Ординатура / Офтальмология / Английские материалы / Ocular Disease Mechanisms and Management_Levin, Albert_2010

history for glaucoma in 16–48% of cases.9,11 These disparate observations might be explained in several ways, including incomplete penetrance of a dominant disorder, multigenic influences, and complex gene–environment interactions, to name a few. Promising findings have reported linkage to loci at chromosomal locations 7q3612 and 18q22.17 Mutations at these loci have not yet been identified. To date, the potential importance of these loci has neither been replicated nor refuted by other groups, leaving their potential role in most cases unclear.

Diagnostic workup

Initial diagnosis of PDS is typically based entirely on slitlamp examination. Classical features contributing to diagnosis include: (1) presence of a Krukenberg’s spindle; (2) dense trabecular pigmentation; (3) presence of Scheie’s stripe; and

(4) radial, slit-like, midperipheral iris transillumination defects. However, many patients will not simultaneously exhibit all four features.9 Posterior bowing of the midperipheral iris, which likely contributes to mechanical abrasion between the iris and lens zonules, is often detectable (Figure 21.4). The extent of pigment dispersion is an important component of diagnosis as small amounts of pigment accumulation within the trabecular meshwork are also part of the normal aging process. Because PDS tends to become quiescent with advancing age, older patients with glaucoma may have only subtle features of PDS and may be misdiagnosed with primary open-angle or normal-tension glaucoma. Diagnosis of PG involves PDS, plus signs of glaucoma. Approximately 17% of PDS cases have PG at the time of

Figure 21.4  Posterior bowing of iris viewed by gonioscopy. Following the trajectory of the slit of light from the pupil toward the periphery highlights the iris concavity frequently observed in pigment dispersion syndrome.

(Courtesy of Dr. WLM Alward, Department of Ophthalmology and Visual Sciences, University of Iowa.)

Pathology

initial diagnosis, suggesting that screening for glaucoma at the initial examination is warranted.9

Clinical signs of PDS and PG have been reported to show pronounced ethnic variability.18,19 For example, the slit-like midperipheral iris transillumination defects identifiable by slit-lamp exam are frequently absent in AfricanAmericans19 and Chinese.18 Variable iris transillumination related to iris color has previously been observed in other populations, with 25% of blue irides and 68% of brown irides lacking slit-like defects.11 Thus, the frequent absence of iris transillumination defects in African-Americans and Chinese might partially be explained by darker iris colors. Use of sensitive infrared cameras to supplement traditional slit-lamp exams helps to detect iris transillumination defects in African-Americans with PDS, though the defects were not always the classic slit-like defects typical of PDS in Caucasians.20 PDS and PG have previously been thought to be quite rare in African-Americans.4,10 However, PDS might instead be underdiagnosed due to the difficulty of detecting these iris transillumination defects in African-Americans.20

Differential diagnosis

Differential diagnosis of PDS and PG includes exfoliation syndrome, uveitis, melanoma, iris and ciliary body cysts, trauma, postoperative conditions, and age-related changes. Exfoliation syndrome in particular involves substantial dispersion of pigment and some eyes have signs of both PDS and exfoliation syndrome.21 The extent of iridocorneal angle pigmentation occurring in exfoliation syndrome can correlate more strongly with presenting IOP than does the amount of exfoliative material on the anterior lens capsule,22 suggesting that the pigment dispersion occurring in exfoliation syndrome is also of pathologic relevance.

Treatment

In the absence of elevated IOP or progressive optic nerve damage, PDS typically does not require any treatment. Regular follow-up is suggested to monitor for possible progression to PG. Treatment for PG follows the same regime as open-angle glaucoma, but with some special considerations.23 Miotics can decrease iridozonular contact and may prevent further progression of the disease, although use of this class of agents is limited because they can also worsen myopia and increase the risk of retinal detachment. Prostaglandin analogs such as latanoprost are effective in managing IOP in PG,24 although these agents often also cause increased iridial pigmentation: in the context of PDS iris disease the long-term consequences of this pigmentation are largely unknown. When treated surgically, PG patients who have trabeculectomy are more likely to have issues with hypotony maculopathy because they tend to be young and myopic.

Pathology

A key finding in current theories of PDS and PG came from Campbell’s 1979 histologic study of human eyes with PG.1 In studying the normal anatomy of anterior zonules among

eyebank eyes, Campbell noted that zonules inserted on the anterior lens surface were not in single strands, but were organized into packets of zonules. The number of these packets ranged from 65 to 80, precisely the same as the number of slit-like iris transillumination defects observed in PG. This led to the hypothesis that if contact between the zonules and iris were to occur, this abrasion could account for the iris transillumination defects and dispersion of pigment observed in PDS and PG. In testing this by studying the pathology of eyes with radial slit-like iris transillumination defects, it was indeed found that wherever there was a slit-like transillumination defect, there was also a closely apposed packet of zonules. Combined with an observation that patients with PG frequently show peripheral iris concavity, this work led to the theory that mechanical rubbing between anterior packets of lens zonules and the IPE in predisposed eyes is the cause of pigment dispersion in PDS and PG.

