array of multidomain macromolecules such as collagens, cell-binding glycoproteins, and proteoglycans. The macromolecules link together covalently or noncovalently to form a structurally stable composite material. Recent studies have highlighted the view that ECM is not merely an inert supporting material for scaffolding of cells, it is rather a dynamic entity of key importance in all biological systems, determining and controlling the behavior and biologic characteristics of the cells.^[32,48]

The ECM materials in the JCT in normal human eyes were categorized by Lutjen-Drecoll and Rohen^[49] into amorphous basement membrane-like material and sheath derived (SD) plaques. The former is observed primarily underneath the inner wall endothelial lining, and the latter includes the electron dense core that forms with elastic-like fibers, and the surrounding sheath with clusters of banded materials embedded within. In other TM areas, there are discontinuous basement membranes underneath TM cells. Collagen fibers, ground substance, as well as elastic-like fibers make up the core of trabecular beams. Long spacing collagen, an across-banded structure with ~100-120 nm periodicity, has also been described.^[49] ECM components such as collagen,^{[12,15,17,18,36,50-53]} proteoglycan-glycosaminoglycan,^[13,16,54] fibronectin,^{[11,12,50,52,53]} laminin,^[50,52,53] other glycoproteins,^[55,56] and elastin^[14,52,53] are distributed on the various structures (Tables 192.1 and 192.2) in TM tissues.^[52,53]

TABLE 192.1 -- Immunogold Labeling of ECM Components and Myocilin in the Trabecular Lamellae of the Corneoscleral Meshwork

	Basement Membrane	Collagen Fibers and Ground Substances	Core of Elastic-like Fibers	Sheath Material of Elastic-like Fibers	Long-Spacing Collagens
Fibronectin	++	+	+	++	+++
Vitronectin	±	+	++	+	+
Laminin	+++	±	±	+	±
Tenascin	±	+	++	+	+
Elastin	±	±	+++	+	±
Fibrillin-1	±	+	+++	++	++
MAGP-1	±	+	++	+	++
Versican	±	±	++	+	+
Decorin	±	+	+++	++	+++
Hyaluronic acid	±	+	++	+	±
Collagen type I	±	+++	±	±	±
Collagen type III	±	+	+	++	+
Collagen type IV	+++	±	±	±	±
Collagen type V	±	++	±	±	±
Collagen type VI	±	±	+++	+	++
Myocilin	±	+	+	+++	+++

From Ueda J, Yue BYJT: Distribution of myocilin and extracellular matrix components in the corneoscleral meshwork of human eyes. Invest Ophthalmol Vis Sci 2003; 44:4772-4779. Copyright 2003 Association for Research in Vision and Ophthalmology.

The intensity of immunogold labeling was graded in each ECM structure or plaques from ± to +++, with ± representing minimal, and +++, intense staining.

TABLE 192.2 -- Immunogold Labeling of ECM Components and Myocilin in the JCT

	Amorphous Basement Membrane-Like Materials	Core of SD Plaques	Sheath of SD Plaques	Banded Materials in the Periphery
Fibronectin	++	+	++	+++
Vitronectin	±	++	++	+
Laminin	+++	±	+	+
Tenascin	±	++	++	+
Elastin	±	+++	±	±
Fibrillin-1	±	±	+	++
MAGP-1	±	±	+	++
Versican	±	++	+	+
Decorin	±	+++	++	+++
Hyaluronic acid	±	++	++	+
Collagen type I collagen	±	±	±	±
Collagen type III collagen	±	±	++	+
Collagen type IV collagen	+++	±	±	±
Collagen type V collagen	±	±	±	±
Collagen type VI collagen	±	+++	+	++
Myocilin	±	±	++	+++

Modified from Ueda J, Wentz-Hunter K, Yue BYJT: Distribution of myocilin and extracellular matrix components in the juxtacanalicular tissue of human eyes. Invest Ophthalmol Vis Sci 2002; 43:1068-1076, 2002. Copyright 2002 Association for Research in Vision and Ophthalmology.

The intensity of immunogold labeling was graded in each ECM structure or plaques from ± to +++, with ± representing minimal, and + to +++, increasingly intense staining.

At least 28 distinct types of collagen have been identified in mammalian tissues and cell cultures to date.^[57] Most tissues and cells contain more than one type of collagen in accordance with their specific structural and functional properties and with their stage of development. Collagen types I, III, IV, V, VI, VIII, and XIII have been found in TM tissues. Immunofluorescence^[12] and immunogold labeling studies^{[17,50,52,53]} have localized interstitial collagen types I and V to the striated collagen fibrils of the trabecular core (Fig. 192.5). Collagen type III is also associated with sheath of the SD plaques. Type IV collagen is in the basement membranes surrounding trabecular beams and in the amorphorous materials in the JCT. Type VI collagen associates with the core of the elastic-like fibers as well as long spacing collagens. Type VIII collagen is known to be responsible for the formation of the hexagonal lattice type of sheet structure such as that found in Descemet's membrane and type XIII collagen is a novel nonfibrillar collagen. How exactly the different types of collagen are assembled in the TM is unclear. In TM cell cultures, similar types of collagen have been identified.^[15,36]

FIGURE 192.5 Immunogold labeling of ECM components in trabecular cores. Trabecular cores were made up of ground substances with collagen fibers as a matrix and elastic-like fibers as a plexiform framework. Collagen fibers were strongly immunolabeled for collagen type I (a) and moderately for collagen type V (b). Core elastic-like fibers (white asterisks) were composed of electron-dense and electron-lucent areas, and were surrounded by microfibrillar sheath materials (black asterisks). Elastin was immunolocalized almost exclusively to the region within or adjacent to the electron-lucent area of the core (c). Minimal staining was observed in mouse IgG negative control (d). TC, trabecular cell. Scale bar = 200 nm. From Ueda J, Yue BYJT: Distribution of myocilin and extracellular matrix components in the corneoscleral meshwork of human eyes. Invest Ophthalmol Vis Sci 2003; 44:4772-4779. Copyright 2003 Association for Research in Vision and Ophthalmology.