Additional insight into the pathology of PDS and PG has come from several studies examining tissue with microscopy. Throughout the iris, there are radial defects of the IPE. Within these defects, cells are either missing or have disrupted membranes that cause extrusion of pigment.1,25,26 The iris stroma in the area of the defects is sometimes affected and is typically infiltrated with pigment-engulfed macrophages.1,25,26 Some studies note the presence of irregular iris melanosomes.26,27 The trabecular meshwork is densely pigmented, with dispersed pigment found free, in macrophages, or phagocytosed by endothelial cells lining the trabecular beams.25–28 In some eyes, it appears that pigment engulfment by trabecular endothelial cells is followed by a loss of cellularity and regional collapse of trabecular sheets.29

Etiology

The etiology of PDS is incompletely understood, but can be explained in part by the unique anatomical and physiological phenomenon of reverse pupillary block.30 According to this hypothesis, irides predisposed to PDS come into close physical contact with the lens and adopt a ball-and- valve-like relationship (Figure 21.5). The close contact of the iris and lens restricts the flow of aqueous humor, except during events such as eye blinks which squeeze aqueous humor into the anterior segment. During eye blinks, aqueous humor is forced into the anterior segment, causing an increase in the volume of aqueous humor in the anterior segment and elevated IOP relative to the posterior segment. This pressure differential promotes a backward bowing of the midperipheral iris against packets of lens zonules. Patients with PDS tend to have more posterior iris insertions31 and deeper anterior chambers,32 which might promote reverse pupillary block. Thus, reverse pupillary block addresses why the iris often becomes bowed posteriorly in PDS to allow mechanical rubbing against the anterior packets of lens zonules.

In recent years, ultrasound biomicroscopy has allowed iris pathology in PG to be studied in new ways.31,33–35 Recurrent findings in these studies are that iris concavity is common33,35 and that the iris tends to have a more posterior insertion31 in eyes of PDS patients compared to

Trabecular

pigment

Figure 21.5  Mechanism of reverse pupillary block contributing to pigment dispersion syndrome. Left image: close physical contact between the iris and lens prevents aqueous humor flow in predisposed eye. During eye blinks, pressure from the eyelids (large arrows) squeezes aqueous humor into the anterior segment (small arrows). Middle image: a pressure differential between the anterior and posterior segment causes posterior bowing of the iris and allows mechanical abrasion between the iris pigment epithelium and anterior packets of lens zonules. Right image: mechanical rubbing causes pigment dispersion. Dispersed pigment tends to accumulate in the trabecular meshwork, on the corneal endothelium as a Krukenberg’s spindle (KS), and at the junction of posterior lens capsule and zonules as a Scheie’s stripe (SS). (Redrawn from the figure kindly provided by Dr. WLM Alward, Department of Ophthalmology and Visual Sciences, University of Iowa.)

controls. A similar trend holds true in ultrasound studies of patients with asymmetric PDS; the eye with worse pigment dispersion tends to have significant differences in iris concavity and have a more posterior insertion.33 Although midperipheral iris concavity and posterior iris insertions are not observed in all patients that have been examined, these findings are consistent with the etiology suggested by Campbell1 and Karickoff30 that pigment dispersion often involves mechanical irideozonular abrasion in predisposed eyes.

When PDS progresses to PG, the etiology of the resulting glaucoma is related to increased IOP. One of the greatest risk factors for predicting conversion of PDS to PG is IOP at initial examination.9 Among PG patients, elevations in IOP typically correlate to the amount of trabecular meshwork pigmentation.1,8,11 Therefore, it appears that directly, or indirectly, pigment accumulations within the irideocorneal angle are the primary insult initiating this stage of disease.

Pathophysiology

Liberation of iris pigment

The combined work of Campbell1 and Karickhoff30 leads to a “mechanical rubbing hypothesis” stating that mechanical irideozonular abrasion in predisposed eyes underlies the pathophysiology of pigment dispersion. Some of the strengths often ascribed to the mechanical rubbing hypoth-

esis are that it explains why PDS is often associated with myopia (increased iris–lenticular contact), why PDS often regresses with age (loss of accommodation with the onset of presbyopia), why some PDS patients have relatively deep anterior chambers with posteriorly located irides (anatomical variations that would promote rubbing), and why PDS is typically described as more common among males (males tend to have comparatively larger irides which may be more prone to backward bowing). Thus, the scenarios suggested by the mechanical rubbing hypothesis could logically explain many associations of PDS and PG.

However, experimental and clinical support for the mechanical rubbing hypothesis has not been universal and much remains unknown.35–39 PDS does not develop in every­ one with a relatively posterior iris insertion, concave iris configuration, or myopia. There are also other clinical features not at all accounted for in the mechanical rubbing hypothesis, including a propensity for retinal lattice degeneration.10 Furthermore, even in the PDS cases associated with anatomical or physiological conditions likely to promote mechanical rubbing, the genetic or environmental factors leading to these circumstances are completely unknown. In sum, additional risk factors predisposing the iris toward pigment dispersion likely remain to be identified and many important questions remain to be studied.

Recent genetic studies conducted with mice have suggested new hypotheses contributing to PDS pathophysiology. Among published reports, pigment dispersion in mice has been observed in a small number of different mouse strains,40–43 including several with mutations influencing melanosomes.40,41 For example, DBA/2J mice exhibit pronounced accumulations of dispersed iris pigment (Figure 21.6) and progressive transillumination defects.41,44,45 Genetic experiments have clearly defined that pigment dispersal in DBA/2J mice results from the digenic interaction of two mutations in genes encoding melanosomal proteins, Gpnmb and Tyrp1.41 These genes appear to trigger iris disease through a mechanism involving aberrant melanosomal processes related to pigment biosynthesis.41 This hypothesis is further supported by the recent identification of pigment dispersion in several additional strains of mice with mutations influencing melanosomes.40 Combined, these findings identify an additional insult capable of predisposing irides to pigment dispersion. The extent to which these processes might contribute to human PDS is currently unknown.

Pigment-related insults to intraocular pressure

Once pigment is liberated from the IPE, some eyes progress toward elevated IOP, while in others, PDS can persist for extended periods of time without further incident. Despite its importance, the pathophysiology influencing these different outcomes remains only partially understood. The simplest explanation for pigment-related changes in IOP is that dispersed pigment accumulated within the trabecular meshwork physically blocks aqueous humor outflow. In support of this, in some patients vigorous exercise can briefly lead to temporary “pigment storms” and elevations in IOP.46,47 The rapid and temporary nature of these linked events is consistent with physical obstruction to aqueous outflow caused by accumulations of pigment within the trabecular meshwork.

Pathophysiology

Figure 21.6  Iris disease and pigment dispersion in DBA/2J mouse. As a consequence of a pigment-dispersing iris disease, DBA/2J mice

develop a form of pigmentary glaucoma. Indices of disease present in this eye of a 13-month DBA/2J mouse include dispersed pigment across the lens and iris, the presence of multiple pigment-engulfed clump cells, and iris atrophy that is particularly pronounced near the pupillary margin.

This eye also has peripheral anterior synechiae that are causing the pupil to be acentric.

Contrasting this, modeling studies suggest that the amount of pigment chronically present in the juxtacanalicular tissue would likely have a negligible influence on trabecular meshwork permeability.48 Thus, while dispersed pigment can acutely influence IOP through simple physical blockade, this does not seem likely to be the major mechanism causing progression of PDS to PG in most human eyes with chronic pigment dispersion.

Animal studies of PDS suggest that the IOP response to dispersed pigment is a multistep process. Experimental studies in living cynomolgus monkeys have found that injecting pigment into the anterior chamber will result in trabecular meshwork pigmentation, but no lasting changes in outflow facility.49 This inducible model results in PDS, but not PG. Likewise, changes in genetic background can genetically convert the DBA/2J mouse model of PG into a model of only PDS.50 These experiments demonstrate that progression of PDS to PG can be genetically modified. Together, these animal studies indicate that PG is not typically caused solely by physical obstruction to aqueous humor outflow. Rather, the damaging aspects of pigment dispersion appear to result from complex reactions to the dispersed pigment. In the future, identification of the mechanisms mediating these responses will likely bring important new insight into the pathophysiology of PDS.

In conclusion, many aspects of PDS pathophysiology can be explained by mechanical abrasion of the IPE against anterior packets of lens zonules. Iris concavity allowing

mechanical irideozonular rubbing is likely promoted by the process of reverse pupillary block. However, additional risk factors likely also exist. The detrimental influences of dispersed pigment appear to involve a complex reaction to pigment. Defining the precise events dictating whether or not PDS progresses to PG remains a significant challenge to

our understanding of these diseases. In years to come, technological advances such as high-density genome-wide arrays will bring new opportunities to study the heredity of PDS and PG in humans, while newly described mouse models will allow complementary mechanistic studies into the molecular events influencing PDS and PG.