Proteoglycans are macromolecules consisting of a core protein to which glycosaminoglycan side chains are covalently attached. This class of molecules has been implicated in the maintenance of resistance to aqueous humor outflow ever since Barany,^[58] in the 1950s, demonstrated that perfusion of the anterior chamber with testicular hyaluronidase greatly reduced the outflow resistance in enucleated bovine eyes. Injection of chondroitin sulfate into animal eyes also induced IOP elevation.^[59] Although in dispute,^[60] introduction of hyaluronidase^[61] and chondroitinase ABC,^[62] both glycosaminoglycan-degrading enzymes, was shown to decrease IOP in some studies.

In the TM tissue, proteoglycans form gel-like networks that may function as a gel filtration system. The major types identified include chondroitin, dermatan, and heparan sulfate proteoglycans.^{[13,16,36,45,54]}These proteoglycans may represent decorin, biglycan, versican, perlecan, and syndecan.^[54,63,64] Different isoforms of versican have recently been reported to exist in TM tissues.^[65] The most predominant form, V1, has been noted to promote expression of epithelial-like adherens junctions.^[66]

The relative amounts of each type of glycosaminoglycan in the TM tissue have been determined.^[13,45] Chondroitin-dermatan sulfates are the major constituents, and heparan sulfate and keratan sulfate are present in much smaller amounts. Hyaluronic acid composes ~10-20% of the total glycosaminoglycans. Biochemical analyses^[36,67] have also shown that the same types of glycosaminoglycans are produced by TM cells in culture. High levels of hyaluronic acid are observed in explant cultures. However, as the cells adapt to culture conditions, the content of hyaluronic acid returns to the typical 10-20% level.^[13,45,67] Both chondroitin sulfate and hyaluronic acid have been shown to contribute to flow resistance and influence flow rate in vitro.^[68]

With aging, there seems to be a tendency for loss of the collagen associated chondroitin-dermatan sulfate proteoglycans in normal and glaucomatous tissues.^[69] Conversely, a depletion in hyaluronic acid and an accumulation of chondroitin sulfates and undigestible glycosaminoglycan material have been associated with POAG conditions.^[70] Interestingly, the level of an ectodomain fragment of hyaluronic acid receptor CD44 (sCD44) was found to be elevated in the aqueous humor of POAG patients.^[71] sCD44 is cytotoxic to TM cells but the toxicity can be blocked by hyaluronic acid.^[72] The decreased hyaluronic acid may thus result in diminished protective capacity and further deterioration in POAG conditions.

Fibronectin, laminins and vitronectin, the multidomain and multibinding adhesive glycoproteins in the ECM, have been shown to be products of TM cells^[15,36] and are localized in the TM.^{[11,12,52,53]} These glycoproteins are crucial in biologic processes such as cell attachment and spreading, cell differentiation, migration, and wound healing. Overexpression of fibronectin inhibits permeability of TM cells.^[73] A fragment of fibronectin called the heparin II (Hep II) domain increases outflow facility in perfusion cultures of human anterior segments, indicating that fibronectin mediated interactions may have a role in modulating the aqueous hydrodynamics.^[74] Matricellular proteins that include tenascin,^[52,53] SPARC^[56] and thrombospondin-1,^[55] are also present in the TM. Thrombospondin-1, a known activator of TGF-?, is speculated to act as an endogenous activator in the aqueous humor dynamics, and mediate the TGF-? effect on the outflow.^[55]

Elastin is localized to the central core of SD plaques or elastic-like fibers in the TM (Fig. 192.5).^[14,52,53] Fibrillin-1, a component of microfibrils, has been found in both the core and the surrounding sheath of the elastic-like fibers. Within the microfibrillar framework, fibronectin, decorin, collagen type VI and versican have also been identified.^[52,53] It is believed that the collagen fibers and elastic-like fibers are organized in the TM to accommodate resilience and tensile strength, providing a mechanism for reversible deformation in response to cyclic hydrodynamic loading.^[39] Microfibrillar elements including fibrillin-1 and type VI collagen are also constituents of long-spacing collagens (Fig. 192.6).^[52,53] In trabecular lamellae and in JCT regions, accumulation of long spacing collagens and SD plaques has been documented in POAG and aged eyes.

FIGURE 192.6 In the eyes of 58- and 72-year-old donors, long spacing collagens (white stars) were observed both adjacent to the sheath (white asterisks) of elastic like fibers and/or embedded in the ground substances. This structure was specifically immunolabeled by fibronectin (a), fibrillin-1 (b), decorin (c), and collagen type VI (d). TC, trabecular cells. Scale bar: = 200 nm. From Ueda J, Yue BYJT: Distribution of myocilin and extracellular matrix components in the corneoscleral meshwork of human eyes. Invest Ophthalmol Vis Sci 2003; 44:4772-4779. Copyright 2003 Association for Research in Vision and Ophthalmology.

The ECM is a dynamic structure that is constantly modified by the surrounding cells through enzymes and protease inhibitors. The enzymes involved in the remodeling or turnover of the ECM components include the plasminogen activator, plasmin and the matrix metalloproteinase (MMP) families. Both types of enzymes^[10,75] and their inhibitors, plasminogen activator inhibitor-1 (PAI-1)^[75] and tissue inhibitor for matrix metalloproteinase (TIMP),^[10] have been found in the TM. Ongoing ECM turnover, initiated by one or more of MMPs, appears to be essential for maintenance of the aqueous outflow homeostasis. For instance, MMP-2 and MMP-14 may be important initiators in the process of IOP homeostasis.^[10,76,77] MMP-3 and possibly also MMP-9 may be responsible for the efficacy of laser trabeculoplasty, a common alternative treatment in reducing IOP in patients with glaucoma.^[78] Addition or induction of MMP-3 in perfused human anterior segment organ cultures increases,^[76] whereas blocking the endogenous activity of the MMPs in the TM reduces, the aqueous humor outflow facility.^[77]

In addition to the degradation process, the ECM may also be remodeled in response to exogenous stimuli such as glucocorticoids^[79] and oxidative stress.^[80] Mechanical stretch caused an increase in MMP-1 and MMP-3 activities and alteration of ECM molecules including proteoglycans and matricellular proteins.^[81,82] After a physiologic process such as phagocytosis, reduced amounts of fibronectin and laminin and an accompanying disruption of the fibronectin-laminin network were observed.^[6] TM tissues in perfusion organ cultures contained higher levels of fibronectin, laminin, and collagen type I after treatment with ascorbic acid.^[83]

The ECM in the TM is modulated by cytokines. The most studied cytokine is TGF-?, which is a component of the aqueous humor. In patients with POAG, TGF-?2 is found to be present in the aqueous humor at an elevated level.^[84] Overnight treatment of human TM cells with TGF-?2, upregulated ECM-related genes, and increased secretion of fibronectin and PAI-1.^[85] In perfusion cultures of human anterior segments, focal accumulation of fine fibrillar extracellular material was observed in TGF-?2-perfused TM tissues. TGF-?2 perfusion also reduced outflow facility and elevated IOP.^[85] These results suggest that an increased level of TGF-?2 in the aqueous humor may be related to the pathogenesis of glaucoma. Other cytokines such as interleukin-1? (IL-1?) and tumor necrosis factor-? (TNF-?) also modulate the ECM, probably via regulation of MMP and TIMP expressions.^[10,76,86]

A recent proteomic analysis revealed that cochlin is present in TM tissues of glaucomatous but not in normal human eyes.^[87] Cochlin is a major noncollagenous ECM protein in the inner ear. It is also found in the TM in DBA/2J mice with elevated IOP but not in other strains without IOP elevations.^[88] The cochlin deposits in the glaucomatous TM appear to increase with age and are associated with proteoglycans. Such deposits have been proposed to contribute to the increase of ECM resistance to outflow and the POAG pathology.^[88]

CELL ADHESION

Cell-to-cell and cell-to-matrix adhesion is critical for the assembly of individual cells into three-dimensional tissues. Members of cell adhesion molecules such as integrins, cadherins, immunoglobulins, and selectins are usually trans-membrane glycoproteins that mediate binding interactions at the extracellular surface and determine the specificity of cell-to-cell and cell-to-ECM recognitions. Studies of integrins, other cell adhesion molecules, and cell junctions in the TM system are highly relevant. TM cells that line the trabecular beams are continually subjected to flows of the aqueous humor and IOP fluctuations. The lining integrity against stress is achieved by adhesion of cells to the matrices through cell surface receptors along with cell junctions between the cells. Disruption in these adhesions would possibly lead to cell loss, denudation of the beams, and pathologic consequences.

Integrin receptors are known to connect ECM network with actin cytoskeletal components, including vinculin, talin, tensin, and ?-actinin. They function not only as a structural link but also as sites of communication between the cell and the extracellular environment, coordinating signals from outside with intracellular events.^[89] The major type of cell-matrix adhesion structures in cultured human TM cells are focal adhesions associated with the ends of actin- and myosin II-containing stress fibers, small dot-like focal complexes and tensin-positive fibrillar adhesions (Fig. 192.7).^[90] Human TM cells in culture have been shown to possess integrins ?₁, ?₂, ?₃, ?₄, ?₅, ?₆, ?_v, ?₅?₁, and ?_v?₃.^[91] Antibody blocking experiments indicated that these integrins have functional roles in the adhesion of TM cells to fibronectin, laminin, and vitronectin. The cell adhesion is, at least in part, dependent on the Arg-Gly-Asp sequence. Adhesion of TM cells to ECM proteins such as fibronectin induces a time-dependent tyrosine phosphorylation. The phosphotyrosine-containing proteins include focal adhesion kinase and paxillin.^[92] The integrin reportedly present in human TM tissues is similar to that found in cultured cells.^[93]

FIGURE 192.7 Typical organization of the actin cytoskeleton and cell-matrix adhesions in control human TM cells. (a) Phalloidin staining reveals polymerized actin (act) in long, straight actin filament bundles (stress fibers); (b) staining with antibody to myosin light chain (myo) reveals myosin II striated distribution along the stress fibers in the same cell. (c) Phalloidin staining shows actin (act) in stress fibers and peripheral lamellipodia; (d) vinculin antibody staining (vin) visualizes numerous elongated focal adhesions associated with the ends of stress fibers and small dot-like focal complexes associated with lamellipodia at the cell edges. (e) Staining with tensin antibody (ten) shows focal adhesions at the cell periphery and fibrillar adhesions in the central part of the cell; (f) focal, but not fibrillar adhesions are enriched in tyrosine-phosphorylated proteins visualized by phosphotyrosine (PY) antibody staining. Scale bars = 20 ?m. From Grosheva I, Vittitow JL, Goichberg P, et al: Caldesmon effects on the actin cytoskeleton and cell adhesion in cultured HTM cells. Exp Eye Res 2006; 82:945-958.

SC cells have been shown to extend cytoplasmic processes into the JCT space while JCT processes attach also to the trabecular-lamellae processes.^[2] Desmosomes capable of sustaining cellular stress are present between cell process attachments.^[94] Freeze-fracture and electron microscopic studies have described three different morphological types of cell junctions in the outflow pathway: adherens junctions, gap junctions, and tight junctions.^[94,95] These junctional structures in SC cells become less complex with increasing pressure.^[95]

Proteins that form adherens junctions such as intercellular cell adhesion molecule (ICAM)-1, N-CAM, and N-cadherin including intercellular cell adhesion molecule (ICAM)-1 are expressed in TM cells or tissues.^[96,97] SC cells express vascular endothelial adherence proteins, platelet-endothelium cell adhesion molecule-1 (PECAM-1) and vascular endothelial (VE)-cadherin, further suggesting that SC cells are vascular in origin.^[98] ICAM-2, ICAM-3, vascular cell adhesion molecule-1, and platelet-endothelium cell adhesion molecule-1, are known to be involved in heterophilic cell-to-cell interactions. These molecules all contain repetitive extracellular immunoglobulin-like domains.

Selectins such as E-selectin (endothelial leukocyte adhesion molecule-1 or ELAM-1), L-selectin, and P-selectin are cell adhesion molecules characterized by conserved cysteine residues, a calcium-dependent lectin-like domain that mediates interactions with complex carbohydrate moieties, and tandem repeat sequences similar to those found in complement-binding proteins. E- and P-selectins recognize sialylated glycans such as sialyl-Lewis X- and sialyl-Lewis A-containing oligosaccharide molecules. Upregulation of these selectins has been observed in TM tissues in POAG eyes.^[99,100] The upregulation could be related to stress response of the cells.