1.Campbell DG. Pigmentary dispersion and glaucoma. A new theory. Arch Ophthalmol 1979;97:1667–1672.

5.Ritch R, Steinberger D, Liebmann JM. Prevalence of pigment dispersion syndrome in a population undergoing glaucoma screening. Am J Ophthalmol 1993;115:707–710.

6.Farrar SM, Shields MB, Miller KN, et al. Risk factors for the development and severity of glaucoma in the pigment dispersion syndrome. Am J Ophthalmol 1989;108:223–229.

9.Siddiqui Y, Ten Hulzen RD, Cameron JD, et al. What is the risk of developing pigmentary glaucoma from pigment dispersion syndrome? Am J Ophthalmol 2003;135:794–799.

10.Ritch R. A unification hypothesis of pigment dispersion syndrome. Trans Am Ophthalmol Soc 1996;94:381–405; discussion 409.

12.Andersen JS, Pralea AM, DelBono EA, et al. A gene responsible for the

pigment dispersion syndrome maps to chromosome 7q35–q36. Arch Ophthalmol 1997;115:384–388.

22.Shuba L, Nicolela MT, Rafuse PE. Correlation of capsular pseudoexfoliation material and iridocorneal angle pigment with the severity of pseudoexfoliation glaucoma. J Glaucoma 2007;16:94–97.

25.Fine BS, Yanoff M, Scheie HG. Pigmentary “glaucoma.” A histologic study. Trans Am Acad Ophthalmol Otolaryngol 1974;78:OP314–OP325.

26.Kampik A, Green WR, Quigley HA, et al. Scanning and transmission electron microscopic studies of two cases of pigment dispersion syndrome. Am J Ophthalmol 1981;91:573–587.

30.Karickhoff JR. Pigmentary dispersion syndrome and pigmentary glaucoma: a new mechanism concept, a new treatment, and a new technique. Ophthalm Surg 1992;23:269–277.

36.Balidis MO, Bunce C, Sandy CJ, et al. Iris configuration in accommodation in

pigment dispersion syndrome. Eye 2002;16:694–700.

39.Krupin T, Rosenberg LF, Weinreb RN. Pigmentary glaucoma: facts versus fiction. J Glaucoma 1994;3:273–274.

40.Anderson MG, Hawes NL, Trantow CM, et al. Iris phenotypes and pigment dispersion caused by genes influencing pigmentation. Pigment Cell Melanoma Res 2008;21:565–578.

48.Murphy CG, Johnson M, Alvarado JA. Juxtacanalicular tissue in pigmentary and primary open angle glaucoma. The hydrodynamic role of pigment and other constituents. Arch Ophthalmol 1992;110:1779–1785.

49.Epstein DL, Freddo TF, Anderson PJ, et al. Experimental obstruction to

aqueous outflow by pigment particles in living monkeys. Invest Ophthalmol Vis Sci 1986;27:387–395.

Clinical background

Primary open-angle glaucoma (POAG) is the most common type of glaucoma, particularly in populations with European and African ancestry. This disease is the leading cause of blindness in African-Americans. The major risk factors for POAG include intraocular pressure (IOP) elevation and aging. The prevalence of POAG increases from 0.02% at ages 40–49 to 2–3% for persons over the age of 70,1 and the incidence of ocular hypertension increases from 2% to 9% over the same time span.2

Pathophysiology

Anatomical and physiological background

The trabecular meshwork (TM), a specialized tissue at the chamber angle, is the major site for regulation of the normal bulk flow of the aqueous humor.3 It functions as a selfcleaning, unidirectional, pressure-sensitive, low-flow (2 l/ min/mmHg) biologic filter for the aqueous humor, and contributes thereby to control of the IOP.3 The TM tissue is divided into the uveal meshwork, corneoscleral meshwork, and juxtacanalicular connective tissue (JCT) regions (Figure 22.1). In the uveal and corneoscleral meshwork, sheets of trabecular beams that contain lamellae made of connective tissue or extracellular matrix (ECM) materials are lined by TM cells. In the JCT region, the cells reside relatively freely and are embedded in the ECM. In the Schlemm’s canal (SC), there are endothelial cells also referred to as inner wall cells4,5 (Figure 22.2). The aqueous humor flows through the TM and the SC into collector channels and aqueous veins, and the outflow resistance is believed to locate largely in the JCT/SC area.4,5 In 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 various types of glaucoma (Box 22.1).

Effects of disease

To facilitate comparisons and to offer mechanistic clues, biochemical changes such as upor downregulation of

Abnormal trabecular meshwork outflow

Paul A Knepper and Beatrice YJT Yue

genes/proteins that have been reported in POAG in the aqueous humor, TM, optic nerve, and blood are organized into three categories: (1) ECM elements and remodeling; (2) cell signaling molecules; and (3) changes related to stress and aging, and listed respectively in Tables 22.1,6–21 22.2,22–31 and 22.3.32–43

What follows are discussions of cellular mechanisms in the TM system that may affect the aqueous humor outflow pathway. Although discussed under separate headings, the mechanisms that include the ECM composition, turnover, and modulation, cell adhesion, cytoskeletal structure, and intracellular signaling are all interconnected. The effects or influences of aqueous humor components and stressinducing conditions are also described.

Trabecular meshwork and Schlemm’s canal cell profiles

TM cells are unique and have the capacity to perform a variety of functions, including phagocytosis, migration, elaboration of metabolic, lysosomal, and matrix-degrading enzymes, and production of ECM elements.44,45 TM cells incorporate acetylated low-density lipoprotein (LDL),45 as do vascular endothelial cells (VEC) in culture. However, they do not stain for factor VIII antigen, a characteristic VEC marker. Neither do TM cells form an endothelium as tight as that of cultured VEC. TM cells on the other hand phagocytose more avidly than VEC.

TM cells are essential for maintenance of the normal aqueous humor outflow system.44,45 Disturbances in the vitality and functional status by genetic predisposition, aging, or other insults may result in obstruction of the aqueous outflow, leading to IOP elevation and glaucomatous conditions. In vivo, TM cells have limited proliferative activity. A continuous loss of TM cells occurs during adulthood.44,46 In patients with POAG, the cell loss and disruption of the endothelial covering are striking.47 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 POAG.

While TM cells are highly specialized, adaptive, and multifunctional cells, SC cells are pressure-sensitive endothelial cells of vascular origin. SC cells endocytose LDL and acetylated LDL and organize in the presence of Matrigel into

Figure 22.1  Three-dimensional drawing of the aqueous outflow pathway depicting the structural elements of the trabecular meshwork. The uveal meshwork (UM) is a loose lattice of delicate rope-like components in continuity from the base of the iris to Descemet’s membrane (DM). The corneoscleral meshwork (CSM) is more tightly packed and consists of sheets of trabecular lamellae. The juxtacanicular connective tissue (JCT) forms a thin band of connective tissue adjacent to the aqueous collector channel known as Schlemm’s canal (SC). (Adapted from Weddell JE. The limbus. In: Hogan MJ, Alvarado JA, Weddell JE (eds) Histology of the Human Eye. Philadelphia: WB Saunders, 1971:112–182.)

Figure 22.2  Three-dimensional drawing of the corneoscleral meshwork, the juxtacanicular connective tissue (JCT), and Schlemm’s canal (SC). Note the trabecular spaces (ts), intervening endothelial processes, the inner wall (iw), the external wall (ew), and the endothelium (e) of the SC. Giant vacuoles (gv), common on the inner wall of the SC, are pressure-sensitive. The aqueous outflow pathway is complex; there are at least three layers of organization. The first layer is formed by a series of perforated trabecular lamellae. Interposed between the last trabecular lamellae and the inner wall of the SC is a thin cellular zone with an abundant extracellular matrix, the JCT, which is a highly fibrillar, cellular, and glycosaminoglycan-enriched area. (Adapted from Weddell JE. The limbus. In: Hogan MJ, Alvarado JA, Weddell JE (eds) Histology of the Human Eye. Philadelphia: WB Saunders, 1971:112– 182.)

SC

GV

JCT

Box 22.1  Trabecular meshwork cells and glaucoma

•Essential for maintenance of the normal aqueous humor outflow system

•Disturbances by genetic predisposition, aging, or other insults may result in obstruction of the aqueous outflow, leading to intraocular pressure elevation and glaucomatous conditions

•Limited proliferative activity in vivo

•Continuous loss of trabecular meshwork cells occurs during adulthood

CSM

Figure 22.3  Scanning electron micrograph of the Schlemm’s canal (SC) and the juxtacanicular connective tissue (JCT) of a primate eye (Macaca fascicularis). The endothelia of the inner wall of SC are elongated and form transient giant vacuoles (GV) that respond to intraocular pressure changes and serve as a pressure-sensitive marker of pressure between JCT and SC. Note the organization of the corneoscleral meshwork (CSM) lamellae, trabecular spaces, and relative compactness of the JCT as aqueous passes through the interstices of the aqueous pathway. Scale bar, 10 m.

multicellular tubelike structures.48 In situ, cells in the various regions of TM and those in SC appear to be interconnected by cell processes (Figure 22.3).49 The SC/TM configuration is pressure-sensitive. If the IOP is exceedingly elevated, the SC/TM may be forced on to the outer wall, effectively preventing outflow of the aqueous humor.