Gap junction protein, connexin-43 has been localized to sites of contact in adjacent TM cells in culture where the cells communicate with each other.^[101] Tight junctions or zonula occludens formed around the lateral membrane of adjacent cells serve as a physical barrier. They have a highly dynamic structure and their permeability, assembly, and disassembly can be regulated by a variety of cellular and metabolic regulators. TM and SC cells express zonular occludens-1 (ZO-1), an associated protein of calcium-sensitive tight junctions.^[102] Administration of glucocorticoid such as dexamethasone (DEX) resulted in an increased expression of ZO-1, formation of a greater number of tight junctions and an increase in fluid flow resistance.^[103] Antisense oligonucleotides that diminished ZO-1 expression abolished the DEX-induced increase in resistance and the accompanying alteration in cell junctions.^[103]

Numerous studies have in addition shown that the outflow facility is modified via alterations in cell-matrix and/or cell-cell adhesions.^[46] For instance, Bill et al^[104] have reported that depletion of extracellular calcium, by dissociating cell-cell junctions, decreases outflow resistance. Overexpressing of caldesmon^[92,105] or perfusion of the Hep II domain of fibronecton^[97] in human anterior segment organ cultures increases outflow facility perhaps by mechanisms that involve disassembly of actin filaments, loss of focal adhesions and/or disruption of adherens junctions in TM cells.

CYTOSKELETAL ORGANIZATION, CONTRACTILITY, AND VOLUME REGULATION

Key Features

. Actin-disrupting agents that affect cell-matrix and cell-cell adhesions increase the aqueous outflow facility . Change in the actin cytoskeleton is linked to change in the TM cell contraction/relaxation status and cell volume . Glucocorticoid such as dexamethsone alters F-actin architecture and promotes CLAN formation in TM cells . CLANs are also seen in glaucomatous eyes

Cytoskeletal structures are closely connected to adhesion complexes and cell junctions. The cell shape is also determined by the cytoskeleton. The physiologic roles of the cytoskeleton of TM cells in association with outflow facility^[46] and glaucoma have drawn considerable attention. TM cells exhibit three major types of cytoskeletal elements: actin filaments, microtubules, and the intermediate filaments vimentin and desmin.^[106] Agents that affect the actin cytoskeleton such as chelating reagents and cytoskeleton-active drugs including latrunculin A, latrunculin B, and cytocholasin B have been demonstrated to increase the aqueous outflow in relatively short-term perfusion organ culture or in vivo animal studies.^[106] The increase was often associated with morphologic changes, including separation of junctions between TM cells and breaks between SC cells. Sulfhydryl-reactive compounds, including iodoacetamide, N-ethyl maleimide, and ethacrynic acid,^[106] also can influence the outflow system in both enucleated and living eyes. A common mechanism seems to involve changes in cell shape and cell adhesion through cytoskeletal elements. Significantly, glucocorticoids such as DEX alter F-actin architecture and promote cross-linked actin network (CLAN) formation (Fig. 192.8) in human TM cells and tissues.^[107] These CLANs, also seen in glaucomatous eyes, are thought to be an important factor in the disease development.^[108] Moreover, the F-actin architecture in SC and JCT cells in situ has been found to differ between normal and glaucomatous eyes, with the latter showing an overall more disordered actin structure.^[108] These studies collectively underscore the notion that the actin cytoskeleton architecture is an important mediator of the aqueous outflow pathway.

FIGURE 192.8 Whole mount transmission electron micrographs of the cytoskeleton of control TM cells (a) and TM cells exposed to 10?7 M DEX for 14 days (b). The stress fibers in the control cells are arranged in normal linear arrays while DEX-treated microfilaments are grouped into 90-120 nm bundles radiating from electron dense vertices. Scale bar = (a) 0.5 ?m; (b) 2 ?m; (?, inset) 0.5 ?m From Clark AF, Wilson K, McCartney MD, et al: Glucocorticoid-induced formation of CLANs in cultured human TM cells. Invest Ophthalmol Vis Sci 1994; 35: 281-294. Copyright 1994 Association for Research in Vision and Ophthalmology.

Changes in the actin cytoskeleton may also be linked to cellular contraction and relaxation as well as cell swelling or shrinkage.^[107,109] The TM has been characterized as a smooth muscle-like tissue, expressing major contractility regulating proteins featuring contractile mechanisms.^[26,110-112] Contraction and relaxation for smooth muscle are known to be regulated through the phosphorylation status of myosin light chain (MLC) controlled by MLC kinase and phosphatase. MLC phosphorylation promotes cellular contraction while dephos-phorylation leads to relaxation. In the TM system, inhibitors to MLC kinase such as H-7 that influence the actomyosin-driven contractility appear to decrease outflow resistance by cell relaxation, expansion of the TM and SC, and changes in the overall geometry without significantly affecting intracellular adhesion.^[106] Likewise, BDM (3-butanedione 2-monoxime) and blebbistatin, compounds that interfere with actomyosin function through the myosin adenosine triphosphatase (ATPase) to induce actin and/or morphologic changes in TM cells, increase the outflow facility.^[106,102]

Cell volume regulation has also been reported to participate in regulation of the outflow facility. The resting cell volume is a function of coordinated activities and expression of ion channels and transporters that determine the water and solute flux at the plasma membrane. TM cells express water channel protein aquaporin-1.^[113] Alteration in the level of aquaporin-1 has been shown to lead to changes in resting volumes of TM cells and their paracellular permeability. Aquaportin-1 is suggested to act in conjunction with ion channels and/or transporters to exert its effect on the resting volume of TM cells. The Na-K-Cl co-transporter has been proposed to be one of such molecular bases for TM cell volume regulation.^[114] Inhibition of this cotransporter activity causes a reduction in intracellular volume and an increase in TM cell permeability. Potassium and chloride channels may also be involved as their blockade has been reported to impair outflow recovery after cell swelling.^[109,115] Corroborating the cell volume theory, epinephrine, isoproterenol, and others have been shown to cause cell shrinkage and decrease the outflow resistance.^[116] These agents are known to increase the intracellular cyclic adenosine monophosphate (cAMP) levels that are tightly linked to ion channels and transporters.^[116]