172

ECM composition, turnover, and modulation

The ECM components in the TM are essential for maintenance of the normal aqueous humor outflow.45,50–52 In the TM of POAG eyes, excessive, abnormal accumulations of certain ECM materials as well as decreases of other ECM

materials (Table 22.1) have been documented.6–21,45,50–52 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. Recent studies have also revealed that ECM is a dynamic entity of key importance

in all biological systems, determining and controlling the behavior and biologic characteristics of the cells.

One key component of the TM is proteoglycans, which 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,53

in the 1950s, demonstrated that perfusion of the anterior chamber with testicular hyaluronidase greatly reduced the outflow resistance in enucleated bovine eyes. 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.45,50 These proteoglycans may represent decorin, biglycan, versican, perlecan, and syndecan.45,50

The relative amounts of each type of glycosaminoglycan in the TM tissue have been determined.45,50 Hyaluronic acid and chondroitin-dermatan sulfates are the major constituents, and heparan sulfate and keratan sulfate are present in much smaller amounts. A depletion in hyaluronic acid and an accumulation of chondroitin sulfates and undigestible glycosaminoglycan material have been associated with POAG conditions.8,45 Both chondroitin sulfate and hyaluronic acid have been shown to contribute to flow resistance and influence flow rate in vitro54 (Figure 22.4). The flow rate was decreased when hyaluronic acid and chondroitin sulfate were used at POAG concentrations.56 Delays to achieve steady-state level were also observed with increased pressure in in vitro studies.54 Of note, the level of an ectodomain fragment of hyaluronic acid receptor CD44 (sCD44) was found to be elevated (Table 22.2) in the aqueous humor of POAG patients27 and the concentration was correlated with visual field loss.27 sCD44 is cytotoxic to TM cells, but the toxicity can be blocked by hyaluronic acid.55 The decreased hyaluronic acid may thus result in diminished protective capacity and further deterioration in POAG conditions.

Fibronectin, laminin, and vitronectin, and matricellular proteins that include tenascin, SPARC, and thrombospondin-1 have been localized in the TM.16,45,50 These glycoproteins are crucial in biologic processes such as cell attachment, spreading, and cell differentiation. Overex-

174

pression of fibronectin and laminin as well as collagen type IV resulted in a decrease in the TM cell monolayer permeability.45,56 The expression of thrombospondin-1 has in addition been shown to be increased16 in the TM of POAG eyes (Table 22.1).

Elastin is localized to the central core of sheath-derived plaques or elastic-like fibers in the TM.45,50 Fibrillin-1, a component of microfibrils, has been found in both the core and the surrounding sheath of the elastic-like fibers. Fibrillin-1 and type VI collagen are also constituents of long-spacing collagens found in the TM.45,51 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. In trabecular lamellae and in JCT regions, accumulation of long spacing collagens and sheathderived plaques has been documented in POAG and aged eyes.45,51

The ECM is constantly modified by the surrounding cells through enzymes such as matrix metalloproteinase (MMP) family member and inhibitors such as tissue inhibitors for matrix metalloproteinase (TIMPs) found in the TM.45,50 Ongoing ECM turnover, initiated by MMPs, appears to be essential for maintenance of the aqueous outflow homeo­ stasis. MMP-3, and possibly also MMP-9, may be responsible for the efficacy of laser trabeculoplasty, an alternative treatment to reduce IOP in patients with glaucoma.45,50 Addition or induction of MMP-3 in perfused human anterior-segment organ cultures increases, whereas blocking the endogenous activity of the MMPs in the TM reduces, the aqueous humor outflow facility.45

The ECM in the TM may also be remodeled in response to exogenous stimuli such as glucocorticoids and oxidative stress.45 Mechanical stretch caused an increase in MMP-1 and MMP-3 activities and alteration of ECM molecules including

Flow rate (ml/min)

A

0.07

JCT Normal 10 mmHg 0.06 JCT Normal 20 mmHg

JCT Normal 40 mmHg

0.05

0.04

0.03

0.02

0.01

Time (hours)

Pathophysiology

Box 22.2  Extracellular matrix components in the

trabecular meshwork

•Important for normal aqueous humor outflow

•Proteoglycans help maintain resistance to aqueous humor outflow by forming a gel-like network that may function as a gel filtration system