INTRACELLULAR SIGNALING

Key Features

. Signal transduction via members of the small GTPase Rho family is vital to the aqueous outflow . Inactivation or inhibition of RhoA or ROCK results in reduced MLC phosphorylation and enhanced outflow facility . Other signaling molecules/pathways such as protein kinase C may also be important

Signal transduction via members of the Rho family of small guanosine triphosphatases (GTPase) has been shown to be of vital importance in the outflow system. Similar to all members of the Ras proteins, Rho GTPases cycle between inactive (GDP-bound) and active (GTP-bound) forms. They exist predominantly in the GDP-bound dormant status, presumably associated with GDP-dissociation inhibitor (GDI). In response to a stimulus such as growth factors or integrin engagement, small GTPases are recruited to specific sites, where activation takes place. Both processes are facilitated by exchange factors (GEF). Inactivation is facilitated by GTPase-activating proteins (GAP) that help the GTPases to hydrolyze their bound GTP into GDP and phosphate. In the active GTP-bound state, Rho GTPases interact with and activate downstream effectors such as Rho kinase to control the assembly of actin filaments into morphologically distinct structures.^[117] RhoA, for example, regulates formation of stress fibers and focal adhesions to coordinate cellular processes including adhesion, migration, and morphologic changes. It also stimulates contractility mediated by activation of complexes of actin and myosin and modulates ECM production. In TM cells, a decrease in actin stress fibers and focal adhesions has been shown to occur with treatment of Rho kinase inhibitors such as Y-27632 and H-1152,^[111] and gene transfer of dominant negative RhoA,^[118] dominant negative Rho-binding domain of Rho kinase,^[46] and exoenzyme C3 transferase^[119] that inactivates Rho by ADP-ribosylation. These cellular changes are all associated with reduced MLC phosphorylation, and/or enhanced outflow facility (Fig. 192.9).^[111] On the other hand, lysophospholipid growth factors including lysophosphatidic acid and sphengosine-1-phosphate, that activate Rho/Rho kinase signaling pathway through G-protein coupled receptors, promote MLC phosphorylation, and in turn decrease the aqueous humor outflow facility.^[120] Similarly, agonists of G-protein coupled receptors such as endothelin-1 and angiotensin II activate Rho GTPase signaling, trigger activation of MLC phosphorylation, and affect IOP.^[111,121]

FIGURE 192.9 Modulation of MLC phosphorylation in TM and its potential involvement in the regulation of aqueous humor outflow facility. Increased MLC phosphorylation evoked by external agonists and by activation of intracellular mechanisms including Rho/Rho-kinase, PKC, CPI-17, an inhibitor of MLCP, and MLCK may lead to TM contraction and modulate outflow facility negatively as shown in the left panel. On the other hand, decreased MLC phosphorylation caused by pharmacological inhibition of Rho-kinase, PKC, and MLCK may lead to relaxation of TM and influence aqueous outflow facility positively as shown in the right panel. MLC, myosin light chain; MLCK, MLC kinase; MLCP, MLC phosphatase; MLC-P, phosphorylated MLC. From Rao PV, Deng P, Sasaki Y, Epstein DL: Regulation of myosin light chain phosphorylation in the TM: role in aqueous humor outflow facility. Exp Eye Res 2005; 80:197-206.

Hallmarks of tissue relaxation that include decreased MLC phosphorylation and loss of actin stress fibers and focal adhesions have in addition been observed with protein kinase C (PKC) inhibitors such as GF109203X.^[122] Pharmacological activators of PKC including phorbol-12-myristate 13-acetate conversely produced an opposing effect. This implicates a role of PKC also towards the outflow regulation (Fig. 192.9).^[111]

PKC has additionally been shown to be involved in upregulation of MMP-3 and MMP-9 levels that is mediated by cytokines IL-1? and TNF? after laser trabeculoplasty.^[123] Extracellular signal-regulated kinase (ERK), mitogen activated protein kinase (MAPK)^[124] and c-Jun N-terminal kinase (JNK)^[86] signaling pathways are players as well. In TM cells, activating ERK pathways by platelet-derived growth factor has been shown to promote the secretion of MMP-2.^[125] IL-1? stimulation of TM cells also activates PKC, MAPK, and p38 signaling, thus leading to activation of AP-1 transcription and upregulation of MMP-3.^[126]

MAPK, p38 and JNK kinases^[127] have been shown in a recent report to be constitutive pathways in both normal human and glaucomatous TM cells in culture. Notably, an increase in activity of these kinases was observed in normal cells after treatment with exogenous IL-1?, but not in the glaucomatous counterparts.

Mechanical stretch causes normal human TM cells to elongate and to alter their diffuse actin network to exhibit complex geodesic patterns, similar to CLANs observed following DEX induction. Mechanotransduction through the MAPK signaling pathway was depressed.^[128] It is speculated that mechanical forces from IOP variations may influence TM cell morphology or activity, and that in glaucoma, homeostatic mechanisms may be impaired.^[128]

GLAUCOMA PARADIGM: MYOCILIN

Key Features

. Myocilin is located at both intra- and extracellular sites in TM cells, forming homomultimers and interacting with ECM and other proteins . Myocilin has a de-adhesive activity, compromising adhesion of TM cells to the ECM . Myocilin mutants with mutations at the olfactomedin domain may be misfolded and misprocessed, accumulating intracellularly into insoluble aggregates to cause cytotoxicity and cell death

Myocilin, formerly termed TM-induced glucocorticoid response protein (TIGR), is the product of the GLC1A gene linked directly to juvenile- and adult-onset POAG.^[129] This glaucoma gene is believed to hold the key for elucidation of glaucoma disease mechanisms even if the myocilin mutations are found associated only with a small percentage (~3-4%) of the patient population.^[129,130]

Myocilin was initially identified as a major 55/57 kDa protein secreted into the media of TM cultures after induction with glucocorticoids such as DEX.^[131,132] Myocilin gene has been cloned from human and other species^[133-135] and characterized as containing 3 exons. The human gene encodes a protein of 504 amino acids. There is a nonmuscle myosin-like domain near the amino (N)-terminus and an olfactomedin-like domain at the carboxyl (C)-terminus.^[132] In between is an undefined linker region. The N-terminus has two initiation sites, a signal peptide,^[132,134] and within the myosin-like domain, a leucine zipper motif and a coiled-coil region. The leucine zipper and the olfactomedin domain are well conserved. At the secondary level, the N-terminal region is primarily ?-helical and the C-terminal consists mostly of ?-strands.^[136]

The myocilin transcript or protein has been detected not only in the TM, but also in the retina, cornea, optic nerve head, ciliary body, iris, and nonocular tissues including skeletal muscle and heart.^[133] The regulation and distribution of myocilin, however, appears to be unique in TM cells. For instance, myocilin expression is highly upregulated by DEX in TM cells but not in other cell types including corneal fibroblasts.^[131,133] The myocilin expression in TM cells can be induced by mechanical stretch, TGF-?, and H₂O₂.^[133] It may represent a stress response protein.

Treatment of myocilin from TM cell extracts with endoglycosidase H and PN-glycosidase F results in a shift in electrophoretic mobility, indicating that myocilin is asparagine-glycosylated.^{[133,134,137]} Myocilin can form homomultimers. The myocilin-myocilin interaction occurs mainly at the N-terminus in the leucine zipper/coiled coil region.^[138,139] Disulfide bond formation is important as well.^[140] The leucine zipper/coiled coil domain has also been shown to be the site of myocilin interaction with other proteins.^{[138,139,141]}

The signal sequence at the N-terminal of myocilin has been suggested to target the protein for secretion.^[132-134] Evidence has also been presented that in TM cells, myocilin associates with exosome-like vesicles and is secreted by a nonconventional pathway.^[142] Myocilin protein in addition has been localized to both intra- and extracellular sites. In TM cells, immunofluorescence showed that the intracellular form of myocilin is distributed in the cytoplasm including perinuclear regions,^[133] in association with endoplasmic reticulum (ER), Golgi apparatus, and mitochondria.^[46,133] The mitochondrial association was further confirmed by immunoelectron microscopy.^[143] Immunogold labeling also illustrated the extracellular localization of myocilin.^[52,53] In the JCT and CS meshwork of normal human eyes, extracellular form of myocilin is found associated mostly with microfibrillar architecture in SD plaques and with long-spacing collagens (Tables 192.1 and 192.2) where changes have been documented to occur in aging eyes and in eyes of POAG patients.^[47,49] Myocilin interacts with ECM molecules such as fibronectin and fibrillin-1, but not with elastin and tenascin.^[52,144] The Hep II domain of fibronectin in particular has been found to be the interacting site for myocilin with fibronectin.^[145]

The crucial aspects of myocilin function(s), particularly those in the TM in relation to outflow resistance and glaucoma, still remain unclear. Myocilin is present in the aqueous humor.^[146] Perfusion of recombinant myocilin, especially when supplemented with the aqueous humor, increased outflow resistance in human anterior segment cultures.^[147] The C-terminal olfac-tomedin domain of myocilin shows little effect,^[148] whereas the entire N-terminus plus 98 amino acids of the olfactomedin domain in perfusion human cultures causes an increase in the outflow facility.^[137] In animal experiments, overexpression of wild-type myocilin in the aqueous humor of mice did not induce IOP elevations.^[149,150] Targeted disruption of the myocilin gene in knock out mice did not produce any phenotype either.^[151]

The effects of extracellular form of myocilin have been examined in vitro by plating TM cells on bacterial or eukaryotic recombinant myocilin or fibronectin/myocilin mixtures. Myocilin is a very poor substrate for TM cells. It blocks the TM attachment to fibronectin, causes a dramatic reduction in actin stress fibers and focal adhesions (Fig. 192.10), and triggers TM cells to assume a stellate morphology with microspikes (Fig. 192.11).^[152] The myocilin phenotype appears to be largely due to the N-terminal half of the protein.^[152] Similarly, overexpressing myocilin intracellularly by transfecting wild-type myocilin^[144]or transducing myocilin fusion protein^[153] into TM cells results in an altered actin architecture, loss of focal adhesion, and/or a reduced level of MLC phosphorylation. Adhesion of myocilin transfected cells to fibronectin and collagen is also compromised. These results suggest a de-adhesive activity for myocilin, similar to that described with matricellular proteins such as thrombospondin-1 and SPARC. The de-adhesion process, on a long-term chronic basis, may render TM cells vulnerable. Such a vulnerability, rather than myocilin per se, is proposed to be the necessary factor, although additional stress may still be needed for the development of glaucoma. Consistent with this hypothesis, myocilin-overexpressing cells display an increased susceptibility to apoptotic challenge.^[144]

FIGURE 192.10 Phase-contrast micrographs of human TM cells. The cells were plated for 1 h on wells coated with fibronectin (FN, 5 ?g/ml), fibronectin/BSA (FN/BSA, 5/5 ?g/ml), fibronectin/bacterial full length myocilin (FN/Myoc, 5/5 ?g/ml), fibronectin/myocilin 1-270 (FN/Myoc-N, 5/2.5 ?g/ml), or fibronectin/myocilin 271-504 (FN/Myoc-C, 5/2.5 ?g/ml). Original magnification, ×10. Scale bar = 50 ?m. From Park BC, Shen X, Fautsch MP, et al: Optimized bacterial expression of myocilin proteins and functional comparison of bacterial and eukaryotic myocilins. Mol Vis 2006; 12:832-840. Copyright 2006 Molecular Vision.

FIGURE 192.11 Actin and paxillin staining patterns in human TM cells. The cells were plated for 4 h on fibronectin (FN, 5 ?g/ml), or fibronectin (FN, 5 ?g/ml) mixed with BSA (5 ?g/ml), bacterial full length myocilin (Myoc, 5 ?g/ml), myocilin 1-270 (Myoc-N, 2.5 ?g/ml), or myocilin 271-504 (Myoc-C, 5 ?g/ml). Green fluorescence, actin; red fluorescence, paxillin. Nuclei were stained with DAPI in blue. The merged images for actin (green) and paxillin (red) are also shown. Original magnification, ×40. Scale bar = 20 ?m. From Park BC, Shen X, Fautsch MP, et al: Optimized bacterial expression of myocilin proteins and functional comparison of bacterial and eukaryotic myocilins. Mol Vis 2006; 12:832-840. Copyright 2006 Molecular Vision.