•Glycoproteins are involved in cell attachment, spreading, and cell differentiation

•Modification via enzymes, e.g., matrix metalloproteinases and their inhibitors

•Remodeling in response to stretch, glucocorticoids and oxidative stress, and cytokines

0.01

Figure 22.4  Reconstitution of hyaluronic acid and chondroitin sulfate at the normal (A) and primary open-angle glaucoma (B) juxtacanicular connective tissue (JCT) concentration and the effect of 10, 20, and 40 mmHg pressure on the flow rate. The flow rate was measured using an in vitro microtest chamber. The initial flow rate of the normal JCT at 10 mmHg was 6.7 l/min and the lag time required to obtain the steady-state flow was 26 hours. In contrast, the initial flow rate of the primary open-angle glaucoma (POAG) JCT at 10 mmHg was 3.7 l/min and the lag time required to obtain steady-state flow rate was 46 hours. As hydrostatic pressure was increased to 20 or 40 mmHg, the flow rate and the lag time of the POAG JCT were markedly slower than the normal JCT. (Reproduced from Knepper PA, Fadel JR, Miller AM, et al. Reconstitution of trabecular meshwork GAGs: influence of hyaluronic acid and chondroitin sulfate on flow rates. J Glaucoma 2005;14:230–238.Copyright 2005 J Glaucoma.)

proteoglycans and matricellular proteins.45,57 The ECM is modulated by cytokines. The most studied cytokine in the TM is TGF-ß. A higher than normal level28 of TGF-ß2 was found in the aqueous humor of patients with POAG (Table 22.1). TGF-ß2 upregulated ECM-related genes in TM cell cultures. In TGF-ß2-perfused organ cultures, focal accumulation of fine fibrillar extracellular material was observed in TM tissues. Furthermore, TGF-ß2 perfusion reduced outflow facility and elevated IOP. These results suggest that the increased TGF-ß2 level in the aqueous humor may be related to the pathogenesis of glaucoma. Other cytokines such as interleukin-1α (IL-1α) and tumor necrosis factor-α (TNF-α) also modulate the ECM, probably via regulation of MMP and TIMP expressions.45,58 The cochlin deposits in the glaucomatous TM (Table 22.1) 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 (Box 22.2).7

Cell adhesion

Cell adhesion molecules including integrins, cadherins, and selectins mediate binding interactions at the extracellular surface and determine the specificity of cell-to-cell and cell- to-ECM recognitions. 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 pathology. TM cells in culture express a variety of integrins and form focal adhesions upon attachment to ECM proteins.45

SC cells have been shown to extend cytoplasmic processes into the JCT space (Figure 22.3), while JCT processes attach also to the trabecular lamellae processes.4,5,49 Freeze-fracture and electron microscopy studies have described adherens junctions, gap junctions, and tight junctions in the outflow pathway.45 Proteins that form adherens junctions such as intercellular cell adhesion molecule (ICAM)-1, N-CAM and N-cadherin are expressed in TM cells or tissues.45,59,60 SC cells express platelet-endothelium cell adhesion molecule-1 (PECAM-1) and vascular endothelial (VE)-cadherin.48 Gap junction protein connexin43 has also been localized in TM cells for communication with each other. Both TM and SC cells also express zonular occludens-1, an associated protein of calcium-sensitive tight junctions. Administration of glucocorticoid such as dexamethasone resulted in an increased expression of ZO-1, formation of a greater number of tight junctions, and an increase in fluid flow resistance.45

Selectins such as E-selectin (endothelial leukocyte adhesion molecule-1 or ELAM-1) are cell adhesion molecules that mediate interactions with complex carbohydrate moieties. Upregulation of selectins has been observed in TM tissues in POAG eyes.42,60 The upregulation could be related to transcription factor NF-κB42 (Table 22.3), reflecting stress response of the cells.

A number of studies have shown that the outflow facility is modified via alterations in cell-matrix and/or cell-cell adhesions.45,52 For instance, depletion of extracellular calcium by dissociating cell-cell junctions decreases outflow resistance.45,52 Perfusion of the Hep II domain of fibronectin59 in

human anterior-segment cultures increases outflow facility perhaps by mechanisms that involve loss of focal adhesions and/or disruption of adherens junctions in TM cells.