More than 70 mutations of myocilin have been detected in different population groups.^[130] Most of the mutations linked with glaucoma have been mapped to the third exon (olfactomedin domain) of the myocilin gene.^[130,133] It is thought that haploinsufficiency or loss of function may not be causative for myocilin mutation-associated glaucoma, and that pathology is actually due to a gain of function. This point of view is derived from the knock out data^[151] and from the observation that homozygous nonsense Arg46Stop mutation, while yielding severely truncated myocilin, did not result in glaucoma.^[133,141] A consensus has also been reached in the field that many olfactomedin domain mutants of myocilin are not secreted, but are instead retained within cells.^{[133,137,141,154,155]} These mutants in addition suppress the endogenous wild-type myocilin secretion. Mutants including Gln368Stop, Pro370Leu, and Tyr437His, when transiently transfected into HEK 293 and COS-7 cells, are found Triton X insoluble. Notably, the degree of the mutant Triton-insolubility has been correlated to glaucoma phenotypes.^[156]

Studies from myocilin mutants with mutations at the olfactomedin domain further support the theory that these mutants may be misprocessed in the ER, may not be secreted due to misfolding, and accumulate intracellularly into insoluble aggregates to cause cytotoxicity and cell death.^{[141,157-159]} Culturing cells at lower temperatures (e.g., 30°C), a condition known to facilitate protein folding, promotes secretion of the mutants and prevents cells death.^[141,158] The pathogenic mechanism of such mutation-associated glaucoma may hence be related to protein misfolding and ER stress, analogous to that reported for conformational diseases including ?^[1]-antitrypsin deficiency and Alzheimer's disease.^[160]

For myocilin mutations at the N-terminal half, especially those in the leucine zipper/coiled coil domain, the pathogenic mechanisms may be altogether distinct. Mutants such as Arg82Cys and Leu95Pro remain fully secreted but their adhesion to the ECM and/or cell surface seems to be affected. Altered interactions of the secreted mutants with the ECM and/or cell surface elements are speculated to be the basis as to how these mutants may affect the aqueous outflow.^[141]

AQUEOUS HUMOR COMPONENTS AND GLUCOCORTICOIDS

The aqueous humor produced by the ciliary body contains albumin as a major constituent. Other components encompasses ascorbic acid, hydrogen peroxide (H₂O₂), growth factors such as TGF-?, and molecules including MMPs, proteinase inhibitors, fibronectin, sCD44, myocilin, and hyaluronic acid.^[161,162] Increased levels of ascorbic acid, TGF-?2, sCD44, endothelin-1, and angiotensin II, and a decreased level of hyaluronic acid have been reported in the aqueous humor of POAG eyes.^{[84,85,121,161,163-166]} Since TM cells are in constant contact with the aqueous humor, it is unsurprising that altered levels and/or activities of aqueous humor components have an impact on the behavior and activities of these cells. Prominent examples include TGF-?^[84,85] and ascorbic acid,^[83,161] both have been shown, when added to TM cells, to affect the ECM production and composition. Hypophosphoryolated sCD44 found in the POAG aqueous humor has high cytotoxicity and low hyaluronic acid-binding affinity and is suggested to represent a pathophysiologic feature of the disease process.^[163] Normal aqueous humor stimulates TM cell migration and fibronectin may be one of the chemoattractants contained therein.^[7]Furthermore, addition of aqueous humor rather than the standard fetal bovine serum to monolayers of TM cultures decreases cell proliferation, and produces changes in cellular and molecular characteristics to mimic more closely the TM physiologic profiles in situ.^[162] Efforts are now underway to identify factors in the aqueous humor that are important for maintaining TM cells in a homeostatic state.

Glucocorticoids, when administrated either systemically or topically, can lead to the development of ocular hypertension in a subset of the population.^[167] The histologic changes in steroid glaucoma bear a resemblance to those seen in POAG.^[32,131] In light of this, much attention has been paid to the glucocorticoid effects on TM cells. One mechanism through which gluco-corticoid exerts its effect may be ECM modulations. It is known that in tissue culture, treatment of glucocorticoids alters the expression of collagen, glycosaminoglycans, elastin, and fibronectin in TM cells.^[15,79] The levels of tissue plasminogen activator and stromelysin are reduced.^[168] In perfusion cultures of human eyes, DEX causes a similar pressure elevation and accumulation of ECM materials.^[169] Glucocorticoids in addition inhibit phagocytosis, increase secretory activity, decrease hydraulic conductivity, upregulate myocilin expression and reorganize actin into CLANs in cultured human TM cells.^{[32,36,131,167]} TM cells possess both ? and ? isoforms of glucocorticoid receptor (GF? and GF?) and glucocorticoid sensitivity in glaucoma has been attributed to differences in the expression of these two receptors.^[170] Glaucomatous TM cells are documented to express higher levels of GF? than those from normal individuals. Overexpression of GF? in glaucomatous cells inhibits DEX induction of myocilin (Fig. 192.12) and fibronectin. The level of GF? is hypothesized to be the underlying basis for glucocorticoid responsiveness and ocular hypertension.^[170]

FIGURE 192.12 Overexpression of GR? inhibited DEX-induced expression of myocilin in primary glaucomatous TM cells. SGTM152-99 cells were transiently transfected with a control vector pCMX or GR? expression vector pCMX-hGR?. After transfection, cells were incubated with either vehicle control (ethanol) or 100 nM DEX in serum-free DMEM for 24 h. The effects of increased GR? on DEX-induced expression of myocilin were examined by confocal immunofluorescence microscopy, Western blot analysis, and quantitative PCR. (a) Confocal immunofluorescent microscopy. Cells were incubated with primary rabbit anti-GRb and sheep antimyocilin antibodies and subsequently with the secondary antibodies Alexa Flour 633 goat anti-rabbit IgG to label GR? (red) and Alexa Flour 488 donkey anti-sheep IgG to label myocilin (green). (b) Western Blot analysis: SGTM152-99 cells were transiently transfected with a control vector pCMX or GR? expression vector pCMX-hGR?. After transfection, cells were incubated with either the vehicle control or 100 nM DEX for 24 h. Either whole-cell lysates were then subjected to Western blot analysis (b) with anti-GR? and antimyocilin or (c) total cellular mRNA was isolated and subjected to quantitative PCR to detect myocilin expression. Lane 1: pCMX+Con; lane 2: pCMX+DEX; lane 3: pCMX-hGR?+Con; and lane 4: pCMX-hGR?+DEX. (*P > 0.05 pCMX+DEX versus pCMX+vehicle control; t-test). From Zhang X, Clark AF, Yorio T: Regulation of glucocorticoid responsiveness in glaucomatous TM cells by glucocorticoid receptor-?. Invest Ophthalmol Vis Sci 2005; 46:4607-4616.Copyright 2005 Association for Research in Vision and Ophthalmology.