Cytoskeletal structure

The physiologic roles of the actin cytoskeleton of TM cells in association with outflow facility52,61 and glaucoma have drawn considerable attention. Agents that affect the actin cytoskeleton, such as chelating reagents and cytoskeletonactive drugs including cytochalasin B and latrunculin A and B have been demonstrated to increase the aqueous outflow in relatively short-term perfusion organ culture or in vivo animal studies.45,52,61 The increase was often associated with morphologic changes, such as separation of junctions between TM cells and breaks between SC cells. Sulfhydrylreactive compounds, including iodoacetamide, N-ethyl maleimide, and ethacrynic acid,61 can also influence the outflow system. A common mechanism seems to involve changes in cell shape and cell adhesion through cytoskeletal elements. Significantly, glucocorticoids alter F-actin architecture and promote cross-linked actin network (CLAN) formation in human TM cell and tissues.62 Moreover, glaucomatous eyes, compared to normals, displayed an overall more disordered F-actin architecture in SC and JCT cells.63 These studies collectively underscore the notion that the actin architecture is an important mediator of the aqueous outflow pathway (Box 22.3).

Intracellular signaling

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.52,64 In the active GTP-bound state, Rho GTPases interact with and activate downstream effectors such as Rho kinase to modulate the assembly of actin structures. RhoA, for example, regulates formation of stress fibers and focal adhesions to coordinate cellular processes including adhesion, migration, and morphologic changes. In TM cells, a decrease in actin stress fibers and focal adhesions has been shown to occur with treatment of Rho kinase inhibitors Y-27632 and H-1152, and gene transfer of dominant negative RhoA and dominant negative Rho-binding domain of Rho kinase.52,64 These cellular changes are associated with reduced myosin light-chain (MLC) phosphorylation, and/or enhanced outflow facility.52 Conversely, molecules including sphengosine-1-phosphate and endothelin-1 that activate Rho/Rho kinase signaling pathway through G-protein-coupled receptors promote MLC phosphorylation, and in turn decrease the aqueous humor outflow facility.52,61,64

Box 22.3  Effects on cytoskeletal structure

•Modulating actin cytoskeleton with chelating reagents, sulfhydryl-reactive compounds, and cytoskeleton-active drugs increases aqueous outflow

•Mechanism is likely changes in cell shape and cell adhesion through cytoskeletal elements

Signaling molecule such as protein kinase C (PKC) also has a role in the outflow regulation.45,64 PKC has additionally been shown to be involved in upregulation of MMP-3 and MMP-9 levels mediated by cytokines IL-1ß and TNF-α after laser trabeculoplasty. Extracellular signal-regulated kinase (ERK), mitogen-activated protein kinase (MAPK), c-Jun N-terminal kinase (JNK), and p38 MAPK pathways are also necessary in addition for the signal transduction.65

Aqueous humor components

The aqueous humor contains albumin as a major constituent. Other components encompass hydrogen peroxide (H2O2), ascorbic acid, growth factors such as TGF-ß, heptocyte growth factor and vascular endothelial growth factor, and molecules including MMPs, proteinase inhibitors, sCD44, and hyaluronic acid.66,67 A recent study has demonstrated that addition of normal 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.67

Increased levels of TGF-ß2, sCD44, endothelin-1, IL-2, phospholipase 2, thymulin (Table 22.2), glutathione, ascorbic acid (Table 22.3), and a decreased level of hyaluronic acid (Table 22.1) have been reported in the aqueous humor of POAG eyes. Since TM cells are in constant contact with the aqueous humor, it is expected that altered levels and/or activities of aqueous humor components would have an impact on the behavior and activities of these cells. The sCD44 found in the POAG aqueous humor is hypophosphorylated.68 The hypophosphorylated form has high cytotoxicity and low hyaluronic acid-binding affinity and is suggested to represent a pathophysiologic feature of the disease process.68

Aging, oxidative stress, and other insults

TM cellularity is reduced with aging.46 Morphologic studies have also revealed thickened basement membranes and accumulation of sheath-derived plaques and long spacing collagens in the TM of aged eyes.51 Cultured TM cells from old donors show a decline in proteasome activity and acquisition of the senescence phenotype including reduced proliferative capacity and enlarged cell morphology.69 A decrease of TM cellularity and an accumulation of ECM have also been documented in POAG eyes.47,51 The number of senescent cells which stain positive for senescence-associated ß-galactosidase is increased (Table 22.3) in the TM of POAG eyes,43 supporting further that POAG is an age-related disease.70

Oxidative damage has been implicated to contribute to the morphologic and physiologic alterations in the aqueous outflow pathway in aging and glaucoma.71 The TM is known to be exposed to 20–30 µM H2O2 present in the aqueous humor and is subjected to chronic oxidative stress.45 Enzymes that are involved in the protection against oxidative damage, including catalase, glutathione reductase, superoxide dismutase, and glutathione peroxidase, have been studied in the TM. The specific activity of superoxide dismutase, but not catalase, was shown to decline with age in human TM tissues.72 TM cells also synthesized a specific set of proteins,