AGING, OXIDATIVE STRESS, AND OTHER INSULTS

Key Features

. TM cellularity is reduced with aging . In POAG eyes, cellularity is decreased but the number of senescent cells is increased in the TM . Oxidative damage is implicated to contribute to the morphologic and physiologic alterations in the aqueous outflow pathway in aging and glaucoma . The level of ELAM-1 is found to be increased in glaucomatous tissues, representing perhaps a cellular stress response

TM cellularity is reduced with aging^[33] Morphologic studies have also revealed thickened basement membranes and accumulation of SD plaques and long spacing collagens in the TM of aged eyes.^[49,164]Cultured TM cells from old donors show a decline in proteasome activity and the acquisition of the senescence phenotype including reduced proliferative capacity and enlarged cell morphology.^[171] In vitroreplication of TM cells from younger donors is also associated with decreased proteasome activity and accumulation of oxidized proteins.^[171] Similarly, a decrease of TM cellularity and an accumulation of ECM have also been documented in POAG eyes.^[34,54,56] The number of senescent cells that stain positive for senescence-associated ?-galactosidase was also reported to be increased in the TM of POAG eyes.^[166] These observations further reinforce that POAG is an age-related disease.^[164]

Oxidative damage has been implicated to contribute to the morphologic and physiologic alterations in the aqueous outflow pathway in aging and glaucoma.^[164] Analysis of proteins in calf and cow TM suggested that an aggregation of proteins occurring with aging may be a result of oxidation processes^[172] The loss of proteasome function may likewise be related to oxidation of proteasome components or inhibition by cross-linked protein aggregates.^[170] TM is known to be exposed to 20-30 ?M of H₂O₂ present in the aqueous humor and is subjected to chronic oxidative stress.^[36] Additional H₂O₂ and other reactive oxygen products may also be generated by light-catalyzed reactions, metabolic pathways, and phagocytic or inflammatory processes.^[166]

Enzymes that are known to be involved in the protection against oxidative damage, including catalase, glutathione reductase, superoxide dismutase, and glutathione peroxidase, have been studied in the TM.^[36] The specific activity of superoxide dismutase, but not catalase, was shown to decline with age in human TM tissues.^[173] TM cells also synthesized a specific set of proteins, such as ?B-crystalline, that may act as molecular chaperones to prevent oxidative or heat shock damage.^[174]

In tissue culture, H₂O₂ is capable of inducing gross morphologic changes such as blebbing of the plasma membrane and cell rounding.^[36] Under mild, nonlethal H₂O₂ condition, the adhesion of TM cells to ECM is compromised and the level of transcription factor NF-?B is enhanced.^[80] After 10 days of exposure to hyperoxic O₂ (40% O₂), TM cells showed a marked decline of proteasome activity, premature senescence and decreased cell viability.^[175]

Markers of oxidative damage,^[176] and diminished blood levels of oxidant scavengers glutathione and reduced glutathione are found in POAG patients.^[177] It appears that oxidative stress that exceeds the capacity of TM cells for detoxification is involved in damaging the cells and alteration of the aqueous humor outflow.

The TM is also subjected to other types of stress. As discussed above, the level of ELAM-1 is found increased in glaucomatous tissues.^[99,127] Upregulation of this gene is shown to be a result of sustained activation of an IL-1? feedback loop regulated through transcription factor NF-?B, representing a cellular stress response.^[99] TM cells also sense IOP fluctuations as mechanical stress. In response, they reorganize actin cytoskeleton^[128] and change expression of a variety of genes including TGF-?1^[178] and MMPs.^[77,82]

CONCLUSIONS AND PERSPECTIVES

This chapter summarizes the current knowledge regarding major cellular mechanisms in the TM that may influence the aqueous humor outflow pathway. Not included are the roles of serum proteins, immune response, adrenergic receptors and other important intracellular or intercellular regulators such as nitric acid and prostaglandins. As noted throughout this chapter, TM cells are highly specialized, adaptive and multifunctional cells and SC cells are pressure sensitive endothelial cells of vascular origin. Both the cells and the ECM are altered in glaucoma.

Our understanding of the various TM/SC mechanisms has vastly increased in the past several years; although to varying degrees, the precise roles of these mechanisms and their direct links to the outflow resistance still remain to be established. Expeditious progress is to be expected, since cell cultures of TM and SC cells and perfusion organ cultures are now readily available. Recently developed noninvasive in vivo imaging techniques, coupled with molecular biology manipulations,^[179] will also be extremely powerful, facilitating the visualization and elucidation of the outflow pathways. In addition, gene expression type of studies^[82,180] are underway and TM or SC-specific or enriched promoters such as promoter fragments from the matrix Gla, VE-cadherin and chitinase 3-like 1 genes are being identified.^[181,182] Exciting new tools including the use of lentivirus^[178] or adeno-associated virus^[183,184] for gene delivery, fusion proteins for protein transduction,^[153] and RNA interference for gene silencing are being applied to the TM field. Individual genes for proteins or receptors can thus be overexpressed, deleted, mutated or reconstituted in conventional and 3-dimensional cell cultures, organ cultures, and in vivo systems for assessment of their effects, functions, or roles in the outflow pathway. These investigations will undoubtedly provide new insights into the cellular mechanisms operative in the TM under normal homeostasis and those directly involved in the disease development. Such information will not only advance the field but also allow design of novel gene therapies or other treatment modalities for glaucoma.

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